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RNA polymerase II holoenzyme-mediated regulation of GAL4 Hirst, John Martin 2000

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RNA POLYMERASE IIHOLOENZYME-MEDIATED REGULATION OF G A L 4 by John Martin Hirst B.Sc. (Hons Biochem.), The University of British Columbia, 1994 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n FACULTY OF GRADUATE STUDIES (Department of Biochemistry and Molecular Biology) I accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH COLUMBIA July 1999 © Martin Hirst, 1999 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 ^ \ c x J ^ o M . . C W f rt. The University of British Columbia Vancouver, Canada D a t e — T > 0 2^ W \ DE-6 (2/88) Abstract Much of the current understanding of eukaryotic transcriptional regulation is based on insights gained through the use of model systems. The premier transcriptional model system is galactose metabolism in the yeast Saccharomyces cerevisiae, regulated by the transcriptional activator GAL4. Regulation by GAL4 involves a number of mechanisms that act to ensure that galactose is catabolized when available, but that glucose is used preferentially. One such mechanism involves phosphorylation of GAL4 at serine 699. Phosphorylation at S699 is required for efficient galactose-inducible transcription of the GAL genes, but occurs as a consequence of transcriptional activation by GAL4. This has led to the hypothesis that phosphorylation occurs through interaction with RNA polymerase II holoenzyme components. The experiments described in this thesis determine the role of RNA polymerase II holoenzyme-associated kinases in phosphorylation of GAL4, and investigate the mechanism through which phosphorylation at S699 affects induction. Data presented here demonstrate that GAL4 S699 is a substrate for the cyclin dependent protein kinase SRBTO. S699 phosphorylation requires SRB10 in vivo, and this site is phosphorylated by purified SRB10/ SRB11 in vitro. RNA polymerase II holoenzymes purified from WT yeast phosphorylate GAL4 at sites observed in vivo, whereas holoenzymes from srblO yeast are incapable of phosphorylating GAL4 at S699. SRB10 is required for efficient GAL induction and is shown to be epistatic to S699 phosphorylation. To investigate interactions between GAL4 and it's regulator GAL80, a two-hybrid based strategy was devised and used to demonstrate that S699 phosphorylation may regulate interaction between GAL4 and GAL80 in vivo. These ii data define a function for the SRB10 holoenzyme-associated CDK8 that involves regulation of transactivators by phosphorylation during transcriptional activation. iii TABLE OF CONTENTS A B S T R A C T i i T A B L E O F CON T E N T S iv LIST O F T A B L E S v i i LIST O F FIGURES v i i i LIST O F ABBREVIATIONS x A C K N O W L E D G M E N T S x i 1. INTRODUCTION 1 1.1 OBJECTIVE 1 1.2 EUKARYOTIC TRANSCRIPTIONAL REGULATION 2 1.2.1 Promoter Structure 2 TATA elements 2 Initiator elements 3 UAS and URS Elements 4 Poly (dA-dT) Elements 7 1.2.2 RNA Polymerase II 7 Carboxy-Terminal Repeat Domain 8 RNA Polymerase II Holoenzymes 10 1.2.3 General Transcription Factors 12 Tata Binding Protein 13 TFIIB 14 TFIIF 14 TFIIE 16 TFIIH 16 1.2.4 Transcriptional Coactivators 18 iv TBP-Associated Factors 19 Mediator 20 TFIIA 24 Acetyltransferases 24 Chromatin-Remodeling Complexes 26 1.3 THE GAL GENES AS A MODEL FOR EUKARYOTIC TRANSCRIPTION....27 1.3.1 GAL2 28 1.3.2 GAL3 30 1.3.3 GAL80 30 1.3.4 GAL4 31 DNA Binding 33 Transcriptional Activation 34 Gal80 Interaction 35 Central Region 36 Phosphorylation 37 1.3.5 Induction by galactose 38 1.3.6 Repression by glucose 39 1.3.7 Two-hybrid systems 40 2. MATERIALS AND METHODS 45 2.1 PLASMIDS AND YEAST MANIPULATIONS 45 2.2 ANTIBODIES AND RECOMBINANT PROTEINS 47 2.3 METABOLIC LABELING, TRYPTIC PHOSPHOPEPTIDE ANALYSIS, AND IN VITRO KINASE ASSAYS 48 3. RESULTS 51 3.1 GAL4 PHOSPHORYLATION 51 3.1.1 A single C-terminal GAL4 phosphorylation is requiredfor GAL gene induction 51 3.1.2 Mutations to RNA Polymerase II holoenzyme components affect GAL4 phosphorylation in vivo 54 3.1.4 TFIIKpredominately phosphorylates GAL4 at S83 7 in vitro 63 3.1.5 SRB10 specifically phosphorylates GAL4 at S699 in vitro 66 3.1.6 GAL4 S699 is phosphorylated by SRBlO-containing RNA Pol II holoenzymes in vitro 77 3.1.7 The requirement ofSRBW for GAL induction is epistatic to GAL4 S699 phosphorylation 84 3.2 S699 PHOSPHORYLATION REGULATES THE AFFINITY OF GAL4 FOR GAL80 IN VIVO 90 3.2.1 The Repressed Transactivator Assay 92 3.2.2 The N terminus of TUP 1 represses transcription as a fusion in a two hybrid system 96 3.2.3 SRB10 is requiredfor repression by the N terminus ofTUPl 97 3.2.4 Repression of GAL transcription by TUP1-GAL80 requires the C-terminal 30 residues of GAL4 99 3.2.5 S699 inhibits the GAL4/GAL80 interaction in vivo 102 v 4. DISCUSSION 107 4.1 FUNCTION OF THE R N A POLYMERASE II HOLOENZYME-ASSOCIATED CDKS . . .107 4.2 REGULATION OF R N A POLYMERASE HOLOENZYME CDK FUNCTION 109 4.3 REGULATION OF G A L 4 ACTIVITY BY S699 PHOSPHORYLATION 112 4.4 SIGNIFICANCE OF G A L 4 S699 PHOSPHORYLATION FOR GAL GENE REGULATION 113 4.5 REPRESSED TRANSACTIVATOR ASSAY 114 4.6 FUTURE DIRECTIONS 115 4.7 CONCLUSION 116 REFERENCES 118 vi LIST OF TABLES 1. Mediator complex components 21 2. Yeast strains 50 vii LIST OF FIGURES 1. Model for regulation of transcription by RNA polymerase II.... 6 2. The Leloir Pathway 29 3. Functional domains on GAL4 32 4. Induction of the GAL genes 42 5. Schematic diagram of WT and A683 GAL4 derivatives 52 6. Mutation of S699 to alanine or glutamate impairs GAL induction by GAL4 53 7. Inactivation of KIN28 reduces overall phosphorylation of GAL4 57 8. Deletion of SRB10 eliminates specific phosphorylations of GAL4 59 9. Inactivation of RPB1 reduces overall phosphorylation of GAL4 61 10. SRB10 phosphorylates GAL4 at S699 in vivo 65 11. Immunopurified complexes containing HA-KIN28 and HA-SRB10 phosphorylate GAL4 in vitro 68 12. Immunopurified HA-KIN28, HA-SRB10, and purified TFIIH phosphorylate GAL4 at S699 and S837, S699, and S837 respectively 70 13. Immunopurified HA-SRB10 phosphorylates a tryptic peptide spanning S699 more efficiently than HA-KIN28 73 14. Immunopurified HA-SRB10 and HA-KJN28 are recovered at similar levels 74 15. GAL4 phosphorylated by immunopurified HA-KIN28 isolated from srblO yeast show a marked decrease in phosphorylation at S699 76 16. Recombinant SRB10/ SRB11 complexes specifically phosphorylate GAL4 79 viii 17. Purified recombinant SRB10/ SRB11 complexes phosphorylate GAL4 at S699., 81 18. SRB10 is present in RNA pol II holoenzyme preparations 83 19. Purified RNA pol II holoenzymes phosphorylate GAL4 but not GAL80 86 20. SRB10 is required for phosphorylation of GAL4 at S699 by purified RNA pol II holoenzymes 88 21. The requirement of SRB10 in GAL gene induction is epistatic to S699A 89 22. Schematic representation of the RTA system 94 23. The N-terminus of TUP1 represses transcription in the context of a two-hybrid interaction 98 24. Repression mediated by the N-terminus of TUP 1 is dependent on SRB10 101 25. Repression of GAL transcription by TUP1-GAL80 requires the C-terminal 30 residues of GAL4 104 26. S699 functions by modifying the GAL4 / GAL80 interaction 106 27. Model of RNA pol II holoenzyme mediated regulation of GAL4 111 ix LIST OF ABBREVIATIONS GTF General Transcription Factors HATs Histone Actetyltransferase PIC Preinitiation Complex Cdk Cyclin Dependent Kinase pol II Polymerase II GAL Galactose Inr Initiator UAS Upstream Activating Sequence URS Upstream Repression Sequence CTD Carboxy Terminal Domain NER Nucleotide Excision Repair CAK Cdk activating Kinase mRNA Messenger ribonucleic acid DNA Deoxyribonucleic acid PCR Polymerase chain reaction RSC Restructures Chromatin HIV-1 Human Immunodeficiency Virus (Subtype 1) SAGA Spt-Ada-Gcn5 -actetyltransferase LTA Long Term Adaptation AA Amino Acid RTA Repressed Transactivator TBP TATA Binding Protein SD Synthetic Dropout BP Base Pair KB Kinase Buffer ATP Adenosine Triphosphate TRP Tryptophan HIS Hjstidine LEU Leucine URA Uracil 5-FOA 5'-Fluoro-orotic acid PTEF-b Positive transcription elongation factor b TAF TBP associated factor CPM Counts per minute Mab Monoclonal antibody GRD Glucose Response Domain ID Inhibitory Domain UV Ultraviolet Light DBD DNA binding domain micro-n nano-kDa kiloDalton X A C K N O W L E D G E M E N T S I would like to thank my family for their unlimited support. Ivan for giving me the freedom to pursue my thesis. Michelle for giving me focus. Paul and Sara for their understanding. Mario for the late night adventures. Most of all to Claire and Graeme who gave me light during my darkest moments. xi Introduction 1. INTRODUCTION 1.1 OBJECTIVE Eukaryotic genes are regulated by interactions between transacting factors bound to DNA at enhancers or upstream activating sequences, and components of the general transcription factor (GTF) machinery which assemble near the site of transcriptional initiation. Transactivator proteins have been shown to interact with RNA polymerase II holoenzyme and TFIID components (for Review see Ptashne and Gann, 1997). These interactions are thought to catalyze assembly of preinitiation complexes (PIC) through the passive recruitment of GTFs. However, yeast and mammalian RNA polymerase II (pol II) holoenzymes, and mammalian TFIID complexes, are known to contain protein kinases, whose presence implies that the GTF machines may themselves be targets for signals that modulate gene expression. The RNA pol II holoenzyme contains two cyclin-dependent protein kinases (cdks) associated with TFIIH and the mediator whose role in controlling specific transcriptional events has yet to be determined (for Review see Carlson, 1997). Mechanisms of eukaryotic transcription are highly conserved. The GAL genes are controlled by well defined positive and negative stimuli and have proven to be invaluable for elucidating many of the basic mechanisms involved in regulation of eukaryotic transcription. The objective of my thesis research was to determine the function of the RNA pol II holoenzyme-associated cdks in regulation of the GAL genes in the yeast Saccharomyces cerevisiae. 