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A genetic and molecular analysis of histone deacetylase one in Drosophila melanogaster : specific missense… Mottus, Randall C. 2003

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A GENETIC A N D M O L E C U L A R ANALYSIS OF HISTONE DEACETYLASE O N E I N DROSOPHILA  MELANOGASTER:  SPECIFIC MISSENSE  r  M U T A T I O N S SUPPRESS POSITION E F F E C T V A R I E G A T I O N by R A N D A L L C L A R K MOTTUS B.Sc.(Hon.), The University of British Columbia, 1979 M . S c , The University of British Columbia, 1983 L L . B . , The University of British Columbia, 1985  A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E O F D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E STUDIES (Department of Zoology) We accept this thesis as conforming to/tt^required star^lar4  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A A p r i l 2003 © Randall Clark Mottus 2003  In presenting this thesis in partial fulfilment  of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or by his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  ^ Q Q ^ 06 V  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  ?/°3  Abstract  E s s e n t i a l l y a l l h i g h e r o r g a n i s m s are m a d e u p of t w o or m o r e types of tissues. T h e specific i d e n t i t y of those tissues is d e p e n d e n t o n the genes that are expressed w i t h i n the cells of the p a r t i c u l a r tissue type. T h e correct set of genes m u s t be e x p r e s s e d a n d genes, that are not part of the set specific to that tissue, m u s t be k e p t s i l e n c e d . I n a d d i t i o n , i n m o s t cells, the d e c i s i o n w h e t h e r a gene w i l l be active or not is m a d e early i n d e v e l o p m e n t a n d therefore m u s t be passed o n to d a u g h t e r cells.  T h e focus of this thesis is a n i n v e s t i g a t i o n i n t o the  m e c h a n i s m o r m e c h a n i s m s e m p l o y e d b y e u k a r y o t e s to s i l e n c e genes a n d to m a i n t a i n that s i l e n c e d state t h r o u g h o u t d e v e l o p m e n t . T h e m o d e l s y s t e m o u r l a b o r a t o r y has b e e n u s i n g to investigate s i l e n c i n g is p o s i t i o n effect v a r i e g a t i o n ( P E V ) i n D. melanogaster.  In P E V a gene is s i l e n c e d i n a certain p r o p o r t i o n of the  cells of a tissue i n w h i c h it is n o r m a l l y e x p r e s s e d d u e to its p r o x i m i t y to a n heterochromatic breakpoint.  T h e d e c i s i o n w h e t h e r a gene w i l l be active or  i n a c t i v e i s m a d e e a r l y i n d e v e l o p m e n t a n d that d e c i s i o n is p a s s e d o n to d a u g h t e r cells w i t h reasonable fidelity. T h u s P E V m i m i c s n o r m a l d e v e l o p m e n t i n m a n y w a y s . T h i s has l e d o u r lab, a n d s e v e r a l others, to try to dissect the m e c h a n i s m s u n d e r l y i n g P E V w i t h the h o p e they w i l l s h e d s o m e l i g h t o n the m o r e general s i l e n c i n g m e c h a n i s m s that occur d u r i n g n o r m a l d e v e l o p m e n t . In C h a p t e r 2 of this thesis I describe the c l o n i n g a n d characterization of a gene i d e n t i f i e d i n a s c r e e n for d o m i n a n t s u p p r e s s o r s a s s o c i a t e d w i t h P E V [Su(var)s].  of the v a r i e g a t i o n  The gene encodes H D A C 1 ,  an histone  deacetylase h o m o l o g o u s to H D A C 1 f r o m m a m m a l s a n d R p d 3 f r o m S. cerevisiae.  ii  Specific mis-sense m u t a t i o n s i n HDAC1 cause s t r o n g d o m i n a n t s u p p r e s s i o n of P E V w h i l e n u l l o r h y p o m o r p h i c m u t a t i o n s h a v e n o effect o n the v a r i e g a t i n g phenotype.  I p r e s e n t a m o d e l p r o p o s i n g that the m i s - s e n s e m u t a t i o n s are  acting as a n t i - m o r p h i c m u t a t i o n s that " p o i s o n " the deacetylase c o m p l e x . T h e l e v e l of v a r i e g a t i o n of a gene subject to P E V i s v e r y sensitive to a w i d e v a r i e t y of factors, some, w h i c h m a y be a c t i n g d i r e c t l y a n d some, w h i c h m a y be a c t i n g i n d i r e c t l y . H D A C 1 l o c a l i z e s to a large n u m b e r of sites o n the p o l y t e n e c h r o m o s o m e s o f D. melanogaster (Pile a n d W a s s e r m a n , 2000) a n d therefore appears to regulate a large n u m b e r of genes. T h u s it is a p o s s i b i l i t y that the Su(var) m u t a t i o n s i n HDAC1 are affecting P E V i n d i r e c t l y . I n C h a p t e r 3 I present data f r o m c h r o m a t i n i m m u n o - p r e c i p i t a t i o n e x p e r i m e n t s ( X - C h I P ) that provides  compelling  evidence that H D A C 1  is a c t i n g d i r e c t l y o n the  e u c h r o m a t i c r e g i o n subject to s i l e n c i n g i n P E V . I p r o p o s e a m o d e l l i n k i n g the histone deacetylase a c t i v i t y of H D A C 1 to the f u n c t i o n of other p r o t e i n s k n o w n to be i n v o l v e d i n the s i l e n c i n g associated w i t h P E V .  iii  T a b l e of C o n t e n t s  Abstract  ii  Table of C o n t e n t s  iv  L i s t o f Tables  vi  L i s t of F i g u r e s  vii  Acknowledgements  viii  C H A P T E R I Literature R e v i e w  1  G e n e s i l e n c i n g a n d p o s i t i o n effect v a r i e g a t i o n  1  C h r o m a t i n structure  2  Heterochromatin and euchromatin  7  H i s t o r y of p o s i t i o n effect v a r i e g a t i o n  9  T i m i n g of P E V  15  Factors that m o d i f y P E V  17  Isolation a n d characterization of d o m i n a n t m u t a t i o n s affecting P E V ... 21  C H A P T E R 2 M u t a t i o n a l analysis of a histone deacetylase i n Drosophila melanogaster. missense m u t a t i o n s s u p p r e s s gene s i l e n c i n g associated w i t h p o s i t i o n effect v a r i e g a t i o n  37  Introduction  38  Materials and methods  42  Results  48  Discussion  67  iv  C H A P T E R 3 C h r o m a t i n I m m u n o p r e c i p i t a t i o n analysis of a r e g i o n subject to p o s i t i o n effect v a r i e g a t i o n i n Drosophila melanogaster  76  Introduction  77  Materials and methods  84  Results  90  Discussion  104  C H A P T E R 4 Summary and General Discussion  110  REFERENCES  123  A P P E N D I X I L i s t of A b b r e v i a t i o n s  146  v  List of Tables  Chapter 2 Table 1  T h e effects of selected Su(var) H D A C 1 m u t a t i o n s o n v a r i o u s genes subject to P E V  50  Table 2  L e t h a l phase a n a l y s i s of m u t a t i o n s i n H D A C 1  58  Table 3  C o m p a r i s o n of a m i n o a c i d s u b s t i t u t i o n s i n the Su(var) H D A C 1 group w i t h conserved regions i n h u m a n s and yeast h o m o l o g u e s  Table 4  62  S u m m a r y o f the effects of v a r i o u s m u t a t i o n s i n HDAC1  o n v i a b i l i t y a n d P E V i n D . melanogaster  vi  68  List of Figures Chapter 2 Figure 1  E x a m p l e s of the eyes f r o m m a l e flies b e a r i n g the In(l)w"' c h r o m o s o m e a n d v a r i o u s t h i r d 4  chromosomes  49  Figure 2  G e n o m i c o r g a n i z a t i o n of the HDAC1  Figure 3  T h e cross e m p l o y e d to attempt to generate a r e c o m b i n a n t b e t w e e n HDAC  328  gene  53  and a possible  second site suppressor of P E V Figure 4  N o r t h e r n analysis of p o l y ( A ) R N A f r o m adults b e a r i n g HDAC1  Figure 5  55  mutations  66  A m o d e l p r o p o s e d to e x p l a i n the p h e n o t y p e s o b s e r v e d i n the v a r i o u s k i n d s of HDAC1  mutations  71  Chapter 3 Figure 1  D i a g r a m m a t i c representation of the g e n o m i c r e g i o n 5' to the w gene i n D . melanogaster  94  +  Figure 2  G r a p h of the r e l a t i o n s h i p b e t w e e n fluorescence (amount of D N A ) a n d P C R cycle i n the D i s t a l Promoter Region  Figure 3  96  H D A C 1 is associated w i t h the P r o x i m a l P r o m o t e r of w i n a l l strains b u t the l e v e l is elevated i n iv'" +  Figure 4  4  H D A C 1 is associated w i t h the D i s t a l P r o m o t e r of the w gene i n w'" +  Figure 5  98  99  4  H D A C 1 is associated w i t h the 5' D i s t a l R e g i o n of the w g e n e mw'" +  100  4  vii  Acknowledgements  I w o u l d l i k e to a c k n o w l e d g e m y s u p e r v i s o r a n d f r i e n d D r . T o m G r i g l i a t t i . H e e n c o u r a g e d m e to r e t u r n to science, for w h i c h I w i l l be a l w a y s be grateful, a n d has p r o v i d e d m e w i t h g u i d a n c e t h r o u g h o u t this w o r k . I w o u l d also l i k e to thank the m a n y p e o p l e over that last few years that h a v e p a s s e d t h r o u g h the lab for their h e l p a n d encouragement. F i n a l l y , I w o u l d l i k e to t h a n k m y wife, K i n d e r M o t t u s , for her patience a n d u n w a v e r i n g s u p p o r t . She e n c o u r a g e d m y r e t u r n to science, despite the significant financial sacrifice, a n d cared for o u r c h i l d r e n w i t h o u t c o m p l a i n t w h e n the lab, a n d this thesis, m a d e m e absent from our home.  viii  Literature Review E u k a r y o t e s are c o m p l e x o r g a n i s m s that m u s t p r e c i s e l y regulate their genes to s u r v i v e .  A s an example, i n a multicellular organism, w i t h many  different tissue types, the i d e n t i t y of a tissue is d e t e r m i n e d b y the c o m b i n a t i o n of genes that are active. T h u s m e c h a n i s m s m u s t exist to activate the correct genes i n a p a r t i c u l a r tissue a n d then r e m e m b e r that p a t t e r n t h r o u g h subsequent cell d i v i s i o n s . O n the other h a n d , it w o u l d be deleterious or lethal, i f genes that w e r e s u p p o s e d to be i n a c t i v e , w e r e expressed, a n d therefore c o m p l e m e n t a r y m e c h a n i s m s m u s t exist to i n a c t i v a t e specific genes a n d m a i n t a i n that i n a c t i v e state t h r o u g h o u t subsequent cell d i v i s i o n s . T h i s thesis w i l l be c o n c e r n e d w i t h the latter p r o b l e m : w h a t is the m e c h a n i s m or m e c h a n i s m s that e u k a r y o t e s e m p l o y to silence genes a n d then to pass this d e c i s i o n o n to d a u g h t e r cells? The m o d e l s y s t e m o u r lab has u s e d to a d d r e s s this q u e s t i o n is p o s i t i o n effect v a r i e g a t i o n i n Drosophila  melanogaster.  Gene Silencing and Position-Effect Variegation (PEV) Classical P E V occurs w h e n a c h r o m o s o m a l rearrangement n o r m a l l y e u c h r o m a t i c r e g i o n of a c h r o m o s o m e to a n breakpoint.  abuts a  heterochromatic  G e n e s l o c a t e d i n the e u c h r o m a t i n , i m m e d i a t e l y adjacent to the  b r e a k p o i n t , often d i s p l a y a m o s a i c p h e n o t y p e i n the tissues i n w h i c h they are normally expressed.  I n s o m e cells the gene or genes are f u l l y active a n d the  cells appear n o r m a l , h o w e v e r , i n others, the gene(s) is s i l e n c e d , a n d the tissue appears m u t a n t .  This silencing phenomenon mimics n o r m a l development i n  that the d e c i s i o n as to w h e t h e r a gene w i l l be active or s i l e n c e d appears to be m a d e e a r l y a n d t h e n is p a s s e d o n to d a u g h t e r c e l l s .  1  S i n c e the genes i n  e u c h r o m a t i n are not m u t a t e d (see b e l o w ) b u t are s i l e n c e d d u e to the p r o x i m i t y of the h e t e r o c h r o m a t i c b r e a k p o i n t , P E V has b e e n e m p l o y e d as a m o d e l s y s t e m for  d i s s e c t i n g the s i l e n c i n g effects of c h r o m a t i n s t r u c t u r e , i n p a r t i c u l a r  h e t e r o c h r o m a t i n . M o r e details o n the nature of P E V w i l l be p r o v i d e d b e l o w . For n o w suffice it to say this a p p r o a c h has b e e n f r u i t f u l a n d the d i s s e c t i o n of P E V has p r o v i d e d t r e m e n d o u s i n s i g h t s i n t o the s t r u c t u r e of h e t e r o c h r o m a t i n a n d s o m e of the m e c h a n i s m s of s i l e n c i n g e m p l o y e d b y e u k a r y o t e s to c o n t r o l gene e x p r e s s i o n . O n e feature of gene e x p r e s s i o n that has b e c o m e apparent i n recent y e a r s is that r e g u l a t i o n of gene e x p r e s s i o n , i n c l u d i n g s i l e n c i n g , is dependent o n c h r o m a t i n structure. T h i s c h a p t e r w i l l p r o v i d e a brief r e v i e w of c h r o m a t i n structure a n d its role i n gene r e g u l a t i o n f o l l o w e d b y a r e v i e w of the p h e n o m e n o n of P E V a n d the role of s o m e of the m o r e w e l l c h a r a c t e r i z e d c h r o m a t i n p r o t e i n s that h a v e been isolated as m o d i f i e r s of P E V . T h e focus of the thesis w i l l be h i s t o n e deacetylase one ( H D A C 1 ) , a p r o t e i n that w a s i d e n t i f i e d i n a s c r e e n for m u t a t i o n s that modify P E V .  C h r o m a t i n Structure M u c h is n o w k n o w n about the basic structure of c h r o m a t i n . C h r o m a t i n is a d y n a m i c a s s e m b l a g e c o n s i s t i n g of a p p r o x i m a t e l y 50% D N A a n d 50% protein.  A b o u t o n e - h a l f of the p r o t e i n s are h i s t o n e s , a g r o u p of five b a s i c  proteins, w h i c h eukaryotes.  are a m o n g the m o s t h i g h l y  conserved proteins i n all  H i s t o n e s H 2 A , H 2 B , H 3 a n d H 4 are k n o w n as the core histones  and they associate to f o r m a n octamer. I n i t i a l l y , H 3 a n d H 4 f o r m h e t e r o d i m e r s that associate to f o r m a tetramer.  T h e t e t r a m e r t h e n associates w i t h t w o  2  h e t e r o d i m e r s of H 2 A a n d H 2 B to f o r m a n octamer that is s h a p e d l i k e a flattened sphere. A p p r o x i m a t e l y 146 base pairs of D N A w i n d about 1.7 times a r o u n d the o u t s i d e of the sphere i n a left-hand s u p e r c o i l . T h i s structure, the n u c l e o s o m e , forms the basic b u i l d i n g b l o c k of a l l e u k a r y o t i c c h r o m a t i n . T h e fifth histone, histone H I , also c a l l e d the " l i n k e r histone", b i n d s to n u c l e o s o m e s a n d protects a p p r o x i m a t e l y 20 a d d i t i o n a l base pairs of D N A o u t s i d e the n u c l e o s o m e ( H o r n a n d P e t e r s o n , 2002; L u g e r et al., 1997; T u r n e r , 2002; W o r k m a n a n d K i n g s t o n , 1998). X - r a y c r y s t a l l o g r a p h i c analysis of the n u c l e o s o m e at a r e s o l u t i o n of 2.8 A has b e e n c o m p l e t e d a n d p r o v i d e s the f o l l o w i n g p i c t u r e of n u c l e o s o m e structure ( L u g e r et a l . , 1997).  T h e core h i s t o n e s c o n t a i n t w o f u n c t i o n a l l y separable  regions, the central "histone fold" d o m a i n , a n d the a m i n o - a n d c a r b o x y - t e r m i n a l tail domains.  T h e h i s t o n e f o l d d o m a i n s of the h i s t o n e s are i n v o l v e d i n  h i s t o n e / h i s t o n e i n t e r a c t i o n s that s t a b i l i z e the n u c l e o s o m e a n d also m a k e c o n t a c t . w i t h the D N A as it w i n d s a r o u n d the n u c l e o s o m e .  W h e r e the D N A  m a k e s contact w i t h the n u c l e o s o m e , its s t r u c t u r e is h i g h l y o r d e r e d .  Those  r e g i o n s of the h i s t o n e s n o t i n contact w i t h D N A , the a m i n o - a n d c a r b o x y t e r m i n a l tails, a p p e a r to be m u c h m o r e flexible a n d e x t e n d out a n d b e t w e e n the D N A gyres. The  apparently  simple  structure  of  the  nucleosome  led  early  i n v e s t i g a t o r s to a s s u m e it w a s a p a s s i v e structure that f o r m e d a scaffold for DNA  architecture but d i d not p l a y a role i n r e g u l a t i n g genetic activity.  H o w e v e r , the first i n d i c a t i o n s that this w a s far f r o m accurate w e r e r e p o r t e d a l m o s t 40 years ago w h e n A l l f r e y et a l . (1964) o b s e r v e d a c o r r e l a t i o n b e t w e e n a c e t y l a t i o n of h i s t o n e r e s i d u e s a n d active t r a n s c r i p t i o n of genes.  3  W i t h the  emergence of a p o s s i b l e role for histone m o d i f i c a t i o n i n the r e g u l a t i o n of gene activity, the search for further histone m o d i f i c a t i o n s r e v e a l e d that histones are subject to a s u r p r i s i n g n u m b e r of p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s .  These  m o d i f i c a t i o n s i n c l u d e l y s i n e acetylation, l y s i n e a n d a r g i n i n e m e t h y l a t i o n , serine p h o s p h o r y l a t i o n a n d u b i q u i n a t i o n of H 2 A ( S p o t s w o o d a n d T u r n e r , 2002). A s n o t e d above, the a m i n o - a n d c a r b o x y - t e r m i n a l tail d o m a i n s of a l l core histones extend out f r o m the core n u c l e o s o m e particle a n d it w a s i n i t i a l l y a s s u m e d that a l l m o d i f i c a t i o n s w o u l d o c c u r i n these d o m a i n s .  However very  recent  e x p e r i m e n t s h a v e r e v e a l e d m o d i f i c a t i o n s o c c u r to the g l o b u l a r , h i s t o n e f o l d d o m a i n , as w e l l ( B r i g g s et al., 2002). c a n n o t be r u l e d out.  T h e existence of further m o d i f i c a t i o n s  A l l o f the m o d i f i c a t i o n s h a v e the p o t e n t i a l to alter the  structure of the n u c l e o s o m e a n d / or the structure of the c h r o m a t i n fiber. It has b e e n suggested the pattern of m o d i f i c a t i o n o n a n u c l e o s o m e forms a "histone code" w h i c h not o n l y regulates w h e t h e r a gene is expressed or not, b u t also m o d u l a t e s e x p r e s s i o n levels of active genes. I n a d d i t i o n , the histone code c o u l d p r o v i d e the e p i g e n e t i c m a r k that a l l o w s a c e l l to pass e x p r e s s i o n patterns o n f r o m one g e n e r a t i o n to the next (Strahl a n d A l l i s , 2000). T h e large n u m b e r of p o t e n t i a l m o d i f i c a t i o n s c o m b i n e d w i t h the w i d e v a r i e t y of sites a v a i l a b l e for m o d i f i c a t i o n creates the o p p o r t u n i t y for a n e x t r e m e l y c o m p l e x code to be created o n a n u c l e o s o m e ; i n fact, s e v e r a l t h o u s a n d different patterns are p o s s i b l e (Turner, 2002). H i s t o n e H 3 alone c a n be acetylated at six lysines, m e t h y l a t e d at five l y s i n e s ( a n d this c a n be m o n o - , d i - o r t r i m e t h y l a t i o n ) , m e t h y l a t e d at o n e a r g i n i n e a n d p h o s p h o r y l a t e d at s e r i n e 10.  A similar  c o m p l e x i t y of m o d i f i c a t i o n s exists o n H 4 , s l i g h t l y less o n H 2 B a n d still less o n H 2 A . In a d d i t i o n , since the n u c l e o s o m e is a n octamer c o n t a i n i n g t w o of each of  4  the  core  histories,  it i s p o s s i b l e  that  each  histone  maybe  modified  independently. F i n a l l y , there is the p o t e n t i a l for, a n d the l i k e l i h o o d of, i n t e r - n u c l e o s o m a l interactions  that are  a l s o affected  by histone  modifications. Complete  e l u c i d a t i o n of the h i s t o n e code, i f i n d e e d it does exist, w i l l r e q u i r e that m a n y genes be p r o b e d for e v e r y possible m o d i f i c a t i o n a n d the pattern o b s e r v e d m u s t be c o r r e l a t e d w i t h t r a n s c r i p t i o n a l a c t i v i t y , or the p o t e n t i a l for a c t i v i t y , a n d t r a n s c r i p t i o n levels. T h e pattern o b s e r v e d at one l o c u s m u s t t h e n be c o m p a r e d to that of other genes to determine s i m i l a r i t i e s a n d differences. H o w e v e r , this is o n l y the first l e v e l of analysis. T h e pattern m a y also c o n t r o l the p o s i t i o n i n g or l o c a t i o n of a gene w i t h i n specific c o m p a r t m e n t s of the n u c l e u s (see b e l o w ) or p l a y a role i n h e r i t a b i l i t y of the e x p r e s s i o n p a t t e r n a n d t h u s cell lineages w i l l also h a v e to be e x a m i n e d before a c o m p l e t e p i c t u r e w i l l be generated.  While  this task m a y a p p e a r d a u n t i n g , this area of research is one of the most active i n m o l e c u l a r b i o l o g y a n d s o m e c o n s e n s u s , r e g a r d i n g the effect of h i s t o n e m o d i f i c a t i o n s , is e m e r g i n g . F o r e x a m p l e m e t h y l a t i o n of h i s t o n e H 3 l y s i n e 4 appears to be a w i d e l y u s e d m a r k for gene a c t i v i t y , w h i l e m e t h y l a t i o n of l y s i n e 9 o n H 3 appears to m a r k a gene for repression (see b e l o w ) . The n u c l e o s o m e is o n l y the first l e v e l of c h r o m a t i n p a c k a g i n g . A t y p i c a l c e l l m a y c o n t a i n a b o u t one meter of D N A that m u s t be p a c k a g e d i n t o a n organelle as s m a l l as the n u c l e u s a n d thus c h r o m a t i n c a n not exist as a s t r i n g of n u c l e o s o m e s . E x a c t l y w h a t the h i g h e r o r d e r structure of c h r o m a t i n is has been the subject of i n t e n s i v e s t u d y a n d r e m a i n s c o n t e n t i o u s ( H o r n a n d Peterson, 2002). In vitro c h r o m a t i n r e c o n s t i t u t i o n studies suggest that, as the d i v a l e n t c a t i o n c o n c e n t r a t i o n is i n c r e a s e d to p h y s i o l o g i c a l l e v e l s , the n u c l e o s o m e first  5  condenses i n t o a 30 n m fiber a n d then i n t o h i g h e r o r d e r i r r e g u l a r aggregates. T h e f o r m a t i o n of these h i g h e r order aggregates in vitro requires the histone tails to be present o n the core histones (Hansen, 2002; L u g e r et al., 1997). T h e h i g h e r o r d e r i r r e g u l a r aggregates w e r e m a d e m o r e r e g u l a r , s t a b i l i z e d a n d c o m p a c t e d b y the a d d i t i o n of a l i n k e r histone, s u c h as H 5 ( C a r r u t h e r s a n d H a n s e n , 2000). In vivo, s t u d i e s that f o l l o w e d d e c o n d e n s a t i o n of the c h r o m o s o m e s  following  m i t o s i s suggest they first d e c o n d e n s e to a 100 - 130 n m fiber that m a y further d e c o n d e n s e to a 60 - 80 n m fiber for short i n t e r v a l s .  O n l y occasional, very  short, stretches o f a 30 n m fiber w e r e o b s e r v e d ( B e l m o n t a n d B r u c e , 1994). T h i s o b s e r v a t i o n suggests that the basic structural u n i t of c h r o m a t i n is a 100 n m fiber ( T u m b a r et al., 1999) that m a y present a c o n s i d e r a b l e obstacle to factors that m u s t m o d i f y the histones o n a n u c l e o s o m e to regulate gene expression. H o w then, d o the factors that regulate t r a n s c r i p t i o n g a i n access to a gene? T h i s is a c c o m p l i s h e d w i t h t w o types of c e l l u l a r c o m p l e x e s , one w h i c h relies o n a d e n o s i n e t r i p h o s p h a t e  ( A T P ) to p r o v i d e the e n e r g y to r e m o d e l  c h r o m a t i n a n d one w h i c h m o d i f i e s the histones to stabilize a n active or inactive state ( N a r l i k a r et a l . , 2002).  T h r e e t y p e s of A T P d e p e n d e n t r e m o d e l i n g  c o m p l e x e s h a v e b e e n classified based o n the k i n d of A T P a s e e m p l o y e d b y the c o m p l e x : the S W I 2 / S N F 2 f a m i l y ; the I S W 1 f a m i l y ; a n d , the M i - 2 f a m i l y . E a c h family m a y have several different  c o m p l e x e s that d i f f e r i n the  proteins  associated w i t h the A T P a s e . A l t h o u g h each f a m i l y increases the accessibility of n u c l e o s o m a l D N A , they a p p e a r to d o so b y s l i g h t l y different m e c h a n i s m s that m a y reflect the different c h r o m o s o m a l context i n w h i c h the genes they regulate are located ( N a r l i k a r et al., 2002).  6  ATP  dependent remodeling complexes w o r k i n conjunction w i t h  c o m p l e x e s that, o n one h a n d , c o n t a i n h i s t o n e acetyltransferases  ( H A T s ) that  h y p e r a c e t y l a t e h i s t o n e tails c r e a t i n g a c h r o m a t i n s t r u c t u r e c o r r e l a t e d w i t h t r a n s c r i p t i o n a l a c t i v a t i o n ( R e i d et al., 2000; V o g e l a u e r et a l . , 2000).  O n the  other, they either c o n t a i n , as i n the case of the N u R D c o m p l e x , or w o r k w i t h c o m p l e x e s that c o n t a i n , h i s t o n e deacetylases  ( H D A C s ) that  hypoacetylate  histones c r e a t i n g a s t r u c t u r e c o r r e l a t e d w i t h r e p r e s s i o n of gene a c t i v i t y a n d heterochromatin  ( X u e et al., 1998; Z h a n g et a l . , 1998).  E x a c t l y h o w these  c o m p l e x e s are targeted to a specific site is not k n o w n for certain b u t evidence is a c c u m u l a t i n g that at least some sequence specific factors c a n r e c o g n i z e their cognate sequences i n the context of the c h r o m a t i n fiber a n d recruit b o t h a n A T P d e p e n d e n t r e m o d e l i n g c o m p l e x a n d a histone m o d i f y i n g c o m p l e x ( N a r l i k a r et a l , 2002).  Heterochromatin and Euchromatin E a r l y c y t o l o g i s t s s t u d y i n g e u k a r y o t i c cells i d e n t i f i e d t w o t y p e s of chromatin: heterochromatin and euchromatin.  E u c h r o m a t i n , w h i c h becomes  diffuse a n d l i g h t l y s t a i n i n g i n the interphase cell, contains most, b u t not a l l , of the active genes. H e t e r o c h r o m a t i n , o n the other h a n d , r e m a i n s d a r k l y s t a i n i n g t h r o u g h o u t the cell cycle, contains r e l a t i v e l y few genes a n d is f o u n d p r i m a r i l y associated w i t h the centromeres a n d telomeres. Subsequent w o r k has s h o w n that h e t e r o c h r o m a t i n i n characterized b y a n u m b e r of s t r u c t u r a l features ( H e n i k o f f , 2000; R i c h a r d s a n d E l g i n , 2002). Heterochromatin  contains  a n a b u n d a n c e of r e p e t i t i v e  D N A sequences,  i n c l u d i n g satellites sequences, d e r i v a t i v e s of v i r u s e s a n d t r a n s p o s o n s .  7  It is  often c h a r a c t e r i z e d as b e i n g g e n e t i c a l l y inert, b u t it is n o t e n t i r e l y d e v o i d of genes. A p p r o x i m a t e l y 40 to 50 genes h a v e b e e n i d e n t i f i e d i n the p e r i c e n t r i c h e t e r o c h r o m a t i n of D . melanogaster ( E b e r l et al., 1993; W e i l e r a n d W a k i m o t o , 1995).  These  genes  are  also  subject  to  P E V but  react i n a  manner  c o m p l e m e n t a r y to e u c h r o m a t i c l o c i (see b e l o w ) . H e t e r o c h r o m a t i n d i s p l a y s l o w levels of m e i o t i c r e c o m b i n a t i o n a n d replicates late i n S phase. H e t e r o c h r o m a t i n has also b e e n c h a r a c t e r i z e d ,  to a l i m i t e d  extent,  b i o c h e m i c a l l y . It has a l o w e r e d accessibility to nucleases, s u g g e s t i n g it has a n altered, closed, f o r m of p a c k a g i n g . nucleosomal arrays  A n a l y s i s o f e u c h r o m a t i n suggests the  are i r r e g u l a r a n d c o n t a i n n u c l e o s o m e - f r e e ,  nuclease  h y p e r s e n s i t i v e sites associated w i t h active genes. I n contrast, the n u c l e o s o m e s in heterochromatin  are r e g u l a r l y s p a c e d o v e r l a r g e r e g i o n s a n d a h i g h e r  p r o p o r t i o n of the D N A is associated w i t h the n u c l e o s o m a l core ( G r e w a l a n d E l g i n , 2002; S u n et al., 2001).  T h e h i s t o n e s of h e t e r o c h r o m a t i c r e g i o n s are  h y p o a c e t y l a t e d r e l a t i v e to those f o u n d i n e u c h r o m a t i n . g e n e is i n a c t i v a t e d i n e u c h r o m a t i n ,  Interestingly, w h e n a  i n a c t i v i t y is o f t e n  h y p o a c e t y l a t i o n of the n u c l e o s o m e s at or near the p r o m o t e r .  associated  with  M o r e recently it  has b e e n d i s c o v e r e d that a n o t h e r h i s t o n e m o d i f i c a t i o n is s t r o n g l y associated w i t h the h e t e r o c h r o m a t i c state. T h e m e t h y l a t i o n of l y s i n e 9 o n histone H 3 ( H 3 m K 9 ) is f o u n d i n the p e r i c e n t r i c h e t e r o c h r o m a t i n of Drosophila l a r g e l y h e t e r o c h r o m a t i c f o u r t h c h r o m o s o m e (Jacobs et a l . , 2001).  a n d o n the Chromatin  i m m u n o - p r e c i p i t a t i o n e x p e r i m e n t s i n Schizosaccharomyces pombe s h o w the silent m a t i n g locus is e n r i c h e d i n H 3 m K 9 w h i l e f l a n k i n g e u c h r o m a t i c regions contain little or n o H 3 m K 9 ( N o m a et a l , 2001). T h e m e t h y l a t i o n H 3 K 9 is also f o u n d on  facultative heterochromatin,  s u c h as the i n a c t i v e X c h r o m o s o m e  8  in  m a m m a l i a n females (Boggs et al., 2002; H e a r d et al., 2001; Peters et al., 2002). H o w e v e r , this m o d i f i c a t i o n is not restricted to h e t e r o c h r o m a t i n .  It has also  been associated w i t h the s i l e n c i n g of genes i n e u c h r o m a t i n ( H w a n g et al., 2001) but unlike i n heterochromatin,  w h e r e H 3 m K 9 is w i d e l y d i s p e r s e d , i n  e u c h r o m a t i n it occurs at a single n u c l e o s o m e at the p r o m o t e r of the i n a c t i v a t e d gene ( N i e l s e n et al., 2001). A n o t h e r b i o c h e m i c a l m a r k e r frequently f o u n d i n h e t e r o c h r o m a t i n is the presence of m e t h y l a t i o n of cytosine residues i n the D N A . v e r y l o w levels i n Drosophila  It is o n l y f o u n d at  b u t i n most other h i g h e r o r g a n i s m s it is the most  c o m m o n f o r m of D N A m o d i f i c a t i o n . T h e m o d i f i c a t i o n is also f o u n d at some silenced l o c i located i n e u c h r o m a t i n . N o t o n l y is it i m p o r t a n t for the stability of the p e r i c e n t r i c h e t e r o c h r o m a t i n ( B a c h m a n et al., 2001; O k a n o et a l , 1999; X u et al., 1999) b u t it also p l a y s a r o l e i n m a i n t a i n i n g the e p i g e n e t i c e x p r e s s i o n p a t t e r n b o t h i n h e t e r o c h r o m a t i n a n d e u c h r o m a t i n (Jones a n d T a k a i , 2001; M a r t i e n s s e n a n d H e n i k o f f , 1999).  History of Position-Effect Variegation Position-effect v a r i e g a t i o n ( P E V ) w a s first d o c u m e n t e d o v e r 70 years ago w h e n M u l l e r (1930) o b s e r v e d m o s a i c e x p r e s s i o n i n the c o l o u r of eyes of D . melanogaster that h a d b e e n e x p o s e d to X - r a y s . N o r m a l l y the eyes of this species are b r i g h t r e d , b u t i n these m u t a n t lines, the eyes w e r e a p a t c h w o r k of n o r m a l r e d eye cells a n d c o l o u r l e s s eye cells. M o s a i c gene e x p r e s s i o n , as a result of P E V , has since b e e n o b s e r v e d i n vertebrates (Cattanach, 1974), l o w e r eukaryotes ( C l u t t e r b u c k a n d Spathas, 1984; E k w a l l et a l . , 1997) a n d p l a n t s (Catcheside, 1947).  T h u s it o c c u r s i n r e p r e s e n t a t i v e s  9  from all eukaryotic kingdoms.  H o w e v e r , since it has b e e n most e x t e n s i v e l y s t u d i e d i n Drosophila ( r e v i e w e d i n (Baker, 1968; G r i g l i a t t i , 1991; H e n i k o f f , 1994; S p o f f o r d , 1976; S p r a d l i n g a n d K a r p e n , 1990), t h i s r e v i e w w i l l f o c u s o n t h e w o r k d o n e i n f r u i t  flies.  A c c o r d i n g l y , unless otherwise specified, a l l studies of P E V described herein e m p l o y e d D . melanogaster as the e x p e r i m e n t a l o r g a n i s m . P E V t y p i c a l l y o c c u r s w h e n , as the r e s u l t o f c h r o m o s o m e breakage, a normally  euchromatic  region  heterochromatic breakpoint.  of the  chromosome  is rejoined  to a  A gene or, i n s o m e cases s e v e r a l genes, i n the  e u c h r o m a t i n i m m e d i a t e l y adjacent to the h e t e r o c h r o m a t i c b r e a k p o i n t , often display a mosaic phenotype  i n the tissues i n w h i c h t h e y are n o r m a l l y  expressed. I n some cells the gene is o n , a n d the tissue appears w i l d - t y p e , w h i l e i n others, the gene i s off, a n d the cells d i s p l a y a m u t a n t p h e n o t y p e . V i r t u a l l y a l l genes that d i s p l a y a cell a u t o n o m o u s p h e n o t y p e a n d h a v e b e e n tested, c a n be m a d e subject to P E V .  H o w e v e r , exceptions h a v e b e e n n o t e d : ebony (Brosseau,  1970) a n d the b i t h o r a x c o m p l e x (E.B. L e w i s , cited i n H e n i k o f f , 1990). C l a s s i c a l P E V i n v o l v e s the a s s o c i a t i o n of a e u c h r o m a t i c gene w i t h h e t e r o c h r o m a t i n b u t the c o m p l e m e n t a r y s i t u a t i o n also occurs. V a r i e g a t i o n for genes located i n ^ - h e t e r o c h r o m a t i n  also  occurs  r e a r r a n g e m e n t s juxtapose t h e m to e u c h r o m a t i c D N A .  when  chromosomal  T h e light gene, located  i n the (3-heterochromatin at the base of the left a r m o f c h r o m o s o m e 2, a n d the cubitus interruptus  +  gene, located o n the l a r g e l y h e t e r o c h r o m a t i c  fourth  c h r o m o s o m e , w i l l variegate w h e n m o v e d to a e u c h r o m a t i c e n v i r o n m e n t ( H e a r n et a l , 1991; Hessler, 1958; Stern a n d K o d a n i , 1955).  10  T h e r e i s n o w o v e r w h e l m i n g e v i d e n c e that the m o s a i c  expression  o b s e r v e d i n P E V is n o t the result of m u t a t i o n of the v a r i e g a t i n g gene, b u t is the result  of the n e w association  between  the e u c h r o m a t i c  h e t e r o c h r o m a t i n l o c a t e d at the i l l i c i t b r e a k p o i n t .  region and  T h e first of these studies  d e m o n s t r a t e d that the v a r i e g a t i n g gene w a s still intact b y m o v i n g it a w a y f r o m the h e t e r o c h r o m a t i c b r e a k p o i n t , either b y r e c o m b i n a t i o n ( J u d d , 1955) or b y further c h r o m o s o m a l r e a r r a n g e m e n t  ( H i n t o n a n d G o o d s m i t h , 1950).  These  studies c o n f i r m e d that the v a r i e g a t i n g gene h a d n o t b e e n m u t a t e d a n d c o u l d be restored to f u l l e x p r e s s i o n b y r e m o v i n g it f r o m the v i c i n i t y of h e t e r o c h r o m a t i n . F u r t h e r e v i d e n c e that it w a s p r o x i m i t y to the h e t e r o c h r o m a t i c b r e a k p o i n t that  was causing  mosaic  expression  came  from  studies  involving  rearrangements i n w h i c h the expression pattern of m o r e t h a n o n e gene c o u l d be o b s e r v e d . It w a s n o t e d that the gene closest to the b r e a k p o i n t w a s i n a c t i v a t e d at a h i g h e r f r e q u e n c y t h a n genes l o c a t e d further a w a y . T h i s w a s p a r t i c u l a r l y e v i d e n t i n the T(l;4)w '  258 21  strain, w h i c h variegates for white", a gene r e q u i r e d for  the n o r m a l r e d e y e c o l o u r o f D . melanogaster a n d f o r roughest*, a gene w h i c h causes d i s o r g a n i z e d eye facets w h e n s i l e n c e d . T h e s e genes are v e r y t i g h t l y l i n k e d a n d i n t h i s s t r a i n the t r a n s l o c a t i o n p l a c e s roughest  c l o s e r to t h e  b r e a k p o i n t t h a n white*. C a r e f u l analysis of the eye r e v e a l e d that white* clones w e r e a l w a y s s m a l l e r a n d c o m p l e t e l y c o n t a i n e d w i t h i n roughest clones. There w e r e n o e x a m p l e s r e p o r t e d of clones i n w h i c h white* w a s s i l e n c e d b u t roughest* was active.  T h i s s u g g e s t s that i n a c t i v a t i o n i s s p r e a d i n g o u t f r o m t h e  h e t e r o c h r o m a t i c b r e a k p o i n t . ( D e m e r i c a n d S l i z y n s k a , 1937; c i t e d i n ( C o h e n , 1962)). S p r e a d i n g a p p e a r s to o c c u r at h e t e r o c h r o m a t i c genes as w e l l since a  11  s i m i l a r s p r e a d i n g effect has b e e n r e p o r t e d for s u c h l o c i i n Drosophila hydei (Hess, 1970). C y t o l o g i c a l observations also seem to s u p p o r t the m o d e l that s i l e n c i n g is a p o l a r p h e n o m e n o n that emanates f r o m the h e t e r o c h r o m a t i n at the b r e a k p o i n t . In  polytene  chromosome  preparations  from  variegating  strains,  the  e u c h r o m a t i c r e g i o n adjacent to the b r e a k p o i n t often a d o p t s a m o r p h o l o g y s i m i l a r to (3-heterochromatin, d a r k l y s t a i n i n g a n d u n b a n d e d G o l d s t e i n , 1967). T h e translocation T(l;4)wm '  258 21  (Hartmann-  variegates for white*, located at  b a n d 3 C 2 a n d for notch*, located at 3 C 7 . I n this s t r a i n the b a n d c o n t a i n i n g white is c l o s e r to the b r e a k p o i n t t h a n the b a n d c o n t a i n i n g notch*. I n p o l y t e n e p r e p a r a t i o n s f r o m this strain, one either sees b o t h b a n d s , the b a n d at 3 C 7 o r neither b a n d . T h e b a n d at 3 C 2 w a s never v i s i b l e w h e n the b a n d at 3 C 7 w a s n o t (Schultz, 1936). T h i s n o t i o n has b e e n recently c h a l l e n g e d b y a careful a n a l y s i s of several X c h r o m o s o m e i n v e r s i o n s that variegate for white* a n d roughest* (Talbert a n d H e n i k o f f , 2000). T h e authors f o u n d that i n t w o strains, In(l)w'  n5W  a n d In(l)w" , ,Mc  patches of e y e cells c o u l d be f o u n d that w e r e m u t a n t for roughest* gene b u t n o r m a l l y p i g m e n t e d s u g g e s t i n g that the white* gene w a s f u l l y active. I n these strains white* is closer to the h e t e r o c h r o m a t i c b r e a k p o i n t t h a n roughest*, a n d therefore this appears to be a n e x c e p t i o n to the n o t i o n that s i l e n c i n g occurs i n a p o l a r f a s h i o n e x t e n d i n g o u t f r o m the h e t e r o c h r o m a t i c b r e a k p o i n t .  However,  u n k n o w n to these authors, there is e v i d e n c e that, o v e r t i m e , these t w o strains h a v e a c q u i r e d m o d i f i e r s that r a d i c a l l y affect e x p r e s s i o n of the white* gene. I n lab stocks of these strains the eyes are v e r y r e d , a l m o s t i n d i s t i n g u i s h a b l e f r o m w i l d - t y p e eyes, a n d therefore the white* gene is n o t s i l e n c e d i n v e r y m a n y cells.  12  V. Lloyd  (personal communication) outcrossed  the  l a b s t o c k s for  five  generations a n d f o u n d that the eye c o l o u r b e c a m e a l m o s t c o m p l e t e l y w h i t e , i n d i c a t i n g the lab stocks h a d a c q u i r e d m o d i f i e r s that either p r e v e n t e d s i l e n c i n g of the white* gene or r e a c t i v a t e d it i n m o s t cells after it h a d b e e n s i l e n c e d b y P E V . If the latter is the case then a n alternative e x p l a n a t i o n for the observations of T a l b e r t a n d H e n i k o f f is that the m o d i f i e r s reactivate white* i n s o m e cases w i t h o u t r e a c t i v a t i n g roughest*. A l t e r n a t i v e l y , it m a y s i m p l y be the case that the factors that s i l e n c e genes d o s p r e a d o u t f r o m the h e t e r o c h r o m a t i n at the breakpoint, but d o not necessarily silence every gene they encounter.  A  p a r t i c u l a r p r o m o t e r m a y be s t r o n g e n o u g h to o v e r c o m e the repression. F i n a l l y , it is p o s s i b l e the s i l e n c i n g p r o c e s s m a y o c c a s i o n a l l y s k i p r e g i o n s i n the e u c h r o m a t i n a n d therefore genes i n that r e g i o n w i l l escape repression. The  p o l a r i t y d i s p l a y e d b y n e i g h b o u r i n g genes w i t h respect to the  b r e a k p o i n t l e d S c h u l t z (1939) to p r o p o s e that a n i n a c t i v a t i o n process spreads out f r o m the h e t e r o c h r o m a t i c b r e a k p o i n t c a u s i n g P E V . I n s o m e cases the i n a c t i v a t i o n process spreads far e n o u g h to silence a gene w h i l e i n other cells the process does n o t s p r e a d as far as the gene a n d it retains its n o r m a l f u n c t i o n . T h i s theory, the " S p r e a d i n g M o d e l " , has e n d u r e d for o v e r 60 years, b u t other models have been proposed. A n alternative theory, w h i c h i n i t i a l l y generated a lot of attention, posits that it is the p o s i t i o n i n the n u c l e u s that determines w h e t h e r a v a r i e g a t i n g gene w i l l b e o n o r off. heterochromatic  T h i s t h e o r y is b a s e d  regions  of c h r o m o s o m e s  o n the occupy  o b s e r v a t i o n that distinct regions  the or  c o m p a r t m e n t s i n the nucleus. T h e c h r o m o s o m e s a d o p t a c o n f o r m a t i o n w i t h the h e t e r o c h r o m a t i c r e g i o n s s u r r o u n d i n g the c e n t r o m e r e a n d at the  13  telomeres  o c c u p y i n g the p e r i p h e r y of the n u c l e u s w h i l e e u c h r o m a t i n , c o n t a i n i n g most of the active genes, o c c u p i e s the i n t e r i o r r e g i o n (Ferreira et al., 1997; R a b l , 1885; S a d o n i et a l , 1999). A l o c u s subject to P E V c a n be l o c a t e d i n a e u c h r o m a t i c c o m p a r t m e n t , w h i c h contains a l l the factors r e q u i r e d for t r a n s c r i p t i o n , w h e r e it is expressed n o r m a l l y . H o w e v e r , d u e the to n e a r b y h e t e r o c h r o m a t i n it can also be m i s - l o c a t e d to a h e t e r o c h r o m a t i c c o m p a r t m e n t o n the n u c l e a r p e r i p h e r y . In this c o m p a r t m e n t the factors necessary for t r a n s c r i p t i o n are either absent or i n v e r y short s u p p l y a n d therefore the v a r i e g a t i n g gene is not expressed (Sass a n d H e n i k o f f , 1999). T h e e v i d e n c e for this m o d e l is, for the m o s t part, c i r c u m s t a n t i a l . It is b a s e d p r i m a r i l y o n the c o r r e l a t i o n b e t w e e n the n u c l e a r l o c a l i z a t i o n of a v a r i e g a t i n g g e n e a n d its l e v e l of e x p r e s s i o n (Sass a n d H e n i k o f f ,  1999).  H o w e v e r , studies o n the l o c a t i o n of d e v e l o p m e n t a l l y r e g u l a t e d genes i n B a n d T l y m p h o c y t e s also d e m o n s t r a t e d a n e x p r e s s i o n d e p e n d e n t l o c a t i o n i n the nucleus;  when  the  genes  were  inactive  they  were  associated  with  h e t e r o c h r o m a t i n at the n u c l e a r p e r i p h e r y ( B r o w n et a l . , 1999; B r o w n et al., 1997). H o w e v e r , s u b s e q u e n t studies i n d i c a t e that, e v e n t h o u g h the s i l e n c e d gene w a s associated w i t h the pericentric h e t e r o c h r o m a t i n , it w a s not p a c k a g e d as h e t e r o c h r o m a t i n (Sabbattini et al., 2001). T h u s , w h i l e it m a y i n d e e d be the case that a s i l e n c e d gene is l o c a t e d i n a specific c o m p a r t m e n t at the n u c l e a r p e r i p h e r y , there is n o e v i d e n c e to d i s t i n g u i s h w h e t h e r this is the cause of s i l e n c i n g or the result of s i l e n c i n g . I n a n y event these m o d e l s are not m u t u a l l y e x c l u s i v e a n d P E V m a y i n fact be the result of c o n t r i b u t i o n s f r o m b o t h m o d e l s . F o r e x a m p l e , as p u t a t i v e s i l e n c i n g factors b e g i n to s p r e a d out f r o m the h e t e r o c h r o m a t i c b r e a k p o i n t they  14  m i g h t increase the l i k e l i h o o d the s u r r o u n d i n g r e g i o n w i l l be l o c a l i z e d to the n u c l e a r p e r i p h e r y w h e r e the concentration of s i l e n c i n g factors is increased a n d the c o n c e n t r a t i o n of t r a n s c r i p t i o n a l activators is decreased.  This i n turn could  increase the l i k e l i h o o d that the v a r i e g a t i n g gene is s i l e n c e d . F a i l u r e to relocate to the n u c l e a r p e r i p h e r y w o u l d reduce the s p r e a d of "heterochromatic factors" a n d increase the l i k e l i h o o d the gene w a s expressed.  T i m i n g of P E V C a r e f u l e x a m i n a t i o n of the m o s a i c p h e n o t y p e a s s o c i a t e d w i t h P E V suggests that the i n i t i a l d e c i s i o n as to w h e t h e r a v a r i e g a t i n g locus w i l l be o n or off is m a d e e a r l y i n d e v e l o p m e n t a n d the d e c i s i o n , once m a d e , is t h e n passed on  to d a u g h t e r  rearrangements,  cells w i t h reasonable  f i d e l i t y ( J a n n i n g , 1970).  In  some  the fields of cells that are either m u t a n t or w i l d - t y p e w e r e  large a n d r o u g h l y f o l l o w e d b o u n d a r i e s s i m i l a r to cell lineages. S e v e r a l other s t u d i e s i n d i c a t e the o n / o f f  d e c i s i o n is m a d e e a r l y .  The strain,  IniDsd , 1  variegates for the r i b o s o m a l D N A genes. I n X/0 males, n e w l y h a t c h e d larvae a l r e a d y h a d l e v e l s of r D N A 14% l o w e r t h a n t h e i r w i l d - t y p e  counterparts  i n d i c a t i n g that s i l e n c i n g h a d o c c u r r e d p r i o r to h a t c h i n g (Puckett a n d S n y d e r , cited i n S p o f f o r d , 1976). S i m i l a r results w e r e f o u n d for the v a r i e g a t i n g peach* gene i n Drosophila virilis (Baker, 1967). E v i d e n c e f r o m v a r i e g a t i o n of the yellow* gene a n d white* gene i n D. melanogaster suggest the i n i t i a l d e t e r m i n a t i v e event occurs at the t i m e of b l a s t o d e r m f o r m a t i o n (Baker, 1967; J a n n i n g , 1970). P E V is sensitive to t e m p e r a t u r e (see b e l o w ) a n d this s e n s i t i v i t y has b e e n e m p l o y e d to d e t e r m i n e the temperature sensitive p e r i o d (tsp) of the v a r i e g a t i n g phenotype.  T e m p e r a t u r e shift studies r e v e a l t w o m a i n t e m p e r a t u r e sensitive  15  periods: one d u r i n g early embryogenesis a n d a second d u r i n g p u p a t i o n (Spofford, 1976). T h e early tsp w a s the m o s t sensitive, a g a i n p o i n t i n g to early e m b r y o g e n e s i s as the t i m e for the i n i t i a l d e t e r m i n a t i v e d e c i s i o n (Spofford, 1976). Finally,  flies  reared  on Na-butyrate  or p r o p i o n a t e  show  strong  s u p p r e s s i o n o f P E V ( M o t t u s , 1979; M o t t u s , 1983) a n d see b e l o w ) . S t u d i e s i n w h i c h e m b r y o s , l a r v a e a n d p u p a e w e r e r e a r e d o n m e d i a c o n t a i n i n g these c o m p o u n d s for d e f i n e d d e v e l o p m e n t a l p e r i o d s also s h o w e d definite t i m e s d u r i n g d e v e l o p m e n t that w e r e s e n s i t i v e to the effects of these c h e m i c a l s . A g a i n , the m o s t sensitive p e r i o d w a s i n e m b r y o g e n e s i s , a l t h o u g h d e v e l o p i n g flies w e r e also sensitive at other p e r i o d s d u r i n g their d e v e l o p m e n t that r o u g h l y f o l l o w e d the cell d i v i s i o n patterns i n the eye i m a g i n a l d i s k ( M o t t u s , 1983). These data  s u g g e s t that b u t y r a t e  and propionate  affect b o t h the  early  d e t e r m i n a t i v e d e c i s i o n a n d the maintenance of that d e c i s i o n , p e r h a p s t h r o u g h a common mechanism. Is there a n event that occurs at b l a s t o d e r m f o r m a t i o n that c o u l d be this d e t e r m i n a t i v e event? A f t e r f e r t i l i z a t i o n Drosophila e m b r y o s u n d e r g o 13 r o u n d s of r a p i d n u c l e a r d i v i s i o n w i t h o u t c e l l d i v i s i o n ( L a w r e n c e , 1992). e m b r y o n i c g e n e s are n o t e x p r e s s e d a n d the c h r o m o s o m e s are s t a i n i n g a n d d o not a p p e a r to be p a c k a g e d as h e t e r o c h r o m a t i n .  The  uniformly  H o w e v e r , at  about the t i m e of b l a s t o d e r m f o r m a t i o n , the e m b r y o n i c g e n o m e b e g i n s to f u n c t i o n , the c h r o m o s o m e s u n d e r g o a c h a n g e i n m o r p h o l o g y a n d d i s t i n c t regions of e u c h r o m a t i n a n d h e t e r o c h r o m a t i n a p p e a r . Since P E V is o b v i o u s l y closely  linked  to  heterochromatin,  this  suggests  the  formation  of  h e t e r o c h r o m a t i n as a l i k e l y candidate for the early d e t e r m i n a t i v e event. If this  16  is the case, t h e n o n e w o u l d e x p e c t factors t h a t affect the f o r m a t i o n of heterochromatin w o u l d modify P E V . W h i l e this i n i t i a l d e c i s i o n is p a s s e d o n w i t h reasonable f i d e l i t y i n m o s t r e a r r a n g e m e n t s , i n others one c a n r e a d i l y o b s e r v e cells or patches of cells of wild-type  tissue  observations).  within  a large  clone of mutant  cells  (unpublished  T h u s it a p p e a r s the e a r l y d e c i s i o n c a n be u n s t a b l e a n d the  v a r i e g a t i n g l o c u s m a y be subject to r e a c t i v a t i o n . I n a d d i t i o n , w h e n v a r i e g a t i n g strains are k e p t i n l a b stocks w i t h o u t s e l e c t i o n for l o n g p e r i o d s of t i m e , the n u m b e r o f cells i n w h i c h the gene is s i l e n c e d b e c o m e s r e d u c e d , s o m e t i m e s markedly.  O u t c r o s s i n g the s t r a i n for s e v e r a l g e n e r a t i o n s r e t u r n s the l e v e l of  s i l e n c i n g to i n i t i a l l e v e l s ( V . L l o y d , p e r s o n a l c o m m u n i c a t i o n ) , s u g g e s t i n g that the strains h a v e a c q u i r e d m o d i f i e r s that decrease the l i k e l i h o o d a gene w i l l be silenced. T h i s p h e n o m e n o n has not b e e n w e l l c h a r a c t e r i z e d a n d it is not clear w h e t h e r the m o d i f i e r s are a c t i n g o n the i n i t i a l d e t e r m i n a t i v e event(s), are affecting the m a i n t e n a n c e of that d e c i s i o n or b o t h .  Factors that m o d i f y P E V O v e r the last 60 years m a n y factors h a v e b e e n i d e n t i f i e d that m o d i f y P E V . S o m e , a p p a r e n t l y disparate m o d i f i e r s , a p p e a r to act t h r o u g h a c o m m o n route, d e v e l o p m e n t a l rate. It appears, b r o a d l y s p e a k i n g , that m o s t factors that s l o w d e v e l o p m e n t cause e n h a n c e m e n t of P E V , as seen b y a n increase i n the n u m b e r of cells i n w h i c h the v a r i e g a t i n g gene is s i l e n c e d . T e m p e r a t u r e w a s o n e of the first m o d i f i e r s o f P E V i d e n t i f i e d .  Rearing  flies at h i g h t e m p e r a t u r e suppresses P E V , that is, decreases s i l e n c i n g , w h i l e l o w temperature has the o p p o s i t e effect a n d enhances P E V ( G o w e n a n d G a y , 1934).  17  F r u i t flies, l i k e a l l insects, are c o l d - b l o o d e d a n d therefore at h i g h e r temperature d e v e l o p at a m u c h faster rate t h a n at c o l d t e m p e r a t u r e s .  F o r e x a m p l e , flies  reared at 25°C d e v e l o p f r o m e g g to a d u l t i n a p p r o x i m a t e l y 12 days, w h i l e flies reared at 18°C r e q u i r e 21 days. H i n t o n (1949) first n o t e d that flies r e a r e d i n c r o w d e d c u l t u r e s s h o w e d e n h a n c e d v a r i e g a t i o n r e l a t i v e to n o n - c r o w d e d c u l t u r e s .  C o m p e t i t i o n for  nutrients i n c r o w d e d cultures s l o w s d e v e l o p m e n t . M a n y c h e m i c a l s h a v e b e e n tested for their effect o n P E V . P r e d i c t a b l y , most,  if not  development.  a l l , c h e m i c a l s that are  added  to g r o w i n g c u l t u r e s  slow  F o r e x a m p l e a n u m b e r of D N A s y n t h e s i s i n h i b i t o r s c a u s e d  d e l a y e d d e v e l o p m e n t a n d e n h a n c e d P E V ( S c h u l t z , 1956) a n d r e a r i n g flies i n a c i d i c c u l t u r e c o n d i t i o n s of p H 2.6 also d e l a y e d d e v e l o p m e n t a n d e n h a n c e d P E V ( M i c h a i l i d i s et al., 1988). H o w e v e r , one e x c e p t i o n o c c u r r e d w h e n flies w e r e reared o n N a - b u t y r a t e or p r o p i o n a t e . These c h e m i c a l s w e r e tested for their effects o n P E V because it w a s s u s p e c t e d that h i s t o n e p r o t e i n s , the b a s i c b u i l d i n g b l o c k s of c h r o m a t i n , m i g h t be i n v o l v e d i n the m e c h a n i s m of P E V . It h a d b e e n s h o w n that e x p o s u r e of F r i e n d l e u k e m i a cells to butyrate, w h i c h w a s associated w i t h a n increase i n the l e v e l of acetylated histones i n the cell, caused t h e m to differentiate (Reeves a n d Cserjesi, 1979). I n a d d i t i o n , d e l e t i o n of the h i s t o n e gene cluster c a u s e d s u p p r e s s i o n P E V . I n s p i t e of the fact b o t h c h e m i c a l s c a u s e d p r o l o n g e d d e v e l o p m e n t , they s t r o n g l y s u p p r e s s e d P E V ( M o t t u s , 1983; M o t t u s et a l , 1980; R u s h l o w et a l , 1984). It w a s suggested that butyrate's effects w e r e the result of i n h i b i t i o n of an, as yet u n i d e n t i f i e d , histone deacetylase ( C a n d i d o et al., 1978; M o t t u s et al., 1980). H o w e v e r , e x p o s u r e to b u t y r a t e w a s s h o w n to h a v e a large  18  n u m b e r of effects o n other c e l l u l a r processes (Boffa et a l 1981; C h r i s t m a n et al., v  1980) a n d it m i g h t h a v e b e e n butyrate's effect o n these that caused s u p p r e s s i o n of P E V .  M o r e recent w o r k has c o n f i r m e d that b u t y r a t e does affect c h r o m a t i n  structure ( A n n u n z i a t o et a l . , 1988) a n d c o n f i r m e d it is a p o t e n t i n h i b i t o r of certain classes of histone deacetylases ( B a r l o w et al., 2001; E m i l i a n i et al., 1998). W h y w o u l d a n increase i n d e v e l o p m e n t t i m e cause a n increase i n the n u m b e r of cells i n w h i c h a gene is silent? Z u c k e r k a n d l (1974) p r o p o s e d that the s i l e n c i n g o b s e r v e d i n P E V is d e p e n d e n t u p o n the f o r m a t i o n of m a c r o m o l e c u l a r complexes.  D e l a y e d d e v e l o p m e n t a l l o w s m o r e t i m e for these c o m p l e x e s for  f o r m a n d thereby increases s i l e n c i n g . S u r p r i s i n g l y , this h y p o t h e s i s , or perhaps a s l i g h t l y m o r e s o p h i s t i c a t e d v e r s i o n of it, still seems p l a u s i b l e today. S e v e r a l genetic factors h a v e also b e e n s h o w n to m o d i f y the v a r i e g a t i n g p h e n o t y p e i n c l u d i n g the a m o u n t , a n d p e r h a p s the k i n d , of h e t e r o c h r o m a t i n . T h e Y c h r o m o s o m e i n Drosophila is a l m o s t c o m p l e t e l y h e t e r o c h r o m a t i c .  An  extra Y c h r o m o s o m e , for e x a m p l e i n X Y Y males, suppresses P E V ( G o w e n a n d G a y , 1934) w h i l e loss of the Y c h r o m o s o m e (X0 males) enhances P E V .  It has  b e e n r e p o r t e d the s t r e n g t h of this effect is p r o p o r t i o n a l to the a m o u n t of Y c h r o m o s o m e m a t e r i a l ( D i m i t r i a n d P i s a n o , 1989) h o w e v e r there is s o m e e v i d e n c e f r o m m o r e d e t a i l e d studies, e m p l o y i n g s m a l l fragments of the Y c h r o m o s o m e , that s o m e r e g i o n s of the Y exert a s t r o n g e r effect o n P E V t h a n others (T. G r i g l i a t t i , p e r s o n a l c o m m u n i c a t i o n ) . T h a t this effect is d u e to the h e t e r o c h r o m a t i n of the Y c h r o m o s o m e is s u p p o r t e d b y s t u d i e s that s h o w d u p l i c a t i o n s a n d deficiencies of a u t o s o m a l h e t e r o c h r o m a t i n m o d i f y v a r i e g a t i o n i n a s i m i l a r m a n n e r (Spofford, 1976).  19  H i s t o n e s are one of the basic b u i l d i n g b l o c k s o f c h r o m a t i n , a n d since P E V a p p e a r s to be related to c h r o m a t i n structure, this l e d some g r o u p s to ask w h e t h e r m o d i f y i n g the dosage of the histone genes w o u l d h a v e a n y effect o n P E V . T w o g r o u p s r e p o r t e d that deficiencies for the h i s t o n e gene cluster caused s t r o n g s u p p r e s s i o n o f the white* gene i n the s t r a i n ln{l)w"  A  (Khesin and  L e i b o v i t c h , 1978; M o o r e et al., 1979). It w a s o r i g i n a l l y p r o p o s e d that h a p l o i d y for the h i s t o n e cluster w o u l d cause a r e d u c t i o n i n the c e l l u l a r h i s t o n e p o o l . Since h e t e r o c h r o m a t i n replicates late i n the cell cycle, at a t i m e w h e n histone p r o t e i n s m i g h t be l i m i t i n g , their short s u p p l y w o u l d i m p e d e the f o r m a t i o n o f the s i l e n c i n g structure ( M o o r e et al., 1979). S u r p r i s i n g l y , h o w e v e r , m o r e recent w o r k h a s s h o w n that a d e f i c i e n c y for the h i s t o n e c l u s t e r a c t u a l l y increases t r a n s c r i p t i o n f r o m the r e m a i n i n g genes a n d the cell a c c u m u l a t e s m o r e histone m R N A s t h a n n o r m a l ( N e r et a l . , 2002).  T h i s i n c r e a s e d t r a n s c r i p t i o n of the  histones m a y l e a d to a s l i g h t increase i n p r o t e i n levels a n d this m a y f a v o u r the f o r m a t i o n of e u c h r o m a t i n at the v a r i e g a t i n g l o c u s t h e r e b y s u p p r e s s i n g P E V . A l t e r n a t i v e l y , m i s - r e g u l a t i o n of h i s t o n e m e t a b o l i s m c a u s e d b y deficiencies of the h i s t o n e cluster m a y be a c t i n g i n d i r e c t l y o n P E V to s u p p r e s s v a r i e g a t i o n (Ner et al., 2002). T h e P o l y c o m b G r o u p (PcG) of proteins m a i n t a i n s i l e n c i n g of H O X genes i n m u l t i c e l l u l a r o r g a n i s m s ( B r e i l i n g et al., 2001). T h e f o u n d i n g m e m b e r of that g r o u p , P O L Y C O M B , a n d a k n o w n s u p p r e s s o r of P E V , H P 1 , h a v e a d o m a i n i n c o m m o n , t h e c h r o m o d o m a i n (see b e l o w ) .  T h e o b s e r v a t i o n that b o t h  P O L Y C O M B a n d H P 1 c o n t a i n c h r o m o d o m a i n s c o u p l e d w i t h the k n o w n f u n c t i o n o f the P c G suggested the P c G m i g h t also be i n v o l v e d i n s i l e n c i n g at v a r i e g a t i n g l o c i . H o w e v e r , most P c G proteins d o n o t h a v e a m a r k e d effect o n  20  P E V ( S i n c l a i r et a l  v  1998) w i t h o n l y Enhancer  s u p p r e s s i o n a n d Additional  of Polycomb  s h o w i n g strong  sex combs s h o w i n g s t r o n g e n h a n c e m e n t of P E V .  T h u s it appears that, w h i l e there m a y be some v e r y l i m i t e d i n t e r a c t i o n b e t w e e n these s i l e n c i n g p h e n o m e n a , for the m o s t part, t h e y r e p r e s e n t t w o d i s t i n c t s i l e n c i n g m e c h a n i s m s i n the cell.  Isolation and Characterization of Dominant Mutations Affecting P E V D u r i n g the spontaneous  c o u r s e of i n v e s t i g a t i n g P E V o v e r the y e a r s , s e v e r a l  mutations  were identified  that d o m i n a n t l y m o d i f i e d  the  v a r i e g a t i n g p h e n o t y p e (Spofford, 1976). T h e fact that s i n g l e site m o d i f i e r s of P E V c o u l d be i s o l a t e d , c o u p l e d w i t h the r e l a t i o n s h i p b e t w e e n P E V a n d c h r o m a t i n structure, l e d several labs to u n d e r t a k e large scale genetic screens to isolate a n d i d e n t i f y d o m i n a n t m u t a t i o n s that e i t h e r s u p p r e s s , S u ( v a r ) s or enhance, E(var)s, the v a r i e g a t i n g p h e n o t y p e . T h e h o p e w a s that s u c h screens w o u l d i d e n t i f y factors i n v o l v e d i n c h r o m a t i n structure a n d s h e d light, n o t o n l y o n the m e c h a n i s m u n d e r l y i n g P E V b u t p r o v i d e s o m e i n s i g h t i n t o gene regulation generally.  A l a r g e n u m b e r of s i n g l e site m o d i f i e r s h a v e b e e n  d e s c r i b e d ( L o c k e et al., 1988; M o t t u s , 1983; R e u t e r a n d W o l f f , 1981; S i n c l a i r et al., 1983) a n d o v e r 40 E(var)s a n d m o r e t h a n 140 Su(var)s are c u r r e n t l y listed o n Flybase (http:/ /flybase.bio.Indiana.edu:82/). number  of g e n e s  T h i s n u m b e r closely matches the  o f these classes t h a t w e r e  d u p l i c a t i o n / d e f i c i e n c i e s studies of the Drosophila  p r e d i c t e d to e x i s t  from  g e n o m e ( L o c k e et a l , 1988;  R e u t e r et a l . , 1987; R e u t e r a n d Spierer, 1992; W u s t m a n n et al., 1989). A l t h o u g h most h a v e b e e n r e c o m b i n a t i o n a l l y m a p p e d the vast majority of these m u t a t i o n s have not b e e n c l o n e d . T h i s is despite n u m e r o u s attempts to use c o n v e n t i o n a l P  21  e l e m e n t gene t a g g i n g to c l o n e Su(var)s ( L o c k e et a l 1994). O n l y a s i n g l e gene, Su(var)3-9  v  1988; T s c h i e r s c h et a l . ,  has b e e n c l o n e d u s i n g this m e t h o d . T h e  reasons for t h i s h a v e n o t b e e n clear.  D u r i n g the c o u r s e of these screens  n u m e r o u s n e w S u ( v a r ) m u t a t i o n s w e r e r e c o v e r e d , b u t w i t h the e x c e p t i o n of Su(var)3-9,  n o n e of t h e m c o n t a i n e d P elements. A n a l y s i s of the n e w l y i n d u c e d  m u t a t i o n s i n d i c a t e d they w e r e d u p l i c a t i o n s or deficiencies (Locke et al., 1988), s u g g e s t i n g that P e l e m e n t s w e r e i n d e e d t r a n s p o s i n g i n t o genes that w e r e i n v o l v e d i n P E V , b u t it w a s o n l y w h e n they i m p r o p e r l y e x c i s e d , that a v i s i b l e m u t a t i o n w a s created.  T h e i n a b i l i t y to c l o n e these genes has b e e n a major  i m p e d i m e n t to a d v a n c i n g o u r u n d e r s t a n d i n g of P E V . Since there are a large n u m b e r of m u t a t i o n s that m o d i f y P E V a n d o n l y a few h a v e b e e n c l o n e d a n d c h a r a c t e r i z e d , I w i l l l i m i t m y r e v i e w to those that h a v e b e e n c l o n e d a n d characterized to the extent that their role i n s i l e n c i n g has been partly elucidated.  In a d d i t i o n , m o s t of the r e s e a r c h i n this area has  concentrated o n the Su(var) class of m u t a t i o n s . T h i s w a s a reflection of o p i n i o n about the m e c h a n i s m of P E V . T h e evidence o v e r w h e l m i n g l y indicates that P E V is a s i l e n c i n g p h e n o m e n o n related to the n e w association w i t h h e t e r o c h r o m a t i n . A c c o r d i n g l y , m u t a t i o n s that d i s r u p t the f o r m a t i o n of h e t e r o c h r o m a t i n w o u l d be expected to decrease s i l e n c i n g a n d therefore be Su(var)s. T h e E(var) class of m u t a t i o n s w e r e t h o u g h t to r e p r e s e n t m u t a t i o n s i n factors that affect f u n c t i o n or s t r u c t u r e of e u c h r o m a t i c l o c i .  the  T h i s m i g h t i n c l u d e a v a r i e t y of  t r a n s c r i p t i o n factors ( b o t h g e n e r a l a n d s p e c i f i c ) as w e l l as t r a n s c r i p t i o n associated r e g u l a t o r y proteins. O b v i o u s l y these factors are interesting i n their o w n right but w o u l d not s h e d any l i g h t d i r e c t l y o n the s i l e n c i n g associated w i t h P E V . T h i s v i e w m a y h a v e been short-sighted. T h e recent c l o n i n g of  22  E(var)93D,  also k n o w n as mod(mdg4), is a case i n p o i n t . It encodes a factor i n v o l v e d i n the r e g u l a t i o n o f n u m e r o u s genes a n d is b e l i e v e d to act at e u c h r o m a t i c b o u n d a r y elements w h e r e it f u n c t i o n s i n m a i n t a i n i n g a n o p e n c h r o m a t i n c o n f o r m a t i o n ( G e r a s i m o v a a n d C o r c e s , 2001). T h e failure of this p r o t e i n to act at a b o u n d a r y element m a y a l l o w s i l e n c i n g c o m p l e x e s to s p r e a d m o r e efficiently f r o m the heterochromatic breakpoint and thereby enhance P E V . T h u s E(var)s and Su(var)s m a y w o r k a n t a g o n i s t i c a l l y a n d thus a c o m p l e t e u n d e r s t a n d i n g of P E V w i l l require c h a r a c t e r i z a t i o n of b o t h classes of m o d i f i e r s . A p p r o x i m a t e l y a d o z e n Su(var)s h a v e n o w b e e n c l o n e d but, for most, their i n v o l v e m e n t i n P E V has r e m a i n e d unclear. A c c o r d i n g l y , the r e m a i n d e r of this r e v i e w w i l l focus o n s e v e r a l Su(var) genes that h a v e b e e n c l o n e d a n d for w h i c h a p o t e n t i a l role i n P E V has b e e n d e t e r m i n e d . w a s Su(var)3-7.  T h e first Su(var) c l o n e d  A s m a l l d e f i c i e n c y , that o n l y r e m o v e d t w o c o d i n g regions,  e n a b l e d Rueter et a l . (1990) to clone the gene u s i n g g e r m - l i n e transformation. It codes for a p e p t i d e of 932 a m i n o acids w i t h 7 w i d e l y s p a c e d , a t y p i c a l z i n c fingers ( C l e a r d et a l . , 1995).  D o m a i n a n a l y s i s of S U ( V A R ) 3 - 7 r e v e a l e d the  p r o t e i n consists of t w o c o m p l e m e n t a r y d o m a i n s . T h e N - t e r m i n a l d o m a i n , that contains the s e v e n a t y p i c a l z i n c fingers, confers D N A b i n d i n g w i t h a preference for the repeat sequences of satellite D N A located i n p e r i c e n t r i c h e t e r o c h r o m a t i n ( C l e a r d a n d S p i e r e r , 2001).  T h e C - t e r m i n a l p o r t i o n of the p r o t e i n p r o m o t e s  d i m e r i z a t i o n t h r o u g h a B E S S m o t i f (Jaquet et al., 2002). I m m u n o p r e c i p i t a t i o n studies suggest that S U ( V A R ) 3 - 7 interacts w i t h h e t e r o c h r o m a t i c p r o t e i n 1 ( H P I , see b e l o w ) , a l t h o u g h this i n t e r a c t i o n is p r o b a b l y i n d i r e c t (Jaquet et a l . , 2002) a n d the d o m a i n r e q u i r e d for the interaction has not b e e n characterized.  23  I m m u n o s t a i n i n g o f Drosophila  p o l y t e n e c h r o m o s o m e s r e v e a l s that  S U ( V A R ) 3 - 7 is p r i m a r i l y associated w i t h p e r i c e n t r i c h e t e r o c h r o m a t i n , a l t h o u g h a few e u c h r o m a t i c sites are also detected b y i m m u n o s t a i n i n g (Delattre et al., 2000). T h i s s t a i n i n g pattern is almost i d e n t i c a l to that o b s e r v e d for H P 1 (James et al., 1989). T h e s i m i l a r s t a i n i n g pattern a n d the i m m u n o p r e c i p i t a t i o n studies suggest S U ( V A R ) 3 - 7 a n d H P 1 m a y be a c t i n g together i n the s i l e n c i n g that occurs i n P E V .  G i v e n the D N A b i n d i n g p r e f e r e n c e  of S U ( V A R ) 3 - 7 for  p e r i c e n t r i c s a t e l l i t e sequences, i t is t e m p t i n g to s p e c u l a t e that S U ( V A R ) 3 - 7 b i n d s to h e t e r o c h r o m a t i n a n d r e c r u i t s o t h e r p r o t e i n s , i n c l u d i n g H P 1 , to e s t a b l i s h a s i l e n c i n g c o m p l e x . H o w e v e r , the f a i l u r e to d e m o n s t r a t e a direct i n t e r a c t i o n b e t w e e n H P 1 a n d S U ( V A R ) 3 - 7 (Jaquet et a l . , 2002) suggests other proteins m u s t exist that b r i d g e the gap b e t w e e n S U ( V A R ) 3 - 7 a n d H P 1 . T h e best c h a r a c t e r i z e d s u p p r e s s o r of P E V is h e t e r o c h r o m a t i c p r o t e i n 1, H P 1 , o r i g i n a l l y i d e n t i f i e d i n Drosophila (Eissenberg a n d E l g i n , 2000; James a n d E l g i n , 1986). HP1  w a s c l o n e d b y r a i s i n g a n t i b o d i e s to p r o t e i n s e n r i c h e d i n  h e t e r o c h r o m a t i n a n d , u s i n g reverse genetics, the gene w a s l o c a l i z e d to b a n d 2 9 A , a site w h e r e the suppressors, Su(var)205 ( M o t t u s , 1983; S i n c l a i r et al., 1983) a n d Su(var)2-5  (Reuter a n d W o l f f , 1981), h a d b e e n i n d e p e n d e n t l y l o c a l i z e d .  