1 Introduction 1.2 EUKARYOTIC TRANSCRIPTIONAL REGULATION 1.2.1 Promoter Structure The eukaryotic promoter, defined as a site where RNA polymerase binds to and initiates transcription, can be divided into core and regulatory elements that act to nucleate the cellular machinery required for the initiation of transcription. The core element defines the site of assembly for the pre-initiation complexes (PICs) and is comprised of the TATA element, found upstream of the transcriptional start site, and the initiator sequence (Inr) encompassing the transcriptional start site. Regulatory elements can be divided into two main classes: upstream activation sequences (UAS) and upstream repression sequences (URS). As their name implies, these sequences are involved in recruiting specific factors to promoters which act to either activate or repress transcription by modifying initiation rates from promoters. In addition, a third regulatory element characterized by poly(dA-dT) sequences functions to facilitate constitutive gene expression. TATA elements In Saccharomyces cerevisiae the TATA element is located 40 to 120bp upstream of the transcriptional start site in contrast to most other eukaryotes, including Schizosaccharomyces pombe, where it is almost always found 25 to 30bp from the 2 Introduction transcriptional start site (for review see Struhl, 1995). The TATA sequence is bound by the TATA binding protein (TBP) which functions to nucleate the PIC (Klein and Struhl, 1994). Extensive mutational analysis, combined with random selection for functional TATA elements, defined the sequence "TATAA" as the consensus TATA-box sequence in yeast (Chen and Struhl, 1988; Singer et al., 1990; Wobbe and Struhl, 1990). However, there appears to be no strict requirement for a single consensus TATA element in promoters; promoters containing multiple TATA elements (Li and Sherman, 1991), mutant TATA elements (Wobbe and Struhl, 1990), or no TATA element, have been described (Cormack and Struhl, 1992). Regardless of the nature of the promoter, all are dependent on TBP, including TATA-less promoters which employ additional DNA binding components in the PIC to aid in recruitment (Burke and Kadonaga, 1997; Burke and Kadonaga, 1996; Cormack and Struhl, 1992). Initiator elements Inr elements define sequences encompassing the transcriptional start site. Unlike the TATA element, no consensus Inr sequence has been identified although some sequences appear to be preferred (Furter-Graves and Hall, 1990). In higher eukaryotes, Inr elements in both TATA-containing and TATA-less promoters function to nucleate the assembly of PICs through the binding of a host of proteins including RNA pol II itself (Carcamo et al., 1991; Kaufmann et al., 1998; Kaufmann et al., 1996; Roy et al., 1993; Usheva and Shenk, 1994). In yeast it is still unclear whether Inr elements are able to nucleate PIC assembly; however, studies of the GAL80 promoter have revealed a Inr-3 Introduction dependent transcriptional start site which appears to recruit an Inr-binding protein (Sakurai et al., 1994). UAS and URS Elements UAS elements function as DNA targets for specific transcriptional activators. They are analogous to metazoan enhancers and can function in either orientation and at variable distances from the start site. Unlike their metazoan counterparts however, UAS elements do not function downstream of the TATA element (Guarente and Hoar, 1984; Struhl, 1984). Once bound to their cognate elements, transcriptional activators facilitate the assembly of the PIC either through direct contact with general transcription factors (GTF) or indirectly through coactivators. Yeast promoters also contain URS elements, which recruit specific transcriptional repressors. Once bound to URS elements, transcriptional repressors inhibit transcription through a variety of mechanisms: sterically inhibiting the association of transcriptional activators, inhibiting formation of the PIC, or interacting with components which organize repressive chromatin and/or modify existing chromatin through the action of deacetylases (for review see Johnson, 1995). An example of chromatin-mediated repression is represented by the TUP1 repressor. TUP1 is recruited to a wide variety of promoters through its association with specific DNA binding partners, and acts to organize repressive chromatin (see below). Histone deacetylation-mediated repression is exemplified by UME6 which recruits the deacetylation factor RPD3 to meiotic-specific promoters (Kadosh and Struhl, 1997). 4 Introduction Figure 1. A simplified model of transcriptional regulation by the RNA polymerase II holoenzyme. (A) The repressed or "off state of a promoter bound by histones in chromatin. (B) Recruitment of TFIID by a transcriptional activator is the rate limiting step in activation and involves the displacement of repressive chromatin and the bending of DNA flanking the TATA box. TFIIB and TFIIA act to stabilize the TFIID/ DNA complex . (C) Recruitment of the holoenzyme involves interactions (denoted by arrows) between the transactivator and its GTF (dark grey) and coactivator (light grey) targets. Other coactivators whch have been found to be associated with the RNA polll holoenzyme have been left out for simplicity (see text). 5 6 Introduction Poly (dA-dT) Elements Poly (dA-dT) tracts are found upstream of a wide variety of yeast promoters and are required for normal transcription in a subset of these (for review see Iyer and Struhl, 1995). Poly (dA-dT) sequences form structures which are refractory to nucleosome assembly and stability, suggesting that they cause transcriptional activation independent of sequence-specific transcription factors (Chen et al., 1987). Support for this model comes from the poly (dA-dT) element which accentuates transcription in a length-dependent manner located upstream of the GCN4 binding site in the HIS3 promoter (Iyer and Struhl, 1995). In addition, examination of the S. cerevisiae genome has revealed that poly (dA-dT) elements are abundant, and occur at unit nucleosomal length both upstream and downstream of open reading frames, implying a role in nucleosome positioning (Raghavan et al., 1997). 1.2.2 RNA Polymerase II Transcription of all mRNA in eukaryotes is performed by a complex of 12 polypeptides which are collectively known as RNA polymerase II (RNA pol II)(Woychik and Young, 1994). 6 subunits of yeast RNA pol II can be functionally replaced by their respective human orthologs (McKune et al., 1995). The two largest subunits RPB1 (~200kDa) and RPB2 (~150kDa) are the most highly conserved and are both structurally and functionally related to the p' and P subunits of bacterial RNA 7 Introduction polymerase (Sweetser et al., 1987; Woychik and Young, 1994; Young, 1991). In addition, RPB3 is related to the a-subunit in that they are both present at two copies per polymerase, are of similar size, and contain partial sequence similarity. No subunits appear to be related to the a-subunit although some similiarities are noted in certain GTFs (see below). RNA poll and III have a high degree of similiarity to RNA pol II, and share five subunits, RPB5, RPB6, RPB8, RPB10, and RPB12. Furthermore both polymerases contain homologs to RPB1, RPB2, RPB3, and RPB11. Only RPB4, RPB7, and RPB9 are unique to RNA pol II. Of the 12 genes encoding the subunits of RNA polll, 10 are essential for cell viability. RPB4 and RPB9 are dispensible, but deletion of their corresponding genes confers conditional growth phenotypes (Woychik et al., 1991; Woychik and Young, 1989). Carboxy-Terminal Repeat Domain The largest subunit of RNA pol II (RPB1) is distinct from its homologs found in RNA pol I, pol III, and bacterial (3' by the presence of tandem repeats of a heptapeptide sequence at its carboxy-terminus called the C-terminal domain (CTD). This repeated sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser, is highly conserved amongst eukaryotes, but the number of repeats appears to increase with increasing genome complexity: 26-27 in yeast, 34 in C. eiegans, 43 in Drosophila, and 52 in humans (Hampsey, 1998). The CTD is essential for viability; yeast with only 8/ 27 repeats are viable but are cold-sensitive and display a number of other phenotypes associated with altered gene regulation (Nonet and Young, 1989; West and Corden, 1995). The cold-sensitive phenotype was exploited by 8 Introduction Young and his colleagues to identify extragenic suppressors of CTD truncations, collectively known as the SRBs (suppressor of RNA polymerase B) (Nonet and Young, 1989). These genes were subsequently found to encode components of the mediator complex required for transcriptional activation (Bjorklund and Kim, 1996; Nonet and Young, 1989). The fact that the SRB alleles suppress CTD-truncation defects implies that they are involved in negative regulation of RNA pol II function. This is supported by numerous genetic screens which have identified SRB genes in transcriptional regulation (see below). RBP1 exists as two isoforms in vivo designated IIO and IIA. IIO is extensively phosphorylated on the CTD and is present in elongating complexes, whereas IIA is unphosphorylated and is preferentially recruited to the PIC (reviewed in Dahmus, 1995). Phosphorylation of IIA occurs concomitantly with the transition from initiation to elongation, implicating the CTD in the transition of RNA Pol II from a promoter recognition to an elongation-competent complex (Lu et al., 1991; O'Brien et al., 1994). A wide variety of protein kinases in yeast and mammalian cells are capable of phosphorylating the CTD. These include the kinase subunit of TFIIH (Feaver et al., 1991; Lu et al., 1992; Serizawa et al., 1995), CTK1 (Lee and Greenleaf, 1991), SRB10/11 (Liao et al., 1995), mammalian CDC2 (Cisek and Corden, 1989), and mammalian P-TEFb (Marshall et al., 1996). The catalytic subunit of human P-TEFb (positive transcription elongation factor b), also known as PITALRE / CDK9, associates with the HIV-1 transactivator TAT to potentate the transition from abortive to active elongating complexes (Marshall et al., 1996; Zhu et al., 1997). In contrast, experiments with recombinant SRB 10/11 suggest that the repressive effect mediated by SRB 10 in vivo is 9 Introduction associated with premature CTD phosphorylation (see below)(Hengartner et al., 1998). Thus, kinases which phosphorylate the CTD appear to play both positive and negative roles in regulation of RNA pol II function. A CTD phosphatase (CTDP), which is regulated through the actions of TFIIB and TFIIF (see below), has been identified (Chambers and Kane, 1996; Chambers et al., 1995). Phosphatase activity is stimulated by the RAP74 subunit of TFIIF but is inhibited by TFIIB (Archambault et al., 1997; Chambers and Kane, 1996). Since assembly of the PIC requires dephosphorylation of the CTD (Dahmus, 1995), it has been suggested that the phosphatase acts in concert with TFIIB and TFIIF to regulate RNA pol II recycling (Hampsey, 1998). The CTD has also been implicated in pre-mRNA processing (for review see Steinmetz, 1997). Splicing is inhibited in vivo (Yuryev et al., 1996) and in vitro (Du and Warren, 1997) by the addition of excess recombinant CTD repeat polypeptide, and anti-CTD antibodies inhibit splicing in vitro (Yuryev et al., 1996). A model has been proposed whereby the hyperphosphorylated form of IIO, being highly negatively charged, interacts with the positively charged regions of some splicing factors (Greenleaf, 1993). Thus the CTD may play a role in splicing by providing a platform for the assembly of pre-mRNA processing factors. RNA Polymerase II Holoenzymes Initial studies of the general transcription factors for RNA pol II involved purification of individual factors. Subsequently, reassembly of transcription factor complexes in vitro identified a defined order of GTF assembly to the RNA pol II core 10 Introduction (Buratowski et al., 1989; Maldonado et al., 1990). The identification of the SRBs by Young and colleagues led to the purification of a complex which contained not only the SRBs but also several GTFs (Koleske and Young, 1994). This complex, unlike core RNA pol II, responded to activators in in vitro transcription reactions. A holoenzyme complex was also independently identified by Kornberg and colleagues through its association with the mediator complex (Kim et al., 1994). Both holoenzymes contain comparable subunits, although the former contained TFIIB, TFIIF, and TFIIH (Koleske and Young, 1994) whereas the latter contained TFIIF as the only GTF (Kim et al., 1994). A variety of holoenzymes have subsequently been purified from both yeast and mammalian sources. In yeast, holoenzymes containing SRB 10/11 (Hengartner et al., 1995; Liao et al., 1995) as well as the SWT/ SNF chromatin remodeling complex have been reported (Wilson et al., 1996) . In addition, another form of a RNA pol II holoenzyme has recently been isolated through its association with immobilized CTD, containing TFIIB, TFIIF, TFIIS, and GAL11 but not the SRB/ mediator components (Shi et al., 1997; Wade et al., 1996). Novel components of this complex include PAF1, CDC73, CCR4, and HPR1 (Chang and Jaehning, 1997; Shi et al., 1997; Wade et al., 1996). This form of the holoenzyme affects expression of a subset of genes distinct from those affected by SRB/ mediator-containing holoenzymes (Shi et al, 1996). This raises the possibility that different classes of RNA pol II holoenzyme forms may be present in vivo, and are either recruited to, or assembled at promoters by specific transcription factors in response to different stimuli. Mammalian holoenzymes which are able to respond to activators in vitro have also been identified (Chao et al., 1996; Maldonado et al., 1996; Ossipow et al., 1995; Pan et al., 1997) . Like their yeast counterparts, they are found associated with GTFs as well as 1 1 Introduction other factors, including DNA repair proteins (Maldonado et al., 1996), splicing and polyadenylation factors (McCracken et al., 1997), and the breast cancer tumor suppressor BRAC1 (Scully et al., 1997). In addition, some mammalian RNA pol II holoenzyme preparations contain TFIID and as such require no additional factors to initiate transcription in vitro (Pan et al., 1997). 1.2.3 General Transcription Factors The general transcription factors were originally identified biochemically from mammalian cell extracts as factors required for accurate transcriptional initiation by RNA polymerase II, and include the factors TFIID (TBP plus the TAFs, see below),TFIIB, TFIIE, TFIIF, and TFIIH (for review see Roeder, 1996). Subsequent fractionation of yeast nuclei similarly identified five comparable factors designated; a (TFIIE), b (TFIIH), d (TBP), e (TFIIB), and g (TFIIF) (Sayre et al., 1992). Once fractionated, order of addition experiments demonstrated that PIC assembly is nucleated in vitro by TFIID binding to the TATA element, followed by the sequential binding of TFIIB, RNA pol II-TFIIF, TFIIE, and TFIIH (Buratowski et al., 1989). The in vivo significance of this finding was challenged by the discovery of holoenzyme complexes (see above). However, it remains unclear whether holoenzymes actually represent free complexes, or whether these purified complexes are formed on promoter elements and subsequently stripped of DNA during purification. In fact, Kimura et al. have recently performed quantitation studies on holoenzymes and they observed less than 3 percent of unengaged polymerases present in holoenzyme sized complexes (Kimura et al., 1999). 1 2 Introduction Tata Binding Protein TATA Binding Protein (TBP) is required for initiation by all three eukaryotic RNA polymerases, and as such has been called the universal transcription factor (Hernandez, 1993). TBP was identified as a subunit of TFIID, the large (~750kDa) multisubunit complex composed of TBP and TBP-associated factors (TAFs)(Hoey et al., 1990; Peterson et al., 1990). In in vitro transcription reactions, metazoan TBP functions in basal transcription, while TFIID is required for response to transcriptional activators (Burley and Roeder, 1996). Yeast TBP is a 27kDa monomer which can functionally replace mammalian TBP in in vitro transcription reactions (Buratowski et al., 1988). Initial cloning and sequence analysis of yeast TBP revealed that it was identical to SPT15 isolated as a suppressor of the his4-9178 mutation (Eisenmann et al., 1989). This mutation, arising from the integration of a Ty-retrotransposon 8 element upstream of the HIS4 gene, shifts the initiation of transcription to within the 8 element, resulting in abnormally long non-functional transcripts (Hahn et al., 1989). The SPT genes were isolated as suppressors of this phenotype which shift initiation back downstream, resulting in functional H1S4 transcripts (Hahn et al., 1989). Further analysis of SPT15 demonstrated that it is essential and required for the expression of many, if not all genes, in vivo (Eisenmann et al., 1989). The crystal structures of Arabidopsis and yeast TBP have been solved and reveal a novel structure containing a saddle-like DNA binding fold that sits astride the DNA, introducing kinks at either end of the TATA element (Kim et al., 1993; Kim et al., 1993; Nikolov et al., 1992). Recruitment of TBP has been identified as the rate-limiting step in transcriptional activation and as such is a target of both transcriptional activators and repressors (Auble et al., 1994; Klein and Struhl, 1994; 13 Introduction Melcher and Johnston, 1995). In addition, TBP interacts with both TFIIA and TFIIB which appear to assist in TBP's recruitment to promoters (Killeen et al., 1992) TFIIB Yeast TFIIB is a 38kDa protein, encoded by SUA7, originally identified in a screen for suppressors of a translational defect arising from an aberrant ATG codon that initiates a short non-functional open reading frame in the CYC1 transcribed leader region (Pinto et al., 1992). Recessive sua7 mutations shift the transcriptional start site downstream of normal, eliminating the translational impediment, thus implicating TFIIB in start site selection (Pinto et al., 1994). TFIIB interacts directly with TBP (Nikolov et al., 1995) and RNA pol II (Malik et al., 1993; Sun et al., 1996) as well as other GTFs, including the TFIIF subunits RAP30 (Ha et al., 1993) and RAP74 (Fang and Burton, 1996), and the TAF„40 subunit of TFIID (Goodrich et al., 1993). TFIIB is also the target of numerous transcriptional activators (Baniahmad et al., 1993; Colgan et al., 1993; DeFalco and Childs, 1996; Haviv et al., 1998; Haviv et al., 1996; Kim and Roeder, 1994; Roberts etal., 1993). TFIIF TFIIF is composed of two subunits, RAP30 and RAP74, originally defined by their affinity for RNA Pol II (Burton et al., 1988). TFIIF was not identified as one of the four HeLa cell chromatographic fractions (TFIIA,TFIIB, TFIID, and TFIIE) required for accurate initiation in vitro (Sawadogo and Roeder, 1985); however, subsequent purification resolved TFIIE into E and F (Flores et al., 1989). There is some sequence similarity between the RAP30 subunit of TFIIF and bacterial a factor (McCracken and 14 Introduction Greenblatt, 1991), and TFIIF shares a number of functional characteristics with a factor, including suppression of non-specific binding of Pol II to DNA, stabilization of the PIC, and strong affinity for RNA pol II (Conaway and Conaway, 1993). This functional similiarity is extended by the observation that human TFIIF can bind to E. coli RNA 70 polymerase and is displaced by a (McCracken and Greenblatt, 1991). TFIIF has been implicated in both the initiation and elongation of mRNA transcripts. During initiation, TFIIF helps to stabilize the PIC as demonstrated by photo-cross-linking studies which have identified a TBP-TFIIB-TFIIF-RNA pol II-TFIIE complex (Coulombe et al., 1994; Robert et al., 1996). During elongation, TFIIF suppresses transient pausing of RNA polymerase (Bengal et al., 1991). In addition, TFIIF has been associated with dephosphorylation of the CTD through its interaction with CTD specific phosphatases (see above), suggesting it also plays a role in RNA pol II recycling (Archambault et al., 1998; Chambers and Kane, 1996). Yeast TFIIF (factor g) is found stably associated with RNA pol II and is required for specific initiation at all promoters tested. It is comprised of three subunits of 105kDa, 54kDa, and 30 kDa encoded by TFG1, TFG2, and TFG3 respectively (Henry et al, 1994; Henry et al., 1992). TFG1 and TFG2 are orthologs of RAP74 and RAP30, essential for viability, and are present in single copy in the yeast RNA pol II holoenzyme (Henry et al, 1994; Sun and Hampsey, 1995). TFG3 has no known mammalian homolog and is identical to ANC1, which was identified based on genetic interactions with ACT1 (Sun and Hampsey, 1995). TFG3 is only weakly associated with TFIIF and is also found in TFIID (TAF30)(Poon et al., 1995) and the SWI/ SNF complex (SWP29)(Cairns et al., 1996). TFG3 is the only non-essential GTF; however, there is a homolog in the 1 5 Introduction yeast genome (YOR213c), a subunit of the RSC chomatin remodeling complex (see below), which may functionally replace it (Hampsey, 1998). TFIIE Mammalian TFIIE (yeast factor a) has been shown to interact directly with the unphosphorylated version of RNA pol II (IIA), both subunits of TFIIF, and with TFIIH (Flores et al., 1988; Maxon et al., 1994). TFIIE recruits TFIIH to the PIC, stimulates TFIIH-dependent phosphorylation of the CTD (Lu et al., 1992; Ohkuma et al., 1995), and has been identified as a target of transcriptional activators (Sauer et al., 1995; Zhu and Kuziora, 1996). Purification of yeast factor a yielded two subunits of 66 and 43kDa which were later cloned as the essential genes TFA1 and TFA2, respectively (Feaver et al., 1994a; Sayre et al., 1992). TFA1 physically interacts with GAL11, a component of the yeast SRB/ mediator complex, and stimulation of transcription by GAL11 is dependent on TFIIE (Sakurai and Fukasawa, 1998). TFIIH Yeast factor b was originally identified as a three subunit complex of 85, 73, and 55kDa which restored transcriptional acitvity to heat-inactivated nuclear extracts supplemented with TBP (Feaver et al., 1991). The 73kDa subunit was subsequently used to purify a five-subunit core-TFIIH complex composed of 85, 73, 55, 50, and 38kDa polypeptides (Feaver et al., 1993). This complex could complement heat inactivated factor b in crude in vitro reactions, but was inactive in highly purified systems, providing an assay for the isolation of a TFIIH holoenzyme (Svejstrap et al., 1994). TFIIH 1 6 Introduction holoenzyme was found to contain, in addition to core TFIIH, SSL2, and two additional subunits of 47/ 45 and 33kDa which, through their tight association formed a subcomplex denoted TFIIK (Svejstrup et al., 1994). The TFIIK subcomplex of TFIIH includes a cyclin-dependent protein kinase encoded by KIN28 and a cyclin H homolog, encoded by CCL1 (Feaver et al., 1994b; Svejstrup et al, 1996). KIN28 is homologous to M015/ CDK7, the catalytic subunit of human TFIIH and a member of the p34/ CDC2/ CDC28 family of cyclin-dependent protein kinases (Roy et al., 1994). A temperature-sensitive kin28 allele causes rapid loss of CTD phosphorylation at the restrictive temperature, implicating KIN28 as the principle kinase of the CTD in vivo (Valay et al., 1995). The human MO 15/ Cyclin H subcomplex of TFIIH also includes MAT1, and this tripartite complex comprises the cdk-activating kinase (CAK) in humans implicated in cell cycle progression (Adamczewski et al., 1996; Fisher and Morgan, 1994; Shiekhattar et al., 1995). In yeast the MAT1 homolog is encoded by TFB3 and is part of the core TFIIH. TFB3 and KIN28 interact, likely providing a bridge between TFIIK and core-TFIIH (Feaver et al., 1997). CAK in yeast is encoded by CAK1I CIVI and is not a component of TFIIH (Espinoza et al., 1996; Kaldis et al., 1996; Thuret et al., 1996). A role for TFIIH in nucleotide excision repair (NER) was demonstrated through the identification of p89, the largest subunit of human TFIIH, as ERCC3 (excision repair cross-complement), which complements the DNA repair deficiency associated with the XPB gene defect (Schaeffer et al., 1994; Svejstrup et al., 1996). In yeast, the purification of two separate forms of TFIIH suggested that yeast TFIIH, like its mammalian ortholog, functions in two separate pathways. Consistent with this, TFIIK is dispensible for NER which requires only the core TFIIH, SSL2, and all other known NER proteins (Sung et al., 1 7 Introduction 1996). The largest subunit of TFIIH was subsequently identified as RAD3, the yeast homolog of ERCC2, a 5'- 3' DNA helicase shown previously to function in NER (Feaver et al., 1993). RAD3 also affects transcription; however, a mutation which eliminates ATPase/ helicase activity and NER has no effect on transcription, suggesting that its critical role in transcription is structural rather than functional in nature (Bardwell et al., 1994; Feaver et al, 1993; Gustafsson et al., 1997). SSL1 (50kDa subunit) and SSL2 were identified in a screen looking for suppressors of an artificial stem loop structure introduced into the transcribed leader region of the HIS4 gene (Feaver et al., 1993; Gulyas and Donahue, 1992; Yoon et al., 1992). Both ssll and ssl2 mutations led to increased UV sensitivity and SSL2 was cloned independently based on hybridization with a human ERCC3 probe and designated RAD25 (Parketal., 1992). 