S u b s e q u e n t a n a l y s i s d e m o n s t r a t e d the m u t a t i o n s w e r e i n the same gene a n d h a d created single base p a i r substitutions i n H P 1 (James a n d E l g i n , 1986). H P 1 h o m o l o g u e s h a v e b e e n i d e n t i f i e d i n o r g a n i s m s f r o m yeast to h u m a n s .  Most  o r g a n i s m s h a v e three c l o s e l y related H P l - l i k e p r o t e i n s c o d e d for b y different genes. F o r e x a m p l e , H P l a , b, a n d c are f o u n d i n Drosophila, a n d H P l a , |3 a n d y in humans.  S. pombe, o n the other h a n d , a p p e a r s to h a v e o n l y one m e m b e r ,  Siui6 ( E i s s e n b e r g a n d E l g i n , 2000).  A l l contain an amino-terminal chromo  24  domain, a hinge region and a carboxy-terminal chromo shadow domain. The c h r o m o d o m a i n is a n a p p r o x i m a t e l y 44 a m i n o a c i d r e g i o n s h a r e d b y the POLYCOMB  p r o t e i n i n Drosophila  ( P a r o a n d H o g n e s s , 1991).  Domain  s w a p p i n g s t u d i e s ( M e s s m e r et al., 1992; P l a t e r o et a l . , 1999) i m p l i c a t e d the c h r o m o d o m a i n i n p r o t e i n - p r o t e i n interactions, h o w e v e r , m o r e recent studies h a v e s h o w n the c h r o m o d o m a i n s of S w i 6 , H P l a a n d M 3 1 ( H P l a f r o m mouse) are capable of r e c o g n i z i n g the tail of histone H 3 b u t o n l y w h e n it is m e t h y l a t e d at l y s i n e 9 ( H 3 m K 9 ) (Bannister et al., 2001; Jacobs et al., 2001; L a c h n e r et a l , 2001; N a k a y a m a et a l , 2001; N i e l s e n et al., 2001).  I n t e r e s t i n g l y , the p r o t e i n  responsible for m e t h y l a t i n g H 3 K 9 is S U ( V A R ) 3 - 9 (see b e l o w ) a n d S U ( V A R ) 3 - 9 interacts w i t h b o t h H P I a n d S U ( V A R ) 3 - 7 (Schotta et al., 2002). T h e h i n g e a n d c h r o m o s h a d o w d o m a i n s a p p e a r to be responsible for the c h r o m o s o m a l t a r g e t i n g of the H P I f a m i l y ( S m o t h e r s a n d H e n i k o f f , 2000) to different l o c a t i o n s o n the c h r o m o s o m e . Studies i n h u m a n s a n d m o u s e suggest H P l a a n d (3 are e n r i c h e d i n h e t e r o c h r o m a t i n w h i l e H P l y is f o u n d e x c l u s i v e l y i n e u c h r o m a t i n ( H o r s l e y et a l . , 1996; M i n e et a l . , 1999).  These studies  extended  (2001) w h o  and confirmed by Smothers and Henikoff  were  created  antibodies specific for each of the f a m i l y m e m b e r s . T h e y d e m o n s t r a t e d that, i n Drosophila,  H P l c is l o c a l i z e d e x c l u s i v e l y to e u c h r o m a t i n , H P l b is f o u n d i n b o t h  h e t e r o c h r o m a t i n a n d e u c h r o m a t i n a n d H P l a , the f o u n d i n g m e m b e r , l o c a l i z e s p r i m a r i l y to h e t e r o c h r o m a t i n . I n d o m a i n s w a p p i n g e x p e r i m e n t s they w e n t o n to s h o w the H P l a h i n g e a n d c h r o m o s h a d o w d o m a i n s c a n separately target h e t e r o c h r o m a t i n , w h i l e the H P l c c h r o m o s h a d o w d o m a i n e x c l u s i v e l y targets e u c h r o m a t i n . T h u s , a l t h o u g h the c h r o m o d o m a i n is capable of b i n d i n g to H 3  25  m K 9 , this b i n d i n g c a p a c i t y is not r e s p o n s i b l e for l o c a l i z i n g the H P 1 p r o t e i n s . T h e targeting f u n c t i o n of the H P 1 f a m i l y m e m b e r s is c o n t a i n e d i n the h i n g e a n d c h r o m o s h a d o w d o m a i n s (Smothers a n d H e n i k o f f , 2000). S e v e r a l g r o u p s , e m p l o y i n g a w i d e v a r i e t y of techniques, h a v e i d e n t i f i e d m o r e t h a n 40 p r o t e i n s that interact w i t h H P 1 . T h e p r o t e i n s are f r o m almost a l l aspects  of  chromosomal  metabolism  including:  transcriptional  r e g u l a t i o n / c h r o m a t i n m o d i f y i n g proteins; D N A r e p l i c a t i o n a n d repair; n u c l e a r architecture; a n d , other c h r o m o s o m e - a s s o c i a t e d p r o t e i n s ( L i et al., 2002). T h u s it a p p e a r s the H P 1 f a m i l y of p r o t e i n s are i n v o l v e d i n a w i d e v a r i e t y of processes i n the n u c l e u s w h i c h m a y account for the presence of three different HP1 genes.  W h a t t h e n is its f u n c t i o n w i t h respect to s i l e n c i n g a n d P E V ?  No  m u t a t i o n s are a v a i l a b l e for H P l b a n d c, h o w e v e r d e f i c i e n c y studies i n d i c a t e that absence of these H P 1 f a m i l y m e m b e r s does not h a v e a n y d o m i n a n t effect o n P E V ( G r e g D o h e n e y , p e r s o n a l c o m m u n i c a t i o n ) . I n contrast a d u p l i c a t i o n for H P l a enhances P E V w h i l e a deficiency suppresses ( W u s t m a n n et al., 1989) a n d , as n o t e d a b o v e , the o n l y m u t a t i o n s that affect P E V h a v e b e e n r e c o v e r e d i n H P l a , despite extensive screening, (Locke et al., 1988; M o t t u s , 1983; Reuter a n d W o l f f , 1981; S i n c l a i r et a l . , 1983). T h i s suggests that o n l y H P l a is i n v o l v e d i n the s i l e n c i n g associated w i t h P E V . Immunostaining  studies  with  a monoclonal antibody  for  HP1  d e m o n s t r a t e d H P 1 is associated w i t h the e u c h r o m a t i n s i l e n c e d d u e to P E V . T h i s association w a s a b o l i s h e d i n HP1 m u t a n t s ( B e l y a e v a et al., 1993). A s n o t e d above, a n e u c h r o m a t i c r e g i o n subject to s i l e n c i n g d u e to P E V , d i s p l a y s a r e d u c e d a c c e s s i b i l i t y to n u c l e a s e s .  H o w e v e r , i n strains bearing an H P 1  m u t a t i o n that s u p p r e s s e s P E V , the r e g i o n s h o w s i n c r e a s e d a c c e s s i b i l i t y to  26  nuclease attack ( C r y d e r m a n et al., 1998). These e x p e r i m e n t s suggest H P l a is a c t i n g d i r e c t l y at the heterochromatic  variegating locus and  participates  i n creating  a  e n v i r o n m e n t that is m o r e c o m p a c t a n d less accessible to  c h r o m o s o m a l proteins, l i k e t r a n s c r i p t i o n factors. T h i s has l e d to the n o t i o n that H P l a is a " b i f u n c t i o n a l cross-linker, p e r h a p s o r g a n i z i n g h i g h e r o r d e r c h r o m a t i n structure b y l i n k i n g or a n c h o r i n g c h r o m a t i n s u b u n i t s " ( E i s s e n b e r g a n d E l g i n , 2000). There is n o w a d d i t i o n a l evidence that this m a y i n d e e d be the case. H P I interacts w i t h S U ( V A R ) 3 - 7 a n d S U ( V A R ) 3 - 9 (Schotta et a l . , 2002) a n d  the  c h r o m o d o m a i n of H P I b i n d s H 3 m K 9 . T h e o n l y Su(var) that has b e e n c l o n e d i n a screen for P e l e m e n t - i n d u c e d m u t a t i o n s is Su(var)3-9  ( T s c h i e r s c h et a l . , 1994).  A n a l y s i s of the  protein  r e v e a l e d that it c o n t a i n e d a c h r o m o d o m a i n , s i m i l a r to the ones f o u n d i n H P I and P O L Y C O M B . was  found  in  I n a d d i t i o n , it contains a S E T d o m a i n so c a l l e d because it  SU(VAR)3-9 and  two other  k n o w n chromatin  proteins,  E N H A N C E R O F Z E S T E a n d T R I T H O R A X , ( T s c h i e r s c h et a l , 1994). T h e S E T d o m a i n has n o w b e e n f o u n d i n a n u m b e r of c h r o m a t i n p r o t e i n s ( J e n u w e i n et al., 1998).  S U ( V A R ) 3 - 9 is h i g h l y c o n s e r v e d i n h o m o l o g u e s f r o m yeast to  h u m a n s as is its d i s t r i b u t i o n pattern i n the n u c l e u s . I n a l l o r g a n i s m s e x a m i n e d S U ( V A R ) 3 - 9 is associated p r i m a r i l y w i t h h e t e r o c h r o m a t i n . found  at  the  chromocenter  and  in a banded  pattern  h e t e r o c h r o m a t i c f o u r t h c h r o m o s o m e (Schotta et al., 2002).  In Drosophila at  the  it is  largely  I n S. pombe it is  associated w i t h the regions f l a n k i n g the centromere a n d w i t h the silent m a t i n g locus ( H a l l et al., 2002; N a k a y a m a et al., 2001; N o m a et a l . , 2001) a n d i n m i c e at the pericentric h e t e r o c h r o m a t i n (Peters et al., 2001). H o w e v e r , its l o c a l i z a t i o n is not e x c l u s i v e l y h e t e r o c h r o m a t i c a n d it has b e e n s h o w n to l o c a l i z e to a n d  27  regulate e u c h r o m a t i c genes i n Drosophila ( H w a n g et a l 2001; N e r et al., 2002) v  a n d h u m a n cell lines ( N i e l s e n et al., 2001). It has b e e n s h o w n i n a n u m b e r of o r g a n i s m s that S U ( V A R ) 3 - 9 , a n d its h o m o l o g u e s , are the p r i m a r y e n z y m e s r e s p o n s i b l e for the m e t h y l a t i o n of l y s i n e 9 o n h i s t o n e H 3 ( H 3 m K 9 ) (Rea et a l , 2000) w h i c h creates a site for H P 1 binding.  S. pombe a n d Drosophila  h a v e a s i n g l e gene, clr4 a n d  Su(var)3-9  r e s p e c t i v e l y ( N o m a et al., 2001; Schotta et al., 2002), w h i l e i n m i c e a n d h u m a n s there are t w o v e r y closely related genes, SUV39H1  a n d SUV39H2  ( N i e l s e n et al.,  2001; Peters et a l . , 2001). S i n g l e k n o c k - o u t s of the S. pombe gene, a n d d o u b l e k n o c k - o u t s of the m a m m a l i a n genes, l e a d s to c h r o m o s o m e i n s t a b i l i t y a n d m i t o t i c defects.  S u r p r i s i n g l y , n u l l m u t a t i o n s of Drosophila  Su(var)3-9  are  h o m o z y g o u s v i a b l e a n d s h o w n o s e g r e g a t i o n defects ( T s c h i e r s c h et al., 1994). P e r h a p s , i n Drosophila,  there m a y be a second, as yet u n i d e n t i f i e d ,  Su(var)3-9-  like gene. H o w e v e r , i n Su(var)3-9 n u l l m u t a t i o n s , H 3 m K 9 at the chromocenter is s e v e r e l y r e d u c e d s u g g e s t i n g this is the major h i s t o n e  methyltransferase  ( H M T ) specific for H 3 K 9 (Schotta et al., 2002). M u t a t i o n a l a n a l y s i s i n Drosophila a n d S. pombe has c o n f i r m e d the S E T d o m a i n is r e s p o n s i b l e for S U ( V A R ) 3 - 9 ' s H M T a c t i v i t y ( N a k a y a m a et a l , 2001; Schotta et al., 2002) w h i l e the c h r o m o d o m a i n , the S E T d o m a i n a n d the cysteiner i c h d o m a i n adjacent to the S E T d o m a i n a l l p a r t i c i p a t e i n h e t e r o c h r o m a t i c targeting to H 3 (Rea et a l , 2000; Schotta et a l , 2002). A s n o t e d above S U ( V A R ) 3 - 9 associates w i t h H P 1 a n d S U ( V A R ) 3 - 7 . T h e d o m a i n that interacts w i t h b o t h proteins m a p s to the a m i n o t e r m i n u s of H P 1 i n Drosophila  (Schotta et a l . , 2002).  These i n t e r a c t i o n s t u d i e s w e r e d o n e i n the  yeast d i h y b r i d s y s t e m s u g g e s t i n g a d i r e c t i n t e r a c t i o n b e t w e e n S U ( V A R ) 3 - 9 ,  28  H P I a n d S U ( V A R ) 3 - 7 . H o w e v e r , this result has not b e e n c o n f i r m e d e m p l o y i n g a m o r e d i r e c t t e c h n i q u e , s u c h as G S T p u l l - d o w n assays.  I n a d d i t i o n to these  a s s o c i a t i o n s , i m m u n o p r e c i p i t a t i o n of S U ( V A R ) 3 - 9 f r o m e m b r y o extracts i n Drosophila  also p r e c i p i t a t e d a n histone deacetylase a c t i v i t y w h i c h p r o v e d to be  the h i s t o n e deacetylase, H D A C 1 .  H o w e v e r , S U ( V A R ) 3 - 9 - G S T fusions d i d n o t  p r e c i p i t a t e H D A C 1 s u g g e s t i n g the a s s o c i a t i o n b e t w e e n these p r o t e i n s is i n d i r e c t ( C z e r m i n et a l . , 2001). Interestingly, the a c t i v i t y o f C l r 4 , the S U ( V A R ) 3 9 h o m o l o g u e i n S. pombe, is dependent o n the activities of t w o H D A C s , C l r 3 a n d C l r 6 . C l r 6 is the S. pombe h o m o l o g u e o f H D A C 1 a n d C l r 3 is the h o m o l o g u e of the S. cerevisiae h i s t o n e deacetylase, H d a l ( N a k a y a m a et a l . , 2001).  T h u s it  a p p e a r s that S U ( V A R ) 3 - 9 is i n a c o m p l e x w i t h t w o other s u p p r e s s o r s of P E V a n d w i t h one or p e r h a p s t w o histone deacetylase e n z y m e s . W h i l e S U ( V A R ) 3 - 7 has not b e e n s h o w n to h a v e h o m o l o g u e s o u t s i d e Drosophila,  H P I a n d S U ( V A R ) 3 - 9 are c o n s e r v e d f r o m yeast to h u m a n s .  In S.  pombe the roles of a n d r e l a t i o n s h i p s b e t w e e n these p r o t e i n s has b e e n a n a l y z e d i n c o n s i d e r a b l e d e t a i l . I n S. pombe the silent m a t i n g - t y p e r e g i o n o c c u p i e s about twenty  kilobases  heterochromatin.  of  D N A and  has  many  of  the  characteristics  of  T h e h i s t o n e tails are h y p o a c e t y l a t e d , the D N A s h o w s  r e d u c e d a c c e s s i b i l i t y to nucleases, r e c o m b i n a t i o n is s u p p r e s s e d a n d reporter genes i n s e r t e d i n t o the r e g i o n are s i l e n c e d ( N a k a y a m a et al., 2001; N o m a et al., 2001). T h e r e g i o n i n c l u d e s the mat! a n d mat3 l o c i a n d a n i n t e r v a l b e t w e e n t h e m k n o w n as the K - r e g i o n . M u t a t i o n a l analysis h a d i d e n t i f i e d several genes w h i c h , w h e n m u t a t e d , r e s u l t e d i n e x p r e s s i o n of a reporter gene that h a d b e e n s i l e n c e d because it w a s i n s e r t e d i n the m a t i n g - t y p e r e g i o n .  T h e s e genes i n c l u d e d  H D A C s , clr3 a n d clr6, the Su(var)3-9 h o m o l o g u e , clr4, a n d the HPI h o m o l o g u e ,  29  swi6, ( G r e w a l et al., 1998). S e v e r a l l a n d m a r k p a p e r s i n the past t w o years h a v e e m p l o y e d m u t a t i o n a l studies a n d X - C h I P a n a l y s i s to p r o v i d e e v i d e n c e for a m o d e l that e x p l a i n s , n o t o n l y h o w genes are s i l e n c e d w h e n they are i n or near h e t e r o c h r o m a t i n , b u t offers a n e x p l a n a t i o n about h o w this o n / o f f d e c i s i o n is established a n d t h e n p a s s e d o n b y w a y of a n epigenetic m a r k ( H a l l et al., 2002; N a k a y a m a et al., 2001; N o m a et al., 2001). T h e m o d e l p r o p o s e s that a r e q u i r e m e n t f o r h e t e r o c h r o m a t i n ( a n d h e t e r o c h r o m a t i c s i l e n c i n g ) i n S. Pombe is that H 3 K 9 b e m e t h y l a t e d b y C l r 4 , the S U ( V A R ) 3 - 9 h o m o l o g u e . H o w e v e r , if, o n the H 3 tail, K 14 o r K 9 is acetylated, H 3 K 9 m e t h y l a t i o n is i n h i b i t e d . A c c o r d i n g l y , a necessary step i n the process is d e a c e t y l a t i o n of K 1 4 b y C l r 3 , the H d a l h o m o l o g u e a n d d e a c e t y l a t i o n of K 9 b y C l r 6 , the H D A C 1 h o m o l o g u e .  A t this p o i n t , C l r 4 is r e c r u i t e d to the certfi  repeats, l o c a t e d i n the K - r e g i o n , a n d C l r 4 m e t h y l a t e s H 3 K 9 . C o n c o m i t a n t l y , either d i r e c t l y o r w i t h other partners, C l r 4 recruits S w i 6 that b i n d s to H 3 m K 9 t h r o u g h its c h r o m o d o m a i n . T h i s r e c r u i t m e n t i s r e c i p r o c a l a n d l o c a l i z a t i o n of C l r 4 a n d S w i 6 are m u t u a l l y d e p e n d e n t .  T h i s p r o v i d e s the b a s i c s for the  h e t e r o c h r o m a t i c structure a n d i t spreads f r o m the cenh repeats u n t i l i t reaches s p e c i a l i z e d b o u n d a r y elements that p r e v e n t the s i l e n c i n g f r o m s p r e a d i n g i n t o the adjacent e u c h r o m a t i n . demonstrated  The m u t u a l recruitment of C l r 4 a n d Swi6 was  b y r e m o v i n g the b o u n d a r y  elements.  W h e n Clr4 was  overexpressed, it s p r e a d into the adjacent e u c h r o m a t i n as d i d S w i 6 . W h e n S w i 6 w a s o v e r e x p r e s s e d it s p r e a d i n t o the n e i g h b o u r i n g e u c h r o m a t i n , as d i d C l r 4 ( N o m a et a l , 2001). W h i l e s i l e n c i n g of a reporter gene is c o m p l e t e w h e n it is i n s e r t e d at most places i n the m a t i n g - t y p e r e g i o n , w h e n a r e p o r t e r gene r e p l a c e s the c e n H  30  repeats i n the K - r e g i o n it d i s p l a y s a metastable state, the reporter can be o n or off,  a n d this d e c i s i o n is stable t h r o u g h m i t o s i s a n d m e i o s i s .  T h i s m a y be  a n a l o g o u s to the e a r l y d e c i s i o n m a d e r e g a r d i n g a v a r i e g a t i n g l o c u s i n P E V (see above). A c c o r d i n g l y , i n b o t h cases, there m u s t be a m e c h a n i s m that passes the d e c i s i o n o n to d a u g h t e r cells. S e v e r a l g r o u p s h a v e s u g g e s t e d this epigenetic m a r k is s i m p l y m e t h y l a t i o n of K 9 o n H 3 ( C z e r m i n et al., 2001; R i c h a r d s a n d E l g i n , 2002; S c h o t t a et al., 2002; T u r n e r , 2002).  H o w e v e r , H a l l et al., (2002)  p r o v i d e c o m p e l l i n g e v i d e n c e that b o t h C l r 4 a n d S w i 6 are r e q u i r e d for accurate m a i n t e n a n c e of the d e c i s i o n a n d suggest the epigenetic m a r k also requires the function of b o t h proteins. H a l l et a l . (2002) t h e n w e n t o n to ask h o w the i n i t i a l d e c i s i o n w a s m a d e i n S. Pombe.  R e c e n t w o r k i n t h e i r l a b h a d s h o w n t h a t the f o r m a t i o n of  h e t e r o c h r o m a t i n at c e n t r o m e r i c repeats r e q u i r e d a f u n c t i o n a l R N A interference ( R N A i ) s y s t e m ( V o l p e et al., 2002). T h e y e x t e n d e d this a n a l y s i s b y e x a m i n i n g the effect of d e l e t i o n of any one of three genes i n v o l v e d i n R N A i o n s i l e n c i n g of a r e p o r t e r gene i n t r o d u c e d at the m a t i n g - t y p e r e g i o n .  R e p r e s s i o n of the  reporter gene w a s a b o l i s h e d i n the d e l e t i o n strains a n d the m a t i n g - t y p e r e g i o n c o u l d not recruit a n d / o r m a i n t a i n S w i 6 a n d m e t h y l a t i o n at H 3 K 9 . H o w e v e r , if they u s e d genetic crosses to i n t r o d u c e a reporter gene, f r o m a w i l d - t y p e s t r a i n i n w h i c h s i l e n c i n g h a d b e e n established, i n t o strains b e a r i n g m u t a t i o n s i n the R N A i genes, s i l e n c i n g r e m a i n e d intact. T h u s the R N A i genes w e r e not r e q u i r e d to m a i n t a i n r e p r e s s i o n once it h a d b e e n established b u t p e r h a p s w e r e r e q u i r e d to i n i t i a t e f o r m a t i o n of h e t e r o c h r o m a t i n .  T o c o n f i r m this result they treated  strains b e a r i n g a reporter inserted i n the m a t i n g - t y p e r e g i o n w i t h T r i c h o s t a t i n A ( T S A ) , a p o t e n t i n h i b i t o r of h i s t o n e deacetylases.  31  E x p o s u r e to T S A h a d  p r e v i o u s l y b e e n s h o w n to erase the s i l e n c i n g of genes r e p r e s s e d b y i n s e r t i o n into c e n t r o m e r i c h e t e r o c h r o m a t i n ( E k w a l l et al., 1997) or the m a t i n g - t y p e r e g i o n ( G r e w a l et a l . , 1998). A f t e r e x p o s u r e to T S A for ten generations, s i l e n c i n g of the reporter i n w i l d - t y p e strains a n d i n strains b e a r i n g deletions for the R N A i genes was abolished.  T h e y r e m o v e d the T S A a n d a l l o w e d the cells to g r o w for  a n o t h e r 10 g e n e r a t i o n s .  I n the w i l d - t y p e cells, s i l e n c i n g w a s c o m p l e t e l y  reestablished. H o w e v e r , i n the R N A i d e l e t i o n strains o n l y a few cell w e r e able to reestablish s i l e n c i n g . T h e y speculate that the c e n H r e g i o n , a r e g i o n w h i c h can r e c r u i t C l r 4 i n a n H D A C d e p e n d e n t m a n n e r , p r o d u c e s t r a n s c r i p t s that are p r o c e s s e d b y R N A i . T h e p r o c e s s e d transcripts are r e q u i r e d to r e c r u i t H D A C s a n d C l r 4 to the mat l o c u s to initiate the f o r m a t i o n of h e t e r o c h r o m a t i n . How  d o e s this m o d e l relate to P E V ?  T h e p a r a l l e l s are o b v i o u s a n d  c o m p e l l i n g a n d s e v e r a l g r o u p s h a v e suggested the m o d e l o u t l i n e d above, w i t h m i n o r v a r i a t i o n s , is a p p l i c a b l e to P E V a n d p e r h a p s  to the f o r m a t i o n of  h e t e r o c h r o m a t i n i n g e n e r a l ( C z e r m i n et a l . , 2001; R i c h a r d s a n d E l g i n , 2002; Schotta et a l . , 2002; T u r n e r , 2002). I h a v e t a k e n the l i b e r t y of e x t e n d i n g the m o d e l to a c c o m m o d a t e m o r e recent w o r k .  T h e u n u s u a l z i n c fingers of  S U ( V A R ) 3 - 7 b i n d D N A w i t h a preference for the satellite sequences f o u n d i n pericentric heterochromatin.  A c c o r d i n g l y , the z i n c f i n g e r s o f S U ( V A R ) 3 - 7  target it, a n d the c o m p l e x that contains it, to the c e n t r o m e r i c h e t e r o c h r o m a t i n . T h a t c o m p l e x c o n t a i n s H P 1 , S U ( V A R ) 3 - 9 a n d o n e or m o r e H D A C s .  The  H D A C s deacetylate the histone tails creating the h y p o a c e t y l a t e d tails f o u n d i n h e t e r o c h r o m a t i n a n d c l e a r i n g the w a y for m e t h y l a t i o n of H 3 K 9 b y S U ( V A R ) 3 9. T h i s creates a b i n d i n g site for H P 1 a n d some or a l l m e m b e r s of this c o m p l e x i n i t i a t e the  f o r m a t i o n of h e t e r o c h r o m a t i n  32  and  propagate  it a l o n g  the  c h r o m o s o m e i n t o the e u c h r o m a t i n , s i l e n c i n g a n y gene i n its p a t h . T h e process c o n t i n u e s u n t i l it reaches s o m e as yet u n k n o w n b o u n d a r y e l e m e n t o r u n t i l the c o m p o n e n t s of the c o m p l e x b e c o m e l i m i t i n g . T h i s i n i t i a l s p r e a d i n g occurs i n Drosophila at b l a s t o d e r m f o r m a t i o n w h e n h e t e r o c h r o m a t i n first forms. W h e t h e r i n i t i a t i o n i n v o l v e s R N A m o l e c u l e s processed b y the R N A i s y s t e m has yet to be e x a m i n e d i n p u b l i s h e d w o r k . H o w e v e r , p r e l i m i n a r y results w i t h a m u t a t i o n i n one of the c o m p o n e n t s of the R N A i system, argonaute, i n d i c a t e s it is a s t r o n g s u p p r e s s o r of P E V (S. N e r , p e r s o n a l c o m m u n i c a t i o n ) . O n c e the extent of the i n i t i a l s p r e a d i n g is e s t a b l i s h e d at b l a s t o d e r m , the d e c i s i o n is p a s s e d o n b y a n epigenetic m a r k , either m e t h y l a t i o n of H 3 K 9 , the presence of S U ( V A R ) 3 - 9 , the presence of H P 1 or a c o m b i n a t i o n of some or a l l of these p o t e n t i a l m a r k e r s . T h i s m o d e l for P E V is h i g h l y s p e c u l a t i v e , b u t d o e s m a k e  some  p r e d i c t i o n s that are testable. O n e p r e d i c t i o n is that one or m o r e H D A C s s h o u l d be i n v o l v e d i n e s t a b l i s h i n g a n d m a i n t a i n i n g the s i l e n c i n g associated w i t h P E V . T h i s p r e d i c t i o n is also s u g g e s t e d b y the o b s e r v a t i o n that P E V is s t r o n g l y s u p p r e s s e d w h e n flies are g r o w n o n m e d i a s u p p l e m e n t e d w i t h b u t y r a t e , a potent i n h i b i t o r of some H D A C s ( M o t t u s , 1979; M o t t u s , 1983). T h i s thesis presents the c l o n i n g a n d c h a r a c t e r i z a t i o n of the Drosophila HDAC1  gene a n d p r o v i d e s e v i d e n c e that it is d i r e c t l y i n v o l v e d i n the s i l e n c i n g  associated w i t h P E V .  T h e starting p o i n t for this a n a l y s i s w a s the c l o n i n g a n d  c h a r a c t e r i z a t i o n of a set of s t r o n g S u ( v a r ) m u t a t i o n s that f o r m e d a s i n g l e c o m p l e m e n t a t i o n g r o u p that h a d b e e n i d e n t i f i e d i n o u r o r i g i n a l screen for d o m i n a n t s u p p r e s s o r s of P E V ( M o t t u s , 1983; Sinclair et a l , 1983). In o r d e r to d o so, I d e v e l o p e d a n e w m e t h o d for c l o n i n g e s s e n t i a l S u ( v a r ) m u t a t i o n s i n Drosophila w h i c h s h o u l d be of w i d e s p r e a d u t i l i t y . A s n o t e d above, the gene I  33  c l o n e d w i t h t h i s t e c h n i q u e w a s the Drosophila  HDAC1  gene.  T h i s data is  presented i n C h a p t e r 2 a n d has been p u b l i s h e d ( M o t t u s et al., 2000). The H D A C s i n e u k a r y o t e s are a s u r p r i s i n g l y large g r o u p of p r o t e i n s . T a u n t o n et a l . (1996) w e r e the first to isolate a n d c h a r a c t e r i z e a m a m m a l i a n histone deacetylase, H D A C 1 a n d s h o w e d that it h a d a h i g h degree of h o m o l o g y to a w e l l - k n o w n t r a n s c r i p t i o n a l repressor i n S. cerevisiae, R p d 3 . T h i s d i s c o v e r y p a v e d the w a y for the i d e n t i f i c a t i o n of a large n u m b e r of p r o t e i n s w i t h histone deacetylase a c t i v i t y that h a v e b e e n d i v i d e d i n t o three classes: C l a s s I H D A C s , w h i c h i n c l u d e H D A C 1 , that are h o m o l o g o u s to R p d 3 ; C l a s s II H D A C s , that are h o m o l o g o u s to the S. cerevisiae H D A C , H d a l ; a n d , C l a s s III H D A C s , that are h o m o l o g o u s to Sir2, a n N A D d e p e n d e n t H D A C first i d e n t i f i e d i n S. cerevisiae. C l a s s I a n d C l a s s II H D A C s are related i n sequence b u t the Sir2-like H D A C s d o not s h o w a n y s t r o n g relatedness to the other classes, d e m o n s t r a t i n g that at least t w o d i s t i n c t m e c h a n i s m s h a v e e v o l v e d to deacetylate h i s t o n e s .  T h i s leaves  o p e n the p o s s i b i l i t y there m a y be a d d i t i o n a l classes of p r o t e i n s w i t h the ability to deacetylate histones that h a v e yet to be i d e n t i f i e d . I n m o s t e u k a r y o t e s there are m u l t i p l e m e m b e r s i n e a c h class, for example, i n h u m a n s five C l a s s I a n d six C l a s s II H D A C s are k n o w n ( H o o k et a l , 2002). I n D . melanogaster, three C l a s s I H D A C s ( H D A C 1 a k a R P D 3 , H D A C 3 a n d CG10899),  t w o C l a s s II H D A C s ( H D A C 4 a n d H D A C 6 ) , a n d five C l a s s III  H D A C s (SIR2, CG5085, CG11305, CG3187, a n d CG6284) h a v e b e e n i d e n t i f i e d based  on  sequence  similarity  and  listed  on  Flybase  (http:/ / f l y b a s e . b i o . i n d i a n a . e d u : 8 2 / ) . A n o b v i o u s q u e s t i o n is w h y w o u l d s u c h a d i v e r s i t y of H D A C s h a v e e v o l v e d ? If a "histone code" does i n d e e d exist, it m i g h t r e q u i r e a l a r g e n u m b e r of e n z y m e s that target s p e c i f i c r e s i d u e s or  34  specific h i s t o n e s .  A l t e r n a t i v e l y , n u c l e a r H D A C s are a l w a y s f o u n d i n large  m u l t i - p r o t e i n c o m p l e x e s a n d the H D A C s c o u l d be p r o m i s c u o u s w i t h targeting d i r e c t e d b y other m e m b e r s of the c o m p l e x . These alternatives are not m u t u a l l y e x c l u s i v e a n d e v i d e n c e exists that a c o m b i n a t i o n of the t w o m a y be at w o r k . In S. cerevisiae, X - C h i p studies, w i t h antibodies to acetylated l y s i n e residues, h a v e s h o w n that R p d 3 is r e s p o n s i b l e for deacetylating a l l four core histones ( K a d o s h a n d S t r u h l , 1998; R u n d l e t t et a l , 1998; S u k a et al., 2001) i n d i c a t i n g it is r e l a t i v e l y promiscuous.  S i m i l a r studies w i t h H d a l suggest its a c t i v i t y is restricted to  histones H 3 a n d H 2 B ( W u et al., 2001). The subject of this s t u d y , H D A C 1 , is a C l a s s I H D A C .  I n m o s t cases,  C l a s s I H D A C s are 500-600 a m i n o acids i n l e n g t h , share a s i m i l a r structure a n d are r e l a t e d b y t h e i r sequence s i m i l a r i t y to S. cerevisiae,  Rpd3.  The amino-  t e r m i n a l 200 - 300 a m i n o acids are h i g h l y c o n s e r v e d a n d s i m i l a r to R p d 3 . T h i s r e g i o n c o n t a i n s the d o m a i n r e q u i r e d for h i s t o n e d e a c e t y l a t i o n . T h e c a r b o x y t e r m i n a l h a l v e s of the p r o t e i n s d o n o t s h a r e a h i g h d e g r e e of  sequence  s i m i l a r i t y a n d it is p r e s u m e d they are i n v o l v e d i n p r o t e i n / p r o t e i n interactions w i t h m e m b e r s of the H D A C complexes ( K h o c h b i n a n d Wolffe, 1997). H D A C 1 is a t r a n s c r i p t i o n a l repressor u s e d b y a w i d e v a r i e t y of c e l l u l a r s y s t e m s (for m o r e d e t a i l s see C h a p t e r 2) a n d therefore it is p o s s i b l e that m u t a t i o n s i n this gene s u p p r e s s P E V i n d i r e c t l y . C h a p t e r 3 presents e v i d e n c e that H D A C 1 is n o t n o r m a l l y a s s o c i a t e d w i t h the white* gene of  Drosophila.  H o w e v e r , w h e n the white* gene is subject to s i l e n c i n g d u e to P E V , H D A C 1 is present i n a b u n d a n c e at the white* gene r e g u l a t o r y r e g i o n s . T h i s association is a b o l i s h e d i n the m u t a t i o n s that suppress P E V . I interpret these results to m e a n  35  that H D A C 1 is a c t i n g d i r e c t l y at the v a r i e g a t i n g l o c u s a n d p l a y s a n i n t e g r a l role i n m a i n t a i n i n g the gene s i l e n c i n g o b s e r v e d i n P E V .  36  Chapter 2  Mutational Analysis of a Histone Deacetylase in Drosophila  melanogaster:  Missense Mutations Suppress Gene Silencing Associated with Position Effect Variegation  The f o l l o w i n g C h a p t e r is essentially the same as that p u b l i s h e d u n d e r the same title:  • M o t t u s , R., R . E . Sobel a n d T. A . G r i g l i a t t i Genetics 154: 657-668 (2000)  37  Introduction T h e b a s i c u n i t of c h r o m a t i n is the n u c l e o s o m e  w h i c h c o n s i s t s of  a p p r o x i m a t e l y 146 b p of D N A w r a p p e d a r o u n d the four core histones a r r a n g e d i n a n octamer.  T h e a m i n o t e r m i n a l tails of the histones, i n p a r t i c u l a r H 3 a n d  H 4 , are h i g h l y c o n s e r v e d a n d c o n t a i n four l y s i n e (K) r e s i d u e s w h i c h can be r e v e r s i b l y acetylated (Felsenfeld, 1996; W o r k m a n a n d K i n g s t o n , 1998). It w a s first n o t e d o v e r 30 years ago that there is a c o r r e l a t i o n b e t w e e n a c e t y l a t i o n of histones a n d t r a n s c r i p t i o n a l a c t i v i t y or the p o t e n t i a l for t r a n s c r i p t i o n a l a c t i v i t y ( A l l f r e y et al., 1964), b u t the significance of this o b s e r v a t i o n has o n l y b e c o m e apparent i n recent years. It has n o w b e e n d e m o n s t r a t e d that s o m e t r a n s c r i p t i o n a l activators a n d m e m b e r s of the t r a n s c r i p t i o n a l m a c h i n e r y , i n c l u d i n g G C N 5 ( B r o w n e l l et al., 1996; W a n g et al., 1997), P C A F ( Y a n g et a l , 1996b), p 3 0 0 / C B P ( O g r y z k o et a l , 1996) a n d T A F 2 3 0 / 2 5 0 ( M i z z e n et a l , 1996) are capable of a c e t y l a t i n g H 3 a n d n  H 4 b o t h in vitro a n d in vivo.  These h i s t o n e a c e t y l transferases ( H A T s ) are  m e m b e r s of l a r g e p r o t e i n c o m p l e x e s w h i c h are t a r g e t e d to the genes they regulate b y m e m b e r s of the c o m p l e x w h i c h h a v e D N A b i n d i n g a c t i v i t y (Grant et a l , 1997). Conversely, histone  h y p o a c e t y l a t i o n is g e n e r a l l y  correlated  transcriptional inactivity, telomeric and centromeric heterochromatin  with and  s i l e n c e d areas of the g e n o m e s u c h as the d o n o r m a t i n g - t y p e l o c i i n yeast (Turner, 1998; W o r k m a n a n d K i n g s t o n , 1998). A s is the case w i t h the H A T s , h i s t o n e deacetylases  ( H D A C s ) also exist as m e m b e r s of large m u l t i - p r o t e i n  c o m p l e x e s . H o w e v e r , a n u n e x p e c t e d f i n d i n g w a s that some H D A C complexes,  38  i n b o t h yeast ( R u n d l e t t et a l 1996) a n d m a m m a l s ( H a s s i g et al., 1998), c o n t a i n v  m o r e t h a n o n e d e a c e t y l a s e , s u g g e s t i n g that e a c h d e a c e t y l a s e m a y h a v e a specific target a n d that f u l l repression m a y require the activity of m o r e than one H D A C ( K u o a n d A l l i s , 1998). T h e H D A C s i s o l a t e d thus far d o not appear to have any D N A b i n d i n g activity a n d therefore  t a r g e t i n g o f the  HDAC  c o m p l e x e s to the genes they regulate appears to d e p e n d o n a s s o c i a t i o n w i t h D N A b i n d i n g co-repressor proteins, s u c h as M A D ( L a h e r t y et al., 1997), U M E 6 ( K a d o s h a n d S t r u h l , 1997), Y Y 1 ( Y a n g et al., 1996a), S M R T ( N a g y et a l , 1997), N - C o R ( A l l a n d et a l , 1997; H e i n z e l et al., 1997) a n d R B ( B r e h m et a l , 1998; L u o et al., 1998; M a g n a g h i - J a u l i n et al., 1998), that h a v e the a b i l i t y to b i n d to specific target loci. It has b e e n s u g g e s t e d that a c e t y l a t i o n of the l y s i n e s i n the N - t e r m i n a l tails of the histones m a y f u n c t i o n b y o p e n i n g u p c h r o m a t i n structure because it eliminates  p o s i t i v e charges  which  may  nucleosome/nucleosome interactions  reduce  nucleosome/DNA  (Workman and  Kingston,  or  1998).  