1.2.4 Transcriptional Coactivators Transcriptional coactivators, mediators, or adapters are proteins required for transcriptional activation. Coactivators are distinct from the GTFs in that they are dispensible for basal level transcription in vitro, and distinct from activators in that most do not directly bind DNA and none appear to bind DNA in a sequence-specific manner (Hampsey, 1998). Yeast coactivators can be roughly divided into 5 functionally distinct classes including: the TAF components of TFIID; the SRB/ mediator complex; TFIIA; SAGA, and related complexes that catalyze nucleosomal histone acetylation; and the SWI/ SNF, and related, chromatin-remodeling complexes. Coactivators function either by providing a link between activators and the holoenzyme or by modifying repressive chromatin structure. 1 8 Introduction TBP-Associated Factors Although TBP is referred to as the universal transcriptional factor and is required for transcription by all three RNA polymerases, it is in each case found associated with a disinct set of polymerase-specific factors. Mutations in the class of SPT genes which includes TBP (SPT15), gave a related set of pleiotropic phenotypes, and the product of one of these genes SPT3, was found to physically associate with TBP (Eisenmann et al., 1992; Eisenmann et al., 1989). Subsequent purification of cellular TBP produced a large complex containing the majority of TBP, and immunoprecipitation resulted in the copurification of nine polypeptides (Poon and Weil, 1993). Initial characterization of this complex identified BRF1, a subunit of the RNA Pol Ill-specific factor TFIIIB (Poon and Weil, 1993) and MOT1 (TAF170) a RNA pol II-specific inhibitor of TBP (Poon et al., 1994). Further purification of polypeptides associated with this complex yielded three TAFs; TAF130, TAF90, TAF60 and TSM1 cloned previously based on physical linkage to the M4riocus (Poon et al., 1995; Ray et al., 1991). Using TBP as an affinity ligand also yielded a TFIID complex from yeast required for activated transcription in vitro (Reese et al., 1994). Two components of this complex were identified as TAF145 and TAF90, and turned out to be identical to the previously identified TAF130 and TAF90 purified by immunoprecipitation (Poon et al., 1995). Computer searches of the yeast genome yielded additional TAFs based on their homology to human and Drosophila TAFs, confirmed by coimmunoprecipitation with TBP (Moqtaderi et al., 1996). In total, 12 yeast TAFs have been identified and without exception have metazoan homologs 1 9 Introduction (Klebanow et al., 1997; Klebanow et al., 1996; Moqtaderi et al, 1996; Poon et al., 1995; Reese etai., 1994). The exact function of TAFs remains unclear. They are assumed to interact with transcriptional activators to mediate activation (reviewed in Pugh and Tjian, 1992). However, depletion or inactivation of several TAFs including the scaffold TAF130/145 does not compromise activation in vivo from promoters containing consensus TATA boxes, but does affect transcription from promoters lacking a consensus TATA box (Moqtaderi et al., 1996; Walker et al., 1996). Thus, the essential function of TAFs likely relates to their role in transcription of a subset of genes. In fact, yeast containing a tafl45's allele block transcription of the Gl/ S cyclin genes at the restrictive temperature (Walker et al., 1997). There also appears to be a cell cycle connection in mammalian cells where TAF250 was identified as the product of the CCG1 gene required for passage through the Gl phase of the cell cycle (Ruppert et al., 1993). Mediator The first evidence of a mediator of transcriptional activation came from "squelching experiments" which demonstrated that overexpression of one activator could inhibit activation dependent on a second activator (Flanagan et al., 1991; Gill and Ptashne, 1988; Kelleher et al., 1990). This effect could be demonstrated in vitro and rescued by a partially purified yeast fraction but not by excess GTFs. Purified mediator is functionally defined by three activities: (i) stimulation of basal transcription; (ii) response to transactivators; and (iii) stimulation of pol II CTD phosphorylation in the presence 20 Introduction Table 1. Mediator Complex Components1 COMPONENT ESSENTIAL GENE(S) COMMENTS SRB2 NO SRB2 Interacts with SRB4, SRB5, TBP SRB4 YES SRB4 Interacts with SRB2,SRB6,MED6,GAL4 SRB5 NO SRB5 Interacts with SRB2 SRB6 YES SRB6 Interacts with SRB4 SRB7 YES SRB7 ROX3 YES ROX3, SSN7 Involved in transcriptional repression MED8 YES MED8 Tightly associated with ROX3 MED6 YES MED6 Interacts with SRB4 SRB8 NO SRB8, SSN5, ARE2 Involved in transcriptional repression SRB9 NO SRB9, SSN2, UME2 Involved in transcriptional repression SRB10 NO SRB10, SSN3, UME5, ARE] CDK interacts with GAL4 and SRB11 SRB11 NO SRB11, SSN8, UME3 Cyclin C, interacts with SRB10 RGR1 YES RGR1 Involved in glucose repression ROX3 YES ROX3, SSN7 Involved in glucose regulated expression MEDIO YES MEDIO / NUT2 Required for GCN4 dependent HIS4 expression. MED11 YES MED 11 Required for MFA1 transcription GAL11 NO GAL11, SPT13, SDS4, RAR3 Required for growth on GAL and non-ferm Carb NUT1 NO NUT1 Involved in transcriptional repression SIN4 NO SIN4, SSN4, TSF3 Complexed with GAL11, RGR1, PGD1, MED1 NO MED1 MED2 MED2 NO MED2 Interacts with MED2, neg. regulated by SRB10 MED3 NO MED3, PGD1, HRS1 Interacts with MED1, SRB4, reg. for GAL4 act. MED9 NO MED9/CSE2 Complexed with GAL11, RGR1, SIN4, MED2 Involved in Chrom. Seg. and required, for basal HIS4 expression MED4 YES MED4 MED7 YES MED7 of TFIIH (Hampsey, 1998; Kim et al., 1994; Myers et al., 1998). In contrast to the TAFs, many of the mediator components were identified in genetic screens including; suppression of CTD truncations (SftB)(Nonet and Young, 1989), suppression of ' Mediator components are presented in functional groupings 2 1 Introduction constitutive glucose repression (SSN)(Carlson et al., 1984), suppression of Ty insertions (SP7)(Fassler and Winston, 1988), suppression of aberrant chromosomal segregation (CSE2)(Xiao et al., 1993), derepression of the HO gene (W)(Stillman et al., 1994), derepression of the GAL1 gene (T£F)(Chen et al., 1993), derepression of meiotic genes (L^ ME)(Strich et al, 1989), derepression of MFA2 (^i?£)(Wahi and Johnson, 1995), derepression of SUC2 (Sakai et al., 1988), derepression of the CYC7 gene (ROX) (Rosenblum-Vos et al., 1991), derepression of a URS2 element (A^ i77)(Tabtiang and Herskowitz, 1998) and repression of the GAL genes (GAL1 i)(Nogi and Fukasawa, 1980). Thus, components of the mediator are involved in the regulation of a wide variety of genes and play key roles in both activation and repression of transcription. Mediator genes can be grouped into functional classes, and for ease of understanding, they are referred to by their SRB designations except for GAL11, SIN4, RGR1, ROX3, NUT1, NUT2, and the MED genes for which no such designation exists (Table 1). SRB4, SRB6, and SRB7 are all essential for viability, and temperature-sensitive mutations in srb4 and srb6 cause a rapid decline in mRNA levels upon shift to the non-permissive temperature (Thompson and Young, 1995). In addition, SRB4 affects the transcription of nine specific genes and has been identified as a direct target for transcriptional activation by GAL4 (Koh et al., 1998). SRB4 and SRB6 also form another functional grouping based on the fact that they, along with SRB2 and SRB5, were found as dominant mutations. Structural evidence for this complex has come from cross-linking studies which identified a subcomplex composed of SRB2, SRB4, SRB5, and SRB6 (Koh etai., 1998). 22 Introduction SRB 8 -SRB11 also form a functional grouping. They were all identified as recessive alleles that displayed, in addition to suppression of CTD truncation phenotypes, slow growth, flocculence, and temperature sensitivity (Hengartner et al., 1995; Liao et al., 1995). SRB10 and SRB11 encode a cyclin dependent kinase/ cyclin pair homologous to the CDK8/ cyclin C pair in metazoans (Hengartner et al., 1995; Liao et al., 1995; Surosky et al., 1994) which contributes to CTD phosphorylation in vitro (Liao et al., 1995). In addition to the designation as SRBs, and unlike SRB2 - SRB7, this group of genes has been identified in a miriad of genetic screens, implicating these proteins in the regulation of a wide variety of regulatory responses. A third group of mediator proteins consists of GAL11, SIN4, RGR1, ROX3 , MED3, and MED8. The genes encoding these proteins are distinguished from the SRB8-11 group by the fact that they were not identified as SRB alleles. Furthermore, they differ genetically from the SRB genes in that alleles of gall 1, sin4, rgrl, and rox3 all elevate transcription from UAS-less reporters whereas srb8-ll mutations have little effect on such reporters (Chen et al., 1993; Chen et al., 1993; Covitz et al., 1994; Jiang et al., 1995; Jiang and Stillman, 1992; Song et al., 1996; Wahi and Johnson, 1995). NUT1 and NUT2 were identified as negative regulators of a URS2 element along with SIN4 and ROX3 and may also be components of this group (Tabtiang and Herskowitz, 1998). A fourth and final grouping consists of the MED genes which were identified by purification of mediator complexes devoid of SRB8-11 (Han et al., 1999; Myers et al., 1998)]. As mentioned, MED3 and MED8 are found associated with the GAL11 subcomplex. MED6 is an essential gene encoding a protein which appears to reside in a complex distinct from both the SRB8-11 and GAL11 subcomplexes in that it does not 23 Introduction affect uninduced transcription (Lee et al., 1997). Both human and C. elegans orthologs of MED6 and MED7 have been identified, suggesting that they are universal mediator components (Gu et al., 1999; Jiang et al., 1998; Sun et al., 1998). TFIIA TFIIA was originally classified as a GTF due to its requirement for specific transcription in vitro (Matsui et al., 1980). Subsequent studies have revealed that it is dispensable for basal transcription, but is involved in stimulating in vitro transcription mediated by TFIID (Cortes et al., 1992; DeJong et al., 1995; Hansen and Tjian, 1995; Sayre et al., 1992). TFIIA associates with the PIC through interactions with TBP, and has been shown to stabilize TBP binding to the TATA box (Imbalzano et al., 1994). Yeast TFIIA was identified by complementation of a mammalian in vitro transcription system, and co-purified as two proteins (Hahn et al., 1989; Ranish and Hahn, 1991). Partial sequencing of the components led to cloning and the designation of TAOl and TA02 encoding essential proteins of 32 kDa and 13.5 kDa, respectively (Ranish et al., 1992). TFIIA is dispensible for activated transcription in vitro; however, TBP mutants defective in TFIIA-binding show activation defects in vivo (Stargell and Struhl, 1995). Acetyltransferases Five genes, ADA1, ADA2, ADA3, GCN5, and ADA5, were identified in a screen for suppressors of the toxic effects caused by overexpression of the GAL4 DNA binding domain fused to the activation domain of VP 16 (Berger et al., 1992; Horiuchi et al., 1997; Marcus et al., 1996; Marcus et al., 1994; Pina et al., 1993; Roberts and Winston, 1996). 2 4 Introduction GCN5 was originally identified for it's requirement for transcriptional activation by GCN4 (Georgakopoulos and Thireos, 1992). Subsequently ADA2, AD A3 and GCN5 were shown to be required for full activation of a subset by transcription factors (Barlev et al., 1995). The idea that the ADA and GCN5 genes functioned together as a complex was hinted at by epistasis studies, and was confirmed when ADA2, AD A3, and GCN5 were found to physically interact (Candau and Berger, 1996; Georgakopoulos et al., 1995; Horiuchi et al., 1995; Marcus et al., 1994). However, the function of these proteins was still unclear until the discovery of a histone acetylase gene from Tetrahymena which had significant homology to GCN5 (Brownell and Allis, 1995; Brownell etal., 1996). Since then, a number of laboratories have purified four distinct complexes containing GCN5 and ADA2 (Grant et al., 1997). One of these complexes with a molecular mass of 1.8 MDa copurified with ADA2, SPT3, SPT7, and SPT20/ADA5. This complex has since been named SAGA (Spt-Ada-Gcn5-actetyltransferase) and provides a link between nucleosomal histone acetylation and transcriptional activity (Grant et al., 1997). Other nucleosomal HAT complexes have been purified, including a 200kDa and 170kDa complex distinct from those described above (Ruiz-Garcia et al., 1997; Saleh et al., 1997). Histone acetylation occurs at the e amino group of lysines located on the histone H3 and H4 tails, and has been correlated with gene activation. Acetylation of histones appears to increase the affinity of DNA for transcription factors and GCN5, along with its binding partner ADA2, have been shown to associate with activation domains and TBP. Thus, transcriptional activator proteins appear to regulate gene expression by recruitment of both the GTFs and HATs. 25 Introduction Chromatin-Remodeling Complexes Other complexes have been described which facilitate chromatin remodeling through nucleosomal structure but do not catalyze histone acetylation. These include yeast, human, and Drosophila SWT/ SNF and Drosophila NURF complexes (for review see Bjorklund et al., 1999; Pazin and Kadonaga, 1997). Components of the yeast SWI/ SNF complex were intially identified because mutations in their genes caused defects in mating-type switching (SWT)(Stem et al., 1984) and utilization of sucrose (SNF) (Neigeborn and Carlson, 1984). A connection between the two sets of genes was made when SNF2 and SWI2 turned out to be identical. The connection to chromatin was made when suppression studies of snf and swi mutants lead to the identification of SSN20 and SIN2, which turned out to be identical to SPT6 and HHT1, respectively (Neigeborn et al. 1986, Sternberg et al., 1987). SPT6 belongs to a class of genes which include SPT4, SPT5, SPT11/HTA1, SPT12/HTB1, and SPT16/CDC68 which either affect chromatin structure or, like HHT1, encode histones. The SWI/ SNF chromatin remodeling machine has been purified as a 2-MDa complex and shown to have DNA binding properties similar to HMG-box-containing proteins (Cairns et al., 1994; Cote et al., 1994; Quinn et al., 1996). Subunits include SWI1, SWI2/SNF2, SWI3, SNF5, SNF6, SNF11, SWP29, SWP59, SWP61, SWP73, and SWP82 (Burns and Peterson, 1997). The only component with enzymatic activity in this complex is SWI2/ SNF2, a DNA-dependent ATPase (Laurent et al., 1993). The SWP29 subunit is identical to the TFG3 subunit of TFIIF and to the TAF30 subunit of TFIID, providing a link between the SWI /SNF complex and the RNA pol II holoenzyme (Henery et al., 1994). The SWI/ SNF complex is not a universal transcription factor as it 2 6 Introduction regulates only a subset of promoters including HO, SUC2, Ty, ADH1, ADH2, INOl, and STA1 (Peterson and Herskowitz, 1992; Yoshimoto and Yamashita, 1991) but not PH05, URA3, LYS2, CLN1, CLN2, CLN3, or HSC26 (Peterson and Herskowitz, 1992; Pollard and Peterson, 1997). Based on homology to the SWI/ SNF complex, another chromatin remodeling complex, RSC (remodels the structure of chromatin), has been identified in yeast (Cairns et al., 1996). Like the SWI/ SNF complex, RSC contains a DNA-dependent ATPase activity; however, RSC is more prevalent in the cell, found to be approximately ten fold more abundant than SWI/SNF (Cairns et al., 1996). Unlike SWI/ SNF, components of the RSC complex are essential for cell viability (Cairns et al., 1996). This is likely related to involvement of the RSC complex in regulating cell cycle genes; inactivation of two of its subunits causes G 2/ M cell cycle arrest (Cao et al., 1997; Hampsey, 1998). Thus it appears that SWI/ SNF and RSC function through similiar mechanisms but are distinct in the promoters which they target. 1.3 THE GAL GENES AS A MODEL FOR EUKARYOTIC TRANSCRIPTION The study of galactose catabolism in yeast began over 40 years ago with the identification of mutants unable to utilize galactose as a carbon source. (Robichon-Szulmajster, 1958). This mutant screen, and others performed subsequently, in combination with hybridization techniques (St John and Davis, 1979) led to the identification of the genes required for conversion of galactose into glucose-1-phosphate, known as the Leloir pathway (Fig. 2) (Frey, 1996). The transactivator GAL4, which binds to D N A elements (UASQAL) upstream of the TATA box, is solely responsible for 27 Introduction activating transcription of the GAL genes encoding all the enzymes involved in the Leloir pathway, with the exception of GALS (Douglas and Hawthorne, 1966; Klar and Halvorson, 1976; Klar and Halvorson, 1974; Reece and Ptashne, 1993). GAL4 is inhibited in the absence of galactose by interaction with the negative regulator GAL80, which is thought to mask GAL4's activation domains in non-inducing conditions. Galactose enters the cell via a specific transporter encoded by GAL2, and causes activation of GAL4 through another regulatory protein GAL3. However, in the presence of glucose, the preferred carbon source, GAL4 is inactive and the GAL genes are repressed. 13 .1 G A L 2 Galactose enters the cell through the action of GAL2, a high and low affinity facilitated-diffusion transmembrane transporter (Ramos et al., 1989; Tschopp et al., 1986). Transcription of GAL2 is induced by GAL4 in the presence of galactose however, there is a low level oiGAL2 expression during growth on glycerol, providing a mechanism for initial galactose entry (Huibregtse et al., 1993). 28 Introduction C H g O H 1$L-}L G l u . C H 2 O H H O / | — Q " O H G a l ( O U t ) opgi i l r thk iaao G A L 2 p e r m e a s e C H 2 O H H O / I — P G a l ( in) G A L 1 k i n a s e C H 2 O H G a l - 1 - P C H 2 O H G A L 7 t r a n s f e r a s e U D P - G l u -UDP o - U D P ^® U D P - G a l G l u - 1 - P G A L 5 m u t a s e C H j O H H O J — O K?" .> O H M e l i b i o s e G A L 1 0 e p i m e r a s e ' O H O H G l u - 6 - P G l y c o l y s i s Figure 2 . The Leloir Pathway 2 9 Introduction 1.3.2 GAL3 GAL3 is critical for rapid induction. Cells lacking GAL3 still induce GAL transcription in response to galactose, but a significant level of induction occurs after several days, rather than hours, in a process known as "long term adaptation" (LTA) (Spiegelman et al., 1951; Winge and Roberts, 1948). GAL3 contains sequence homology to the galactokinase GAL1 but is unable to phosphorylate galactose (Bhat et al., 1990). GAL1, when expressed at a high level, is able to functionally replace GAL3 (Bhat and Hopper, 1992; Bhat et al., 1990). Initial studies of GAL induction hinted at the possibility of a second messenger, postulated to be phosphorylated galactose (Bajwa et al., 1988). Recently however, a number of laboratories have shown that a galactose-GAL3 complex acts as the inducer (Blank et al., 1997; Suzuki-Fujimoto et al., 1996; Yano and Fukasawa, 1997). The GAL3/ galactose complex has been shown to bind directly to GAL80 in a ATP-dependent process, forming a terniary complex with GAL4 which is capable of activating transcription (Piatt and Reece, 1998). 1.3.3 GAL80 GAL80 is the negative regulator of GAL4, responsible for inhibiting GAL transcription in the absence of galactose. Mapping studies have defined a number of functional domains on GAL80, including two nuclear localization domains, and an induction domain flanked by a bipartite inhibition domain (Nogi and Fukasawa, 1989). The induction domain is a likely target for GAL3/ galactose, as mutations in this region prevent induction in vivo and block GAL3 binding in vitro (Nogi and Fukasawa, 1989; 30 Introduction Yano and Fukasawa, 1997). Unlike the general repressor TUP1, which causes active repression of transcription, independent of the upstream activator (see below), GAL80 functions as a weak activator when tethered to DNA (Tzamarias et al., 1994, unpublished), suggesting that the bipartite repression domain is passive and likely functions solely by inhibiting GAL4. Mapping studies of GAL4 have defined a single domain required for its primary interaction with GAL80 (see below), located at the C-terminus of GAL4's major activation domain AR2. The proximity of this interaction to AR2 has led to a simple model to explain how GAL80 inhibits GAL4, whereby GAL80 "masks" critical residues of AR2 involved in interaction with targets in the RNA pol II holoenzyme (Ma and Ptashne, 1987a; Perlman and Hopper, 1979) 1.3.4 GAL4 GAL4 was one of the first eukaryotic transcription factors cloned and as such was extensively characterized at a time when very little was known about the mechanism of eukaryotic transcription (Johnston and Hopper, 1982; Laughon and Gesteland, 1982). GAL4 was shown to be transcribed as a 2.8 kilobase transcript encoding an 881 amino acid protein with a molecular weight of approximately 110 kDa. (Laughon and Gesteland, 1984). Once cloned, GAL4 and the genes it regulates became the platform for much of the pioneering work in the field of eukaryotic transcriptional regulation. The GAL genes were used to investigate the ability of plasmid-borne transcripts to be regulated normally (Baker et al., 1984), to determine that activation was a conserved mechanism amongst all eukaryotes (Fischer et al., 1988; Kakidani and Ptashne, 1988; Webster et al., 1988), and importantly, to demonstrate the modular nature of eukaryotic transcriptional regulators 3 1 Introduction GAL4 Functional and Structural Features DNA-Binding Specificity Dimer Formation Transcriptional Activation Inhibition Glucose Response GAL80 Interaction Cooperative DNA-Binding Phosphorylations 100 200 300 400 500 600 700 800 900 I 1 1 1 1 1 1 1 1 1 m ID1 ID2 ID3 GRD MM • • • D — • -% Identified Phosphorylations O Predicted Phosphorylations Figure 3. GAL4 Functional Domains. DNA (AA1-100) required for D N A binding, AR1 (AA149-238) required for activation by full length GAL4, ID1,2,3 (AA239-585) inhibit GAL4's ability to bind D N A in the presence of glucose, GRD (AA600-767) regulates the action of the inhibitory domain, and AR2 (AA768-881) required for induction by full length GAL4. 