A c c o r d i n g l y , one w o u l d p r e d i c t that m u t a t i o n s i n H A T s or m e m b e r s of their complexes, s h o u l d result i n r e d u c e d histone acetylation, a n d thus i m p a i r gene a c t i v a t i o n ( G r u n s t e i n , 1997). T h i s p r e d i c t i o n appears to be true. G C N 5 was first i d e n t i f i e d as a t r a n s c r i p t i o n a l activator before its H A T f u n c t i o n w a s e l u c i d a t e d because m u t a t i o n s i n the gene r e d u c e a c t i v a t i o n o f target l o c i . C o n v e r s e l y , m u t a t i o n s i n a n H D A C or m e m b e r s of its c o m p l e x , s h o u l d i m p a i r deacetylation of the h i s t o n e s at target genes, a n d thus result i n d e - r e p r e s s i o n of the targets. T h i s also a p p e a r e d to be the case. M u t a t i o n a l a n a l y s i s i n S. cerevisiae i d e n t i f i e d R P D 3 as a g l o b a l r e p r e s s o r before its f u n c t i o n i n h i s t o n e d e a c e t y l a t i o n w a s known.  39  H o w e v e r , a n u m b e r of u n e x p e c t e d o b s e r v a t i o n s h a v e b e e n d o c u m e n t e d i n HDAC n u l l m u t a t i o n s . I n RPD3 d e l e t i o n lines of S. cerevisiae, a s m a l l subset of genes w e r e m o r e s t r o n g l y r e p r e s s e d rather t h a n a c t i v a t e d . careful  a n a l y s i s of the genes n o r m a l l y subject  In addition,  to r e g u l a t i o n b y  RPD3  d e m o n s t r a t e d that w h e n they w e r e activated i n the RPD3 n u l l strains, the l e v e l of t r a n s c r i p t i o n of target genes w a s l o w e r t h a n i n w i l d - t y p e strains ( V i d a l a n d Gaber, 1991). F i n a l l y , the s t r a i g h t f o r w a r d p r e d i c t i o n that m u t a t i o n s i n HDACs w o u l d result i n d e - r e p r e s s i o n of s i l e n c e d genes w a s c o n f o u n d e d w h e n it w a s reported, i n b o t h S. cerevisiae a n d Drosophila, that m u t a t i o n s w h i c h r e d u c e d or e l i m i n a t e d a h i s t o n e deacetylase r e s u l t e d i n t r a n s c r i p t i o n a l s i l e n c i n g of genes subject to t e l o m e r i c a n d h e t e r o c h r o m a t i c  p o s i t i o n effect v a r i e g a t i o n (De  R u b e r t i s et a l , 1996).  P o s i t i o n effect v a r i e g a t i o n ( P E V ) m o s t often occurs w h e n a c h r o m o s o m a l rearrangement  a b u t s a n o r m a l l y e u c h r o m a t i c r e g i o n of a  c o n t a i n i n g a c t i v e genes, to a b r e a k p o i n t i n c e n t r o m e r i c  chromosome,  heterochromatin  ( G r i g l i a t t i , 1991; H e n i k o f f , 1992; Reuter a n d Spierer, 1992). I n tissues w h e r e the r e l o c a t e d e u c h r o m a t i c genes are u s u a l l y active, s o m e cells express the genes n o r m a l l y , w h e r e a s i n n e i g h b o r i n g cells, the genes are t r a n s c r i p t i o n a l l y silent, r e s u l t i n g i n a m o s a i c p a t t e r n of gene e x p r e s s i o n .  A n a n a l o g o u s s i t u a t i o n is  t h o u g h t to o c c u r i n the p h e n o m e n o n of t e l o m e r i c p o s i t i o n effects ( T P E V ) . T h i s occurs w h e n a reporter gene is inserted i n or near to the h e t e r o c h r o m a t i n of the telomeres of S. cerevisiae c h r o m o s o m e s (De R u b e r t i s et al., 1996; G r e w a l et al., 1998; G r u n s t e i n , 1998).  I n s o m e cells the r e p o r t e r is t r a n s c r i p t i o n a l l y silent  w h i l e i n o t h e r s the g e n e is t r a n s c r i b e d n o r m a l l y . I n b o t h s y s t e m s there is a c o r r e l a t i o n b e t w e e n p o s i t i o n r e l a t i v e to the h e t e r o c h r o m a t i c m a t e r i a l a n d  40  s i l e n c i n g . I n T P E V a n d P E V the l i k e l i h o o d of s i l e n c i n g is d e p e n d e n t o n h o w c l o s e the r e p o r t e r  i s to the t e l o m e r e  or c e n t r o m e r i c  heterochromatin  respectively; i f i n s e r t e d closer it is m o r e often silent. M o s a i c gene expression i n b o t h cases is b e l i e v e d to reflect differences i n c h r o m a t i n structure; w h e n the gene is active, it is p a c k a g e d n o r m a l l y , h o w e v e r , w h e n the gene is inactive, it is p a c k a g e d m o r e l i k e h e t e r o c h r o m a t i n a n d is therefore t r a n s c r i p t i o n a l l y silent. H e r e w e report the i s o l a t i o n a n d characterization of six n e w m u t a t i o n s i n the HDAC1  gene o f D . melanogaster. T h i s is the first instance of a m u t a t i o n a l  a n a l y s i s of a n H D A C i n a m u l t i - c e l l u l a r e u k a r y o t i c o r g a n i s m .  I n contrast to  p r e v i o u s f i n d i n g s , w e report that specific mis-sense m u t a t i o n s i n the structural gene of HDAC1  s u p p r e s s s i l e n c i n g a n d increase the e x p r e s s i o n of a w  +  gene  subject to P E V . W e p r o p o s e that these mis-sense m u t a t i o n s are a c t i n g as antim o r p h i c m u t a t i o n s that p o i s o n the deacetylase c o m p l e x , w i t h o u t e l i m i n a t i n g it, a n d that this i n t u r n causes h y p e r a c e t y l a t i o n of histones a n d a c t i v a t i o n of genes n o r m a l l y subject to s i l e n c i n g as a result of P E V .  F u r t h e r m o r e , w e s h o w that  n u l l , or v e r y severe h y p o m o r p h i c m u t a t i o n s , h a v e n o s i g n i f i c a n t effect o n P E V . W e further p r o p o s e that the u n e x p e c t e d observations n o t e d above i n the RPD3 d e l e t i o n strains i n S. cerevisiae, the P i n s e r t i o n l i n e i n D . melanogaster a n d the phenotypes  of o u r mis-sense, h y p o m o r p h i c a n d n u l l m u t a t i o n s  explained b y a m o d e l based  o n the o b s e r v a t i o n s  h o m o l o g u e s , are m e m b e r s of a s t r u c t u r a l l y  that H D A C 1 ,  can be a n d its  related, m u l t i - d o m a i n f a m i l y of  proteins w h i c h f o r m s part of a large m u l t i - p r o t e i n c o m p l e x . F i n a l l y , w e argue that this m o d e l w i l l be relevant i n a w i d e v a r i e t y of b i o l o g i c a l a p p l i c a t i o n s a n d as s u c h suggests a n e e d for the i s o l a t i o n a n d c h a r a c t e r i z a t i o n o f d o m i n a n t mutations.  41  MATERIAL AND METHODS  Fly Stocks Flies w e r e reared o n s t a n d a r d D r o s o p h i l a m e d i a at 22°. G e n e t i c m a r k e r s u s e d here are d e s c r i b e d i n the text or c a n be f o u n d i n L i n d s l e y a n d Z i m m (1992). T h e p u t a t i v e h i s t o n e deacetylase d e s c r i b e d h e r e i n has a h i g h l e v e l of sequence s i m i l a r i t y to R P D 3 f r o m S. cerevisiae a n d H D A C 1 f r o m h u m a n s a n d o t h e r m a m m a l s ( D e R u b e r t i s et a l . , 1996).  The initial report and  some  subsequent reports (for e x a m p l e ( M a n n e r v i k a n d L e v i n e , 1999)) r e g a r d i n g the Drosophila  h i s t o n e deacetylase r e l i e d o n the s i m i l a r i t y to the yeast gene a n d  c a l l e d the Drosophila  h o m o l o g u e a n R P D 3 - l i k e d e a c e t y l a s e o r the  RPD3  h o m o l o g u e . I n yeast, R P D 3 (for r e d u c e d p o t a s s i u m d e p e n d e n c y 3) w a s n a m e d p r i o r to the d i s c o v e r y that it has histone deacetylase a c t i v i t y a n d describes o n l y one of the p h e n o t y p e s associated w i t h l e s i o n s i n the gene ( V i d a l a n d Gaber, 1991). F o r this r e a s o n w e prefer the m a m m a l i a n n o m e n c l a t u r e :  H D A C , for  histone deacetylase, f o l l o w e d b y a n u m b e r i n d i c a t i n g to w h i c h , of the several s i m i l a r deacetylases that exist i n each o r g a n i s m , it is m o s t s i m i l a r ( T a u n t o n et al., 1996). A c c o r d i n g l y , since the D r o s o p h i l a deacetylase d e s c r i b e d here has the h i g h e s t d e g r e e of s i m i l a r i t y to H D A C 1 f r o m m a m m a l s w e prefer the n a m e D r o s o p h i l a H D A C 1 for D r o s o p h i l a h i s t o n e deacetylase one a n d w i l l use that n o m e n c l a t u r e i n this m a n u s c r i p t The H D A C 1 m u t a t i o n s that s u p p r e s s P E V (hereafter c a l l e d the Su(var) H D A C l s ) d e s c r i b e d here w e r e i n d u c e d i n a p r e v i o u s l y d e s c r i b e d e t h y l methane sulfonate screen for d o m i n a n t s u p p r e s s o r s of P E V ( S i n c l a i r et a l . , 1983). T h e m u t a t i o n s are m a i n t a i n e d i n stocks b a l a n c e d o v e r TM3 Sb Ser or TM6 Tb. Tb  42  w a s e m p l o y e d because it a l l o w s one to r e a d i l y i d e n t i f y h o m o z y g o u s m u t a n t l a r v a e b y the m o r p h o l o g y of their s p i r a c l e s .  L a t e t h i r d instar h o m o z y g o u s  m u t a n t l a r v a e w e r e selected f r o m c u l t u r e s a n d u s e d to o b t a i n the D N A sequence of HDAC1 i n the v a r i o u s m u t a n t strains. P e l e m e n t i n s e r t i o n strains w e r e o b t a i n e d f r o m the B l o o m i n g t o n Stock C e n t e r a n d w e r e screened for l e t h a l i t y w i t h the Su(var) H D A C l s .  O n e insert  l i n e , 1(3)04556 (hereafter c a l l e d P - U T R ) , w a s a l m o s t c o m p l e t e l y l e t h a l u n d e r n o r m a l c u l t u r e c o n d i t i o n s w i t h a l l m e m b e r of the S u ( v a r ) H D A C 1 g r o u p . H o w e v e r , s i g n i f i c a n t n u m b e r s of m a l e a n d f e m a l e a d u l t s c o u l d be r e a r e d to a d u l t h o o d i f the f l y c u l t u r e s w e r e u n c r o w d e d a n d the m e d i a s u p p l e m e n t e d w i t h l i v e yeast b u t s u r v i v o r s are sterile a n d d i e w i t h i n a few d a y s . T h e Su(var) H D A C 1 g r o u p w a s o r i g i n a l l y l o c a l i z e d because a l l m e m b e r s failed to c o m p l e m e n t a s m a l l d e f i c i e n c y , Df(3L)GN24,  S i n c e , i n a d d i t i o n to  m a n y other l o c i , this d e f i c i e n c y c o m p l e t e l y r e m o v e s the HDAC1  gene it w a s  e m p l o y e d i n the l e t h a l phase analysis as a n u l l allele. M a l e s of the c o n s t i t u t i o n m4  w  j y • Df(3L)GN24  / + were generated b y crossing w  m4  females to + / Y ; Df(3L)GN24  / T M 3 Sb' Ser m a l e s .  I w  m4  ; + /+  F I m a l e s b e a r i n g the  d e f i c i e n c y c h r o m o s o m e w e r e c o l l e c t e d a n d c r o s s e d to 5-7 d a y o l d v i r g i n females of each of the v a r i o u s m u t a n t H D A C 1 strains a n d a l l o w e d to l a y eggs on  petri  plates  overlaid with  s u p p l e m e n t e d w i t h l i v e yeast.  an  agar,  vinegar  and  ethanol  mixture  Eggs were collected b y w a s h i n g w i t h dH20,  batches of a p p r o x i m a t e l y 100 eggs w e r e c o u n t e d out o n c o n s t r u c t i o n paper a n d p l a c e d i n s h e l l v i a l s . A m i n i m u m of five s h e l l v i a l s w a s set u p for each m u t a n t strain. T h e c o n s t r u c t i o n p a p e r w a s r e m o v e d after three d a y s a n d the n u m b e r of  43  u n h a t c h e d eggs c o u n t e d .  U n h a t c h e d eggs t h a t f a i l e d  to d a r k e n  were  c o n s i d e r e d u n f e r t i l i z e d a n d s u b t r a c t e d f r o m the total n u m b e r of eggs.  Eggs  that d a r k e n e d , b u t f a i l e d to h a t c h , w e r e s c o r e d as e m b r y o n i c lethals.  The  n u m b e r of a n i m a l s r e a c h i n g p u p a t i o n a n d a d u l t h o o d w e r e c o u n t e d a n d the l e t h a l i t y at each d e v e l o p m e n t a l stage d e t e r m i n e d f r o m the totals. I n a l l cases the results of e a c h g r o u p w e r e p o o l e d .  I n these crosses, the o n l y a n i m a l s  e x p e c t e d to d i e w e r e those that c a r r i e d the Df(3L)GN24.  mutant H D A C 1 allele a n d  A l l o t h e r g e n o t y p e s w e r e e x p e c t e d to s u r v i v e .  o b s e r v e a n y flies that s u r v i v e d a n d b o r e a m u t a n t DF(3L)GN24.  W e d i d not  HDAC1  allele a n d  These flies w o u l d h a v e b e e n r e a d i l y i d e n t i f i a b l e because of the  suite of defects o b s e r v e d i n h o m o z y g o u s H D A C 1 m u t a n t lines (see Results). In the r e c o m b i n a t i o n e x p e r i m e n t i n w h i c h w e t r i e d to separate the lethal l e s i o n i n HDAC1  i n the H D A C l 3 2 8  strain from  a p o s s i b l e s e c o n d site  s u p p r e s s o r of P E V , the female parents w e r e p r o d u c e d b y c r o s s i n g w  m4  + / + females to w  I Y; HDAC1  m4  the c o n s t i t u t i o n , w  m4  j  w  w  Y ; P-UTR  4  I w ; m4  m4  HDAC1  328  I + , w e r e c o l l e c t e d a n d crossed to  / T M 3 Sb Ser males. A l l flies that d i s p l a y e d s u p p r e s s i o n of  v a r i e g a t i o n w e r e p r o g e n y tested to d e t e r m i n e w h e t h e r t h e y  m4  ;  / TM3 Sb Ser males. V i r g i n F l females of  328  m  I w  r e c o m b i n a n t s o r rare s u r v i v i n g HDAC1  I  328  P-UTR  were  flies.  Determination of the level of variegation T o d e t e r m i n e the l e v e l s of v a r i e g a t e d gene e x p r e s s i o n i n the w 4 a n d m  b w D e 2 strains, eye p i g m e n t assays w e r e p e r f o r m e d e m p l o y i n g p r e v i o u s l y y  p u b l i s h e d t e c h n i q u e s ( S i n c l a i r et a l . , 1983) a n d the a m o u n t of eye p i g m e n t  44  o b s e r v e d i n the v a r i e g a t i n g s t r a i n e x p r e s s e d as a p e r c e n t a g e o f the a m o u n t o b s e r v e d i n the w i l d - t y p e strain, O r e g o n - R . T h e l e v e l o f v a r i e g a t i o n i n the S b strain was determined  b y assaying the percentage o f fourteen  v  bristles  d i s p l a y i n g a Sb p h e n o t y p e as p r e v i o u s l y d e s c r i b e d (Sinclair et al., 1983).  Remobilization of the P element T h e P e l e m e n t i n the P - U T R s t r a i n carries the ry  +  gene a n d therefore  e x c i s i o n of a l l o r p a r t of the P element c a n m o n i t o r e d b y loss of ry .  The P  element i n the P - U T R s t r a i n w a s r e m o b i l i z e d b y c r o s s i n g + / + ; P-UTR  / TM3  +  Sb Sex females to w ryRK Sb e P[ry  +  m 4  / Y ; Ly / TM3 ry  RK  Sb e P[ry+ A2-3] males. T h e TM3  A2-3] c h r o m o s o m e carries a P element transposase source (A2-3)  w h i c h is r e q u i r e d to r e m o b i l i z e the defective P e l e m e n t i n the P - U T R strain. T h e F I + / Y ; P-UTR crossed to + / + ; ry506  / TM3 ry  RK  Sb e P[ry+ A2-3] m a l e s w e r e collected a n d  j y506 females a n d ry~ F 2 m a l e s c o l l e c t e d a n d stocks r  established.  D N A manipulations A l l s t a n d a r d D N A m a n i p u l a t i o n s w e r e p e r f o r m e d as d e s c r i b e d i n S A M B R O O K et al. (1989). P l a s m i d rescue of the D N A s u r r o u n d i n g the i n s e r t i o n of the P element i n P - U T R w a s p e r f o r m e d a c c o r d i n g to p r e v i o u s l y p u b l i s h e d techniques ( K a r p e n a n d S p r a d l i n g , 1992). G e n o m i c D N A for s e q u e n c i n g f r o m each of the H D A C 1 m u t a n t strains w a s o b t a i n e d f r o m cultures i n w h i c h the H D A C 1 m u t a t i o n is b a l a n c e d over the  45  T M 6 T b b a l a n c e r c h r o m o s o m e (see above).  H o m o z y g o u s m u t a n t late t h i r d  i n s t a r l a r v a e w e r e c o l l e c t e d a n d the D N A i s o l a t e d b y s t a n d a r d p r o t o c o l s . Specific fragments  of HDAC1  w e r e a m p l i f i e d u s i n g Pfu p o l y m e r a s e a n d  p r i m e r s w h i c h w e r e d e s i g n e d f r o m the p u b l i s h e d sequence of HDAC1.  The  P C R p r o d u c t s w e r e g e l p u r i f i e d a n d s e q u e n c e d e m p l o y i n g d y e terminators i n a n a u t o m a t e d s e q u e n c i n g facility ( U B C N A P S U n i t ) .  I s o l a t i o n a n d A n a l y s i s of R N A  T o t a l R N A w a s i s o l a t e d f r o m either a d u l t females o r a d u l t m a l e s of each s t r a i n u s i n g the T R I z o l ® Reagent a c c o r d i n g to m a n u f a c t u r e r s i n s t r u c t i o n s (Life Technologies).  P o l y ( A ) R N A w a s s u b s e q u e n t l y i s o l a t e d e m p l o y i n g the  O l i g o t e x m R N A M i n i K i t p r o d u c e d b y Q i a g e n f o l l o w i n g the m a n u f a c t u r e r s instructions. A p p r o x i m a t e l y 1.5 ug of p o l y ( A ) R N A for each gender a n d strain w a s separated o n a f o r m a l d e h y d e agarose d e n a t u r i n g g e l p r e p a r e d a c c o r d i n g to the p r o t o c o l p r o v i d e d b y Q i a g e n i n the O l i g o t e x M i n i K i t . T h e gels w e r e r u n at 7 V / c m , t r a n s f e r r e d to n y l o n m e m b r a n e s a c c o r d i n g to the m a n u f a c t u r e r ' s instructions (Amersham) and probed w i t h D N A labeled w i t h [32p]dATP using Boehringer M a n n h e i m ' s R a n d o m P r i m e d D N A labeling kit. The D N A probe for the HDAC1  m R N A w a s p r e p a r e d b y P C R e m p l o y i n g Pfu p o l y m e r a s e a n d  p r i m e r s for the c a r b o y x l t e r m i n a l c o d i n g r e g i o n s g e n e r a t e d f r o m a c l o n e d c D N A k i n d l y p r o v i d e d to us b y P i e r r e S p i e r e r ' s l a b o r a t o r y .  The relative  a m o u n t s of p o l y ( A ) R N A l o a d e d i n each lane w a s d e t e r m i n e d b y r e - p r o b i n g the N o r t h e r n b l o t s w i t h a p r o b e for the m R N A for the r i b o s o m a l p r o t e i n , D U b 8 0 ( M o t t u s et a l . , 1997). A u t o r a d i o g r a m s w e r e s c a n n e d i n t o a c o m p u t e r a n d the  46  a m o u n t of p o l y ( A ) R N A i n each lane w a s q u a n t i f i e d , r e l a t i v e to D U b 8 0 , u s i n g N I H Image (data not s h o w n )  47  R E S U L T S  Isolation and characterization of mutations in Drosophila HDAC1  that  suppress P E V S e v e r a l g r o u p s , i n c l u d i n g ours, h a v e c o n d u c t e d large genetic screens to isolate S u p p r e s s o r s of p o s i t i o n effect v a r i e g a t i o n or Su(var)s i n D . melanogaster . These screens w e r e b a s e d o n the a s s u m p t i o n that these m u t a t i o n s s h o u l d i d e n t i f y factors i n v o l v e d i n the process of c h r o m a t i n p a c k a g i n g ( L o c k e et al., 1988; R e u t e r a n d Wolff, 1981; Sinclair et al., 1983), either s t r u c t u r a l c o m p o n e n t s of c h r o m a t i n o r factors that m o d i f y c h r o m a t i n s t r u c t u r e .  O u r screen w a s  d e s i g n e d to isolate d o m i n a n t Su(var)s b y selecting p r o g e n y f r o m e t h y l methane sulfonate ( E M S ) m u t a g e n i z e d males i n w h i c h e x p r e s s i o n of the w  +  strain, In(l)w  m4  (w  m 4  ) , was significantly increased.  i n v e r s i o n juxtaposes the w  +  chromosome.  I n the w  m  gene i n the  4 strain, an  gene to the c e n t r o m e r i c h e t e r o c h r o m a t i n of the X  T h i s causes the w  +  gene to be t r a n s c r i p t i o n a l l y i n a c t i v a t e d i n  m o s t p i g m e n t cells i n the fly's eye a n d s i n c e its p r o d u c t is r e q u i r e d for d e p o s i t i o n of p i g m e n t , the eyes of flies i n the w  m  ^ s t r a i n g e n e r a l l y h a v e about  5% to 15% of the w i l d - t y p e l e v e l s of eye p i g m e n t s .  F o u r of the d o m i n a n t  Su(var) m u t a t i o n s i s o l a t e d c o m p r i s e a single c o m p l e m e n t a t i o n g r o u p (hereafter r e f e r r e d to c o l l e c t i v e l y as S u ( v a r ) H D A C l s o r i n d i v i d u a l l y as HDAC1 , 313  HDAC1  326  a n d HDAC1 ). 328  HDACl^,  A l l are s t r o n g d o m i n a n t suppressors of  P E V a n d , i n a d d i t i o n to the d o m i n a n t p h e n o t y p e , a l l four alleles are recessive lethal. I n w  m  4 strains b e a r i n g the Su(var) H D A C l s , p i g m e n t s i n the eyes of  48  Figure 1 Examples of eyes from male flies bearing the In(l)w ^ chromosome m  and third chromosomes of the following constitutions: (A) +/ + ; (B) P-UTR I + ; (C) HDACl f  / + ; (D) HDACl f  de 8  (E) HDAC1  303  (H) HDAC  328  /+;(¥)  HDAC / 313  /+ .  49  /+;  de 24  + ; (G) HDAC 1 326  + and  TABLE 1 The effects of selected Su(var) H D A C 1 mutations on various genes subject to PEV  b  Sex  + /+  F  8+2  38+24  56+20  M  13+3  55+15  69+16  F  83+5  55+4  72+22  M  85±5  55+4  91+9  F  72+8  49±22  72+15  M  88±6  50±8  93+10  HDAC1  326  HDAC1  328  a  bw  w  Genotype  1 +  1 +  vDe2  m4  U  Sb  v  the percentage of eye p i g m e n t s c o m p a r e d to the a m o u n t o b s e r v e d i n the w i l d - t y p e strain, O R - R  b  the percentage of bristles d i s p l a y i n g the Sb p h e n o t y p e  50  b o t h m a l e s a n d females are i n c r e a s e d f r o m 5-15% to 60-90 % o f the p i g m e n t levels o b s e r v e d i n the w i l d - t y p e strain, O R - R (Figure 1 a n d T a b l e 1). In o r d e r to d e t e r m i n e w h e t h e r  the effect o f these m u t a t i o n s w a s  generally a p p l i c a b l e to P E V or specific to the iv  +  of t w o o f the s t r o n g e s t HDAC1 , 328  gene, w e m o n i t o r e d the effects  a l l e l e s o f the S u ( v a r ) H D A C l s , HDAC1  o n t w o other v a r i e g a t i n g rearrangements: In(2R)bw  vDe2  juxtaposes the bw  +  and  326  (bw ) which v  l o c u s to the c e n t r o m e r i c h e t e r o c h r o m a t i n of c h r o m o s o m e 2,  a n d ; T(2,3)Sb ( S b ) w h i c h abuts the d o m i n a n t t h i r d c h r o m o s o m e m u t a t i o n , Sb', v  v  to the c e n t r o m e r i c h e t e r o c h r o m a t i n of c h r o m o s o m e 2 (Table 1). I n females, the m u t a t i o n s c a u s e d s i g n i f i c a n t s u p p r e s s i o n of b o t h b w a n d S b . I n males, S b v  v  v  w a s also s t r o n g l y s u p p r e s s e d b y the m u t a t i o n s , b u t b w w a s either n o t affected v  or s o m e w h a t e n h a n c e d . H e t e r o g e n e i t y i n the response o f genes subject to P E V w h e n e x p o s e d to s u p p r e s s o r m u t a t i o n s is n o t u n c o m m o n . E a c h rearrangement abuts the e u c h r o m a t i c v a r i e g a t i n g gene to a u n i q u e r e g i o n of h e t e r o c h r o m a t i n a n d therefore a v a r i a t i o n i n the l e v e l of response to t r a n s - a c t i n g factors is n o t u n e x p e c t e d ( L l o y d et a l . , 1997). H o w e v e r , it is clear that a l t h o u g h the strength of the s u p p r e s s i o n o f P E V v a r i e s , the S u ( v a r ) H D A C l s s i l e n c i n g a s s o c i a t e d w i t h P E V a n d are n o t m u t a t i o n s specifically m o d i f y the w  +  s u p p r e s s the gene i n factors  which  gene.  M a p p i n g t h e Su(var) H D A C l s W e m a p p e d the recessive l e t h a l i t y associated w i t h the Su(var) H D A C l s to  64B17-64C13-15 e m p l o y i n g deficiencies a n d c o n f i r m e d the Su(var)  51  p h e n o t y p e r e c o m b i n a t i o n a l l y m a p p e d to a p p r o x i m a t e l y the same l o c a t i o n i n a l l four m u t a n t l i n e s .  T h i s p l a c e d the c o m p l e m e n t a t i o n g r o u p v e r y close to a  recently c l o n e d RPD3-like HDAC  (De R u b e r t i s et a l . , 1996). T h i s D r o s o p h i l a  H D A C ( h e r e a f t e r referred to as HDAC1)  w a s c l o n e d as a result o f a P i n s e r t i o n  1.8 kb 5' to the gene w h i c h causes strong d o m i n a n t e n h a n c e m e n t of P E V , b u t is h o m o z y g o u s v i a b l e a n d fertile. H o w e v e r , c o m p l e m e n t a t i o n analysis w i t h the Su(var) H D A C l s a n d the P insert l i n e (hereafter referred to as P-1.8) r e v e a l e d a l l c o m b i n a t i o n s w e r e v i a b l e a n d fertile (data n o t s h o w n ) s u g g e s t i n g that p e r h a p s P-1.8 a n d the Su(var) H D A C l s represented t w o different genes. W e t h e n c r o s s e d the Su(var) H D A C l s to a series of recessive lethal, m o d i f i e d P inserts generated b y the B e r k e l e y D r o s o p h i l a G e n o m e Database a n d l o c a l i z e d to the 64B-64C r e g i o n . The Su(var) H D A C l s w e r e almost c o m p l e t e l y lethal w h e n h e t e r o z y g o u s w i t h the P insert line, 1(3)04556 (hereafter c a l l e d P U T R ) . P l a s m i d rescue of the g e n o m i c D N A s u r r o u n d i n g the i n s e r t i o n p o i n t of the P element r e v e a l e d it h a d i n s e r t e d i n t o the 5' U T R of HDAC1  ( F i g u r e 2).  S u r p r i s i n g l y , w h i l e P - U T R is h o m o z y g o u s l e t h a l a n d l e t h a l w i t h the Su(var) H D A C l s , it has n o d o m i n a n t effect o n v a r i e g a t i o n of w  +  i n the IniDw™  4  strain  (Figure 1 a n d T a b l e 4). Since P - U T R h a d a n i n s e r t i o n i n t o HDAC1, b u t d i d not have a d o m i n a n t affect o n P E V , this raised the p o s s i b i l i t y that the P - U T R strain contained a s e c o n d site m u t a t i o n that w a s c a u s i n g the l e t h a l i t y w i t h the Su(var) H D A C l s . A l t e r n a t i v e l y , it w a s p o s s i b l e that the S u ( v a r ) H D A C l s , i n a d d i t i o n to a recessive l e t h a l l e s i o n i n HDAC1,  c a r r i e d a s e c o n d site m u t a t i o n that w a s  c a u s i n g the d o m i n a n t Su(var) p h e n o t y p e . two ways.  52  W e a d d r e s s e d these p o s s i b i l i t i e s i n  P-1.8  P-UTR  303 and 328  313  326  .8kb-  (-  j  HDACldefS  (- - - - . .  UTR  |  Coding 500 bp  Figure 2  G e n o m i c o r g a n i z a t i o n of the HDAC1 l o c a t i o n of each of the Su(var) HDAC1  gene. T h e a p p r o x i m a t e g r o u p mutatations is s h o w n ,  the i n s e r t i o n p o i n t s of the P elements i n the P-1.8 a n d  P-UTR  strains are i n d i c a t e d b y the triangles, a n d the d a s h e d l i n e indicate the extent of the deficiencies i n the deletions strains.  53  First, i n o r d e r to d e t e r m i n e w h e t h e r P - U T R also c o n t a i n e d a s e c o n d site l e t h a l m u t a t i o n , w e g e n e r a t e d r e v e r t a n t s of P - U T R b y r e m o b i l i z i n g the P element, w h i c h is m a r k e d w i t h ry , +  r e c o v e r e d 25 ry~ revertants.  a n d r e c o v e r i n g m a l e s that w e r e ry~.  We  F o u r of the revertants are h o m o z y g o u s v i a b l e ,  v i a b l e as h e t e r o z y g o t e s w i t h P - U T R a n d v i a b l e as h e t e r o z y g o t e s w i t h a l l m e m b e r s of the Su(var) H D A C l s . Subsequent a n a l y s i s b y P C R i n d i c a t e d that three of the revertants are precise e x c i s i o n s of the-P e l e m e n t w h i l e the f o u r t h retains a s m a l l piece of the P element. Since a precise or n e a r l y precise e x c i s i o n of the P element i n s e r t i o n results i n a h o m o z y g o u s v i a b l e c h r o m o s o m e , the o n l y l e t h a l l e s i o n o n the P - U T R c h r o m o s o m e is c a u s e d b y the i n s e r t i o n of the P e l e m e n t i n t o HDAC1 l e s i o n i n the HDAC1  a n d therefore the S u ( v a r ) H D A C l s also h a v e a l e t h a l gene.  S e c o n d , i n o r d e r to d e t e r m i n e w h e t h e r the Su(var) H D A C l s , i n a d d i t i o n to the l e t h a l l e s i o n i n HDAC1,  c a r r i e d a d o m i n a n t s e c o n d site Su(var) m u t a t i o n ,  w e t r i e d to separate the l e t h a l p h e n o t y p e f r o m the S u ( v a r ) p h e n o t y p e b y recombination. observations  T h e cross is o u t l i n e d i n F i g u r e 3 a n d is b a s e d o n that:  (1) HDAC1  328  is  almost  completely  lethal  the  when  h e t e r o z y g o u s w i t h P - U T R , a n d ; (2) P - U T R does not h a v e a n y d o m i n a n t effect o n P E V . A c c o r d i n g l y , a n y flies that s u r v i v e a n d d i s p l a y e d s u p p r e s s i o n of  w  m4  v a r i e g a t i o n w o u l d be the result of a r e c o m b i n a t i o n event b e t w e e n the l e t h a l l e s i o n i n HDAC1  a n d the p u t a t i v e s e c o n d site S u ( v a r ) .  W e s c o r e d 6125  r e c o m b i n a n t s b u t w e w e r e u n a b l e to separate the l e t h a l p h e n o t y p e f r o m the Su(var) phenotype.  A c c o r d i n g l y , i f the l e t h a l i t y a n d the d o m i n a n t S u ( v a r )  p h e n o t y p e s are caused b y different mutations, t h e n these m u t a t i o n s are less  54  dHDACl  P-UTR  328  wild-type  Figure 3  '  •*  v  T M 3 Sb Ser f - r i T i /T-"*  /-it,  O  T h e cross e m p l o y e d to attempt to generate a r e c o m b i n a n t b e t w e e n the lethal l e s i o n i n the histone deacetylase gene i n the HDAC1  328  strain a n d a possible s e c o n d site s u p p r e s s o r  of p o s i t i o n effect v a r i e g a t i o n . In the F i , HDAC1  328  /P-UTR  is a l m o s t c o m p l e t e l y lethal. A l l non-S£> Ser flies w e r e e x a m i n e d for s u p p r e s s i o n of P E V . S u p p r e s s e d flies are either rare HDAC 1  P-UTR s u r v i v o r s or represent p o t e n t i a l r e c o m -  328  b i n a n t s b e t w e e n a possible second site s u p p r e s s o r of P E V a n d HDAC1 . 328  Despite e x a m i n i n g >6000 r e c o m b i n a n t  c h r o m o s o m e s , w e d i d not isolate a s e c o n d site s u p p r e s s o r of P E V  55  t h a n 1.6 x 10~2 m a p u n i t s apart, a distance r e p r e s e n t i n g a p p r o x i m a t e l y 4 to 7 k b of D N A i n a t y p i c a l r e g i o n of the Drosophila  g e n o m e ( L e f e v r e , 1976). Since  t r a n s c r i p t a n a l y s i s has s h o w n that there are n o o t h e r t r a n s c r i p t s w i t h i n a p p r o x i m a t e l y 8 k b of the 3' e n d or 12 k b of the 5' e n d of HDAC1 (De Rubertis et al., 1996), it is u n l i k e l y that the l e t h a l i t y a n d the Su(var) p h e n o t y p e are caused b y separate m u t a t i o n s . B a s e d o n the results of the r e v e r s i o n e x p e r i m e n t s w i t h P - U T R a n d the failure  to  separate  the  lethal lesion  and  the  Su(var) phenotypes  by  r e c o m b i n a t i o n , w e c o n c l u d e that b o t h p h e n o t y p e s are the result of lesions i n  HDAC1.  Mutant phenotypes associated with lesion in HDAC1 D u r i n g the c o u r s e of the r e c o m b i n a t i o n e x p e r i m e n t w e o b s e r v e d that some P-UTR/HDAC1  a d u l t m a l e flies d i d eclose b u t o n l y s u r v i v e d for a few  328  days.  These a n i m a l s d i s p l a y e d v e r y s t r o n g s u p p r e s s i o n of P E V a n d s e v e r a l  other p h e n o t y p e s . I n o r d e r to further e x a m i n e these p h e n o t y p e s w e generated HDACP /P-UTR  flies.  03  In this cross, u n d e r c a r e f u l l y m a i n t a i n e d c u l t u r e  c o n d i t i o n s , a d u l t m a l e s eclosed at a p p r o x i m a t e l y 40 % of e x p e c t e d a n d females at a p p r o x i m a t e l y 30 % of expected. B o t h sexes o n l y s u r v i v e d for several d a y s a n d the females p r o d u c e d a s m a l l n u m b e r of eggs w h i c h a p p e a r e d to be u n f e r t i l i z e d . These a n i m a l s d i s p l a y e d a suite of defects i n c l u d i n g : v e r y s t r o n g s u p p r e s s i o n of w ; m4  w i n g s that w e r e s e v e r e l y n o t c h e d ; b r i s t l e s that w e r e  smaller, m a l f o r m e d , often c u r v e d a n d d u p l i c a t e d ; a l u l a that w e r e larger t h a n n o r m a l ; a n d , a r e d u c t i o n i n the n u m b e r of sex c o m b s o n the legs of the males  56  f r o m a m e a n of 10.7+ 0.9 to a m e a n of 7.7+ 1.0. T h i s suggests that m u t a t i o n s i n the histone deacetylase, HDAC1, cause defects i n a v a r i e t y of c e l l u l a r systems, a p h e n o t y p e that is consistent w i t h its p r o p o s e d role as a g l o b a l t r a n s c r i p t i o n a l regulator. It also suggests that the Su(var) H D A C l s r e t a i n at least some of their functions, since P - U T R is l e t h a l w h e n h o m o z y g o u s , yet a p p r e c i a b l e n u m b e r s of a d u l t s c a n be r e c o v e r e d w h e n P - U T R is h e t e r o z y g o u s w i t h m e m b e r s of the Su(var) H D A C l s . Since P - U T R a n d the Su(var) H D A C l s are recessive l e t h a l it appears that HDAC1  f u n c t i o n i s e s s e n t i a l for s u r v i v a l i n D . melanogaster, u n l i k e i n S.  cerevisiae, w h e r e n u l l alleles of the RPD3 gene are v i a b l e b u t d i s p l a y a suite of phenotypes.  I n o r d e r to further characterize the r e q u i r e m e n t s for HDAC1 w e  d e t e r m i n e d the d e v e l o p m e n t a l t i m e at w h i c h HDAC1  is r e q u i r e d for s u r v i v a l i n  D . melanogaster. S i n c e P - U T R is a v e r y s t r o n g h y p o m o r p h ( M a n n e r v i k a n d L e v i n e , 1999) a n d w e w e r e u n a b l e to d e t e r m i n e w h e t h e r o r n o t the Su(var) H D A C l s are c o m p l e t e n u l l alleles of the gene a n d r e s i d u a l gene activity w o u l d m a s k the earliest r e q u i r e m e n t for HDAC1,  w e generated n u l l alleles of HDAC1  (see b e l o w for details). T h e results of o u r l e t h a l phase a n a l y s i s are presented i n Table 2. N u l l alleles (HDACl f  de 8  a n d HDACl^f ) 24  of HDAC1  die during larval  stage of life. S u r p r i s i n g l y , i n s p e c t i o n of the stock c u l t u r e s r e v e a l e d that a large percentage of the h o m o z y g o u s m u t a n t l a r v a e s u r v i v e u n t i l v e r y late i n t h i r d instar. These l a r v a e w e r e r e a d i l y i d e n t i f i a b l e because i n the stock cultures the m u t a t i o n s are b a l a n c e d o v e r TM6Tb.  L a r v a e b e a r i n g the balancer c h r o m o s o m e  can be d i s t i n g u i s h e d f r o m l a r v a e h o m o z y g o u s for the HDAC1  mutations  because Tb alters the m o r p h o l o g y of the l a r v a l spiracles. T h i s suggests three  57  TABLE 2 Lethal phase analysis of mutations in HDAC1.  Percentage of animals that  die at the indicated developmental stage  M u t a n t Strain  Embryonic  Larval  Pupal  Male  Tested  Lethality  Lethality  Lethality  Viability  8  6.8  24.7  3.9  93  HDACldef  6.3  25.9  7.6  91  3.9  13.3  17.0  71  3.4  28.4  2.3  51  HDAC1  2.9  30.7  6.5  88  HDAC1  4.1  16.4  16.8  78  HDACl ef d  24  HDAC1  303  HDAC1  313  326  328  a  v i a b i l i t y of males expected to s u r v i v e as c o m p a r e d to their female siblings  58  a  possible scenarios:  1) m a t e r n a l H D A C 1 is p e r d u r i n g u n t i l v e r y late i n  d e v e l o p m e n t ; 2) H D A C l is r e q u i r e d d u r i n g e m b r y o g e n e s i s a n d not r e q u i r e d again u n t i l late i n t h i r d instar a n d m a t e r n a l H D A C l p r o v i d e s sufficient activity for this e a r l y f u n c t i o n ; or, 3) H D A C l is not r e q u i r e d for the e a r l y stages of Drosophila development.  B a s e d o n a recently p u b l i s h e d r e p o r t i n v e s t i g a t i n g  the p h e n o t y p e s associated w i t h P - U T R , w e f a v o u r the s e c o n d o f the above three possibilities. W e also c o n d u c t e d l e t h a l phase analyses of the Su(var) H D A C l s . HDACl ^3  3  d HDACl ^ 32  a  n  a  \  s o  d i e d d u r i n g the l a r v a l p e r i o d . Inspection of the  stock c u l t u r e s r e v e a l e d a large n u m b e r of h o m o z y g o u s m u t a n t l a r v a e at the t h i r d instar stage a n d therefore these alleles cause d e a t h at a p p r o x i m a t e l y the same t i m e as the n u l l alleles. H o w e v e r , o n l y a p p r o x i m a t e l y fifty percent of larvae b e a r i n g HDACl ® 3  3  a n d HDACl  d i e d d u r i n g the l a r v a l p e r i o d w h i l e  328  about 50% s u r v i v e d i n t o p u p a t i o n . T h i s is consistent w i t h the s e q u e n c i n g data (see b e l o w ) w h i c h d e m o n s t r a t e d these m u t a t i o n s are c a u s e d b y i d e n t i c a l base pair substitutions. a n d HDACl 6 32  HDACl  328  T h u s , w i t h r e g a r d to l e t h a l i t y , it a p p e a r s that  HDACl ^3  are i n d i s t i n g u i s h a b l e f r o m n u l l alleles w h i l e HDACl ® 3  3  3  and  r e t a i n some H D A C l activity.  A n u n e x p e c t e d o b s e r v a t i o n f r o m the l e t h a l p h a s e a n a l y s i s w a s that the Su(var) H D A C l s a p p e a r e d to h a v e a d o m i n a n t s e m i - l e t h a l affect o n m a l e s regardless of their g e n o t y p e .  I n the l e t h a l p h a s e a n a l y s i s , three of the four  g e n o t y p e s p r o d u c e d are expected to s u r v i v e (see M a t e r i a l s a n d M e t h o d s ) a n d one of the classes ( + / T M 3 ) does not carry a n y c h r o m o s o m e s w i t h a m u t a t i o n i n HDACl.  I n the crosses w i t h the n u l l alleles, HDACl f  de 8  and  HDACl^f , 24  males a n d females i n the classes expected to l i v e , a p p e a r i n a p p r o x i m a t e l y the  59  same n u m b e r s (Table 2). H o w e v e r , i n the Su(var) H D A C 1 crosses, males of genotypes expected to s u r v i v e , i n c l u d i n g males that h a v e c o m p l e t e l y w i l d - t y p e HDAC1  genes s u r v i v e d at s i g n i f i c a n t l y l o w e r rates t h a n expected. F o r e x a m p l e  males i n the cross i n v o l v i n g HDACl ^3  3  o n l y s u r v i v e d at about 50% the l e v e l of  their g e n o t y p i c a l l y i d e n t i c a l female s i b l i n g s i n the same cross. M a l e s i n crosses i n v o l v i n g the other Su(var) H D A C l s also s u r v i v e d at s i g n i f i c a n t l y l o w e r levels t h a n females.  Since, i n these crosses the m o t h e r s c a r r i e d the Su(var) H D A C 1  m u t a t i o n s , one e x p l a n a t i o n for this o b s e r v a t i o n m a y be that these m u t a t i o n s m a y be e x e r t i n g a d o m i n a n t m a t e r n a l effect o n the d o s a g e c o m p e n s a t i o n m e c h a n i s m . I n D r o s o p h i l a , dosage c o m p e n s a t i o n occurs as a result of h y p e r t r a n s c r i p t i o n of the m a l e X c h r o m o s o m e .  The male X chromosome adopts a  s p e c i a l c o n f o r m a t i o n w h i c h i s b e l i e v e d to b e n e c e s s a r y  for  enhanced  t r a n s c r i p t i o n ( B a s h a w a n d Baker, 1996). A c c o r d i n g l y , if histone deacetylation is a n essential step i n e s t a b l i s h i n g the s p e c i a l i z e d c h r o m a t i n structure r e q u i r e d i n the m a l e , the Su(var) H D A C l s m a y be defective i n this process. A l t e r n a t i v e l y , a l t h o u g h m o s t genes o n the m a l e X c h r o m o s o m e are t r a n s c r i b e d at d o u b l e the n o r m a l rate, there are l o c i that are not subject to d o s a g e c o m p e n s a t i o n a n d therefore  need  to be s i l e n c e d or r e p r e s s e d  o n the  specialized male X  c h r o m o s o m e (Baker et al., 1994). In the Su(var) H D A C 1 strains these l o c i m a y escape r e p r e s s i o n r e s u l t i n g i n r e d u c e d m a l e v i a b i l i t y i n the sons of m u t a n t mothers.  60  S e q u e n c e a n a l y s i s o f the Su(var) g r o u p E M S - i n d u c e d c h a n g e s i n the S u ( v a r ) c o m p l e m e n t a t i o n g r o u p w e r e i d e n t i f i e d b y s e q u e n c i n g the g e n o m i c D N A e n c o d i n g H D A C 1 f r o m the four Su(var) lines a n d f r o m the c h r o m o s o m e w h i c h w a s o r i g i n a l l y e m p l o y e d i n the screen for S u ( v a r ) m u t a t i o n s .  T h e results of this a n a l y s i s are p r e s e n t e d i n  F i g u r e 2 ( A c c e s s i o n N u m . AF086715). T h e g e n o m i c o r g a n i z a t i o n i n o u r strains is s l i g h t l y different t h a n that presented i n the p r e v i o u s l y p u b l i s h e d report (De R u b e r t i s et a l . , 1996) . T h e c o d i n g sequence is i n t e r r u p t e d b y three i n t r o n s rather t h a n t w o a n d the c o n c e p t u a l t r a n s l a t i o n of the p r o t e i n y i e l d s a p r o d u c t of 521 a m i n o a c i d s rather t h a n 520.  T h e extra a m i n o a c i d is p r o d u c e d at the  a d d i t i o n a l i n t r o n / e x o n b o u n d a r y i n o u r sequence.  D N A sequencing revealed  that there is a s i n g l e a m i n o a c i d s u b s t i t u t i o n i n each of the four m u t a n t lines that s u p p r e s s P E V . T h e locations of the a m i n o a c i d s u b s t i t u t i o n s are i n d i c a t e d i n F i g u r e 2.  I n t w o of the strains, HDACP  03  i d e n t i c a l base p a i r substitutions.  a n d HDAC1 , 328  w e observed  These mutations were recovered  from  u n r e l a t e d bottles i n the o r i g i n a l E M S screen a n d therefore m o s t l i k e l y represent i n d e p e n d e n t events. Each single nucleotide substitution resulted i n changing an amino acid that is not o n l y perfectly c o n s e r v e d i n h o m o l o g u e s f r o m Yeast a n d h u m a n , b u t the s u b s t i t u t i o n s are located i n regions of the p r o t e i n that are a l m o s t perfectly c o n s e r v e d i n these d i v e r s e o r g a n i s m s ( T a b l e 3).  T h e f u n c t i o n s of these  p a r t i c u l a r r e s i d u e s a n d the r e g i o n s i n w h i c h t h e y o c c u r h a v e not yet b e e n determined.  H o w e v e r , e v o l u t i o n a r y a n a l y s i s o f the deacetylase p r o t e i n s a n d  some l i m i t e d m u t a t i o n a l analysis suggest that the a m i n o one-half of the p r o t e i n  61  Table 3 Comparison oi amino acid substitutions in the Su(var) H D A C l group with conserved regions in human and yeast homologues.  HDACl  HDACl  313  303  HDACl  326  HDACl  328  R30C D. melanogaster  GHPMKPHRIRM  C98Y FNVGEDCPVFDGL  P204S SFHKYGEYFPGTG  H D A C l Mutant Strain  c  Y  s  H. sapiens H D A C l  R  c  P  S. cerevisiae R P D 3  R  p-c  F-P  -" indicates identity.  62  is the d o m a i n r e s p o n s i b l e for catalytic a c t i v i t y ( K h o c h b i n a n d Wolffe, 1997). It is i n t e r e s t i n g to note that each of the s u b s t i t u t i o n s o c c u r s i n the r e g i o n of the p r o t e i n t h o u g h t to be r e q u i r e d for deacetylase activity.  Interaction with an P-1.8, an E(var) allele T h e HDACl  l o c u s has p r e v i o u s l y b e e n c l o n e d as a d o m i n a n t enhancer of  P E V or E ( v a r ) (De R u b e r t i s et al.  1996).  T h e p h e n o t y p e results f r o m the  i n s e r t i o n of a P element 1.8 k b 5' to the start site of HDACl  w h i c h reduces or  e l i m i n a t e s t r a n s c r i p t i o n o f the gene i n the eye i m a g i n a l d i s k b u t n o t i n other i m a g i n a l d i s k s f r o m the same a n i m a l s . S u r p r i s i n g l y , h e t e r o z y g o u s flies b e a r i n g P-1.8 a n d a n y one of m e m b e r s of the Su(var) H D A C l s w e r e v i a b l e a n d fertile. In a d d i t i o n , i n these heterozygotes, the eyes of flies b e a r i n g io  m4  show a weak  to m o d e r a t e s u p p r e s s i o n of P E V (data not s h o w n ) . Since the eyes a p p e a r to be n o r m a l i n these crosses, w i t h the o n l y a p p a r e n t p h e n o t y p e b e i n g a n effect o n P E V , a n d P-1.8 flies are v i a b l e as h o m o z y g o t e s , these observations suggest that either HDACl  d o e s n o t p e r f o r m a n y essential f u n c t i o n i n the eye d i s k or  alternatively, that P-1.8 m a y be a h y p o m o r p h .  Generation of null alleles W o r k b y M a n n e r v i k a n d L e v i n e (1999) a n d this s t u d y (see b e l o w ) s h o w that P - U T R p r o d u c e s a message at s i g n i f i c a n t l y l o w e r levels t h a n w i l d - t y p e a n d thus is l i k e l y to be a s t r o n g h y p o m o r p h . A s n o t e d above, w e w e r e s u r p r i s e d that P - U T R h a d n o effect o n P E V since it is l e t h a l w h e n h o m o z y g o u s .  One  p o s s i b l e e x p l a n a t i o n is that, a l t h o u g h this m u t a t i o n is a h y p o m o r p h w h i c h is  63  l e t h a l as a h o m o z y g o t e , it p r o d u c e s sufficient a c t i v i t y i n a h e t e r o z y g o t e s u c h that P E V is not affected. If this w e r e the case t h e n one w o u l d p r e d i c t that a n u l l allele of HDAC1  w o u l d h a v e a d o m i n a n t effect o n P E V . A c c o r d i n g l y , w e  g e n e r a t e d n u l l alleles of HDAC1  b y r e m o b i l i z i n g the P e l e m e n t i n P - U T R to  i n d u c e deficiencies of the c o d i n g r e g i o n s of the gene as a result of imperfect e x c i s i o n s of the P element.  T h e P element, w h i c h is m a r k e d w i t h ry ,  was  +  r e m o b i l i z e d b y c r o s s i n g P - U T R to the transposase source, In(3LR)TM3, A2-3 Sb , a n d r e c o v e r i n g m a l e s that w e r e ry~.  F r o m 560 p o t e n t i a l e x c i s i o n events w e  r e c o v e r e d 25 ry~ males, 19 of w h i c h w e r e still l e t h a l o v e r P - U T R a n d the Su(var) H D A C l s a n d therefore  represented potential i m p r o p e r excisions.  DNA  sequence a n a l y s i s has s h o w n w e generated t w o deficiencies w h i c h b e g i n at the i n s e r t i o n p o i n t of P - U T R a n d r e m o v e a m i n o t e r m i n a l c o d i n g regions of HDAC1; HDACl f  de 8  deletes  approximately  a p p r o x i m a t e l y 870 b p (see F i g u r e 2).  440  de 8  de  4  and  HDACl f  de 24  deletes  C o n c e p t u a l t r a n s l a t i o n s f r o m the first  seven A U G c o d o n s r e m a i n i n g i n HDACl f HDACl ft  bp  a n d the first three start codons of  w o u l d p r o d u c e p e p t i d e s that bear n o s i m i l a r i t y to H D A C 1 , a n d  therefore w e b e l i e v e these represent n u l l alleles of the gene. S u r p r i s i n g l y , w e f o u n d that n u l l alleles of HDAC1 have n o d o m i n a n t effect o n s i l e n c i n g of the  w  +  gene i n the w 4 strain (Figure 1). m  Northern Analysis Since w e h a d generated a v a r i e t y of m u t a t i o n s i n the HDAC1  locus it w a s  of c o n s i d e r a b l e interest to d e t e r m i n e h o w the m u t a t i o n s affected the l e v e l of transcription.  Figure 4 shows  N o r t h e r n b l o t s i n d i c a t i n g the l e v e l s of  64  t r a n s c r i p t i o n of HDACl  i n the m u t a n t l i n e s i d e n t i f i e d i n t h i s s t u d y .  To  determine the relative l o a d i n g i n each lane, the N o r t h e r n s w e r e also c h a l l e n g e d w i t h a p r o b e for the r i b o s o m a l p r o t e i n , D U b 8 0 (see M a t e r i a l s a n d M e t h o d s ) . T h e transcript levels of HDAC1 26 3  a  r  e  a p p r o x i m a t e l y the same as that observed  i n the w i l d - t y p e strains i n d i c a t i n g that the Su(var) p h e n o t y p e is not a result of h y p e r - t r a n s c r i p t i o n of the locus. In the P - U T R strain, as is often the case w i t h P insertions, the l e v e l of t r a n s c r i p t i o n is r e d u c e d relative to w i l d - t y p e levels a n d therefore P - U T R is l i k e l y a h y p o m o r p h . T h i s is i n a c c o r d w i t h the f i n d i n g s of M a n n e r v i k a n d L e v i n e (1999), w h o s h o w e d the m a t e r n a l c o n t r i b u t i o n o f HDACl  i n the P - U T R s t r a i n w a s a p p r o x i m a t e l y five f o l d less that w i l d - t y p e . In  a s t r a i n h e t e r o z y g o u s for the d e f i c i e n c y , HDACl^f , 8  the m e s s a g e p r o d u c e d  f r o m the deleted c h r o m o s o m e is r e d u c e d i n a m o u n t a n d e v i d e n t as a w i d e n i n g of the 2.2 k b b a n d p r o d u c e d f r o m the n o n - d e l e t e d h o m o l o g u e . h e t e r o z y g o u s for the deficiency, HDACl^P- , 4  I n the s t r a i n  t w o different s i z e d transcripts  are c l e a r l y v i s i b l e i n d i c a t i n g b o t h h o m o l o g u e s are t r a n s c r i b e d , b u t the a m o u n t of the s m a l l e r transcript p r o d u c e d f r o m the d e l e t e d c h r o m o s o m e is v e r y m u c h reduced.  S i n c e the s m a l l e r t r a n s c r i p t s i n b o t h d e f i c i e n c y strains are v e r y  u n l i k e l y to p r o d u c e f u n c t i o n a l proteins, w e b e l i e v e these m u t a t i o n s represent n u l l alleles.  65  ^BBBB^ ^mHB^ ^(Bd^ ^^•W^  *«m  mmr  ^^^^^^^^ ^^wjP^  "NMRK  HDACl  ^^^^^^  # » t mm  ***  ""™~™*"  DUb80  '  F i g u r e 4 Northern analysis of poly(A) R N A isolated from adults bearing HDACl  mutations. Lanes 1 and 6 are from w i l d -  type female controls and lane 7 is from wild-type male controls. Lanes 2-4 are from females of the constitution HDACl f 1TM3 de 8  Sb Ser, HDACl^f  24  / TM3 Sb Ser and  P-UTR ITM3 Sb Ser, respectively. The approximate amount of poly(A) R N A loaded i n each lane was determined by reprobing the blots with a probe specific for the message for the ribosomal protein DUb80. Loading i n lanes 1-4 is approximately equivalent and show that the levels of total H D A C l message in lanes 1-3 are approximately the same. However, in lane 4 the amount of message is reduced to -50-60% of Lane 1. Lane 5 contains -1.6 times the amount of p o y ( A ) R N A as lane 6, and when taken into account, the levels of H D A C l p o l y ( A ) R N A in HDACl  326  females (lane 5)  and control females (lane 6) are approximately equivalent, as are the amounts i n HDACl  326  (lane 7).  66  males (lane 8) and control males  DISCUSSION  In this s t u d y w e report the i s o l a t i o n a n d c h a r a c t e r i z a t i o n of a n u m b e r of n e w m u t a t i o n s i n the D. melanogaster p u t a t i v e histone deacetylase, HDACl, test their effects o n gene s i l e n c i n g that o c c u r s as a result of P E V .  and  M o d e l s of  gene r e g u l a t i o n , b a s e d o n the c o r r e l a t i o n b e t w e e n h i s t o n e a c e t y l a t i o n a n d gene a c t i v i t y , w o u l d p r e d i c t that m u t a t i o n s i n a n h i s t o n e deacetylase gene, w h i c h r e d u c e o r e l i m i n a t e h i s t o n e deacetylase a c t i v i t y , o u g h t to l e a d to i n c r e a s e d levels of h i s t o n e a c e t y l a t i o n w h i c h , i n t u r n , w o u l d l e a d to d e - r e p r e s s i o n of s i l e n c e d genes. S u r p r i s i n g l y , this s t r a i g h t f o r w a r d p r e d i c t i o n w a s not b o r n out. I n s t e a d the effect o n gene s i l e n c i n g is d e p e n d e n t u p o n the n a t u r e of the m u t a t i o n i n HDACl  (for s u m m a r y see Table 4).  H o w t h e n c a n one e x p l a i n the a p p a r e n t l y c o n t r a d i c t o r y affects o n P E V a n d T P E V of the v a r i o u s k i n d s of m u t a t i o n s i n the h i s t o n e deacetylase genes i n Yeast a n d D r o s o p h i l a ? It m a y be that histone deacetylases b e l o n g to a g r o w i n g class of genes w h i c h h a v e the f o l l o w i n g characteristics: 1) they are m e m b e r s of a closely related gene f a m i l y ; 2) they encode m u l t i - d o m a i n p r o t e i n s , a n d ; 3) n u l l m u t a t i o n s h a v e little or n o o b v i o u s p h e n o t y p i c effect w h i l e p o i n t m u t a n t s h a v e p r o f o u n d , often d o m i n a n t effects. O n e recent e x a m p l e o f this class o f genes i n l o w e r e u k a r y o t e s is the FUS3/KSS1  gene p a i r of S. cerevisiae. N o r m a l l y , these  closely related p r o t e i n s f u n c t i o n i n separate p a t h w a y s . S i n g l e d e l e t i o n strains of either gene are still proficient for m a t i n g because w h e n F u s 3 p is deleted, a n d o n l y w h e n it is d e l e t e d , K s s l p acts as a n i m p o s t o r a n d r e p l a c e s F u s 3 p . H o w e v e r , d e l e t i o n of b o t h p r o t e i n s r e n d e r s the s t r a i n sterile ( M a d h a n i a n d F i n k , 1998; M a d h a n i et al., 1997). E x a m p l e s of this class of gene is certainly not  67  TABLE 4 Summary of the effects of various mutations in HDACl on viability and PEV in D. melanogaster  a  Mutation  Dominant  Homozygous  Heterozygous with  Effect o n P E V  Viability  Su(var) H D A C l s  enhancer  viable  moderate  P-1.8 P element insert 1.8 k b  s u p p r e s s i o n of P E V  5' to gene  s t r o n g semi-lethal; i n  P-UTR P element insert into  n o effect  lethal  strongly suppressed  the 5' U T R  Su(var) H D A C l s point mutants  HDACl  d  e  f  8  HDACl  d  e  f  2  4  strong suppressors  lethal  lethal  n o effect  lethal  lethal  n o effect  lethal  lethal  Deletions  a  rare s u r v i v o r s , P E V  for a complete d e s c r i p t i o n of m u t a t i o n s see text  68  l i m i t e d to l o w e r e u k a r y o t e s . F o r e x a m p l e , gene k n o c k o u t e x p e r i m e n t s i n m i c e h a v e r e v e a l e d a s u r p r i s i n g n u m b e r of genes i n w h i c h the p h e n o t y p e of the h o m o z y g o u s n u l l m u t a t i o n is either not detectable or v e r y m i n o r . A c u r s o r y examination  of  the  Mouse  Knockout  Database  (http:/ / w w w . b i o m e d n e t . c o m / d b / m k m d ) identifies at least 13 s u c h genes. In contrast to the m i l d p h e n o t y p e s of k n o c k o u t alleles, a n a l y s i s of m u t a t i o n s i n some of these genes has s h o w n that p o i n t m u t a t i o n s c a n h a v e v e r y p r o f o u n d , often d o m i n a n t effects.  O n e e x a m p l e is the SRC o n c o g e n e , a m e m b e r of a  c l o s e l y r e l a t e d f a m i l y of p r o t e i n s .  T h e k n o c k o u t causes o n l y m i n o r d e n t a l  a b n o r m a l i t i e s , yet a l m o s t a l l k n o w n p o i n t m u t a t i o n s h a v e severe p h e n o t y p i c consequences, i n c l u d i n g cancer ( L o w e l l a n d S o r i a n o , 1996). R e c e n t l y a m o d e l has b e e n p r o p o s e d to account for the m a i n t e n a n c e of closely related gene f a m i l i e s d u r i n g e v o l u t i o n ( G i b s o n a n d S p r i n g , 1998). B y e x t e n d i n g this m o d e l w e b e l i e v e w e c a n p r o v i d e a n e x p l a n a t i o n for these apparently  contradictory  observations  regarding  relatively  benign  k n o c k o u t / n u l l m u t a t i o n s a n d d o m i n a n t p o i n t m u t a t i o n s w h i c h h a v e severe p h e n o t y p i c consequences.  It is n o w a p p a r e n t that m o s t , i f n o t a l l , o f the  b i o l o g i c a l activities i n the cell are c a r r i e d out b y large, m u l t i - p r o t e i n complexes. A s i n g l e t y p e o f c o m p l e x m a y h a v e m u l t i p l e targets o r f u n c t i o n s that are d e p e n d e n t o n the specific m e m b e r s of the c o m p l e x at a p a r t i c u l a r t i m e d u r i n g the cell cycle or at a p a r t i c u l a r l o c a t i o n i n the cell. If one of the proteins of the c o m p l e x is absent, as i n a n u l l m u t a t i o n , a n d that p r o t e i n is a m e m b e r of a closely related f a m i l y , t h e n another member(s) of the f a m i l y m a y substitute for the m i s s i n g p r o t e i n .  Since they are c l o s e l y related, the i m p o s t o r can p r o v i d e  p a r t i a l a c t i v i t y a n d , as a consequence, a n u l l m u t a t i o n m a y h a v e n o o b v i o u s  69  phenotype.  I n contrast, p o i n t m u t a t i o n s that o n l y alter a s i n g l e d o m a i n , m a y  a l l o w the aberrant p r o t e i n to be i n c o r p o r a t e d i n t o its c o m p l e x ( e s ) . I n cases i n w h i c h the m u t a t i o n o c c u r s i n a d o m a i n r e q u i r e d for a specific f u n c t i o n , the c o m p l e x w o u l d t h e n be c o m p l e t e l y i n a c t i v e for that p a r t i c u l a r f u n c t i o n . A c c o r d i n g l y , a p o i n t m u t a t i o n m a y h a v e a d o m i n a n t n e g a t i v e effect a n d d i s p l a y a m u c h m o r e severe p h e n o t y p e than a n u l l m u t a t i o n (see F i g u r e 5). This m o d e l may D r o s o p h i l a HDAC1  accommodate  our  observations  o f the  various  m u t a t i o n s . I n eukaryotes, the H D A C s are a closely related  f a m i l y of p r o t e i n s that f o r m c o m p l e x e s w i t h o t h e r p r o t e i n s i n c l u d i n g other H D A C s . F o r e x a m p l e i n Yeast, t w o different H D A C s , R P D 3 a n d H D A 1 , h a v e b e e n i s o l a t e d a n d c h a r a c t e r i z e d , a n d sequence a n a l y s i s of the yeast g e n o m e suggests there m a y be at least three a d d i t i o n a l H D A C s .  T w o large m u l t i -  p r o t e i n c o m p l e x e s , H D A a n d H D B , c o n t a i n i n g histone deacetylase activity have b e e n i s o l a t e d a n d a n a l y s i s of H D A has s h o w n that it c o n t a i n s at least t w o H D A C s ( C a r m e n et a l , 1996; R u n d l e t t et al., 1996). S i m i l a r l y , i n m a m m a l s , five different H D A C s h a v e b e e n i d e n t i f i e d a n d a c o m p l e x c o n t a i n i n g the h u m a n R P D 3 - l i k e deacetylase, H D A C 1 , also contains H D A C 2 ( H a s s i g et a l , 1998). I n D r o s o p h i l a , t w o m o r e H D A C s has n o w b e e n i d e n t i f i e d , HDAC2  and  HDAC3  (Johnson et a l , 1998; M a n n e r v i k a n d L e v i n e , 1999). It seems l i k e l y that m o r e c a n d i d a t e deacetylases w i l l be i d e n t i f i e d as the g e n o m e s e q u e n c i n g projects proceed.  A c c o r d i n g l y , the b i o c h e m i c a l a n d sequence a n a l y s i s o f H D A C s i n  Yeast a n d m a m m a l s suggest that H D A C s are m e m b e r s of a related gene f a m i l y and,  m o r e i m p o r t a n t l y to o u r m o d e l , f u n c t i o n as m e m b e r s o f l a r g e p r o t e i n  complexes.  70  8k  .HDACl DNA  C o m p l e x \  Normally H D A C 1 participates in a complex which is targeted to D N A by other members of the complex  B  H D A C family member  However, when H D A C 1 is absent, another member of the H D A C family binds to the complex, is targeted correctly to the D N A but produces an aberrant pattern of histone deacetylation  Su(var) H D A C 1  A Su(var) H D A C 1 mutation, which has only a single amino acid change, w i l l occupy its normal place in the complex and be targeted correctly, however the Su(var) H D A C l w i l l be unable to deacetylate its target histone residues leading to increased acetylation and suppression of PEV.  gure 5  A model to explain the various phenotypes associated with mutations in H D A C l , see text for details.  71  T h e f o r e g o i n g p r o v i d e s the f r a m e w o r k for a m o d e l that m a y e x p l a i n the a p p a r e n t l y c o n t r a d i c t o r y results o b s e r v e d w i t h different k i n d s o f m u t a t i o n s i n this histone deacetylase a n d their effects o n P E V a n d T P E V . I n the RPD3 n u l l m u t a t i o n i n yeast, T P E V is enhanced, i.e., the e x p r e s s i o n of the reporter gene is repressed.  W e p o s t u l a t e that i n the absence of R P D 3 , other H D A C s , w i t h  d i f f e r i n g s p e c i f i c i t i e s , s u b s t i t u t e for R P D 3 i n the m u l t i - p r o t e i n c o m p l e x resulting i n an incorrect histone deacetylation pattern.  The  phenotypic  consequence o f the i n c o r r e c t d e a c e t y l a t i o n p a t t e r n is e n h a n c e m e n t o f T P E V , p o s s i b l y d u e to excess d e a c e t y l a t i o n at the site of the r e p o r t e r gene b y the i m p o s t o r deacetylase. S u b s t i t u t i o n b y other H D A C s has also b e e n suggested b y other a u t h o r s to account for the r e s i d u a l r e p r e s s i o n o b s e r v e d i n RPD3 d e l e t i o n strains ( K a d o s h a n d S t r u h l , 1998). In D r o s o p h i l a the o n l y m u t a t i o n i n  HDAC1  that enhances P E V is P-1.8, a n i n s e r t i o n of a P element 1.8 k b 5' to the c o d i n g r e g i o n . In situ h y b r i d i z a t i o n w i t h a probe for the HDAC1 that, i n the eye d i s k , t r a n s c r i p t i o n of HDAC1 i n the l e g d i s k the HDAC1  m R N A demonstrates  is m a r k e d l y r e d u c e d or absent b u t  t r a n s c r i p t a c c u m u l a t e s to n o r m a l l e v e l s .  One  possible e x p l a n a t i o n for this o b s e r v a t i o n is that the P element has inserted i n t o a n eye d i s k specific e n h a n c e r element r e s u l t i n g i n little or n o t r a n s c r i p t i o n i n the eye d i s k . T h u s , HDAC1  m a y be effectively absent i n the eye d i s k . In its  absence, other H D A C s c o u l d substitute for HDAC1  p r o d u c i n g an incorrect  d e a c e t y l a t i o n p a t t e r n , the consequence of w h i c h is e n h a n c e m e n t of P E V .  In  contrast, the Su(var) H D A C l s described here are capable of p r o d u c i n g a p r o t e i n w i t h o n l y a s i n g l e a m i n o a c i d change i n w h i c h a specific f u n c t i o n has l i k e l y b e e n c o m p r o m i s e d , p o s s i b l y the deacetylase a c t i v i t y . Since o n l y a single a m i n o a c i d has b e e n c h a n g e d , the p r o t e i n w o u l d still associate w i t h its c o m p l e x , b i n d  72  its other c o m p o n e n t s efficiently a n d be targeted to the correct site. H o w e v e r , the c o m p l e x w o u l d be u n a b l e to deacetylate its target h i s t o n e s l e a d i n g to h y p e r a c e t y l a t i o n a n d decreased s i l e n c i n g . In this w a y a p o i n t m u t a t i o n w o u l d act as a d o m i n a n t n e g a t i v e m u t a t i o n a n d w o u l d s u p p r e s s P E V . O n the other hand, null mutations,  s u c h as the d e f i c i e n c i e s d e s c r i b e d here, h a v e  o b s e r v a b l e affect o n P E V b e c a u s e i n h e t e r o z y g o t e s ,  wild-type  no  HDAC1,  p r o d u c e d f r o m the n o n - d e l e t e d h o m o l o g u e , c a n associate n o r m a l l y w i t h the histone deacetylase complexes. HDAC1  T h e other H D A C s c a n o n l y substitute  for  i n its c o m p l e t e absence as is the case w i t h K s s l p a n d F u s 3 p i n Yeast  d e s c r i b e d above. T h i s m o d e l relies o n the s u p p o s i t i o n that a n aberrant f o r m of H D A C 2 is b e i n g p r o d u c e d i n the Su(var) H D A C 1 strains. m a d e for the f o l l o w i n g reasons.  W e b e l i e v e s u c h a p r o t e i n is  First, c o n c e p t u a l t r a n s l a t i o n of the p r o t e i n  p r o d u c e s a f u l l l e n g t h p r o d u c t w i t h o n l y a single a m i n o a c i d change.  Second,  w h e n w e c r o s s e d the m e m b e r s of the S u ( v a r ) H D A C l s to P-1.8, the s t r a i n b e a r i n g the P element i n s e r t i o n 1.8 k b 5' to the HDAC1  gene, flies b e a r i n g b o t h  m u t a t i o n s w e r e v i a b l e a n d fertile a n d s h o w e d a w e a k to m o d e r a t e s u p p r e s s i o n of P E V . Since the P insert l i n e is effectively a n u l l i n the eye d i s k , w e interpret the s u p p r e s s i o n o b s e r v e d i n the h e t e r o z y g o t e s as e v i d e n c e that the Su(var) HDACls  are p r o d u c i n g a p r o d u c t .  T h i r d , i n the c o m p l e m e n t a t i o n  and  r e c o m b i n a t i o n studies, heterozygotes b e a r i n g b o t h the P - U T R c h r o m o s o m e a n d the Su(var) H D A C l s s u r v i v e d at a n a p p r e c i a b l e frequency. i n the I n ( l ) w 4 s t r a i n w a s v e r y s t r o n g l y s u p p r e s s e d m  v i r t u a l l y i n d i s t i n g u i s h a b l e f r o m w i l d - t y p e strains.  73  In these flies, P E V a n d the eyes  were  Since P - U T R is l e t h a l as a  h o m o z y g o t e a n d this l e t h a l i t y is o n l y associated w i t h l e s i o n i n HDAC1,  the  o b s e r v a t i o n that s u c h flies s u r v i v e suggests that the S u ( v a r ) H D A C l s  are  p r o d u c i n g a p r o d u c t w h i c h retains sufficient a c t i v i t y i n the essential function of HDAC1 to rescue the l e t h a l i t y associated w i t h the P - U T R c h r o m o s o m e . F i n a l l y , the o b s e r v a t i o n that the Su(var) H D A C l s d i s p l a y e d a d o m i n a n t m a t e r n a l effect r e d u c t i o n i n the v i a b i l i t y of males, regardless of their p h e n o t y p e , a r e d u c t i o n w h i c h w a s not o b s e r v e d i n crosses w i t h the d e f i c i e n c y strains, i m p l i e s that the Su(var) H D A C s are p r o d u c i n g a p r o t e i n p r o d u c t since this m a t e r n a l effect o b s e r v e d is not seen i n the absence of any p r o d u c t . T h e m o d e l m a y also serve to e x p l a i n o t h e r a p p a r e n t l y o b s e r v a t i o n s i n Y e a s t strains b e a r i n g n u l l m u t a t i o n s i n RPD3.  anomalous  T h e gene w a s  first i d e n t i f i e d as a t r a n s c r i p t i o n a l repressor i n S. cerevisiae because m u t a t i o n s i n the gene r e s u l t e d i n d e - r e p r e s s i o n of the m a j o r i t y of g e n e s it r e g u l a t e d . S u r p r i s i n g l y , further a n a l y s i s of the m u t a n t strains has s h o w n that target genes are also d e f e c t i v e i n the degree to w h i c h t h e y r e s p o n d to a c t i v a t o r s a n d repressors.  R e g u l a t e d genes c a n n o t be a c t i v a t e d as f u l l y , n o r r e p r e s s e d as  c o m p l e t e l y , as i n the w i l d - t y p e s t r a i n ( V i d a l a n d G a b e r , 1991).  Since R P D 3  forms part of a h i s t o n e deacetylase c o m p l e x , w e p r o p o s e that i n the absence of R P D 3 , other H D A C s m a y f i l l i n r e s u l t i n g i n aberrant d e a c e t y l a t i o n patterns at target genes.  A b e r r a n t d e a c e t y l a t i o n patterns m a y result i n d e - r e p r e s s i o n of  m o s t target genes, b u t w o u l d p r o v i d e less t h a n o p t i m a l c o n d i t i o n s for t r a n s c r i p t i o n i n the presence of a n activator a n d w o u l d be l e a k y i n the presence of a repressor.  C o n v e r s e l y , i n some c h r o m o s o m a l contexts, r e c r u i t m e n t of the  w r o n g deacetylase  m a y result i n an aberrant  represses t r a n s c r i p t i o n .  74  deacetylation pattern  that  F i n a l l y , w e e m p h a s i z e that the m u t a t i o n s d e s c r i b e d here w e r e recovered i n a genetic s c r e e n for d o m i n a n t s u p p r e s s o r s of P E V . T h e r e f o r e the s i n g l e a m i n o a c i d changes that w e r e c o v e r e d m a y i d e n t i f y d o m a i n s i n the D r o s o p h i l a H D A C 1 that are i m p o r t a n t for s i l e n c i n g i n h e t e r o c h r o m a t i n r a t h e r  than  a b o l i s h i n g a l l deacetylase activity. In a n y case, since the d o m a i n s are c o n s e r v e d i n Yeast, site-directed mutagenesis s h o u l d p r o v i d e a direct test of the p r o p o s e d model. O n e of the t r a d i t i o n a l g e n e t i c a p p r o a c h e s  to d e t e r m i n i n g p r o t e i n  f u n c t i o n has b e e n to generate n u l l m u t a t i o n s a n d t h e n e x a m i n e the o r g a n i s m for p h e n o t y p i c defects w h i c h c a n be correlated w i t h the n u l l p h e n o t y p e . In fact, this is the basis for creating the k n o c k o u t m u t a t i o n s i n m i c e as p o t e n t i a l m o d e l s for h u m a n s y n d r o m e s .  It is n o w a p p a r e n t that m o s t , i f n o t a l l b i o l o g i c a l  functions i n e u k a r y o t i c cells o c c u r as a result o f the a c t i o n o f p r o t e i n c o m p l e x e s a n d not i n d i v i d u a l p r o t e i n s . If the f o r e g o i n g m o d e l is of g e n e r a l a p p l i c a b i l i t y t h e n this t r a d i t i o n a l a p p r o a c h m u s t be a p p l i e d w i t h c a u t i o n .  If the p r o t e i n  u n d e r s c r u t i n y is a m e m b e r of a gene f a m i l y t h e n i n the absence o f that p r o t e i n , other f a m i l y m e m b e r m a y "fill i n " a n d p r o v i d e p a r t i a l , or e v e n complete, rescue ( u n d e r l a b o r a t o r y c o n d i t i o n s ) of the f u n c t i o n s c o m p r o m i s e d b y the  null  m u t a t i o n . In that case, this type of analysis w i l l be c o m p r o m i s e d a n d the role of the p r o t e i n b e i n g i n v e s t i g a t i o n u n d e r a p p r e c i a t e d .  A more fruitful  strategy  m a y be to create d o m i n a n t m u t a t i o n s , i n the best case c a u s e d b y v e r y s m a l l alterations i n the p r o t e i n s u c h as a single a m i n o a c i d s u b s t i t u t i o n , w h i c h w i l l act i n a d o m i n a n t n e g a t i v e f a s h i o n a n d direct o u r a t t e n t i o n to the p o s s i b l e m a n y roles a p r o t e i n m a y h a v e because of its m e m b e r s h i p i n one or m o r e m u l t i protein machines.  