32 Introduction (Johnston et al., 1986; Keegan et al., 1986; Ma and Ptashne, 1987; Sadowski et al., 1988). GAL4 is constitutively transcribed and GAL4 protein is associated with the UASG of responsive genes in the absence of galactose or glucose (Selleck and Majors, 1987). In this state, GAL4 is poised to induce transcription but is maintained in an inactive state solely through its association with GAL80. With the addition of galactose and subsequent binding of GAL3 to GAL80, GAL4 becomes capable of activating transcription and is phosphorylated concomitant with its activation function (Sadowski et al., 1996). A unique feature of GAL4 is that its mRNA levels are extremely low (estimated at less than one copy per cell) (Laughon and Gesteland, 1982). The addition of galactose does not increase GAL4 levels in the cell; however, in the presence of glucose its transcription is repressed (see below) (Griggs and Johnston, 1993; Griggs and Johnston, 1991; Laughon and Gesteland, 1982). DNA Binding The crystal structure of an N-terminal GAL4 fragment (AA 1-90) revealed what at the time was a novel DNA binding motif, a Zn2Cys6 cluster (Marmorstein et al., 1992). This motif has since been found in many fungal transcription factors involved in the regulation of carbon metabolism and is characterized by a cluster of six cysteine residues coordinating two zinc ions. The crystal structure confirmed earlier work suggesting that GAL4 binds as a dimer (Carey et al., 1989), and demonstrated that the conserved CCG triplet at each end of the DNA site is recognized in the major groove by the Zn2Cys6 cluster. The symmetrical nature of the GAL4 consensus site was also elegantly explained by the crystal strucure which demonstrated that the coiled-coil dimerization domain rises 3 3 Introduction from the center of the dyad, perpendicular to the plane of the DNA. This dimerization domain is connected to the zinc clusters by a linker region which lies along the minor groove spanning 11 base pairs in the case of GAL4. These results were confirmed by the solution structure of the DNA binding domain, solved by NMR, bound as a monomer to one half of a GAL4 dyad cis-element (Baleja et al., 1994). GAL4 binds cooperatively to multiple adjacent sites on DNA (Giniger and Ptashne, 1988; Kang et al., 1993). It has been suggested that the cooperative DNA binding function of GAL4 is mediated through interactions between the DNA binding domain and chromatin (Adams and Workman, 1995). However, this clearly cannot be the sole mechanism since full length GAL4 has been shown to bind cooperatively to naked DNA in vitro (Kang et al., 1993). A number of trancription factors are regulated by inhibiting their ability to bind DNA through subcellular localization (De Vit et al., 1997) or directly through phosphorylation (Lu et al., 1997). This appears also to be true for GAL4, as a central inhibitory region of GAL4 appears to inhibit GAL4's ability to bind DNA in response to glucose (see below) (Perelli-Hentschel, 1995). Transcriptional Activation Transcriptional activation requires the presence of an activation domain and, in the case of GAL4, this function is distinct from the DNA binding domain (Brent and Ptashne, 1985; Keegan et al., 1986). Deletion experiments defined two independent activation domains on GAL4; termed activating region 1 (AR1, residues 149-238) and activating region 2 (AR2, residues 768-881) (Ma and Ptashne, 1987b). Additionally, a 3 4 Introduction third weak activation domain (AR3, residues 94-106) has been identfied which contributes to activation in vitro (Lin et al., 1988). Both AR1 and AR2, GAL4's major activation domain, are characterized by their high acidic content, placing them in the family of so-called acidic activators . Mutations which reduce the number of acidic residues present in AR1 also reduce the potency of activation (Gill and Ptashne, 1987; Gill et al., 1990); however activation by AR2 appears to be dependent on mantainance of the correct structure (Leuther et al., 1993). Transcriptional activation domains are now known to function by contacting one or more of the GTFs and/ or mediator components (see above). AR2 has been shown to interact with TBP, TFIIB, and a component of the yeast mediator complex SRB4 (Koh et al., 1998; Melcher and Johnston, 1995; Wu et al., 1996). Mutations which affect the strength of these interactions also affect the potentancy of activation (Koh et al., 1998; Wu et al., 1996). In the case of TBP, a point mutation in AR2 (Phe to Ala at residue 869) which reduces interaction between AR2 and TBP in vitro, similarly reduces activation in vivo by AR2 (Wu et al., 1996). Furthermore, this point mutation specifically abolishes interaction with SRB4 in vitro and this effect can be suppressed by compensatory mutations to SRB4 (Koh et al., 1998). Gal80 Interaction In addition to its role in recruiting the RNA Pol II holoenzyme, the C-terminus of GAL4 is involved in regulation by its inhibitor GAL80. Early in the study of GAL4 it was demonstrated that GAL80 exerted its repressive effect at least in part through its association with the C-terminal 100 amino acids of GAL4 (Ma and Ptashne, 1987a). 35 Introduction Deletions which remove this region activate transcription constitutively, overexpression of the C-terminus of GAL4 reduces GAL80's inhibition of endogenous GAL4, and a fusion protein containing the C-terminal 100 amino acids is sensitive to inhibition by GAL80 (Ma and Ptashne, 1987a). These experiments supported earlier genetic evidence which indicated that constitutive gal81 mutations represented GAL4 truncations (Matsumoto et al., 1980; Nogi et al., 1977). Further mapping experiments demonstrated that both activation and sensitivity to GAL80 could be mediated by a peptide which spanned the C-terminal 48 amino acids. Activation by this fragment could be inhibited in vivo by GAL80, and in vitro experiments confirmed direct interaction with GAL80 (Wu et al., 1996). The dual role of residues 855-870 in transcriptional activation and GAL80 interaction was shown by studies where deletions to residue 870, but not 868, retained the ability to both activate and interact with GAL80 whereas a deletion to 868 was only capable of activating (Leuther et al., 1993). Recent studies using scanning mutagenesis have refined the role played by residues 855-870, demonstrating that the domain involved in GAL80 interaction is structured, likely forming a P-hairpin structure, and is sensitive to the insertion of proline residues, whereas the underlying activation domain is not (Ansari etai., 1998). Central Region The central region (CR) of GAL4 comprises over 60% of the protein, is multiply phosphorylated, and contains at least 3 inhibitory domains (ID1, ID2, and ID3) controlled by a glucose responsive domain (GRD)(Sadowski et al., 1996; Stone and Sadowski, 1993). These domains, defined through a series of deletion and protein fusion experiments, were originally hinted at by the observation that tethering either ARI or 36 Introduction AR2 to DNA can activate transcription in the absence of the CR (Gill and Ptashne, 1987; Gill et al., 1990). Activation is severely impaired when either AR1 or AR2 are deleted in the context of otherwise full-length GAL4, suggesting an inhibitory role for the CR (Ma and Ptashne, 1987b; Sadowski et al, 1991; Stone and Sadowski, 1993). Subsequent studies demonstrated that the CR can confer glucose inhibition when fused to the constitutive activator LexA-VP16 (Stone and Sadowski, 1993). Of the three inhibitory domains, ID1 is the best characterized and represents a region with a high degree of homology with other fungal transcription factors, including LAC9, PUT3, PPR1, LEU3,NIRA, THI1, and NIT4 (Perelli-Hentschel, 1995). ID1 appears to function by inhibiting DNA binding, likely mediated by self-aggregation of GAL4 dimers. A model has been proposed whereby ID1 is inhibited in the absence of glucose through association with the GRD. With the addition of glucose, ID1 is released, causing self-aggregation of GAL4 dimers ultimately leading to dissociation from DNA. Thus, the ID1 domain, controlled by the GRD, provides for the strong short term glucose regulation observed in vivo (Stone and Sadowski, 1993). Phosphorylation The transcriptional activator protein GAL4 is a phosphoprotein whose phosphorylation state correlates precisely with activation of the GAL genes (Mylin et al., 1989; Mylin et al., 1990; Sadowski et al., 1991). Although GAL4 is phosphorylated on at least four different sites, only one of these phosphorylations at serine 699 is required for full induction of the GAL genes (Sadowski et al., 1996). Previous results have suggested that phosphorylation of GAL4 occurs as a consequence of its transcriptional 37 Introduction activation function, and it was suggested in 1991 that these phosphorylations are mediated by GTF components (Sadowski et al., 1991). 1.3.5 Induction by galactose Although the genes involved in GAL induction were identified and cloned over a decade ago, the exact mechanism of induction has remained unclear. Recent experiments demonstrating the interaction of a GAL3-galactose complex with GAL80 have shed light on the mechanism by which the galactose signal is transduced (Blank et al., 1997; Piatt and Reece, 1998; Suzuki-Fujimoto et al., 1996; Yano and Fukasawa, 1997; Zenke et al., 1996). However, how this signal is received by the GAL80/ GAL4 complex remains unresolved. The fact that GAL80's interaction domain overlaps the major activation region of GAL4 has led to a model of induction involving dissociation of the GAL4/ GAL80 complex. Recent evidence suggests that the mechanism may be more complicated. First, in a two hybrid-assay a mutant GAL4 unable to activate transcription remains associated with GAL80 under inducing conditions (Leuther and Johnston, 1992). Second, a complex of GAL4 associated with GAL80 can be purified from cells grown under inducing conditions (Parthun and Jaehning, 1992). Third, when subjected to EMS A, in vitro transcription reactions which reconstitute the GAL4/ GAL80/ GAL3 "yeast galactose genetic switch" show that a complex of GAL4/ GAL80 remains associated during activation (Piatt and Reece, 1998). Taken together, these observations suggest that induction may involve an alteration in the GAL4/ GAL80 complex that does not necessarily involve dissocation of GAL80. 38 Introduction 1.3.6 Repression by glucose In the absence of glucose, the GAL4 gene is expressed constitutively, and GAL4 is free to activate galactose-inducible transcription. The presence of glucose causes strong and rapid repression of GAL transcription by a combination of mechanisms. Addition of glucose leads to the inactivation of the SNF1/ SNF4/ SIP1/ SIP2/ GAL83 kinase complex and causes REG 1-dependent dephosphorylation of the MIG1 protein (for review see Carlson, 1998). Dephosphorylated MIG1 translocates to the nucleus where it binds its operators within the upstream repression sequences for glucose (URSQ ) , present within most of the GAL promoters, including GAL4 itself (Devit et al., 1997). DNA-bound MIG1 recruits the general repressor complex containing SSN6 and TUP1, which results in direct repression of GAL transcription by organization of repressive chromatin (see above). Repression of GAL4 transcription results in at least a four-fold decrease in GAL4 concentration which, in the longer term, amplifies the effect of glucose by preventing activation from the UASG of the GAL genes (Griggs and Johnston, 1991; Johnston et al., 1994; Kang et al, 1993; Lamphier and Ptashne, 1992; Nehlin et al., 1991). In addition to inhibiting GAL transcription through the URSG, glucose also causes rapid inhibition of GAL4 protein through the central inhibitory domains (see above). Furthermore, glucose causes a rapid reduction in galactose uptake by glucose-induced degradation of GAL2 via an endocytosis and vacuolar proteolysis pathway (Horak and Wolf, 1997). 3 9 Introduction 1.3.7 Two-hybrid systems It is only natural that GAL4 has become one of the most versatile tools for the molecular biologist. The yeast two-hybrid (Fields and Song, 1989) and interaction-trap systems (Golemis et al., 1994) have provided simple and reliable genetic strategies for identification and characterization of interactions between proteins. These systems were developed consequent to the understanding that eukaryotic transcriptional activators have separable DNA-binding (DBD) and transcriptional activation domains (AD) that readily function when fused to heterologous proteins (Brent and Ptashne, 1985). Interaction of an AD "prey" fusion with a DBD "bait" fusion protein produce a functional transactivator complex that can activate reporter genes bearing upstream cz's-elements for the DBD. One limitation of these strategies is that transcriptional activators cannot be used as bait fusions. This has led to the development of many variations on the two-hybrid theme (for review see Vidal and Legrain, 1999). However, no two-hybrid system has been developed for investigating interactions occurring between transactivators and their targets in the context of a RNA pol II dependent promoter. For my experiments investigating interaction between GAL4 and GAL80,1 developed a novel two-hybrid system that can be used with transcriptional activators. 40 Introduction Figure 4. Regulation of the GAL genes. (A) Uninduced state. The GAL genes are transcribed at a low level independent of GAL4 which, is associated with GAL80 and inactive. MIG1 is phosphorylated by the SNF1/ SNF4/ SIP1/ SIP2/ GAL83 kinase complex and exported from the nucleus. (B) Induced state. The GAL genes are transcribed at a high level leading to an increase in the levels of GAL2 and GAL3. GAL4 is active, phosphorylated, and in a tripartate complex composed of GAL4, GAL80, and GAL3. MIG1 remains phosphorylated and exported from the nucleus. (C) Repressed state. The SNF1/ SNF4/ SIP1/ SIP2/ GAL83 kinase complex is inactivated, MIG1 is dephosphorylated and localizes to the nucleus where it binds to its operators and represses transcription through the recruitment of SSN6 and TUP1. GAL4, GAL2, and GAL3 levels are all reduced (see text for details). 4 1 Introduction Introduction 4 3 Introduction Materials and Methods 2. MATERIALS AND METHODS 2.1 Plasmids and Yeast Manipulations All experiments used the yeast Saccharomyces cerevisiae. Strains used in this study are listed in Table 2. DNA manipulations were performed in the E. coli strain DH5a. Plasmid YCpG4 is TRP1, ARS-CEN which expresses GAL4 from its own promoter (Sadowski et al., 1996). pJR006 and pJR007 are equivalent LEU2, ARS-CEN plasmids which express GAL4 and GAL4 S699A (Rohde, 1999). GAL4A683 and GAL4 A683T have been described previously (Sadowski et al., 1996; Stone and Sadowski, 1993). The GAL4 S699E mutation was constructed using the oligonucleotide (5'-GTTTCTCCTGGCGAAGTAGGGCCTTCAC) as described (Sadowski et al., 1996). Plasmid pIS028 is a 2 micron, TRP1 vector which expresses HA-tagged SRB 10 from the ADH1 promoter. pKTN28HA expressing HA-tagged Kin28 was as described (Cismowski et al., 1995). Wild Type full length GAL4 was expressed from the ADH1 promoter on the 2 micron vector pMA210, which has been described previously (Ma and Ptashne, 1987). GAL4 truncations were similiarly expressed from the ADH1 promoter on the 2 micron vectors; pMA230 (expressing AA 1-848), pMA242 (expressing AA l-238::768-881), pKW2 (expressing AA l-238::768-848), pMA246 (expressing AA 1-239), and pMAB17 (expressing AA 1-147 fused to the artificial B17 activation domain) which have been described previously (Ma and Ptashne, 1987a; Ma and Ptashne, 1987b). TUP1-N-terminal-encoding DNA was amplified from genomic DNA derived from the strain YM109 using primers MH50 (5'-GGCGAATTCGTATGACTGCCAGCGTTTCG) and 4 5 Materials and Methods MH51 (5'-GAGCGGCCGCTGCCACGGAAACCTGGGGAGG). The resultant amplified DNA of 600bp was cut with EcoRI and NotI and cloned into the vector YephalAlac (Hung et al., 1997) to create pJMH105. GAL80 was similary amplified using the primers MH54 (5'-GAGCGGCCGCTATGGACTACAACAAGAG) and MH55 (5'-GAGCGG CCGCTT AT A A ACT AT A ATGCG). The amplified DNA of 1.3 kbp was cut with NotI and cloned into either YephalAlac or pJMH105 to generate plasmids pJMH106 and pJMH107, respectively. Plasmid pJMH109 is a LEU2 intergrating vector containing a GAL1-URA3 reporter gene, which was constructed by cloning a Hindlll/ BamHl URA3 fragment, produced by amplification with the primers MH121 (5'-TTCTAAAGCTTATGTCGAAAGCTACATATAAGGAACG) and MH122 (5'-TTATCGGATCCTTAGTTTTGCTGGCCGCATCTTC) into pAOGALl (Olson, unpublished). Cells for GAL4 P-galactosidase assays were grown in synthetic dropout (Guthrie and Fink, 1991) (SD) -Trp (YCpG4, YCpG4S699A) or SD-Leu (pJR006, pJR007) containing non-fermentable carbon, and were induced by adding galactose to 2% from a 40% sterile stock. For the repressed transactivator assay, P-galactosidase assays were performed in the strains indicated grown at 30°C in SD-His, Trp to an OD600 ~ 1 and harvested. P-galactosidase activity was measured by lysing with glass beads as described (Sadowski et al., 1996). Growth assays were performed in the strain yJMHl on SD-His, Trp agar plates, supplemented with the indicated concentration of 5-FOA. Cells were suspended in water, normalized to an O.D. A 6 0o n m = 1 -0 and 10 fold serially diluted in 46 Materials and Methods water. 10 |il of the resultant dilutions were spotted on selective agar plates and incubated for 3 to 5 days at 30°C. 2.2 Antibodies and recombinant proteins Rabbit anti-GAL4 DBD polyclonal antibody has been described (Sadowski et al., 1991). The mouse 8C1 hybridoma producing oc-GAL4 AR2 was isolated from a monoclonal panel produced from mice immunized with WT GAL4 protein (E. Leng and I. Sadowski). Monoclonal antibodies against HA and vimentin were obtained commercially (Santa Cruz). The mouse 8WG16 hybridoma producing oc-RPBl was a kind gift of R. Burgess. Polyclonal antibodies against SRB 10, TFIIB, SRB5, and TFA2 were a kind gift of R. Young. Rabbit anti-TFBl and anti-TFAl polyclonal anitbodies were produced from rabbits immunized with purified GST-TFB1 (aa 1-136) or GST-TFA1 respectively, and affinity purified following standard techniques (Greenblatt, 1998). Rabbit anti-yTBP polyclonal antibody was produced from rabbits immunized with purified yeast TBP (Greenblatt, 1998). Rabbit anti-GAL80 polyclonal antibody was produced from rabbits immunized with purified 6-His-GAL80. Rabbit anti-KIN28 polyclonal antibody was as described (Feaver et al., 1994b). WT GAL4 protein was produced by expression in insect cells (Kang et al., 1993) and was purified by extraction of insoluble material from infected cell lysates with TBS buffer containing 0.02% SDS. GAL80 protein was produced as a 6-His fusion in pRSET-A (Invitrogen) and purified by Ni-chelate affinity chromatography. Recombinant SRB 10/ SRB11 complexes were produced by co-expression in insect cells infected with viruses obtained as gifts from R. Young and 4 7 Materials and Methods purified as described (Hengartner et al., 1998). Purified R N A pol II holoenzymes 2 were isolated as described previously for the purification from human cells (Pan et al., 1997) 2.3 Metabolic labeling, tryptic phosphopeptide analysis, and in vitro kinase assays [ 3 2P]-orthophosphate labeling of yeast, immunoprecipitations, and tryptic phosphopeptide analysis were as described (Hung et al., 1997). Phosphopeptides were resolved by electrophoresis in the horizontal dimension at p H 2.1 and chromatography (butanol: acetic acid: d H 2 0 : pyridine, 75:50:37.5:15.5) in the vertical dimension. Phosphopeptides were detected with a Molecular Dynamics phosphorimager. For in vitro kinase reactions, yeast expressing H A - K J N 2 8 and H A - S R B 1 0 , were grown in S D -Trp containing 2% raffinose to A600 = 1 A and then were induced with 2% galactose for 2 hours. Cells were lysed in kinase lysis buffer ( K L B ) (20mM Tris, 150mM N a C l , I m M E D T A , 10% Glycerol, 0.3% NP-40, I m M P M S F , and protease inhibitors) by vortexing with glass beads. HA-tagged kinases were recovered by immunoprecipitation from 2 mg of clarified lysate with anti-HA M A b and Protein G-Sepharose beads. Immune complexes were washed 3 times with K L B and 2 times in kinase buffer ( K B ) (10 m M M g C l 2 , 50 m M Tris p H 7.5, 1 m M DTT) . Kinase reactions containing HA-tagged kinases or purified immobilized R N A pol II holoenzymes (Pan et al., 1997; Schultz et al., 1991) were performed in 5 ul K B containing 2 pmol y 3 2 P - A T P (3000Ci/mmol) and lOOng of recombinant G A L 4 or G A L 8 0 for 30 mins at 30°C. Kinase reactions containing 2 Holoenzymes were prepared by M . Kobor (Greenblatt Lab) from yeast extracts prepared and shipped on dry ice. Purified holoenzymes were shipped back at 4°C for use in kinase assays 48 Materials and Methods recombinant SRB 10 /SRB11 were performed in 10 ul KB containing 2 pmol y32P-ATP (3000Ci /mmol) with lOOng of recombinant SRB 10 /SRB11 and 50ng of recombinant GAL4 or GAL80 for 30 mins at 30°C. Kinase reactions containing TFIIH were performed as described with purified yeast TFIIH, generously provided by R. Kornberg (Feaver et al., 1994b). Reaction products were resolved on 10% SDS PAGE and detected by exposure to Kodak Biomax film. Synthetic peptides were added to kinase reactions at a concentration of 10 |ig per reaction. Peptides were recovered from the reaction by dilution to 400 ul with dH20, filtration through Micron-10 microconcentrators (Amicon), and then chromatography on Micron-SCX cation exchange columns (Amicon). 4 9 Materials and Methods Table 2. Yeast Strains Strain Genotype Source YT6::171 MATa, gal4, gal80, ura3, his3, adel, adel, lys2, trpl, oral, (Sadowski et al., leu2, met, URA3::GALl-LacZ 1991) YT6G80:171 YT6::\7\, LEU2::GAL80 (Sadowski et al., 1991) H617 MATa, ade2, canl, his3, leu2, trpl, ura3, srblO::HIS3 (Balciunas and Ronne, 1995) W303-1A MATa, ade2, canl, leu2, his3, trpl, ura3 (Thomas and Rothstein, 1989) RP021-1 MATa, ade2, his3, leu2, trpl, ura3, canl, rpo21-l (Himmelfarb et al., 1987) Kin28ts-16 MATa, adel, his2, leu2-3,112, trpl-1, Kin28ts-16::URA3 (Cismowski et al., 1995) YJMH4 MATa, gal4::hisG, his3, ura3, leu2, lys2, URA3::GALl-LacZ This Study YJMH5 MATa, gal4::hisG, his3, ura3, leu2, lys2, ssn3-l(srblO)::HIS3 This Study (srblO), URA3::GALl-LacZ YJMH8 MATa, gal4::hisG, gal80::hisG, his3-200, ura3, leu2, lys2, This Study URA3::GALl-LacZ YJMH9 MATa, gal4::hisG, gal80::hisG, his3, ura3, leu2, lys2, ssn3-This Study l(srblO)::HlS3, URA3::GALl-LacZ YJR10::131 MATa, ade2, canl, leu2, his3, trpl, ura3, gal4::hisG, (Rohde, 1999) URA3::GALl-LacZ YJMH1 MATalpha, Gal4-542, Gal80-538, ura3-52, his3-200, ade2-This Study 101,adel, lys2-80, trpl-901, oral, LEU::GAL1-URA3, met 50 Results 3. RESULTS 3.1 GAL4 PHOSPHORYLATION 3.1.1 A single C-terminal GAL4 phosphorylation is required for GAL gene induction GAL4 is multiply phosphorylated under conditions in which it activates transcription. Sadowski et al. have identified four sites of phosphorylation on GAL4, at serines 691, 696, 699, and 837 (see Fig.5 ), which retard the migration of GAL4 protein in SDS-PAGE (Sadowski et al., 1996; Sadowski et al., 1991) At least one additional phosphorylation that does not alter electrophoretic mobility must reside within the DNA-binding domain (residues 1-147), since the fastest migrating form of most GAL4 derivatives, and the DNA binding domain fragment produced on its own (unpublished), are labeled with 32P-orthophosphate in vivo (see below and Fig. 5). Of the identified sites of phosphorylation, only one at S699 appears to be required for GAL induction. The data in Figure 6 shows that mutation of this residue to alanine or glutamate reduced GAL induction 6 fold in an otherwise wild type yeast strain. In contrast, the phosphorylations at serines 691, 696 and 837 do not appear to be required, since GAL4 bearing individual alanine mutations of these residues (Sadowski et al., 1996; Sadowski et al., 1991), or all three of these serines changed to alanine, induced GAL transcription as efficiently as WT GAL4 (Fig. 6). 