75  Chapter 3 Chromatin Immunoprecipitation Analysis of a Region subject Position Effect Variegation in Drosophila  76  melanogaster  Introduction  One  of the major c h a l l e n g e s i n h e r e n t i n a n y m u t a t i o n a l a n a l y s i s of a  gene is d e t e r m i n i n g w h e t h e r the p h e n o t y p e s o b s e r v e d are a direct result of the defect i n the gene, o r w h e t h e r they are i n d i r e c t , a n d a d o w n s t r e a m consequence of the m u t a t i o n a l t e r i n g other m e t a b o l i c p a t h w a y s that i m p i n g e o n a n d m o d i f y the  phenotype  i n q u e s t i o n . T h i s is e s p e c i a l l y i m p o r t a n t  w h e n one  i n v e s t i g a t i n g m u t a t i o n s i n a gene that is a k n o w n , or s u s p e c t e d , r e g u l a t o r of t r a n s c r i p t i o n .  HDACl  is  general  falls i n t o the class of k n o w n g e n e r a l  r e g u l a t o r s of t r a n s c r i p t i o n a n d f u n c t i o n s as a n e s s e n t i a l m e m b e r of s e v e r a l repressor c o m p l e x e s (see C h a p t e r 2). I n a d d i t i o n , there is s o m e evidence, a n d a g r o w i n g s u s p i c i o n , that this v i e w is too s i m p l i s t i c .  Rather than only being a  repressor, H D A C l m a y also be a n i n t e g r a l part of the s y s t e m that controls the t r a n s c r i p t i o n rate of some active genes (see b e l o w , ( B r e i l i n g et al., 2001)). Since H D A C l regulates a large n u m b e r of l o c i , a n o b v i o u s a n d i m p o r t a n t q u e s t i o n is w h e t h e r the m u t a t i o n s that s u p p r e s s P E V are a c t i n g d i r e c t l y or i n d i r e c t l y to abrogate the s i l e n c i n g n o r m a l l y o b s e r v e d at the v a r i e g a t i n g locus. S e v e r a l m e t h o d s are c u r r e n t l y e m p l o y e d to address this type of question. The oldest m e t h o d is s t a i n i n g p o l y t e n e c h r o m o s o m e s w i t h a n a n t i b o d y specific for the subject p r o t e i n . In the s a l i v a r y g l a n d s of Drosophila, a n d specific tissues of  most  Dipterans,  the  chromosomes  undergo  many  rounds  of  e n d o r e d u p l i c a t i o n . T h e c h r o m o s o m e a r m s r e m a i n associated a n d f o r m giant p o l y t e n e c h r o m o s o m e s w i t h distinct b a n d i n g patterns that are v i s i b l e u n d e r the light microscope. Thus, b y examining well-spread chromosome preparations that h a v e b e e n c h a l l e n g e d w i t h a n a n t i b o d y for a p a r t i c u l a r p r o t e i n , one can  77  d e t e r m i n e the p a r t i c u l a r b a n d or i n t e r b a n d w i t h w h i c h the a n t i b o d y a n d hence the p r o t e i n is associated. T h i s technique has b e e n i n v a l u a b l e i n i d e n t i f y i n g the r e g i o n or r e g i o n s w h e r e a p r o t e i n is l o c a l i z e d , h o w e v e r , the t e c h n i q u e does h a v e l i m i t a t i o n s . O b v i o u s l y , i f the e p i t o p e , to w h i c h the a n t i b o d y reacts, is h i d d e n , w h i c h m a y be the case i n s o m e p r o t e i n / p r o t e i n o r c h r o m a t i n c o m p l e x e s , the a n t i b o d y w i l l f a i l to i d e n t i f y that l o c a t i o n of the p r o t e i n . H o w e v e r , the major l i m i t a t i o n of this t e c h n i q u e is its r e s o l u t i o n . A s n o t e d above, one c a n o n l y d e t e r m i n e if a n a n t i b o d y is s t a i n i n g a b a n d or a n i n t e r b a n d . O n l y a few studies h a v e attempted to d e t e r m i n e h o w m a n y genes are contained i n b a n d s or i n t e r b a n d s a n d h a v e f o u n d the n u m b e r v a r i e s c o n s i d e r a b l y , some b a n d s or i n t e r b a n d s h a v e h i g h gene densities w h i l e others c o n t a i n o n l y a few genes ( F r i e d m a n et al., 1991; H a l l et a l . , 1983; S p i e r e r et a l . , 1983). T h u s , one cannot use this t e c h n i q u e to d e t e r m i n e w h e t h e r a p r o t e i n is associated w i t h a specific gene, or w h e t h e r it is b o u n d at the r e g u l a t o r y or c o d i n g regions of that gene. Pile and  Wasserman  (2000) u s e d  antibody  s t a i n i n g of  polytene  c h r o m o s o m e s to ask w h e r e H D A C 1 w a s l o c a t e d i n the D . melanogaster genome. T h e y f o u n d the a n t i - H D A C l  a n t i b o d y b o u n d t h r o u g h o u t the e u c h r o m a t i c  regions of the g e n o m e , p r i m a r i l y i n the i n t e r b a n d , less c o n d e n s e d , regions of e u c h r o m a t i n . S o m e w h a t s u r p r i s i n g l y , it d i d n o t b i n d to e i t h e r a-  or (3-  h e t e r o c h r o m a t i n . T h e y also stained the same p r e p a r a t i o n s w i t h a n a n t i b o d y to D N A p o l y m e r a s e II to m a r k active genes. T h e p a t t e r n of s t a i n i n g d i d not o v e r l a p w i t h a n t i - H D A C l s t a i n i n g . These o b s e r v a t i o n s l e d t h e m to c o n c l u d e that H D A C l ' s p r i m a r y f u n c t i o n w a s the r e p r e s s i o n of genes l o c a t e d i n the less D N A dense, i n t e r b a n d regions of e u c h r o m a t i n , a n d that it d i d not p l a y a role i n  78  condensing  the m o r e  heterochromatic  compact  r e g i o n s of the  D N A structures found genome.  i n the b a n d s o r  Some doubts regarding  these  c o n c l u s i o n s h a v e b e e n r a i s e d b y the a p p l i c a t i o n of a n e w e r , m o r e sensitive, technique (see b e l o w ) . I n a d d i t i o n , e x a m i n a t i o n o f t h e i r p o l y t e n e p r e p a r a t i o n s stained w i t h a n t i - H D A C l r e v e a l a large, intensely s t a i n i n g b a n d at the base of each c h r o m o s o m e a r m (see F i g u r e 2 of P i l e a n d W a s s e r m a n (2000)) i m m e d i a t e l y adjacent to the c h r o m o c e n t e r .  In strains subject to P E V this a r r a n g e m e n t is  altered b y the f o r m a t i o n of a n illicit h e t e r o c h r o m a t i c / e u c h r o m a t i c j u n c t i o n that m a y create n o v e l H D A C l b i n d i n g sites or p e r h a p s the c e l l u l a r response to s u c h a n i l l i c i t j u n c t i o n is to r e c r u i t H D A C l .  U n f o r t u n a t e l y , the d i s t r i b u t i o n of  H D A C l i n a s t r a i n subject to P E V w a s n o t d e t e r m i n e d . R e c e n t l y , t w o techniques h a v e b e e n d e v e l o p e d w h i c h offer m u c h better resolution.  O n e relies o n the a b i l i t y of the D N A m e t h y l transferase ( D A M )  f r o m E. coli to m e t h y l a t e adenine i n the D N A sequence G A T C ( v a n Steensel et al., 2001; v a n S t e e n s e l a n d H e n i k o f f , 2000). m e t h y l a t e d i n eukaryotes.  T h i s r e s i d u e is n o t n o r m a l l y  T h e D A M p r o t e i n is fused to the p r o t e i n of interest  a n d t r a n s f o r m e d i n t o tissue culture cells or w h o l e o r g a n i s m s a n d the D N A at a specific l o c a t i o n is a n a l y z e d w i t h restriction e n z y m e s that r e c o g n i z e G A T C a n d are either s e n s i t i v e o r i n s e n s i t i v e to a d e n i n e m e t h y l a t i o n .  Differences i n  d i g e s t i o n patterns b e t w e e n controls c o n t a i n i n g the D A M p r o t e i n alone a n d cells t r a n s f o r m e d w i t h the D A M - f u s i o n i m p l y the p r o t e i n u n d e r s t u d y is d i r e c t i n g the f u s i o n p r o t e i n to that p a r t i c u l a r r e g i o n . T h e D A M - f u s i o n methylates G A T C o v e r a 2500 base p a i r r e g i o n t h u s p r o v i d i n g e n h a n c e d r e s o l u t i o n o v e r the s t a i n i n g of p o l y t e n e c h r o m o s o m e s w i t h a n t i b o d i e s .  H o w e v e r , this technique  still has l i m i t a t i o n s . In some cases, i n gene p o o r r e g i o n s of c h r o m o s o m e s , one  79  m a y be able to c o n c l u d e , w i t h confidence, that the subject p r o t e i n is associated w i t h a p a r t i c u l a r gene.  H o w e v e r , i n other regions, the genes are v e r y t i g h t l y  p a c k e d a n d the results m a y be a m b i g u o u s r e g a r d i n g w h i c h of t w o , or perhaps three, g e n e s the p r o t e i n is a s s o c i a t e d .  O b v i o u s l y , its r e s o l u t i o n is further  l i m i t e d b y the existence of G A T C sites that c a n be m e t h y l a t e d . Statistically, these sites s h o u l d o c c u r e v e r y 256 base p a i r s h o w e v e r , i n s o m e sites i n the g e n o m e , these sites m a y o c c u r rarely. A n o t h e r d r a w b a c k is that, i n some cases i n Drosophila, e s p e c i a l l y w i t h some c h r o m a t i n p r o t e i n s , it has b e e n difficult or i m p o s s i b l e to o b t a i n g e r m - l i n e t r a n s f o r m a n t s  of the f u s i o n p r o t e i n s .  For  e x a m p l e , w h i l e G A G A , d S I R 2 ( v a n Steensel et a l . , 2001) a n d S U ( V A R ) 3 - 9 (S. Ner,  personal  communication) DAM-fusion  transformants  have  been  successfully r e c o v e r e d , o n l y one transformant of a n H P 1 f u s i o n w a s o b t a i n e d despite s e v e r a l t h o u s a n d attempts ( v a n Steensel, p e r s o n a l c o m m u n i c a t i o n ) . I h a v e injected o v e r 10,000 Drosophila e m b r y o s i n a n a t t e m p t to generate g e r m line transformants  of e i t h e r a c a r b o x y or a m i n o - t e r m i n a l D A M f u s i o n of  H D A C 1 w i t h o u t o b t a i n i n g a single t r a n s f o r m a n t ( u n p u b l i s h e d observations). H o w e v e r , if a transformant  c a n be p r o d u c e d , t h e n a v a l u a b l e resource is  a v a i l a b l e to q u i c k l y assess w h e t h e r the p r o t e i n associates w i t h p o t e n t i a l n e w targets as they c o m e u n d e r scrutiny. S o m e w h a t s u r p r i s i n g l y , this technique has b e e n little u s e d since its w a s i n i t i a l l y r e p o r t e d ( v a n Steensel a n d H e n i k o f f , 2000). P e r h a p s the d i f f i c u l t y i n o b t a i n i n g g e r m - l i n e t r a n s f o r m a n t s is greater t h a n it a p p e a r s or the l i m i t s of its r e s o l u t i o n h a v e d i s c o u r a g e d its use w h e n a technique w i t h s u p e r i o r r e s o l u t i o n is available. T h e t e c h n i q u e m o s t w i d e l y u s e d to ask w h e t h e r a p r o t e i n is associated w i t h a specific D N A sequence  is f o r m a l d e h y d e c r o s s - l i n k e d C h r o m a t i n 80  Immuno-Precipitation (X-ChIP).  T h i s t e c h n i q u e has b e e n u s e d i n e u k a r y o t i c  cells, f r o m the yeasts, S. cerevisiae (Hecht et al., 1996) a n d S. pombe ( N o m a et al., 2001), to a v a r i e t y of tissue culture cells f r o m v a r i o u s h i g h e r o r g a n i s m s ( N i e l s e n et al., 2001). It has also been successfully a p p l i e d to Drosophila e m b r y o s p r i o r to about 16 h o u r s o f d e v e l o p m e n t . A f t e r 16 h o u r s the a b i l i t y o f the f o r m a l d e h y d e to p e n e t r a t e the  embryo  and  c r o s s - l i n k the  proteins  and  D N A drops  p r e c i p i t o u s l y ( C a v a l l i et al., 1999; O r l a n d o a n d P a r o , 1993). X - C h I P is b a s e d o n the a s s u m p t i o n that f o r m a l d e h y d e c a n r a p i d l y penetrate the n u c l e u s of a cell or s m a l l o r g a n i s m a n d c r o s s - l i n k the proteins a n d DNA  w i t h m i n i m a l d i s r u p t i o n o f the n o r m a l d i s t r i b u t i o n p a t t e r n s of the  proteins i n the n u c l e u s . F o l l o w i n g c r o s s - l i n k i n g w i t h f o r m a l d e h y d e , the D N A is sheared b y s o n i c a t i o n i n t o fragments of an average size of b e t w e e n 500 a n d 1000 base p a i r s .  T h e p r o t e i n / D N A c o m p l e x e s are p r e c i p i t a t e d w i t h a n  a n t i b o d y specific for a p a r t i c u l a r p r o t e i n , the c r o s s - l i n k s r e v e r s e d a n d the D N A that w a s p r e c i p i t a t e d a n a l y z e d either b y P C R or S o u t h e r n B l o t s . T h e r e are t w o further a s s u m p t i o n s u p o n w h i c h this technique is based: one, that a l l regions of the D N A are e q u a l l y susceptible to s h e a r i n g b y s o n i c a t i o n after f i x a t i o n w i t h f o r m a l d e h y d e ; a n d t w o , that the epitope the a n t i b o d y r e c o g n i z e s is available to b i n d the a n t i b o d y a n d is not b u r i e d i n a p r o t e i n / p r o t e i n or p r o t e i n / D N A c o m p l e x . F a i l u r e of either of these a s s u m p t i o n s to be true w i l l result i n a false negative, either because the r e g i o n u n d e r s t u d y is m o r e susceptible to shearing b y s o n i c a t i o n a n d therefore is preferentially s h e a r e d a n d e l i m i n a t e d f r o m the analysis or the a n t i b o d y w i l l not precipitate the p r o t e i n because the e p i t o p e is h i d d e n . H o w e v e r , w h e n this technique does p r o v i d e a p o s i t i v e i n d i c a t i o n that  81  a p r o t e i n is associated w i t h a specific sequence t h e n this is g e n e r a l l y accepted as strong e v i d e n c e that a p r o t e i n is i n d e e d associated w i t h the r e g i o n a n a l y z e d . T h e r e s o l u t i o n of this technique is e x t r e m e l y g o o d w h e n P C R is u s e d to a n a l y z e the i m m u n o - p r e c i p i t a t e d D N A . E m p l o y i n g q u a n t i t a t i v e P C R a n d statistical a n a l y s i s , s o m e h a v e r e p o r t e d r e s o l u t i o n d o w n to the l e v e l of the n u c l e o s o m e , a b o u t 150 base p a i r s ( R u n d l e t t et a l . , 1998).  H o w e v e r , i n most  studies, the reports g e n e r a l l y e m p l o y p r i m e r s that a m p l i f y p r o d u c t s of b e t w e e n 200 a n d 500 base p a i r s . M o r e recently R e a l - T i m e P C R has b e e n e m p l o y e d to a n a l y z e the p r o d u c t s  of X - C h l P .  This technique  p r o m i s e s to b r i n g  the  r e s o l u t i o n d o w n to u n d e r 100 base p a i r s a n d p r o d u c e r e s u l t s that c a n be q u a n t i f i e d p r e c i s e l y since the k i n e t i c s of the entire P C R are m o n i t o r e d a n d q u a n t i f i e d accurately d u r i n g the a m p l i f i c a t i o n process ( M i l n e et al., 2002). X - C h i p a n a l y s i s w i t h a n a n t i - H D A C l a n t i b o d y w a s e m p l o y e d i n one s t u d y to ask w h e t h e r H D A C 1 w a s present at v a r i e t y of p r o m o t e r s a n d c o d i n g regions i n Drosophila S L - 2 cells ( B r e i l i n g et al., 2001). T h e report l o o k e d at t w o genes that w e r e e x p r e s s e d , a n d six genes that w e r e not, i n this cell l i n e .  As  expected, H D A C 1 w a s present at the p r o m o t e r a n d the 5' c o d i n g regions o f a l l six genes that w e r e n o t b e i n g e x p r e s s e d .  U n e x p e c t e d l y , H D A C 1 w a s also  present at the active genes Abdominal-B (Abd-B) a n d the l o c u s that codes for the s u b u n i t of R N A p o l y m e r a s e II w i t h a r e l a t i v e m o l e c u l a r m a s s of 140,000 (RpII140). H o w e v e r , its d i s t r i b u t i o n a p p e a r e d to be s o m e w h a t different f r o m that o b s e r v e d at r e p r e s s e d l o c i . I n the case o f Abd-B, H D A C 1 a p p e a r s to be s t r i c t l y l o c a l i z e d to the c o d i n g r e g i o n of the gene a n d w a s not f o u n d i n the promoter regions.  T h e p r i m e r s e m p l o y e d to a n a l y z e RpII140 o v e r l a p the  p r o x i m a l p r o m o t e r a n d the 5' c o d i n g r e g i o n a n d therefore l o c a l i z a t i o n strictly to  82  the c o d i n g r e g i o n w a s n o t c o n f i r m e d , n o r w a s it r u l e d out. T h e results a p p e a r to c o n f i r m H D A C l ' s role as a general t r a n s c r i p t i o n a l repressor. H o w e v e r , the unexpected  f i n d i n g that H D A C 1 also associates w i t h a c t i v e genes l e d the  authors to suggest that p e r h a p s H D A C 1 p l a y s a role i n the r e g u l a t i o n of active genes.  H D A C 1 m a y regulate transcription either b y acting w i t h  histone  a c e t y l t r a n s f e r a s e s ( H A T s ) to m o d u l a t e the l e v e l o f h i s t o n e a c e t y l a t i o n o r alternatively, to regulate the activity of some of the general t r a n s c r i p t i o n factors (GTFs) that are k n o w n to be acetylated as w e l l . In this s t u d y I e m p l o y e d X - C h I P to ask w h e t h e r H D A C 1 is associated w i t h specific regions  of the white* gene i n Drosophila  and whether  this  a s s o c i a t i o n is altered w h e n the white* gene is subject to s i l e n c i n g as a result of PEV.  I n a d d i t i o n , I a s k e d w h e t h e r the associations o b s e r v e d w e r e altered b y  the presence of a m u t a t i o n i n H D A C 1 that is a s t r o n g s u p p r e s s o r of P E V . The data s h o w that w h e n the white* locus is s i l e n c e d d u e to P E V , H D A C 1 is v e r y s t r o n g l y associated w i t h the 1000 base p a i r r e g i o n i m m e d i a t e l y 5' to the white* c o d i n g r e g i o n . In a d d i t i o n , a 500 base p a i r r e g i o n a p p r o x i m a t e l y 6.0 k b 5' to the t r a n s c r i p t i o n a l start is a l s o v e r y s t r o n g l y a s s o c i a t e d  with H D A C 1 .  The  increased association w i t h H D A C 1 w a s c o m p l e t e l y a b o l i s h e d b y a m u t a t i o n i n H D A C 1 that s u p p r e s s e s P E V a n d the l e v e l s of a s s o c i a t i o n r e t u r n to those o b s e r v e d w h e n white* w a s i n its n o r m a l l o c a t i o n . These results suggest that H D A C 1 is a c t i n g d i r e c t l y at the site of the v a r i e g a t i n g gene a n d is a n essential part of the s i l e n c i n g m e c h a n i s m o b s e r v e d i n P E V .  83  Material and Methods  Drosophila  Strains  T h r e e strains w e r e a n a l y z e d b y f o r m a l d e h y d e c r o s s - l i n k e d c h r o m a t i n i m m u n o - p r e c i p i t a t i o n ( X - C h I P ) : Oregon-R ( O R - R ) , a w i l d - t y p e s t r a i n ; In(l)iu"'  4  ( w ) , a s t r a i n b e a r i n g a n X c h r o m o s o m e i n v e r s i o n w h i c h variegates for the w m4  +  gene; a n d In(l)w"' ; HDAC /TM3 4  326  Sb Ser ( w ™ ; 326), a s t r a i n b e a r i n g the X 4  c h r o m o s o m e i n v e r s i o n a n d h e t e r o z y g o u s for a m u t a t i o n i n HDACl.  All  m u t a t i o n s are d e s c r i b e d either i n the text or c a n be f o u n d i n L i n d s l e y a n d Z i m m (1992). T h e flies w e r e r e a r e d at 2 5 ° C o n s t a n d a r d yeast-sucrose-corn meal-agar m e d i u m to w h i c h a m o l d i n h i b i t o r , Tegosept ( m e t h y l - p - h y d r o x y b e n z o a t e ) , a n d antibiotics were a d d e d .  S e v e r a l t h o u s a n d flies of the a p p r o p r i a t e g e n o t y p e  w e r e a d d e d to p o p u l a t i o n cages a n d e m b r y o s for a n a l y s i s w e r e c o l l e c t e d o n agar p l a t e s s u p p l e m e n t e d w i t h a paste m a d e f r o m l i v e yeast.  I n o r d e r to  e l i m i n a t e a n y eggs r e t a i n e d b y the females for a p r o l o n g e d p e r i o d after fertilization, fresh c o l l e c t i o n plates w e r e a d d e d a n d the flies a l l o w e d to l a y eggs for a p p r o x i m a t e l y three h o u r s a n d this first c o l l e c t i o n w a s d i s c a r d e d . N e w plates w e r e a d d e d a n d the flies w e r e a l l o w e d to l a y eggs for four h o u r s . T h e plates w e r e r e m o v e d a n d h e l d at 25°C for 12 h o u r s a n d t h e n p r o c e s s e d for X C h l P . A c c o r d i n g l y all X - C h I P experiments were conducted o n chromatin from 12 - 1 6 h o u r e m b r y o s .  84  Chromatin Immuno-Precipitation (X-ChIP) Sonication T h e p r o t o c o l I e m p l o y e d for X - C h I P w a s m o d i f i e d f r o m those of C a v e l l i et a l . (1999) a n d M a z o ( u n p u b l i s h e d - p r o v i d e d b y D r . H . B r o c k w i t h the p e r m i s s i o n of D r . M a z o ) .  Approximately  1.0 g r a m o f e m b r y o s  were  d e c h o r i o n a t e d b y w a s h i n g i n 3% N a O C l i n E m b r y o W a s h Buffer ( E W B , 0.03 % T r i t o n X 1 0 0 , 0.4% N a C l ) for three m i n u t e s a n d t h e n e x t e n s i v e l y w a s h e d w i t h E W B . T h e e m b r y o s w e r e transferred to a 50 m l . c o n i c a l t u b e a n d w a s h e d once w i t h 0.01% T r i t o n X 1 0 0 i n p h o s p h a t e - b u f f e r e d s a l i n e (PBS).  The PBS was  r e m o v e d a n d 10 m l . of C r o s s - l i n k i n g S o l u t i o n (1.8% f o r m a l d e h y d e , 50 m M H E P E S , 0.5 m M E G T A , 100 m M N a C l p H 8.0) a n d 30 m l . of h e p t a n e w e r e a d d e d a n d v i g o r o u s l y s h a k e n for 15 m i n . E m b r y o s w e r e p e l l e t e d b y s p i n n i n g at 1000 r p m o n a t a b l e t o p centrifuge a n d the C r o s s - l i n k i n g S o l u t i o n / h e p t a n e w a s r e m o v e d . F i f t y m l . of Stop S o l u t i o n ( P B S , 0.125 M g l y c i n e , 0.01% T r i t o n X100) w e r e a d d e d a n d the tube w a s briefly s h a k e n . T h e e m b r y o s w e r e a l l o w e d to s e d i m e n t w i t h o u t c e n t r i f u g a t i o n a n d the S t o p S o l u t i o n r e m o v e d . T e n m l . o f W a s h S o l u t i o n A ( l O m M H E P E S p H 7.6, 10 m M E D T A p H 8.0, 0.5 m M E G T A p H 8.0, 0.25% T r i t o n X100) w e r e a d d e d a n d the e m b r y o s w e r e w a s h e d for 10 m i n . o n a rotator.  W a s h S o l u t i o n A w a s r e m o v e d a n d r e p l a c e d b y 10 m l . of  W a s h S o l u t i o n B (10 m M H E P E S p H 7.6, 200 m M N a C l , 1 m M E D T A p H 8.0, 0.5 m M E G T A p H 8.0, 0.01% T r i t o n X100) a n d the e m b r y o s w e r e w a s h e d for a n a d d i t i o n a l 10 m i n . o n a rotator.  The embryos, i n W a s h Solution B, were  t r a n s f e r r e d to a r o u n d b o t t o m e d centrifuge tube, a l l o w e d to s e d i m e n t a n d W a s h S o l u t i o n B r e m o v e d . S o n i c a t i o n Buffer (10 m M H E P E S p H 7.6, 1 m M  85  E D T A p H 8.0, 0.5 m M E G T A p H 8.0) w a s a d d e d to 5.5 m l . a n d then 0.5 g m . glass b e a d s ( S i g m a G8893) a n d p r o t e a s e i n h i b i t o r s (1.1 u l of 10 m g / m l A p r o t i n i n , 1.1 u l of 10 m g / m l L e u p e p t i n s , 5.5 jul of 1 m g / m l P e p s t a t i n A , 63.2 jul of 50 m M P M S F , 55 u l o f 100 m M B e n z a m i d i n e ) w e r e a d d e d a n d the tube p l a c e d o n ice. T h e e m b r y o s w e r e s o n i c a t e d o n a S o n i c 300 D i s m e m b r a t o r u s i n g the m i c r o t i p at the m a x i m u m setting of 35%. T h e o p t i m a l s o n i c a t i o n p r o c e d u r e w a s d e t e r m i n e d e m p i r i c a l l y b y m o n i t o r i n g the a v e r a g e s i z e of the g e n o m i c D N A o n agarose gels after a series of 30 sec. s o n i c a t i o n pulses. T h e p r o t o c o l s suggest the o p t i m a l average size for g e n o m i c D N A for i m m u n o - p r e c i p i t a t i o n is b e t w e e n 500 a n d 1000 base p a i r s .  I d e t e r m i n e d that a r e g i m e n of six 30 sec.  s o n i c a t i o n s , w i t h a p a u s e of 90 sec. b e t w e e n e a c h p u l s e , p r o d u c e d g e n o m i c D N A w i t h a n average size of about 1000 base pairs. F u r t h e r 30 sec. pulses d i d not s i g n i f i c a n t l y r e d u c e the average size of the D N A . T h e tube w a s m a i n t a i n e d o n ice t h r o u g h o u t the p r o c e d u r e .  S a m p l e s w e r e transferred to 1.5 m l . tubes  a n d c e n t r i f u g e d at 4 ° C for 15 m i n . at m a x i m u m o n a tabletop centrifuge.  The  s u p e r n a t a n t s w e r e either processed i m m e d i a t e l y o r flash f r o z e n a n d stored at 80°C for n o m o r e t h a n a few d a y s before b e i n g further processed. S a m p l e s w e r e p r e p a r e d for i m m u n o - p r e c i p i t a t i o n b y m i x i n g w i t h a n e q u a l v o l u m e of 6.0 M U r e a a n d d i a l y z i n g at 4 ° C for 4 h o u r s i n 1.0 liter of C h I P D i a l y s i s Buffer (10 m M T r i s - H C l p H 8.0,1 m M E D T A p H 8.0, 0.5 m M E G T A p H 8.0, 10% g l y c e r o l , 1% T r i t o n X100, 0.1% N a - d e s o x y c h o l a t e w / v ) s u p p l e m e n t e d w i t h protease i n h i b i t o r s (11.5 m l . of 50 m M P M S F a n d 10.0 m l . of 100 m M Benzamidine).  T h e D i a l y s i s Buffer w a s r e p l a c e d w i t h fresh D i a l y s i s Buffer,  s u p p l e m e n t e d w i t h protease i n h i b i t o r s , a n d the s a m p l e s d i a l y z e d o v e r n i g h t at 86  4°C. T h e s a m p l e s w e r e centrifuged at m a x i m u m s p e e d o n a tabletop centrifuge at 4°C a n d the'supernatants d i v i d e d into 1.0 m l aliquots a n d stored at -80°C.  Immuno-precipitation T h e s o n i c a t e d extract w a s t h a w e d o n ice a n d 100 u l p e r i m m u n o p r e c i p i t a t i o n r e a c t i o n w a s r e m o v e d to a 1.5 m l t u b e a n d protease i n h i b i t o r s a d d e d ( 2.0 u l of 50 m M P M S F , 0.1 u l of 1.0 m g / m l A p r o t i n i n , 0.2 u l of 1 m g / m l P e p s t a t i n A ) . T h e extract w a s p r e - c l e a r e d b y a d d i n g 10 u l o f a P A S / D N A s l u r r y a n d r o t a t e d for 30 m i n . at 4 ° C . T h e P A S / D N A s l u r r y w a s m a d e b y w a s h i n g 100 m g of p r o t e i n - A - s e p h a r o s e b e a d s ( P A S ) w i t h m i l l i - Q  dH 0, 2  r e m o v i n g the w a t e r a n d a d d i n g 600 u g of s o n i c a t e d h e r r i n g s p e r m D N A , 0.33 m g / m l of B S A a n d T E p H 8.0 to m a k e a f i n a l v o l u m e o f 800 u l . A f t e r preclearing, the e x t r a c t / P A S / D N A s l u r r y w a s centrifuged for 1.0 m i n . at 4000 r p m a n d the s u p e r n a t a n t r e m o v e d to a n e w 1.5 m l tube.  F i v e u l of a n a n t i b o d y  p r o d u c e d against a p e p t i d e i d e n t i c a l to the 20 a m i n o a c i d s at the c a r b o x y t e r m i n a l tail of Drosophila H D A C l ( A b e a m L i m i t e d , a b i 7 6 7 ) w e r e a d d e d to a n extract f r o m e a c h o f the three strains to be tested. I n a d d i t i o n c o n t r o l extracts f r o m e a c h s t r a i n , to w h i c h n o a n t i b o d y w a s a d d e d , w e r e p r o c e s s e d .  The  extracts w e r e rotated o v e r n i g h t at 4°C to a l l o w a n t i b o d y b i n d i n g . T h e i m m u n e c o m p l e x e s w e r e c o l l e c t e d b y a d d i n g 40 u l of P A S / D N A s l u r r y to each reaction, i n c l u d i n g the no a n t i b o d y c o n t r o l , a n d rotating for 2 to 3 h o u r s at 4 ° C . T h e b e a d s w e r e p e l l e t e d b y gentle c e n t r i f u g a t i o n (1000 r p m for 1.0 m i n . ) a n d the supernatants r e m o v e d . T h e s u p e r n a t a n t f r o m the n o a n t i b o d y c o n t r o l t u b e w a s s a v e d a n d s e r v e d as the s o u r c e for the I n p u t D N A for  87  subsequent P C R studies. T h e beads w e r e w a s h e d for 5 m i n . at 4 °C w i t h each of the f o l l o w i n g buffers: L o w Salt I m m u n e C o m p l e x W a s h Buffer = 0.1% S D S , 1.0 % T r i t o n X100, 2.0 m M E D T A p H 8.0, 20 m M T r i s - H C l p H 8.1,150 m M N a C l ; H i g h Salt I m m u n e C o m p l e x W a s h Buffer = 0.1% S D S , 1.0 % T r i t o n X100, 2.0 m M E D T A p H 8.0, 20 m M T r i s - H C l p H 8.1, 500 m M N a C l ; L i C l I m m u n e C o m p l e x W a s h Buffer = 0.25 M L i C l , 1.0% N P 4 0 , 1 . 0 % N a desoxycholate, 1.0 m M E D T A p H 8.0,10 m M T r i s - H C l p H 8.1; T E = 10 m M T r i s - H C l p H 8.0,1.0 m M E D T A p H 8.0; T E = 10 m M T r i s - H C l p H 8.0,1.0 m M E D T A p H 8.0. A f t e r the last w a s h buffer w a s r e m o v e d , 250 u l of freshly m a d e E l u t i o n Buffer (1.0% S D S , 0.1 M N a H C 0 ) w a s a d d e d , the m i x t u r e w a s v o r t e x e d briefly, 3  a n d the i m m u n e c o m p l e x e s w e r e e l u t e d b y r o t a t i n g for 15 m i n . at r o o m temperature.  T h e m i x t u r e w a s c e n t r i f u g e d for 2 m i n . at 9000 r p m a n d the  s u p e r n a t a n t r e m o v e d to a fresh tube.  A second elution was performed w i t h  another 250 pi\ a l i q u o t of E l u t i o n Buffer a n d the eluates c o m b i n e d . In o r d e r to reverse the f o r m a l d e h y d e - i n d u c e d c r o s s - l i n k s , 20 u l of 5 M N a C l w a s a d d e d to each eluate a n d the tubes i n c u b a t e d at 6 5 ° C o v e r n i g h t . T h e DNA  i n the s a m p l e s w a s e t h a n o l p r e c i p i t a t e d , w i t h the a d d i t i o n o f 20 jug  g l y c o g e n as a carrier, a n d r e s u s p e n d e d i n 50 u l T E p H 8.0.  Polymerase Chain Reaction Analysis (PCR) T h e D N A p r e c i p i t a t e d i n the above reactions w a s a n a l y z e d w i t h P C R e m p l o y i n g p r i m e r s pairs for specific regions of the D . melanogaster g e n o m e 5' to the start of w gene t r a n s c r i p t i o n (see F i g u r e 1). T h e p r i m e r p a i r s for the +  88  P r o x i m a l p r o m o t e r were: G ATTCCGGGGCCTG A G ATGAGGTGC /GGTACTTCAAAT ACCCTTGG ATC G ; for the D i s t a l P r o m o t e r were: GTTGTCTGTCACT A G ATCGGCCC / GCACCTCATCTC A G G C C C C G G AATC; a n d , for the 5' D i s t a l R e g i o n w e r e CG A C T C T G C G T C G C T G T C T C G / G T A T G C A G C A G A ATT A G C A G A A G . Two m i c r o l i t e r s of D N A f r o m each s a m p l e w a s a m p l i f i e d a c c o r d i n g to the f o l l o w i n g p r o t o c o l : Step 1 = 94°C for 1 m i n . , Step 2 = 92°C for 30 s e c , Step 3 = 58°C for 30 s e c , Step 4 = 7 5 ° C for 1 m i n . , Step 5 = repeat Step 2 to Step 4 29 times, Step 6 = 75°C for 5 m i n . U n d e r these c o n d i t i o n s a s i n g l e b a n d of D N A , a p p r o x i m a t e l y 500 base p a i r s l o n g , w a s p r o d u c e d b y each p r i m e r p a i r . T h e a m o u n t of D N A p r o d u c e d b y P C R w a s q u a n t i f i e d b y r u n n i n g aliquots of each P C R reaction o n a n agarose gel a n d s t a i n i n g w i t h S Y B R G r e e n 1 a c c o r d i n g to the manufacturer's i n s t r u c t i o n s ( M o l e c u l a r Probes). S Y B R G r e e n 1 is a fluorescent stain that is specific for d o u b l e - s t r a n d e d D N A a n d its s i g n a l is l i n e a r to the a m o u n t of D N A present i n a l l ranges r e p o r t e d .  T h e a m o u n t of  D N A p r o d u c e d after 30 P C R cycles w a s q u a n t i f i e d o n a S t o r m 860 P h o s p h o Imager ( A m e r s h a m P h a r m a c i a Biotech) b y e x c i t a t i o n at 450 n m a n d m e a s u r i n g e m i s s i o n at 520 n m . I n i t i a l e x p e r i m e n t s h a d d e t e r m i n e d that, u n d e r the P C R c o n d i t i o n s e m p l o y e d here, the D N A p r o d u c e d at 30 cycles w a s w i t h i n the linear range of a m p l i f i c a t i o n (see F i g u r e 2). F o r each fly s t r a i n a n d each p r i m e r p a i r the a m o u n t of D N A p r o d u c e d b y P C R i n the m o c k treated, " N o A n t i b o d y " c o n t r o l , i m m u n o - p r e c i p i t a t i o n w a s c o m p a r e d to the a m o u n t p r o d u c e d b y the a n t i - H D A C l a n t i b o d y a n d the results expressed as "fold enhancement".  89  Results A s r e p o r t e d i n C h a p t e r 2, specific a m i n o a c i d substitutions i n Drosophila H D A C l cause s t r o n g d o m i n a n t s u p p r e s s i o n of white* gene v a r i e g a t i o n i n the w'" s t r a i n . 4  H o w e v e r , since H D A C l  is i n v o l v e d i n a l a r g e ( a n d g r o w i n g )  n u m b e r of p r o t e i n c o m p l e x e s a n d b i n d s to, a n d p e r h a p s regulates, a large number  of genes, it w a s of considerable interest  m u t a t i o n s i n HDACl  to d e t e r m i n e  whether  cause s u p p r e s s i o n b y a c t i n g d i r e c t l y o n the v a r i e g a t i n g  locus or w h e t h e r s u p p r e s s i o n is i n d i r e c t a n d the result of d e r e g u l a t i o n of one or m o r e H D A C l - r e g u l a t e d genes. I addressed  this question b y e m p l o y i n g f o r m a l d e h y d e - c r o s s - l i n k e d  c h r o m a t i n i m m u n o - p r e c i p i t a t i o n ( X - C h I P ) . In brief, this technique relies o n the a b i l i t y of f o r m a l d e h y d e to cross-link closely associated p r o t e i n s a n d D N A .  The  c r o s s - l i n k i n g d i s t a n c e of f o r m a l d e h y d e is e s s e n t i a l l y z e r o , since it does not c o n t a i n a l i n k e r , a n d t h u s o n l y proteins i n v e r y close p r o x i m i t y to the D N A w i l l be c r o s s - l i n k e d to it.  A f t e r t r e a t m e n t w i t h f o r m a l d e h y d e , the c r o s s - l i n k e d  proteins a n d D N A are sonicated to shear the D N A i n t o fragments, i d e a l l y of a n average size of b e t w e e n 500 a n d 1000 base p a i r s , w h i c h are t h e n c h a l l e n g e d w i t h an a n t i b o d y to the p r o t e i n of interest. A f t e r a l l o w i n g sufficient time for b i n d i n g , the a n t i b o d y / p r o t e i n / D N A c o m p l e x e s are p r e c i p i t a t e d b y b i n d i n g the a n t i b o d y w i t h P r o t e i n A l i n k e d to agarose beads. G e n t l e centrifugation a l l o w s the entire c o m p l e x to be p r e c i p i t a t e d . A f t e r extensive w a s h i n g the cross-links are r e v e r s e d b y heat treatment a n d the p r e c i p i t a t e d D N A is a n a l y z e d .  One  i n v a r i a b l y i n c l u d e s a m o c k treated s a m p l e ("No A n t i b o d y " ) that w a s e x p o s e d to the P r o t e i n A a g a r o s e b e a d s a l o n e to p r o v i d e a b a s e l i n e for n o n - s p e c i f i c p r e c i p i t a t i o n of D N A b y the p r o t e i n A c o u p l e d to the agarose beads.  90  The  a m o u n t of a specific D N A sequence p r e c i p i t a t e d i n the m o c k treated s a m p l e is compared  to the a m o u n t  p r e c i p i t a t e d b y the a n t i b o d y e m p l o y i n g P C R  a m p l i f i c a t i o n a n d p r i m e r s specific for the r e g i o n of interest.  If the p r o t e i n is  b o u n d to the s e q u e n c e of interest t h e n one s h o u l d o b s e r v e a s e v e r a l f o l d e n h a n c e m e n t i n the a m o u n t of the specific sequence i n the a n t i b o d y treated s a m p l e w h e n c o m p a r e d to the m o c k treated s a m p l e . T h i s t e c h n i q u e has b e e n v e r y successfully a p p l i e d to a w i d e v a r i e t y o f systems, i n p a r t i c u l a r yeast a n d e u k a r y o t i c cell l i n e s , a n d has r e v o l u t i o n i z e d o u r u n d e r s t a n d i n g of c h r o m a t i n structure.  H o w e v e r , there has b e e n a d e a r t h of r e p o r t s o n its a p p l i c a t i o n i n  m u l t i c e l l u l a r o r g a n i s m s . O n e of the o b v i o u s d r a w b a c k s to u s i n g m u l t i c e l l u l a r o r g a n i s m s as a s u b s t r a t e for X - C h I P is that a p a r t i c u l a r p r o t e i n m a y be associated w i t h a specific D N A sequence i n o n l y one tissue t y p e a n d e v e n that m a y o c c u r at o n l y a p a r t i c u l a r t i m e d u r i n g d e v e l o p m e n t .  A c c o r d i n g l y , the  n u m b e r of cells i n w h i c h the p r o t e i n of interest is a s s o c i a t e d w i t h the target sequence m a y be s m a l l a n d w i l l not p r o d u c e a s t r o n g e n o u g h s i g n a l to be s i g n i f i c a n t l y different f r o m the m o c k treated s a m p l e . T h u s , the m o s a i c nature of e p i g e n e t i c states i n a m u l t i c e l l u l a r o r g a n i s m m a y r e n d e r this t e c h n i q u e u n u s a b l e for the analysis of m a n y or most proteins i n intact c o m p l e x organisms. In the present case I t h o u g h t I c o u l d e m p l o y X - C h I P to ask i f H D A C l is associated w i t h the white* gene i n w"' because some of the characteristics of P E V 4  suggest  the  signature  variegating expression  pattern  is c a u s e d  by  a  p h e n o m e n o n o c c u r r i n g t h r o u g h o u t the o r g a n i s m a n d not s o l e l y i n the tissues i n w h i c h the v a r i e g a t i n g gene is expressed. First, i n Drosophila, c l o n a l analysis of v a r i e g a t i o n of the white* gene i n the eye a n d t e m p e r a t u r e shift studies of several genes a l l p o i n t to a v e r y early d e t e r m i n a t i v e event i n e s t a b l i s h i n g the s i l e n c i n g  91  a s s o c i a t e d w i t h P E V (see C h a p t e r 1 for details).  W h i l e this has o n l y b e e n  s t u d i e d for a f e w genes, the results consistently p o i n t to some event o c c u r r i n g about the t i m e of c e l l u l a r b l a s t o d e r m f o r m a t i o n , regardless of the l o c i i n v o l v e d , w h i c h is a p p r o x i m a t e l y the t i m e h e t e r o c h r o m a t i n first a p p e a r s i n Drosophila e m b r y o s a n d the z y g o t i c g e n o m e b e c o m e s t r a n s c r i p t i o n a l l y active ( L a w r e n c e , 1992). T h u s i f P E V is the result of " h e t e r o c h r o m a t i n i z a t i o n " of a v a r i e g a t i n g gene, it m a y o c c u r i n a l l cells at r o u g h l y the s a m e t i m e , a r o u n d b l a s t o d e r m f o r m a t i o n , a n d w e l l before the e m b r y o s are h a r v e s t e d (12 to 16 hours) i n m y experiments.  S e c o n d , m i c r o s c o p y s t u d i e s h a v e e x a m i n e d the e x t e n t o f  s p r e a d i n g of h e t e r o c h r o m a t i n i n the p o l y t e n e c h r o m o s o m e s of  Drosophila.  There w a s a s t r o n g c o r r e l a t i o n b e t w e e n the extent of s p r e a d i n g i n the polytenes a n d the p r o p o r t i o n of cells i n w h i c h a v a r i e g a t i n g g e n e is e x p r e s s e d C h a p t e r 1 for d e t a i l s ) .  This correlation, between polytene  (see  chromosome  m o r p h o l o g y i n the s a l i v a r y g l a n d s late 3 r d instar l a r v a e a n d the e x p r e s s i o n of v a r i e g a t i n g genes i n the cells of the a d u l t , suggests that, once b e y o n d c e l l u l a r blastoderm,  the  architecture  of the  chromatin  surrounding  the  new  h e t e r o c h r o m a t i c / e u c h r o m a t i c j u n c t i o n m a y be the same, or r o u g h l y the same, i n a l l cells regardless of tissue t y p e or d e v e l o p m e n t a l stage. T h i r d , m u t a t i o n s i n HDACl  suppress several different v a r i e g a t i n g genes (see C h a p t e r 2, T a b l e 1). It  is possible that m u t a t i o n s i n HDACl v i a d e r e g u l a t i o n of a d i f f e r e n t  i n d i r e c t l y s u p p r e s s e a c h o f the other genes set of genes, b u t  e x p l a n a t i o n is that m u t a t i o n s i n HDACl  a more  parsimonious  h a v e t h e i r effect v i a a c o m m o n  m e c h a n i s m that o c c u r s t h r o u g h o u t the g e n o m e a n d s u p p r e s s e s these genes regardless of the tissue i n w h i c h they are e x p r e s s e d .  92  T h i s c o u l d be the early  "determinative" event i n P E V that c l o n a l analysis a n d t e m p e r a t u r e shift studies have identified. I e m p l o y e d X - C h I P i n 12 to 16 h o u r Drosophila  embryos and a X-ChIP  q u a l i t y , c o m m e r c i a l a n t i b o d y , specific for D . melanogaster  HDACl  (Abeam  L i m i t e d , a b l 7 6 7 ) to ask i f H D A C l is associated w i t h s p e c i f i c r e g i o n s of the v a r i e g a t i n g white* gene a n d further, i f t h i s a s s o c i a t i o n w a s a l t e r e d i n the HDACl  mutations  that s u p p r e s s e d  PEV.  F i g u r e 1 is a  r e p r e s e n t a t i o n of the g e n o m i c r e g i o n 5' to the white* gene.  diagrammatic  T h e white* gene  p r o m o t e r has b e e n e x t e n s i v e l y c h a r a c t e r i z e d ( D a v i s o n et al., 1985; L e v i s et al., 1985; P i r r o t t a et al., 1985). I chose three r e g i o n s to e x a m i n e for the presence of H D A C l .  T h e first  r e g i o n , the P r o x i m a l P r o m o t e r , is the 500 b p i m m e d i a t e l y 5' to the start of white* gene t r a n s c r i p t i o n a n d c o n t a i n s the m i n i m a l p r o m o t e r f o r the l o c u s .  The  second r e g i o n , the D i s t a l P r o m o t e r , is the 500 base p a i r s i m m e d i a t e l y 5' to the P r o x i m a l P r o m o t e r . F i n a l l y I chose a 500 base p a i r r e g i o n about 6.0 kilobases 5' to the t r a n s c r i p t i o n a l start of white*, the 5 ' D i s t a l R e g i o n . I chose the P r o x i m a l and D i s t a l P r o m o t e r regions because they s h o u l d s h o w whether  HDACl  associates w i t h the r e g u l a t o r y regions of the white* gene w h e n it is subject to PEV.  I chose the 5' D i s t a l r e g i o n because c o m p a r a t i v e a n a l y s i s of the three  r e g i o n s m i g h t p r o v i d e s o m e i n s i g h t i n t o the m o l e c u l a r m e c h a n i s m of P E V . W h i l e the m o l e c u l a r m e c h a n i s m u n d e r l y i n g P E V has n o t b e e n e l u c i d a t e d , the m o s t e n d u r i n g t h e o r y is that s i l e n c i n g s p r e a d s o u t f r o m the h e t e r o c h r o m a t i c b r e a k p o i n t b y a l t e r i n g the structure of c h r o m a t i n . continuous structural change  If " s p r e a d i n g " i n v o l v e s a  i n the c h r o m a t i n e m a n a t i n g  out from  the  b r e a k p o i n t , t h e n one m i g h t expect to see n e w p r o t e i n associations, s u c h as w i t h  93  5' Distal Region  Distal Promoter  Proximal Promoter  primer site _|  Figure 1.  500 base pairs  D i a g r a m a t i c representation of the g e n o m i c r e g i o n 5' to the w+gene i n D . melanogaster. T h e regions a n a l y z e d b y X - C h I P a n d P C R are i n d i c a t e d . T h e f i l l e d a r r o w indicates the start of t r a n s c r i p t i o n a n d the b o x e d r e g i o n s h o w s the first exon.  94  H D A C l , n o t o n l y at the p r o m o t e r of a s i l e n c e d v a r i e g a t i n g gene, b u t also further a w a y f r o m the b r e a k p o i n t , i n this case, further 5' to the white* gene. O n the other h a n d , i f H D A C l ' s n e w associations w e r e just at specific sequences, for e x a m p l e at p r o m o t e r sequences, t h e n a n y m o d e l a t t e m p t i n g to e x p l a i n P E V w o u l d h a v e to a c c o m m o d a t e s u c h selective associations. A c c o r d i n g l y , as a first attempt at e x a m i n i n g the q u e s t i o n of s p r e a d i n g I chose the 5' D i s t a l R e g i o n . F o r c o m p a r a t i v e p u r p o s e s I e x a m i n e d these r e g i o n s f r o m the white* gene i n three strains. First, i n the w i l d - t y p e strain Oregon-R ( O R - R ) , w h e r e the white* gene is i n its n o r m a l l o c a t i o n near the t i p of the X c h r o m o s o m e . S e c o n d , i n the i n v e r s i o n strain, In(l)w"' (w " ). I n this strain, the i n v e r s i o n relocates the white* 4  gene  to  within  1  4  about  25  kilobases  of  the  newly  formed  h e t e r o c h r o m a t i c / e u c h r o m a t i c j u n c t i o n (Tartof et a l . , 1984) a n d as a result the white* gene is subject to P E V . T h e white* gene is s t r o n g l y s i l e n c e d at this l o c a t i o n a n d is o n l y expressed i n a p p r o x i m a t e l y 5-15% of the eye p i g m e n t cells. F i n a l l y , the r e g i o n s w e r e e x a m i n e d i n the i n v e r s i o n s t r a i n w h i c h bears the HDACl  326  m u t a t i o n , In(l)w'" ; HDACl /TM3SbSer 4  326  (w ; 326). P E V is s t r o n g l y  s u p p r e s s e d as a r e s u l t of the m u t a t i o n i n HDACl  m4  a n d the white* gene is  expressed i n 80-90% of the eye p i g m e n t cells (see C h a p t e r 2, T a b l e l ) . I e m p l o y e d P C R to a n a l y z e the p r o d u c t s of X - C h I P reactions. In order to c o m p a r e r e l a t i v e differences b e t w e e n the p r o d u c t s o f P C R reactions, one m u s t e n s u r e the p r o d u c t s are q u a n t i f i e d d u r i n g the l i n e a r p h a s e o f the P C R amplification.  T o d e t e r m i n e the l i n e a r p h a s e o f P C R u n d e r the c o n d i t i o n s I  e m p l o y e d , I u s e d the p r i m e r s s p e c i f i c f o r e a c h r e g i o n a n d q u a n t i f i e d the p r o d u c t p r o d u c e d after a set n u m b e r of P C R cycles. A n e x a m p l e is s h o w n i n F i g u r e 2, w h i c h s h o w s the results of P C R a m p l i f i c a t i o n o f the D i s t a l P r o m o t e r  95  50000  n  40000  -  C  30000  -  ^  20000  -  10000  -  -1 24  1 26  1 28  1 30  1 32  1 34  1 36  P C R Cycles r  Figure 2  2  = 0.981  G r a p h of the r e l a t i o n s h i p b e t w e e n fluorescence ( A m o u n t of D N A ) a n d P C R C y c l e i n the D i s t a l P r o m o t e r R e g i o n (see F i g u r e 1). T h e a m o u n t of D N A w a s q u a n t i f i e d o n a S t o r m 860 P h o s p h o - I m a g e r (see M a t e r i a l s a n d M e t h o d s for details).  96  region.  F o r a l l r e g i o n s u n d e r the c o n d i t i o n s I e m p l o y e d , a m p l i f i c a t i o n w a s  l i n e a r b e t w e e n 26 a n d 32 cycles. A c c o r d i n g l y , i n a l l s u b s e q u e n t e x p e r i m e n t s , the P C R w a s t e r m i n a t e d  after 30 c y c l e s a n d the a m o u n t  of the p r o d u c t  v i s u a l i z e d o n a n agarose gel stained b y e t h i d i u m b r o m i d e a n d q u a n t i f i e d u s i n g S Y B R G r e e n 1 a n d a p h o s p h o - i m a g e r (see M a t e r i a l s a n d M e t h o d s for details). T h e D N A I a n a l y z e d b y P C R came f r o m three sources for each strain. T h e first w a s total g e n o m i c D N A that h a d b e e n c r o s s - l i n k e d , sonicated a n d the cross-links r e v e r s e d (Input D N A ) .  T h e second w a s the D N A p r e c i p i t a t e d f r o m  the I n p u t D N A b y the p r o t e i n A agarose b e a d s alone ( N o A n t i b o d y ) a n d the t h i r d w a s the D N A p r e c i p i t a t e d f r o m the I n p u t D N A b y the a n t i - H D A C l antibody ( a n t i - H D A C l ) .  Each D N A source w a s a n a l y z e d b y P C R u s i n g  p r i m e r s specific for each r e g i o n . F i g u r e 3 presents the results o b t a i n e d f r o m a n a l y s i s of the P r o x i m a l P r o m o t e r r e g i o n , F i g u r e 4 the D i s t a l P r o m o t e r r e g i o n a n d F i g u r e 5 the 5' D i s t a l r e g i o n . It is apparent f r o m the agarose gels (panel A , F i g u r e s 3, 4, a n d 5) that i n each r e g i o n , r e l a t i v e to the N o A n t i b o d y c o n t r o l , there is c o n s i d e r a b l y m o r e P C R p r o d u c t i n the a n t i - H D A C l lane of w'" t h a n 4  there is i n the OR-R lane. It is also apparent that, i n the i n v e r s i o n strain b e a r i n g the H D A C l m u t a t i o n , w'" ;326, the a m o u n t of the P C R p r o d u c t i n the a n t i 4  H D A C l l a n e r e t u r n s to a p p r o x i m a t e l y the l e v e l s o b s e r v e d i n OR-R.  Thus,  u n d e r c o n d i t i o n s w h e r e the white* gene is s i l e n c e d d u e to P E V , H D A C l is s t r o n g l y a s s o c i a t e d w i t h the p r o m o t e r a n d r e g u l a t o r y sequences i n the 1.0 k i l o b a s e i m m e d i a t e l y 5' to the white* gene ( p a n e l A i n F i g u r e 3 a n d 4). T h i s association is n o t restricted to the sequences adjacent white* a n d H D A C l is also f o u n d at the 5' D i s t a l r e g i o n , some 6.0 k i l o b a s e s 5' to white* ( p a n e l A i n F i g u r e 5).  97  No  g Strain  Antibody  Enhanced  2115  11,542  5.5  m4  1154  22,530  19.5  1983  11,025  5.6  w  m4  ;  3  2  6  Proximal Promoter  25 B g  HDACl  Fold  OR-R w  S  Anti  20 15  H io w 2o  5 0 OR-R  w  m4  w  m  ;  4  326  Strain  Figure 3  H D A C l is associated w i t h the P r o x i m a l P r o m o t e r of w  +  b u t the l e v e l is elevated i n w . m 4  i n a l l strains  ( A ) is a s a m p l e agarose gel, (B) is the  a m o u n t fluorescence m e a s u r e d o n a p h o s p h o - i m a g e r a n d (C) s h o w s the l e v e l of H D A C l enhancement i n each strain relative to the " N o A n t i b o d y " controls.  98  A  No Input  anti  Antibody  HDACl  OR-R  w w  m4  m4  ; 326  B No Strain  Anti  Antibody  Fold  HDACl  Enhanced  OR-R  6113  17,163  2.8  m4  2177  30,285  13.9  8763  11,443  1.3  w  w  m  4  ; 326  Distal Promoter  25  ncem  es 20 15  ed  2 10 m "3  5 0 OR-R  w  m4  w  m 4 .326  Strain  Figure 4  H D A C l is associated with the Distal Promoter of the w gene in w +  not w i t h the Distal Promoter of OR-R or w ; m4  m4  but  326. (A) is a sample agarose  gel, (B) is the amount of fluorescence measured on a phospho-imager and (C) shows the level of H D A C l enhancement i n each strain relative to the "No Antibody" controls. 99  No Input  anti  Antibody  HDACl  OR-R w w  m4 m4  ;326  B Anti  No Antibody  Strain  HDACl  Fold Enhanced  OR-R  6382  16,295  2.6  m4  1395  29,965  21.5  3998  10,507  2.6  w  w  m 4  ;  326  5 Distal Region f  25  S  20  s | w  2o  15  5 0  OR-R  w  m4  w  m  4  ;  326  Strain  Figure 5  H D A C l is associated w i t h the 5' D i s t a l R e g i o n of the zv gene i n +  b u t not w i t h the 5 D i s t a l R e g i o n of OR-R or w ; m4  w  m4  326. ( A ) is a s a m p l e  agarose gel, (B) is the a m o u n t of fluorescence m e a s u r e d o n a p h o s p h o i m a g e r a n d (C) s h o w s the l e v e l of enhancement i n each strain at this r e g i o n relative to the " N o A n t i b o d y " controls.  100  In o r d e r to quantify these differences, aliquots o f the P C R reactions w e r e run  o n agarose gels a n d s t a i n e d w i t h S Y B R G r e e n 1.  T h e a m o u n t of the  p r o d u c t s w a s q u a n t i f i e d o n a p h o s p h o - i m a g e r a n d the results are presented i n t a b u l a r f o r m ( p a n e l B , F i g u r e s 3, 4, a n d 5) a n d i n g r a p h i c a l f o r m (panel C , F i g u r e s 3, 4, a n d 5) In OR-R, at the D i s t a l P r o m o t e r r e g i o n ( F i g u r e 4 B a n d C ) a n d the 5' D i s t a l R e g i o n ( F i g u r e 5B a n d C ) there w a s a s l i g h t e n h a n c e m e n t i n the a m o u n t of p r o d u c t p r o d u c e d f r o m the a n t i - H D A C l s a m p l e relative to the N o A n t i b o d y controls, b e t w e e n a 2.6 a n d a 2.8 f o l d enhancement.  I n the P r o x i m a l P r o m o t e r  r e g i o n ( F i g u r e 3B a n d C ) there w a s a 5.5 f o l d e n h a n c e m e n t .  F r o m m y data, I  cannot d e t e r m i n e w h e t h e r this is d u e to n o n - s p e c i f i c p r e c i p i t a t i o n of the D N A sequences b y the a n t i b o d y o r represents a s m a l l b u t s i g n i f i c a n t a m o u n t of H D A C l b o u n d to a l l three regions i n OR-R. H o w e v e r , the increases are v e r y s m a l l at the D i s t a l P r o m o t e r a n d the 5' D i s t a l R e g i o n . I h a v e p e r f o r m e d P C R w i t h p r i m e r s specific for other sequences i n the Drosophila g e n o m e , u n r e l a t e d to the white* gene, a n d a l l are s l i g h t l y i n c r e a s e d i n the s a m p l e f r o m the a n t i H D A C l a n t i b o d y r e l a t i v e to the N o A n t i b o d y c o n t r o l s (data n o t s h o w n ) . I n v i e w of these o b s e r v a t i o n s I b e l i e v e the s l i g h t e n h a n c e m e n t o b s e r v e d at the D i s t a l P r o m o t e r a n d the 5' D i s t a l R e g i o n are l i k e l y the result of n o n - s p e c i f i c p r e c i p i t a t i o n b y the a n t i b o d y .  O n the other h a n d , i n OR-R, the e n r i c h m e n t  o b s e r v e d at the P r o x i m a l P r o m o t e r is s o m e w h a t h i g h e r , i n fact a l m o s t d o u b l e that o b s e r v e d i n the other r e g i o n s .  T h i s m a y r e p r e s e n t a bone fide site o f  H D A C l b i n d i n g a n d suggests H D A C l m a y be i n v o l v e d i n the r e g u l a t i o n of the white* gene i n its n o r m a l l o c a t i o n o n the X c h r o m o s o m e .  101  In contrast, a l l regions of the white* gene i n the w'" s t r a i n s h o w s t r o n g 4  association w i t h H D A C l . I n w " , the a m o u n t of P C R p r o d u c t for each r e g i o n i n 1  4  the a n t i - H D A C l s a m p l e w a s m u c h h i g h e r t h a n that o b s e r v e d i n the N o A n t i b o d y controls, f r o m a 13.9 f o l d e n r i c h m e n t at the D i s t a l P r o m o t e r (Figure 4B a n d C ) to a 21.5 f o l d e n r i c h m e n t at the 5' D i s t a l R e g i o n ( F i g u r e 5B a n d C ) . T h i s represented a n increase of r o u g h l y 5 fold o v e r the levels o b s e r v e d i n OR-R. T h e l e v e l of e n h a n c e m e n t w a s n o t u n i f o r m t h r o u g h o u t the r e g i o n s tested, s u g g e s t i n g that different l e v e l s of H D A C l m a y be a s s o c i a t e d w i t h different regions.  W h i l e t h e differences are s i g n i f i c a n t , as i n t h e case o f the D i s t a l  P r o m o t e r (13.9 fold) as c o m p a r e d to the P r o x i m a l P r o m o t e r (19.5 fold) a n d the 5' D i s t a l R e g i o n (21.5 f o l d ) , it is n o t clear w h a t the i m p l i c a t i o n s of these difference are. A n y c o n c l u s i o n s m u s t await the creation of a m o r e accurate m a p of the d i s t r i b u t i o n of H D A C l i n the white* r e g i o n o f w'" u s i n g P C R p r i m e r s 4  distributed throughout  this i n t e r v a l .  H o w e v e r , w h a t is clear is that the  p r o m o t e r of white*, a n d p e r h a p s the entire r e g i o n c o n t a i n i n g the white* gene, s h o w s a s i g n i f i c a n t increase i n its association w i t h H D A C l w h e n i t is s i l e n c e d due to P E V i n the w"' strain. 4  H o w e v e r , w h e n the w'" s t r a i n carries a m u t a t i o n i n HDACl 4  s u p p r e s s e s P E V , HDACl , 326  the i n c r e a s e d a s s o c i a t i o n o f H D A C l  that  with all  regions tested is a b o l i s h e d . I n fact, the p a t t e r n of H D A C l association becomes i d e n t i c a l to that seen i n OR-R, w i t h the D i s t a l P r o m o t e r ( F i g u r e 4B a n d C ) a n d 5' D i s t a l R e g i o n ( F i g u r e 5B a n d C ) s h o w i n g little or n o a s s o c i a t i o n w i t h H D A C l w h i l e the P r o x i m a l P r o m o t e r ( F i g u r e 3B a n d C ) d i s p l a y s a s l i g h t l y i n c r e a s e d association w i t h H D A C l .  102  T h e s e d a t a p r o v i d e c o m p e l l i n g e v i d e n c e that H D A C l  is s p e c i f i c a l l y  associated w i t h the ivhite gene i n w'" , b u t o n l y w h e n the gene is s i l e n c e d d u e to +  4  P E V . I n the same strain, b e a r i n g a m u t a t i o n i n HDACl,  P E V is suppressed, the  white* gene is active a n d H D A C l is n o l o n g e r associated w i t h the r e g i o n . T h i s p r o v i d e s the first e v i d e n c e that the p r o d u c t of a Su(var) gene is a c t i n g d i r e c t l y at a v a r i e g a t i n g l o c u s a n d suggests H D A C l is a n essential c o m p o n e n t of the s i l e n c i n g m e c h a n i s m at w o r k i n P E V . It is i m p o r t a n t to note that, i n the presence of HDACl , 326  p i g m e n t levels  i n the eye are o n l y i n c r e a s e d to about 85% of the a m o u n t o b s e r v e d i n OR-R. T h i s difference w a s n o t detected b y the X - C h I P e x p e r i m e n t s d e s c r i b e d here a n d the results f r o m OR-R a n d w'" ; 326 a p p e a r a l m o s t i d e n t i c a l . T h e i n a b i l i t y to 4  detect a n y r e s i d u a l H D A C l  at the white* p r o m o t e r i n the w"' ; 326 s t r a i n 4  suggests that, either s i l e n c i n g re-establishes itself after late e m b r y o g e n e s i s , p e r h a p s w i t h o u t a r e q u i r e m e n t for H D A C l , o r that the l i m i t s of s e n s i t i v i t y of this p a r t i c u l a r m e t h o d of X - C h I P h a v e b e e n r e a c h e d a n d it c a n n o t detect r e l a t i v e l y s m a l l differences. If the later is the case t h e n c a u t i o n m u s t be a p p l i e d w h e n a t t e m p t i n g to d r a w c o n c l u s i o n s f r o m s m a l l differences i n a p p a r e n t H D A C l association at the v a r i o u s regions.  103  Discussion T h e p o i n t m u t a t i o n s i n HDACl,  d e s c r i b e d i n C h a p t e r 2, w e r e isolated i n  a g e n e t i c s c r e e n for d o m i n a n t m u t a t i o n s  that s u p p r e s s  the  variegating  p h e n o t y p e a s s o c i a t e d w i t h P E V ( M o t t u s , 1983; S i n c l a i r et a l . , 1983).  These  m u t a t i o n s s u p p r e s s P E V g e n e r a l l y a n d are not specific m o d i f i e r s of the white* gene. There is a large a n d g r o w i n g b o d y of e v i d e n c e that H D A C l is a m e m b e r of several p r o t e i n c o m p l e x e s a n d is i n v o l v e d i n the r e g u l a t i o n of m a n y genes (for e x a m p l e s see C h a p t e r 1 a n d C h a p t e r 2). T h i s raises the q u e s t i o n of w h e t h e r the d o m i n a n t m u t a t i o n s i n HDACl  characterized here are a c t i n g d i r e c t l y o n the  v a r i e g a t i n g l o c u s o r w h e t h e r the s u p p r e s s i o n of P E V is i n d i r e c t , the result of d e r e g u l a t i o n of one or m o r e H D A C l - r e g u l a t e d genes. S e v e r a l other c h r o m a t i n proteins h a v e also b e e n i d e n t i f i e d t h r o u g h screens for d o m i n a n t suppressors of P E V , for e x a m p l e H P 1 ( M o t t u s , 1983; R e u t e r a n d W o l f f , 1981), S U ( V A R ) 3 - 7 (Reuter et al., 1990) a n d S U ( V A R ) 3 - 9 ( T s c h i e r s c h et a l . , 1994).  W h i l e these  proteins h a v e b e e n s h o w n to l o c a l i z e to h e t e r o c h r o m a t i c regions o f the genome, w h e t h e r their role i n s u p p r e s s i n g P E V is direct or i n d i r e c t r e m a i n s speculative. In a d d i t i o n , recent studies w i t h antibodies p r o d u c e d against the D . melanogaster HDACl  p r o t e i n , f a i l e d to f i n d s i g n i f i c a n t h e t e r o c h r o m a t i c l o c a l i z a t i o n of  H D A C l ( B a r l o w et al., 2001; P i l e a n d W a s s a r m a n , 2000). T h u s the q u e s t i o n of whether H D A C l  o r the o t h e r c h r o m a t i n p r o t e i n s p l a y a d i r e c t r o l e i n the  m e c h a n i s m of P E V has r e m a i n e d o p e n . I d e c i d e d to use the t e c h n i q u e of X - C h I P to a d d r e s s this q u e s t i o n directly. T h i s t e c h n i q u e has b e e n e m p l o y e d w i t h great success i n single-celled o r g a n i s m s a n d i n e u k a r y o t i c cells g r o w n i n tissue culture, w h e r e one can easily o b t a i n a r e l a t i v e l y h o m o g e n e o u s p o p u l a t i o n of cells. It h a s b e e n u s e d less  104  frequently i n m u l t i - c e l l u l a r o r g a n i s m s . I n m o s t cases, m u l t i - c e l l u l a r o r g a n i s m s may  n o t be g o o d substrates for X - C h I P since t h e y are c o m p r i s e d of m a n y  different c e l l types, e a c h w i t h their o w n u n i q u e p a t t e r n of gene e x p r e s s i o n . T h i s heterogeneity m a y obscure events o c c u r r i n g i n a s i n g l e cell t y p e a n d thus r e n d e r results f r o m X - C h I P u n i n t e r p r e t a b l e .  T h e e x c e p t i o n s to this r u l e h a v e  o c c u r r e d a l m o s t e x c l u s i v e l y i n studies of Drosophila e m b r y o s that attempted to isolate the b i n d i n g sites of certain r e g u l a t o r y proteins, e s p e c i a l l y m e m b e r of the P o l y c o m b G r o u p of genes, that act i n e a r l y d e v e l o p m e n t ( O r l a n d o a n d Paro, 1993). H o w e v e r , w i t h respect to P E V , there is s o m e e v i d e n c e that the s i l e n c i n g o b s e r v e d i n this p h e n o m e n o n o c c u r s e a r l y i n e m b r y o g e n e s i s i n m a n y , a n d p e r h a p s a l l , cells i n the o r g a n i s m (see C h a p t e r 1). Therefore I w a s of the v i e w that P E V m a y be amenable to X - C h I P analysis. I u s e d X - C h I P a n d a c o m m e r c i a l l y available, X - C h I P q u a l i t y a n t i b o d y to H D A C l , to ask w h e t h e r H D A C l is associated w i t h specific regions of the white* gene i n three strains: Oregon-R n o r m a l l y ; In(l)w'"  4  (OR-R),  i n w h i c h the white* gene is e x p r e s s e d  (w'" ) i n w h i c h the white* gene is s i l e n c e d d u e to P E V a n d is 4  o n l y expressed i n about 5-15% of the eye p i g m e n t cells; a n d , In(l)w"' ; 4  (w ; 326) i n w h i c h the m u t a t i o n i n HDACl m4  HDACl  326  suppresses the s i l e n c i n g d u e to P E V  a n d the white* gene is expressed i n 80-90% of the eye p i g m e n t cells. For this i n i t i a l characterization, I chose to a n a l y z e three regions 5' to the start of t r a n s c r i p t i o n of the white* gene ( F i g u r e 1) i n each strain. T h e first 500 base p a i r r e g i o n , the " P r o x i m a l P r o m o t e r " , is i m m e d i a t e l y 5' to the start of t r a n s c r i p t i o n a n d i n c l u d e s the white* gene m i n i m a l p r o m o t e r ( L e v i s et al., 1985; P i r r o t t a et a l . , 1985). T h e s e c o n d r e g i o n , the " D i s t a l P r o m o t e r " , is the r e g i o n i m m e d i a t e l y 5' to the m i n i m a l p r o m o t e r a n d c o n t a i n s e l e m e n t s i n v o l v e d i n  105  r e g u l a t i n g the white* gene i n a d u l t tissues ( D a v i s o n et al., 1985). In a d d i t i o n , I chose a r e g i o n a p p r o x i m a t e l y 6.0 k i l o b a s e s 5' to the white* gene, the "5' D i s t a l R e g i o n " , w h i c h does n o t c o n t a i n a n y elements k n o w n to regulate white*.  The  first t w o regions w e r e chosen because they c o n t a i n k n o w n r e g u l a t o r y regions of white* a n d therefore m i g h t be e x p e c t e d to s h o w alterations w h e n the gene is silenced. T h e 5' D i s t a l R e g i o n w a s chosen i n o r d e r to d e t e r m i n e w h e t h e r or not any changes o b s e r v e d at the white* p r o m o t e r w e r e p r o p a g a t e d u p s t r e a m of the gene. X - C h I P a n a l y s i s of the association of H D A C l w i t h the white* gene w h e n it is i n its n o r m a l l o c a t i o n at the tip of the X c h r o m o s o m e i n the OR-R  strain  suggest l o w l e v e l s of H D A C l m a y be associated w i t h the P r o x i m a l P r o m o t e r r e g i o n ( F i g u r e 3), b u t that H D A C l is p r o b a b l y n o t associated w i t h either the D i s t a l P r o m o t e r r e g i o n (Figure 4) or the 5' D i s t a l R e g i o n (Figure 5). I n OR-R the l e v e l s of H D A C l  a s s o c i a t i o n at the P r o x i m a l P r o m o t e r d i s p l a y e d a 5 f o l d  increase o v e r b a c k g r o u n d a n d a p p r o x i m a t e l y t w i c e as h i g h as that o b s e r v e d i n either the D i s t a l P r o m o t e r a n d the 5' D i s t a l R e g i o n . It w o u l d not be p a r t i c u l a r l y s u r p r i s i n g to f i n d that H D A C l is n o r m a l l y associated w i t h the white* gene at its p r o m o t e r , w h e r e , p r e s u m a b l y , it w o u l d act as a repressor.  T h e o n l y tissue i n  the larvae i n w h i c h the white* gene is k n o w n to be expressed, is the m a l p i g h i a n t u b u l e s ( L e v i s et al., 1985; P i r r o t t a et al., 1985). Therefore i n a l m o s t a l l cells of the 12-16 h o u r e m b r y o s , w h i c h w e r e the substrate for X - C h I P , the white* gene is inactive. A c c o r d i n g l y , I b e l i e v e the 5-6 f o l d increases o b s e r v e d at the P r o x i m a l P r o m o t e r represent actual sites of H D A C l association. W h e t h e r the 2-3-fold enhancement o b s e r v e d at the D i s t a l P r o m o t e r a n d the 5' D i s t a l R e g i o n r e p r e s e n t a c t u a l sites of H D A C l  106  a s s o c i a t i o n is m o r e  p r o b l e m a t i c . O n e of the difficulties w i t h i n t e r p r e t i n g these results stems f r o m a t t e m p t i n g to d o X - C h I P i n a w h o l e o r g a n i s m , w h e r e i t m a y be difficult, o r i m p o s s i b l e , to f i n d a c h r o m o s o m a l r e g i o n to use as a n e g a t i v e c o n t r o l for association w i t h H D A C l .  However, I have performed  X-ChIP with a  m o n o c l o n a l a n t i b o d y that does not h a v e a n y target p r o t e i n s i n Drosophila, the T7 a n t i g e n a n t i b o d y , a n d it n o n - s p e c i f i c a l l y p r e c i p i t a t e s D N A f r o m several r e g i o n s of the g e n o m e at l e v e l s 2-3 f o l d a b o v e the N o A n t i b o d y c o n t r o l s ( u n p u b l i s h e d observations). Therefore, I t h i n k it l i k e l y that the a 2-3 f o l d levels of e n h a n c e m e n t o b s e r v e d i n OR-R  at the D i s t a l P r o m o t e r a n d the 5' D i s t a l  R e g i o n of OR-R represent non-specific p r e c i p i t a t i o n of D N A sequences b y the a n t i - H D A C l a n t i b o d y a n d not actual sites of H D A C l association. In contrast, the results clearly s h o w that i n w'" , w h e r e the white* gene is 4  s i l e n c e d d u e to P E V , H D A C l s h o w s a s t r o n g association w i t h a l l three regions (Figure 3, 4 a n d 5). T h e levels of H D A C l o b s e r v e d i n w'" at each r e g i o n are far 4  greater t h a n that seen i n OR-R  a n d s h o w at least a 1 4 - f o l d increase i n  association at a l l regions. H o w e v e r , w h e n the d o m i n a n t  suppressor  o f P E V , HDACl , 326  is  i n t r o d u c e d i n t o the w'" strain, the increased H D A C l association n o r m a l l y seen 4  at a l l r e g i o n s i n w'" , is a b o l i s h e d ( F i g u r e s 3, 4 a n d 5). T h e l e v e l s o f H D A C l 4  a s s o c i a t i o n r e t u r n to that o b s e r v e d w h e n the white* gene is at its n o r m a l l o c a t i o n at the t i p o f the X c h r o m o s o m e as i n the OR-R strain. I n fact the levels of H D A C l at each r e g i o n i n w'" ; 326 precisely m i r r o r the levels o b s e r v e d i n OR4  R, i n c l u d i n g the s l i g h t e n r i c h m e n t o b s e r v e d i n the P r o x i m a l P r o m o t e r r e g i o n (Figure 3).  107  T h e h i g h levels of H D A C l associated w i t h the three regions a n a l y z e d at the white* l o c u s w h e n it is s i l e n c e d as a r e s u l t of P E V i n w'" , w h i c h are not 4  present w h e n white* is at its n o r m a l l o c a t i o n i n OR-R, p r o v i d e s s t r o n g evidence for H D A C l ' s direct i n v o l v e m e n t i n P E V . I n a d d i t i o n , the o b s e r v a t i o n that the a s s o c i a t i o n w i t h these r e g i o n s is a b o l i s h e d b y a m u t a t i o n i n HDACl  implies  that H D A C l is a n essential c o m p o n e n t of the s i l e n c i n g m e c h a n i s m at w o r k i n P E V . T h i s is the first evidence of a protein's direct i n v o l v e m e n t i n the s i l e n c i n g associated w i t h P E V . T h e fact that a n H D A C is present at a site does n o t guarantee that it is r e g u l a t i n g the g e n e o r p h e n o m e n o n also p r e s e n t at the site.  For example,  H D A C l has b e e n f o u n d at active genes ( B r e i l i n g et a l . , 2001), w h i c h w a s s u r p r i s i n g , g i v e n H D A C l ' s suspected role as a t r a n s c r i p t i o n a l repressor.  There  c o u l d be m a n y reasons for s u c h a n association, i n c l u d i n g s o m e t h i n g as s i m p l e as H D A C l b e i n g stored, i n a n inactive f o r m , to be q u i c k l y accessible for r a p i d r e p r e s s i o n o f the gene. A c c o r d i n g l y , R o b y r et a l . (2002) h a v e suggested three c r i t e r i a be a d o p t e d to d i s t i n g u i s h w h e t h e r a n H D A C is a c t i n g d i r e c t l y or i n d i r e c t l y o n gene e x p r e s s i o n .  T h e a u t h o r s w e r e s p e a k i n g to g e n o m e w i d e  a n a l y s i s b u t their c o n s i d e r a t i o n s c a n be e a s i l y e x t e n d e d to a p p l y to X - C h I P studies of H D A C s as w e l l . T h e X - C h I P v e r s i o n s of these c r i t e r i a w o u l d be: (1) X - C h I P studies to d e t e r m i n e w h e t h e r the H D A C is associated w i t h c h r o m a t i n at the s u s p e c t e d site; (2) since the H D A C s substrates are the h i s t o n e p r o t e i n s , analysis of the a c e t y l a t i o n p a t t e r n of the histones at the site of association; a n d (3) t r a n s c r i p t i o n a l a n a l y s i s of the suspected H D A C - r e g u l a t e d gene. T h i s thesis has a d d r e s s e d t w o of the three criteria w i t h respect to H D A C l ' s i n v o l v e m e n t i n P E V . T h e X - C h I P studies c o n f i r m H D A C l is associated w i t h the e u c h r o m a t i c 108  r e g i o n that variegates w h e n , a n d o n l y w h e n , it is subject to P E V . S e c o n d l y , m u t a t i o n s i n H D A C l , that s u p p r e s s P E V , a b o l i s h the a s s o c i a t i o n a n d restore t r a n s c r i p t i o n to a l m o s t w i l d - t y p e levels. T h e r e m a i n i n g c r i t e r i o n , a n a l y s i s of the acetylated state of the histone p r o t e i n s i n the c h r o m a t i n s u r r o u n d i n g the white* gene, has not b e e n a p a r t of the s t u d y .  I n spite of this, I s u b m i t the  e v i d e n c e p r e s e n t e d h e r e i n is c o m p e l l i n g that H D A C l p l a y s a n essential role i n the s i l e n c i n g associated w i t h P E V .  109  Chapter 4 Summary and General Discussion  110  General Discussion  Summary The  m o d e l s y s t e m o u r l a b o r a t o r y has b e e n u s i n g to i n v e s t i g a t e gene  s i l e n c i n g is p o s i t i o n effect v a r i e g a t i o n ( P E V ) i n D . melanogaster (Grigliatti, 1991; S c h o t t a et a l . , 2003; S p o f f o r d ,  1976). W h e n P E V o c c u r s , genes n o r m a l l y  expressed i n a p a r t i c u l a r tissue are silenced i n some cells of that tissue because a c h r o m o s o m a l r e a r r a n g e m e n t has p l a c e d the r e g i o n i n w h i c h the genes are located next to a b r e a k p o i n t i n h e t e r o c h r o m a t i n . I n s o m e cells of the tissue, the genes are expressed n o r m a l l y , w h i l e i n others, the genes are silenced, a n d those cells d i s p l a y a m u t a n t p h e n o t y p e .  A c c o r d i n g l y , that t i s s u e is m o s a i c or  v a r i e g a t e d . I m p o r t a n t l y , the d e c i s i o n w h e t h e r a gene w i l l be active or inactive is m a d e e a r l y i n d e v e l o p m e n t a n d that d e c i s i o n is p a s s e d o n to d a u g h t e r cells w i t h reasonable fidelity. T h u s P E V m i m i c s n o r m a l d e v e l o p m e n t i n m a n y w a y s . T h i s has l e d o u r l a b , a n d s e v e r a l others, to try to dissect the m e c h a n i s m s u n d e r l y i n g P E V w i t h the h o p e they w i l l s h e d s o m e l i g h t o n the m o r e general s i l e n c i n g m e c h a n i s m s that occur d u r i n g n o r m a l d e v e l o p m e n t . O n e of the c o n v e n t i o n a l approaches to d i s s e c t i n g a p h e n o m e n o n s u c h as P E V is to isolate m u t a t i o n s that m o d i f y the associated p h e n o t y p e .  Subsequent  a n a l y s i s of the m u t a t i o n s a n d their effects p r o v i d e s s o m e i n s i g h t i n t o that p h e n o m e n o n . I n the case o f P E V , o n l y a few m u t a t i o n s h a v e b e e n c l o n e d a n d a n a l y z e d i n a n y d e t a i l . H o w e v e r , the i n s i g h t s they h a v e p r o v i d e d i n t o P E V , and s i l e n c i n g i n general, h a v e h a d considerable i m p a c t . F o r e x a m p l e , Su(var)39 encodes a n p r o t e i n that c a n m e t h y l a t e l y s i n e 9 o n h i s t o n e H 3 . M a n y h a v e suggested this creates a n epigenetic m a r k that is p a s s e d o n to d a u g h t e r cells to  111  m a i n t a i n s i l e n c i n g , not o n l y i n P E V , b u t also i n other s i l e n c i n g systems i n the cell ( C z e r m i n et al., 2001; R i c h a r d s a n d E l g i n , 2002; Schotta et a l , 2002; T u r n e r , 2002). A c c o r d i n g l y , the c l o n i n g a n d c h a r a c t e r i z a t i o n of a d d i t i o n a l m u t a t i o n s that m o d i f y P E V h o l d s great p r o m i s e for i n c r e a s i n g o u r u n d e r s t a n d i n g of b o t h P E V a n d other s i l e n c i n g m e c h a n i s m s e m p l o y e d b y e u k a r y o t i c cells. C h a p t e r 2 presents the c l o n i n g a n d c h a r a c t e r i z a t i o n of a gene i d e n t i f i e d i n a screen for d o m i n a n t m u t a t i o n s that s u p p r e s s the s i l e n c i n g associated w i t h PEV.  T h e gene is HDACl,  w h i c h encodes a n h i s t o n e deacetylase h o m o l o g o u s  to H D A C l , f r o m m a m m a l s , a n d R p d 3 , f r o m S. cerevisiae. m u t a t i o n s i n HDACl  S p e c i f i c missense  suppress P E V , w h i l e h y p o m o r p h i c or n u l l alleles h a v e no  effect o n the v a r i e g a t i n g p h e n o t y p e . C h a p t e r 3 p r o v i d e s e v i d e n c e that H D A C l is d i r e c t l y i n v o l v e d i n P E V b y d e m o n s t r a t i n g the p r o t e i n is p r e s e n t o n the c h r o m a t i n s u r r o u n d i n g a gene s i l e n c e d as a r e s u l t of P E V . H o w e v e r , the Su(var) mutations  i n HDACl  a b o l i s h the p r o t e i n ' s a s s o c i a t i o n w i t h  the  c h r o m a t i n s u r r o u n d i n g the v a r i e g a t i n g l o c u s a n d restore the a c t i v i t y of the v a r i e g a t i n g gene.  Discussion There are c u r r e n t l y t w o w i d e l y h e l d theories about the m e c h a n i s m at the basis of P E V ( G r i g l i a t t i , 1991; Schotta et al., 2003; S p o f f o r d , 1976). T h e m o s t e n d u r i n g is the " S p r e a d i n g H y p o t h e s i s " . It posits that factors n o r m a l l y f o u n d i n h e t e r o c h r o m a t i n s p r e a d o u t f r o m the n e w c h r o m o s o m a l j u n c t i o n a n d create a c h r o m a t i n e n v i r o n m e n t that suppresses t r a n s c r i p t i o n . T h e distance the factors s p r e a d is i n d e p e n d e n t i n e a c h cell a n d therefore i n s o m e cells the factors w i l l s p r e a d far e n o u g h to i n a c t i v a t e a v a r i e g a t i n g gene, w h i l e , i n other cells,  112  s p r e a d i n g w i l l s t o p b e f o r e r e a c h i n g the g e n e a n d it w i l l be t r a n s c r i b e d normally. M o r e r e c e n t l y , a n a l t e r n a t i v e h y p o t h e s i s , the " C o m p a r t m e n t a l i z a t i o n H y p o t h e s i s " , has b e e n p r o p o s e d .  It p o s i t s that h e t e r o c h r o m a t i c r e g i o n s of  c h r o m o s o m e s are p o s i t i o n e d i n specific l o c a t i o n s o r c o m p a r t m e n t s of the n u c l e u s that e x c l u d e the factors r e q u i r e d for t r a n s c r i p t i o n a n d therefore w h e n a gene is i n this c o m p a r t m e n t it is silenced. T h e m o s a i c p h e n o t y p e of P E V occurs b e c a u s e the v a r i e g a t i n g gene is s o m e t i m e s l o c a l i z e d to a h e t e r o c h r o m a t i c c o m p a r t m e n t , a n d therefore s i l e n c e d , a n d s o m e t i m e l o c a l i z e d to its n o r m a l p o s i t i o n , a n d therefore fully expressed ( C s i n k a n d H e n i k o f f , 1996). These h y p o t h e s e s are not m u t u a l l y e x c l u s i v e i n that l o c i subject to P E V m a y be relocated to h e t e r o c h r o m a t i c c o m p a r t m e n t s w h e r e they are p a c k a g e d as p r o p o s e d b y the " S p r e a d i n g H y p o t h e s i s " .  A l t e r n a t i v e l y , it m a y be that a  v a r i e g a t i n g l o c u s is p a c k a g e d as per the " S p r e a d i n g H y p o t h e s i s " a n d then is relocated to a n h e t e r o c h r o m a t i c c o m p a r t m e n t . If either alternative is true, then a v a r i e g a t i n g gene w o u l d be f u l l y expressed because, either it w a s not relocated to a n h e t e r o c h r o m a t i c c o m p a r t m e n t , or p a c k a g i n g failed to "spread" to the gene i n the h e t e r o c h r o m a t i c c o m p a r t m e n t or b o t h . Any  a t t e m p t to e x p l a i n the r e s u l t s o b s e r v e d h e r e i n t e r m s of these  m o d e l s m u s t a l s o a c c o m m o d a t e the fact that s t r a i n s b e a r i n g the mutations, also carry a w i l d - t y p e c o p y of the HDACl  HDACl  gene a n d therefore at least  50% of the H D A C l i n the n u c l e u s is the w i l d - t y p e p r o t e i n . T h e data presented here c a n n o t d i s t i n g u i s h b e t w e e n these m o d e l s .  H o w e v e r , it is d i f f i c u l t to  interpret m y results b a s e d o n a strict c o m p a r t m e n t a l i z a t i o n m o d e l . H D A C l is n o t f o u n d i n h e t e r o c h r o m a t i n , at least i n c o n c e n t r a t i o n s h i g h e n o u g h to be  113  detected b y a n a n t i b o d y ( B a r l o w et a l . , 2001; P i l e a n d W a s s a r m a n , 2000). A c c o r d i n g l y , it w o u l d seem u n l i k e l y that the presence of H D A C l w o u l d be a s i g n a l to l o c a l i z e a n o r m a l l y h e t e r o c h r o m a t i c r e g i o n to a n h e t e r o c h r o m a t i c compartment.  T h i s i m p l i e s that some other factor(s) m u s t be r e s p o n s i b l e for  l o c a l i z i n g the h e t e r o c h r o m a t i n a s s o c i a t e d w i t h the white* gene i n w  to a  m4  heterochromatic compartment.  H o w e v e r , the fact that H D A C l is f o u n d i n  a b u n d a n c e at the white* gene i n w'" i m p l i e s the a s s o c i a t i o n w i t h H D A C l m u s t 4  occur w h e n the white* gene is m i s - l o c a l i z e d . I n a strict c o m p a r t m e n t a l i z a t i o n m o d e l , it w o u l d be the l o c a l i z a t i o n to the h e t e r o c h r o m a t i c c o m p a r t m e n t that causes s i l e n c i n g a n d n o t the a s s o c i a t i o n w i t h H D A C l . m u t a t i o n s i n HDACl,  Accordingly,  the  that s u p p r e s s P E V , w o u l d cause m i s r e g u l a t i o n of the  factor(s) r e s p o n s i b l e for l o c a l i z i n g h e t e r o c h r o m a t i n to the n u c l e a r p e r i p h e r y w h i c h w o u l d , i n t u r n , result i n the failure of the white* gene to be l o c a l i z e d to the n u c l e a r p e r i p h e r y . w o u l d be i n d i r e c t .  T h u s the effect of the S u ( v a r ) m u t a t i o n s i n  W h i l e this scenario is p o s s i b l e , it seems u n l i k e l y .  HDACl The  presence of H D A C l at the white* gene w h e n it is s i l e n c e d i n w'" a n d its absence 4  w h e n the white* gene is e x p r e s s e d i n the HDACl  S u ( v a r ) m u t a t i o n s suggests a  m o r e direct role for H D A C l . The  d a t a p r e s e n t e d here c a n be m o r e e a s i l y e x p l a i n e d i n terms of the  " S p r e a d i n g H y p o t h e s i s " . T h e s u b s t a n t i a l b o d y of e v i d e n c e that s u p p o r t s this m o d e l w a s r e v i e w e d i n C h a p t e r 1. I n a d d i t i o n , there is a p h e n o m e n o n i n S. cerevisiae, t e l o m e r i c p o s i t i o n effect v a r i e g a t i o n ( T P E V ) that c l o s e l y resembles P E V a n d also a p p e a r s to i n v o l v e the s p r e a d i n g o f s i l e n c i n g c o m p o n e n t s (see C h a p t e r 2). It s h o u l d be n o t e d that T P E V is also d e p e n d e n t o n Sir2, a n N A D d e p e n d e n t C l a s s III h i s t o n e deacetylase, for efficient s i l e n c i n g ( S u k a et al., 2002).  114  A c c o r d i n g l y , the data p r e s e n t e d i n this thesis is c o n s i s t e n t w i t h the following  scenario.  heterochromatin  is  In a chromosomal rearrangement  subject  assembled  the  normally  at  or  near  to P E V ,  new  illicit  h e t e r o c h r o m a t i c / e u c h r o m a t i c j u n c t i o n . T h i s m a y be d e p e n d e n t o n a c o m p l e x that contains S U ( V A R ) 3 - 7 that is targeted to satellite sequences b y the u n u s u a l z i n c fingers c o n t a i n e d i n S U ( V A R ) 3 - 7 .  T h a t c o m p l e x c o n t a i n s or recruits a  c o m p l e x that c o n t a i n s H P 1 , S U ( V A R ) 3 - 9 , H D A C l  a n d l i k e l y s e v e r a l other  p r o t e i n s w h i c h create a n d s t a b i l i z e the s t r u c t u r e r e s p o n s i b l e for s i l e n c i n g variegating loci.  T h e structure c a n s p r e a d i n t o the adjacent e u c h r o m a t i n b u t  requires the a c t i v i t y of each c o m p o n e n t to perpetuate its s p r e a d . H D A C l m u s t deacetylate K 9 of h i s t o n e H 3 , a n d p e r h a p s other l y s i n e residues, w h i c h clears the p a t h for S U ( V A R ) 3 - 9 to methylate H 3 K 9 that i n t u r n creates a b i n d i n g site for H P 1 s t a b i l i z i n g the structure a n d s i l e n c i n g a n y l o c i at that l o c a t i o n . T h i s process w o u l d c o n t i n u e u n t i l the c o n c e n t r a t i o n of the e s s e n t i a l c o m p o n e n t s d r o p p e d b e l o w a critical l e v e l or a b o u n d a r y element, i n the e u c h r o m a t i c r e g i o n of the c h r o m o s o m e , h a l t e d progress. How  t h e n d o m u t a t i o n s i n HDACl  cause s u p p r e s s i o n of P E V ?  I n the  p r o p o s e d m o d e l , H D A C l a c t i v i t y is r e q u i r e d for the s i l e n c i n g m e c h a n i s m to s p r e a d out f r o m the h e t e r o c h r o m a t i c b r e a k p o i n t . I n the m u t a n t strains, 50% of the H D A C l p r o t e i n is w i l d - t y p e a n d s h o u l d f u n c t i o n n o r m a l l y . H o w e v e r , the r e m a i n i n g 50% of H D A C l bears a m u t a t i o n a n d is p r e s u m a b l y defective i n the a b i l i t y to deacetylate h i s t o n e tails. Since the m u t a t i o n is a s i n g l e a m i n o a c i d s u b s t i t u t i o n , the m u t a n t H D A C l m a y still assume its p r o p e r shape a n d take its place i n the p r o t e i n c o m p l e x i n v o l v e d i n c r e a t i n g the s i l e n c i n g structure (see C h a p t e r 2). H o w e v e r , failure of the m u t a n t H D A C l i n the p r o t e i n c o m p l e x to  115  deacetylate its targets w o u l d i n h i b i t the a c t i o n of S U ( V A R ) 3 - 9 w h e n e v e r it e n c o u n t e r e d a n acetylated l y s i n e 9 residue o n H 3 since S U ( V A R ) 3 - 9 ' s activity is b l o c k e d b y a c e t y l a t i o n (Rea et a l . , 2000). A c c o r d i n g l y a b i n d i n g site w o u l d not be c r e a t e d for H P I , the s i l e n c i n g p r o c e s s w o u l d a b o r t a n d P E V w o u l d be suppressed. I n the present case it s h o u l d be n o t e d that the white* gene is still subject to s o m e s i l e n c i n g i n the m u t a n t HDACl  lines ( i n 10-20% o f cells). T h e r e c o u l d  be s e v e r a l reasons for this. T h e process m a y not be a b s o l u t e l y d e p e n d e n t o n H D A C l o r the m u t a n t H D A C l s m a y be h y p o m o r p h s a n d the r e s i d u a l a c t i v i t y a l l o w s s p r e a d i n g a s m a l l percentage of the t i m e .  A l t e r n a t i v e l y , h a l t i n g the  s p r e a d of the s i l e n c i n g structure m a y r e q u i r e the b i n d i n g of s e v e r a l i n a c t i v e d e a c e t y l a s e c o m p l e x e s i n t a n d e m or w i t h i n a c e r t a i n d i s t a n c e a l o n g the chromosome.  O n c e this t h r e s h o l d is met s i l e n c i n g is n o t p r o p a g a t e d b e y o n d  that p o i n t . If that o c c u r s before the white* gene, t h e n the gene is active, i f not, s i l e n c i n g w o u l d s p r e a d t h r o u g h a n d perhaps b e y o n d the white* gene r e n d e r i n g it t r a n s c r i p t i o n a l l y i n a c t i v e . T h e r e s u l t s of X - C h i p f r o m the 5' D i s t a l R e g i o n i n w'" ( F i g u r e 5) 4  demonstrate that, s p r e a d i n g , i f i n d e e d that is the m e c h a n i s m at w o r k , spreads far b e y o n d the white* gene, at least as far as 6.0 kilobases. C y t o l o g i c a l e v i d e n c e suggests s p r e a d i n g can e x t e n d as far as 80 b a n d s o n the p o l y t e n e c h r o m o s o m e s f r o m the b r e a k p o i n t (Spofford, 1976). It is not clear w h a t factors d e t e r m i n e the distance that s i l e n c i n g s p r e a d s .  It does n o t a p p e a r to be d e p e n d e n t o n the  presence of c o d i n g sequences or p r o m o t e r regions, since the r e g i o n c o n t a i n i n g the 5' D i s t a l R e g i o n does n o t c o n t a i n a n y k n o w n genes or r e g u l a t o r y sequences. Silencing  may  spread  until  specialized boundary  116  type  sequences  are  e n c o u n t e r e d or c o m p e t i t i o n w i t h factors c r e a t i n g a n e u c h r o m a t i c e n v i r o n m e n t halts its progress. In C h a p t e r 2 , 1 p r e s e n t e d a m o d e l of H D A C l i n v o l v e m e n t i n P E V b a s e d l a r g e l y o n s p e c u l a t i o n since, at the t i m e of p u b l i c a t i o n , the functions of other p r o t e i n s i n v o l v e d i n the s i l e n c i n g associated w i t h P E V w e r e u n k n o w n .  The  m o d e l p r o p o s e d that, because the Su(var) m u t a t i o n s i n H D A C l are caused b y a s i n g l e a m i n o a c i d change, m u t a n t forms of H D A C l w o u l d s t i l l f o l d p r o p e r l y a n d thus be able to b e c o m e m e m b e r s of their n o r m a l c o m p l e x e s . H o w e v e r , the complexes containing mutant H D A C l  w o u l d be u n a b l e to deacetylate  the  t a r g e t h i s t o n e t a i l s a n d h y p e r a c e t y l a t i o n o f h i s t o n e t a i l s w o u l d l e a d to e x p r e s s i o n of the reporter gene. Since the functions of s o m e of the other Su(var) p r o t e i n s are n o w k n o w n , the m o d e l of h o w s p e c i f i c m u t a t i o n s i n H D A C l suppress P E V requires u p d a t i n g . If P E V d o e s  o c c u r as a r e s u l t of the s p r e a d i n g of factors  from  h e t e r o c h r o m a t i n , t h e n one w o u l d p r e d i c t that factors, w h i c h s u p p r e s s P E V , i n h i b i t or a b o r t the s p r e a d i n g process.  It is n o w k n o w n that S U ( V A R ) 3 - 9  m e t h y l a t i o n of l y s i n e 9 o n H 3 ( H 3 K 9 ) is i n h i b i t e d i f l y s i n e 9 is acetylated a n d therefore H P I w i l l n o t b i n d (Rea et a l . , 2000). I h a v e p r o p o s e d that H D A C l is r e q u i r e d to deacetylate H 3 K 9 a n d clear the w a y for S U ( V A R ) 3 - 9 to m e t h y l a t e that r e s i d u e to create a b i n d i n g site for H P I .  I f u r t h e r p r o p o s e that the  association of S U ( V A R ) 3 - 9 , H P I a n d other factors not o n l y creates a repressive c h r o m a t i n c o n f o r m a t i o n , b u t also recruits a d d i t i o n a l H D A C l w h i c h is r e q u i r e d for p r o p a g a t i o n of the s i l e n c e d c o n f o r m a t i o n . T h u s , the Su(var) m u t a t i o n s i n H D A C l s u p p r e s s P E V because they o c c u p y their place i n the complex(es) b u t  117  cannot deacetylate H 3 K 9 a n d this prevents the association of b o t h S U ( V A R ) 3 - 9 and H P 1 . T h i s m o d e l m a k e s s e v e r a l p r e d i c t i o n s a n d raises s e v e r a l questions that can be tested e x p e r i m e n t a l l y . If s p r e a d i n g does i n d e e d o c c u r t h e n h o w far does the s p r e a d i n g e x t e n d i n the w'" strain? 4  S i n c e the Drosophila  g e n o m e is  sequenced, it is p o s s i b l e to generate p r i m e r s for X - C h I P further 5' to the white* gene a n d d e t e r m i n e h o w far the a s s o c i a t i o n w i t h H D A C l extends.  O n c e the  extent is d e t e r m i n e d a p p r o x i m a t e l y one c a n ask w h e t h e r the association w i t h HDACl  e n d s a b r u p t l y at specific sequences, s u g g e s t i n g the presence of a  b o u n d a r y e l e m e n t , or i f the a s s o c i a t i o n g r a d u a l l y d e c l i n e s , s u g g e s t i n g the s p r e a d is d e p e n d e n t o n the a v a i l a b i l i t y of the c o m p o n e n t s of the s i l e n c i n g mechanism.  The complementary experiments  c a n a l s o be d o n e .  The  h e t e r o c h r o m a t i c / e u c h r o m a t i c j u n c t i o n i n w'" is k n o w n a n d t h u s one c a n ask, 4  using  X-ChIP,  where  spreading  stops  i n an  HDACl  Su(var)  mutant  background. D o c e r t a i n sequences present b o u n d a r i e s to the s p r e a d of P E V ?  The  existence of s o m e t y p e of b o u n d a r y e l e m e n t is s u g g e s t e d b y a n a l y s i s of m u t a t i o n s i n Evar93D [also k n o w n as mod(mdg4)]. M u t a t i o n s i n this gene act as s t r o n g e n h a n c e r s of P E V .  It is s u s p e c t e d the p r o d u c t of this gene b i n d s to  b o u n d a r y elements i n e u c h r o m a t i n creating a n o p e n c h r o m a t i n c o n f o r m a t i o n ( G e r a s i m o v a a n d C o r c e s , 2001).  Thus, i n mutant  l i n e s subject  to P E V ,  h e t e r o c h r o m a t i n m a y s p r e a d m u c h further because b o u n d a r y elements, that w o u l d n o r m a l l y h a l t the s p r e a d , are defective. A n a l y s i s of the d i s t r i b u t i o n of H D A C l or other s i l e n c i n g components, u s i n g X - C h I P , m a y p r o v i d e i n s i g h t into this question.  118  T h e m o d e l also p r o p o s e s that H D A C l is r e q u i r e d to deacetylate  the  histone tails as one of the i n i t i a l steps i n the f o r m a t i o n of s i l e n c e d c h r o m a t i n . A c c o r d i n g l y , u s i n g X - C h I P a n d a n t i b o d i e s for specific acetylated f o r m s of the histones, one c a n d e t e r m i n e the a c e t y l a t i o n state of the h i s t o n e tails. would  predict  that, i n the  n o r m a l w'"  4  s t r a i n , the  histones  One  would  be  h y p o a c e t y l a t e d i n any r e g i o n w h e r e H D A C l is present a n d specifically that H 3 K 9 w o u l d not be acetylated. C o n v e r s e l y , i n zu'" b e a r i n g a Su(var) m u t a t i o n i n 4  HDACl,  one w o u l d p r e d i c t the r e g i o n s u r r o u n d i n g the white gene w o u l d s h o w  i n c r e a s e d a c e t y l a t i o n , e s p e c i a l l y at H 3 K 9 . T h i s w o u l d also a d d r e s s the t h i r d test s u g g e s t e d to c o n f i r m that a n H D A C is a c t i n g at a l o c a t i o n w h e r e X - C h I P indicates it is l o c a l i z e d [see C h a p t e r 3 D i s c u s s i o n ( R o b y r et al., 2002)]. T h e m o d e l also m a k e s specific p r e d i c t i o n s about w h a t proteins s h o u l d be l o c a l i z e d to the s i l e n c e d e u c h r o m a t i c r e g i o n i n w'" a n d further p r e d i c t s that a 4  h i e r a r c h y of interactions m a y occur. In w'" , the s i l e n c e d e u c h r o m a t i n s h o u l d be 4  associated w i t h S U ( V A R ) 3 - 9 , H P 1 a n d p e r h a p s S U ( V A R ) 3 - 7 . A n t i b o d i e s exist to a l l three p r o t e i n s a n d therefore X - C h I P c a n be e m p l o y e d to  determine  w h e t h e r t h e y are p r e s e n t a n d further to a s k i f t h e i r d i s t r i b u t i o n p r e c i s e l y m i r r o r s that of H D A C l . Since d o m i n a n t Su(var) m u t a t i o n s are r e a d i l y a v a i l a b l e for each of the k n o w n p r o t e i n s i n v o l v e d i n the process, one c a n use the m u t a t i o n s to ask q u e s t i o n s a b o u t the n a t u r e o f the s i l e n c i n g c o m p l e x o r c o m p l e x e s .  I n the  m o d e l s s i m p l e s t f o r m one w o u l d p r e d i c t that a m u t a t i o n i n H D A C l s h o u l d be epistatic to m u t a t i o n s i n the other c o m p o n e n t s .  T h u s , i n the Su(var) H D A C l  strains, one s h o u l d f i n d that a l l associations of the other p r o t e i n s are a b o l i s h e d b e c a u s e the a c t i v i t y o f H D A C l  is r e q u i r e d f o r o n e o f the i n i t i a l steps i n 119  s p r e a d i n g . G e n e t i c e x p e r i m e n t s suggest this m a y i n fact be the case. A Su(var) m u t a t i o n i n HDACl  abolishes the enhancer effect of three copies of Su(var)3-9  ( C z e r m i n et a l . , 2001).  If i n d e e d the c o m p o n e n t s are a d d e d i n a s t e p w i s e  f a s h i o n t h e n one w o u l d p r e d i c t a m u t a t i o n i n Su(var)3-9 w o u l d a b o l i s h the a s s o c i a t i o n of H P I because S U ( V A R ) 3 - 9 creates the b i n d i n g site for H P I , b u t not the association of H D A C l . S i m i l a r l y , m u t a t i o n s i n HPI w o u l d not affect the d i s t r i b u t i o n of H D A C l a n d S U ( V A R ) 3 - 9 . H o w e v e r , I t h i n k a s i m p l e stepwise a c c u m u l a t i o n o f factors, w h i c h e v e n t u a l l y creates a s i l e n c e d s t r e t c h of e u c h r o m a t i n , is u n l i k e l y . In S. pombe the s p r e a d of the S U ( V A R ) 3 - 9 h o m o l o g u e , C l r 4 a n d the H P I h o m o l o g u e , S w i 6 , are m u t u a l l y d e p e n d e n t ( N o m a et al., 2001). T h i s is l i k e l y because these factors exist i n c o m p l e x e s w h e r e it appears the a c t i o n of one m e m b e r of the c o m p l e x reinforces a n d abets the function(s) of the other m e m b e r s .  A c c o r d i n g l y i n D . melanogaster,  I p r e d i c t that a s i m i l a r  s i t u a t i o n w i l l be f o u n d . I suspect the four p r o t e i n s s p e c i f i c a l l y m e n t i o n e d a n d several others are m e m b e r s of one or m o r e large c o m p l e x e s . T h e m e m b e r s of the c o m p l e x e s are d e p e n d e n t o n each other for their l o c a l i z a t i o n a n d spread. For e x a m p l e , it a p p e a r s that H D A C l is a m e m b e r of a c o m p l e x that i n c l u d e s S U ( V A R ) 3 - 9 a n d m a y i n c l u d e H P I a n d S U ( V A R ) 3 - 7 ( C l e a r d et a l , 1997; Schotta et al., 2002). T h i s c o m p l e x w o u l d b i n d i n the h e t e r o c h r o m a t i n at o r v e r y near to the h e t e r o c h r o m a t i c / e u c h r o m a t i c b r e a k p o i n t w h e r e H D A C l w o u l d b e g i n the process o f s i l e n c i n g b y d e a c e t y l a t i n g H 3 K 9 i n a n adjacent n u c l e o s o m e .  The  actions of S U ( V A R ) 3 - 9 a n d H P I w o u l d t h e n create c o n d i t i o n s that w o u l d recruit another c o m p l e x w h i c h w o u l d repeat the process u n t i l a b o u n d a r y w a s encountered  o r the c o n c e n t r a t i o n of the c o m p l e x e s f e l l b e l o w a c e r t a i n  threshold.  120  T h e difference b e t w e e n these t w o scenarios is r e a d i l y testable.  I n the  stepwise m o d e l , m u t a t i o n s i n H P 1 s h o u l d not affect the d i s t r i b u t i o n of H D A C l , as m e a s u r e d b y X - C h I P . O n the other h a n d , i f s p r e a d i n g occurs v i a complexes, t h e n a m u t a t i o n i n a n y one of the m e m b e r s of the c o m p l e x s h o u l d alter the distribution of a l l members.  A g a i n , the d i s t r i b u t i o n c a n b e m e a s u r e d b y X -  ChlP. T h e fact that H D A C l a n d q u i t e l i k e l y , S U ( V A R ) 3 - 9 , H P 1 a n d p e r h a p s other proteins, are present at the silenced e u c h r o m a t i n i n 12 to 16 h o u r e m b r y o s suggests t h e i r presence m a y be r e q u i r e d at the site o f s i l e n c i n g t h r o u g h o u t development.  It has b e e n suggested they are p a r t of the e p i g e n e t i c m a r k that  m a i n t a i n s the s i l e n c e d state ( H a l l et al., 2002). T h e m o d e l s u g g e s t e d here also i m p l i e s that these p r o t e i n s are i n v o l v e d i n the i n i t i a l d e t e r m i n a t i v e r e g a r d i n g w h e t h e r a r e p o r t e r gene w i l l be a c t i v e or not.  event  T h e q u e s t i o n of  w h e t h e r these p r o t e i n s f u n c t i o n i n b o t h i n i t i a t i o n a n d m a i n t e n a n c e , or are e x c l u s i v e l y i n v o l v e d i n m a i n t e n a n c e , a n a l o g o u s to the P o l y c o m b G r o u p of proteins ( B r e i l i n g et al., 2001), is a n interesting one that has not b e e n addressed here or e l s e w h e r e .  It m a y be p o s s i b l e to a d d r e s s this q u e s t i o n e m p l o y i n g X -  C h l P a n d carefully staged e m b r y o s . F l y b a s e list o v e r 180 l o c i that, w h e n m u t a t e d , i n f l u e n c e the v a r i e g a t i n g p h e n o t y p e associated w i t h P E V (http:/ / f l y b a s e . b i o . i n d i a n a . e d u : 8 2 / ) . A s n o t e d i n C h a p t e r 1, m a n y external factors, w h i c h affect basic c e l l u l a r m e t a b o l i s m , also affect P E V . T h e s e n s i t i v i t y of the v a r i e g a t i n g p h e n o t y p e to s u c h a large n u m b e r of factors suggests m a n y m a y be acting i n d i r e c t l y . T h u s one of the challenges f a c i n g i n v e s t i g a t o r s is d e t e r m i n i n g w h i c h m o d i f i e r s of P E V to c l o n e a n d characterize, that is, w h i c h are l i k e l y to be m o d i f y i n g P E V d i r e c t l y a n d w h i c h  121  represent i n d i r e c t effects. T h i s thesis presents a n a p p r o a c h to c l o n i n g essential Su(var) a n d E ( v a r ) l o c i .  H o w e v e r , n o c r i t e r i a r e a d i l y present t h e m s e l v e s for  d e t e r m i n i n g w h i c h l o c i to clone a n d characterize first. O u r lab has p u r s u e d l o c i that, w h e n m u t a t e d , h a v e a v e r y s t r o n g effect of the v a r i e g a t i n g p h e n o t y p e . H o w e v e r , a b i o c h e m i c a l a p p r o a c h to i d e n t i f y i n g the m e m b e r s of c o m p l e x e s c o n t a i n i n g k n o w n m o d i f i e r s of P E V s h o u l d also be p u r s u e d . F i n a l l y , the presence of H D A C l a n d p e r h a p s the other p r o t e i n s o n the silenced e u c h r o m a t i n throughout d e v e l o p m e n t suggests they m a y have a greater f u n c t i o n t h a n that s u s p e c t e d f r o m the p u r e l y e n z y m a t i c a c t i v i t y d i s c o v e r e d to date.  T h e s e p r o t e i n s are h i g h l y c o n s e r v e d f r o m yeast  to  m a m m a l s w i t h large tracts of a m i n o a c i d s a l m o s t a b s o l u t e l y c o n s e r v e d (De R u b e r t i s et a l , 1996; E i s s e n b e r g a n d E l g i n , 2000; M o t t u s et al., 2000). It m a y be t h e y are i m p o r t a n t s t r u c t u r a l c o m p o n e n t s  of c h r o m a t i n a n d the  highly  c o n s e r v e d r e g i o n s represent d o m a i n s i n v o l v e d i n c r e a t i n g a n d m a i n t a i n i n g specific c h r o m a t i n c o n f o r m a t i o n s .  S u c h a r o l e for H P I is s u g g e s t e d b y the  studies that d e m o n s t r a t e d altered a c c e s s i b i l i t y to nucleases at the v a r i e g a t i n g l o c u s i n H P I m u t a t i o n s ( C r y d e r m a n et a l . , 1998).  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B i o c h i m i e 56, 937-954.  146  Appendix I  L i s t of A b b r e v i a t i o n s  bw  b r o w n v a r i e g a t e d of D e m e r e c 2  v D e 2  DUb80  D . melanogaster u b i q u i t i n f u s i o n p r o t e i n 80  e  ebony  E(var)s  enhancer of p o s i t i o n effect v a r i e g a t i o n  GCN5  histone acetyltransferases f r o m S. cerevisiae  H3 K9  l y s i n e n i n e of histone H 3  H3 mK9  m e t h y l a t e d l y s i n e n i n e of h i s t o n e H 3  HAT  histone acetyltransferase  HDAC  histone deacetylase  HDACl  histone deacetylase one  HOX  vertebrate h o m e o t i c genes  InCDscf In(l)w  1  m 4  I n ( 3 L R ) T M 3 A2-3 Sb  i n v e r s i o n one scute  sl  i n v e r s i o n one w h i t e m o t t l e d four i n v e r s i o n three left r i g h t b e a r i n g the transposase source A2-3 a n d Stubble  ISW1  chromatin remodeling protein  MAD  m a t r i x associated deacetylase b o d i e s  Mi-2  A T P dependent nucleosome remodeling factor  N-CoR  nuclear receptor corepressor  147  NuRD  nucleosome remodeling complex  P-1.8  a fly strain b e a r i n g a P element inserted 1.8 k b 5' to the start of H D A C l  p300/ C B P  t r a n s c r i p t i o n a l coactivator  PCAF  histone acetyltransferase  PcG  P o l y c o m b G r o u p of p r o t e i n s  PCR  p o l y m e r a s e c h a i n reaction  PEV  p o s i t i o n effect v a r i e g a t i o n  P-UTR  a fly strain b e a r i n g a P element inserted into the 5' u n t r a n s l a t e d r e g i o n of HDACl  RB  retinoblastoma protein  RPD3  r e d u c e d p o t a s s i u m d e p e n d e n c y three  ry  5 0 6  a n allele of the rosy gene f r o m D . melanogaster  ry™  a n allele of the rosy gene f r o m D . melanogaster  Sb  the Stubble gene f r o m D . melanogaster  Sb  v  a v a r i e g a t i n g allele of Stubble  Ser  the Serrate gene f r o m D . melanogaster  SMRT  s i l e n c i n g m e d i a t o r for retinoic a c i d a n d t h y r o i d h o r m o n e receptors  SNF2  sucrose n o n - f e r m e n t i n g t w o f r o m S. cerevisiae  Su(var)s  suppressor of p o s i t i o n effect v a r i e g a t i o n  SWI2  a n A T P d e p e n d e n t helicase  TAF„230/250  transcription associated factor II  Tb  the Tubby gene from D. melanogaster  148  TM3  a multiply inverted third chromosome which suppresses recombination in D. melanogaster  TM6  a multiply inverted third chromosome which suppresses recombination in D. melanogaster  tsp  temperature sensitive period  YY1  yin/yang transcriptional corepressor and activator  149  

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