5 1 Results WT-GAL4 s JS °> (O (O (O </)( / )( / ) inhibitory co CO c/> GAL4A683 1 147 683 881 Figure 5. Schematic representation of WT GAL4 (top) protein indicating the location of phosphorylations and the functional domains; DNA (AA1-100) required for DNA binding, AR1 (AA149-238) required for activation by full length GAL4, Inhibitory (AA239-585) inhibits GAL4's ability to bind DNA in the presence of glucose, GRD (AA600-767) regulates the action of the inhibitory domain, and AR2 (AA768-881) required for induction by full length GALA The GAL4A683 derivative (Sadowski et al., 1996) is indicated below. 5 2 Results 30-, Time (hours) Figure 6. Mutation of S699 to alanine or glutamate impairs GAL induction by GAL4. Yeast strain YT6G80:: 171 bearing a control vector (•), or expressing WT GAL4 (•), GAL4 S699A ( A ) , GAL4 S699E (•), or GAL4T (S691A, S696A, S837A) (•) from plasmid YCpG4 were induced with 2% galactose for the indicated times. GAL induction was measured by assaying (3-galactosidase activity produced from the GALl-LacZ reporter gene as described in section 2.1 Data are the average of 3 independent determinations. 5 3 Results 3.1.2 Mutations to RNA Polymerase II holoenzyme components affect GAL4 phosphorylation in vivo It was previously suggested that GAL4 is phosphorylated during interaction with the general transcriptional machinery (Sadowski et al., 1991). Results supporting this hypothesis include the finding that phosphorylations which produce slower migrating species are only detected on GAL4 derivatives that are capable of both DNA binding and transcriptional activation (Sadowski et al., 1991). Also, the RNA polymerase holoenzyme component GAL11 has been shown to be necessary for at least some of GAL4's phosphorylations (Long et al., 1991; Sadowski et al., 1996). To directly examine this relationship, I determined whether the two RNA polymerase holoenzyme-associated cdks, KIN28 and SRB10, were necessary for GAL4 phosphorylation in vivo. For these experiments I used the GAL4A683 derivative (Sadowski et al., 1996) because I, and others, have found it impossible to reliably analyze phosphorylation of wild type GAL4 by 32P-labeling and phosphopeptide mapping in vivo. GAL4A683 lacks the central inhibitory segment, activates transcription efficiently, retains the major binding site for GAL80 at the C-terminus, and importantly bears all of the known sites of GAL4 phosphorylation (Sadowski et al.; see Fig. 5). I also used a variant of GAL4A683 which bears alanine substitutions at serines 691, 696, and 837 (GAL4A683T, see Fig. 5), leaving S699 as the only phosphorylation which produces a slower migrating electrophoretic species (Sadowski et al., 1996). GAL4A683T also has additional phosphorylations, including at least one in the DNA-binding domain (Fig. 5). Both GAL4A683 and GAL4A683T were efficiently phosphorylated in vivo when labeled with [32P]-orthophosphate, albeit more weakly at higher temperatures (Fig. 7, lanes 2-3 and 5-6). In 54 Results cells bearing a kin28Xs allele there is efficient phosphorylation at the permissive temperature (25°C)(Fig. 7, lanes 8 and 9), but barely detectable phosphorylation of both derivatives at the non-permissive temperature (37°C)(Fig. 7, lanes 11-12) demonstrating that KIN28 is required for most of the phosphorylations on GAL4, apparently including that in the DBD. However, considering that KIN28 is essential and its inactivation leads to a rapid decrease in transcription (see above), the reduction in GAL4 phosphorylation may be only indirectly related to the absence of KIN28. GAL4 phosphorylation was also impaired in cells bearing an srblO disruption (Fig. 8). In the WT background, there is efficient phosphorylation of GAL4 (Fig. 8, lanes 2 and 3). In the srblO strain phosphorylation of GAL4 was reduced, indicated by the lack of a slower migrating species as judged by both 32P-labeling and immunoblotting (Fig.8, lane 5). Note that phosphorylation of GAL4A683 at S837 causes reduced mobility as demonstrated by the presence of a phosphatase sensitive species in SDS-PAGE which can be eliminated by a serine to alanine substitution at 837 (Sadowski et al., 1996). These results demonstrate that each of the holoenzyme-associated cdks are required for some of GAL4's phosphorylations in vivo. To further demonstrate the role of the RNA pol II holoenzyme in GAL4 phosphorylation, GAL4 was examined in cells bearing a temperature-sensitive mutation oiRPBl, which encodes the largest RNA polymerase core subunit. In these experiments there was a significant decrease in phosphorylation of the faster migrating species at the non-permissive temperature (39°C) in rpbl^ cells, as judged by 32P-labeling, and the complete loss of slower migrating phosphorylated species as detected by both immunoblotting and 32P-labeling (Figure 9). Considering that galll mutations have 5 5 Results Figure 7. Inactivation of KIN28 reduces overall phosphorylation of GAL4. WT (lanes 1-6) or kin28ts (lanes 7-12) yeast transformed with a vector control (lanes 1, 4, 7, and 10), GAL4A683 (lanes 2,5,8, and 11) or GAL4A683T (lanes 3,6,9, and 12) expression plasmids were labeled with [32P]-orthophosphate at 25°C (lanes 1-3, 7-9) or 37°C (lanes 4-6, 10-12). GAL4 protein was recovered by immunoprecipitation, separated by SDS-PAGE, and visualized by autoradiography [32P]. Whole cell extracts from parallel mock-labeled samples were separated by SDS-PAGE and western blotted with 8C1 a-GAL4 MAb (GAL4 WB). 56 Results GAL4 [32P] WT 25°C 37°C n kin2ffs n n 25°C 37°C t .5- H •-S H - £ t fl) 6 I Q) • > H GAL4 WB 1 2 3 4 5 6 7 8 9 10 11 12 57 Results Figure 8. Deletion of SRB 10 eliminates specific phosphorylations of GAL4. WT (lanes 1-3) and srblO' (lanes 4-6) yeast transformed a vector control (lanes 1 and 4), GAL4 A683 (lanes 2 and 5) or GAL4A683T (lanes 3 and 6) expression plasmids were were labeled with [32P]-orthophosphate at 25°C as described in section 2.3. GAL4 protein was recovered by immunoprecipitation, separated by SDS-PAGE, and visualized by autoradiography [32P]. Whole cell extracts from parallel mock-labeled samples were separated by SDS-PAGE and western blotted with 8C1 cc-GAL4 MAb (GAL4 WB). 58 Results 59 Results Figure 9. Inactivation of RPB1 reduces overall phosphorylation of GAL4. WT (lanes 1-6) or rpblts (lanes 7-12) yeast transformed with a vector control (lanes 1 ,4,7, and 10), GAL4A683 (lanes 2,5,8, and 11) or GAL4A683T (lanes 3,6,9, and 12) expression plasmids were labeled with [32P]-orthophosphate at 25°C (lanes 1-3, 7-9) or 39°C (lanes 4-6, 10-12). GAL4 protein was recovered by immunoprecipitation, separated by SDS-PAGE, and visualized by autoradiography [32P]. Whole cell extracts from parallel mock-labeled samples were separated by SDS-PAGE and western blotted with 8C1 a-GAL4 MAb (GAL4 WB). 6 0 Results WT rpo21te 25°C 39°C 25°C 39°C Q) Q) 0 Q) t % H % h .9- i- .S-GAL4 [32P ] G A L 4 _ ^ ^ WB r 1 2 3 4 5 6 7 8 9 10 11 12 6 1 Results previously been shown to affect GAL4 phosphorylation (Long et al., 1991; Sadowski et al., 1996), this latter result suggests that mutations affecting RNA Pol II holoenzyme components may generally cause inhibition of one or more GAL4 phosphorylations. These findings strengthen the argument that GAL4 is phosphorylated by the RNA-polymerase holoenzyme-associated cdks but also complicate interpretation of whether GAL4 acts as a direct substrate for these enzymes. KTN28 in particular is required for transcription of many genes, and therefore loss of its function may indirectly prevent GAL4 phosphorylation. In contrast, cells bearing srblO disruptions are viable but have selective defects in transcriptional regulation (see below; Kuchin and Carlson, 1998; Kuchin et al., 1995; Liao et al., 1995 ) 3.1.3 SRB 10 is required for phosphorylation of S699 in vivo To determine which specific GAL4 phosphorylated residues were affected by srblO disruption, the in vivo 32P-labeled GAL4A683 protein was subjected to tryptic phosphopeptide analysis after purification by immunoprecipitation as described in section 2.3. Protein isolated from WT cells labeled in media containing galactose consistently generated eight phosphopeptides which were designated 1-8 (Fig. 10A). That S699 was found on phosphopeptide 1 was demonstrated by the fact that a synthetic peptide representing the predicted tryptic fragment containing GAL4 S699 phosphorylated in vitro (see below) (Fig. 10C) co-migrated with phosphopeptide 1 from in vz'volabeled GAL4 protein (Fig. 10D). Co-migration of in vivo labeled GAL4, isolated by immunoprecipitation, with an in vitro labeled GST-AR2 (residues 768-881) fusion protein demonstrated that phosphopeptide 5 contains S837 (not shown). 6 2 Results Phosphopeptide 2 is likely derived from the DNA binding domain as it is the predominant phosphopeptide from in vivo labeled GAL4 DBD produced on its own (not shown). Tryptic phosphopeptide maps of GAL4A683 protein labeled in cells bearing an srblO disruption (Fig. 10B) lacked several of the phosphopeptides observed in wild type cells, including those designated 1,4, 5, and 6 (compare Fig. 10A and B). The lack of phosphopeptides representing serines 699 and 837 is consistent with the observation that the srblO disruption eliminates the slower migrating electrophoretic GAL4 species (Fig. 8, lane 5). Although there appears to be some phosphorylation of GAL4A683 protein labeled in cells bearing a kin28ts allele, I was unable to obtain enough counts to produce a peptide map. I concluded from these experiments that mutations to SRB10 selectively eliminated a subset of GAL4 phosphorylations in vivo, including those at serines 699 and 837, whereas mutations to KIN28 or RBP1 globally inhibited GAL4 phosphorylation. 3.1.4 TFIIK predominately phosphorylates GAL4 at S837 in vitro To determine whether the RNA Pol II holoenzyme-associated cdks were capable of directly phosphorylating GAL4, recombinant wild type GAL4 protein (Kang et al., 1993) was used as substrate for in vitro kinase reactions with immunopurified HA-tagged SRB10 and KTN28. Both HA-KIN28 and HA-SRB10 were capable of phosphorylating GAL4 in vitro (Fig. 11, lanes 2 and 6), but neither phosphorylated recombinant GAL80 (lanes 3 and 7). Tryptic phosphopeptide analysis of the in vitro phosphorylated GAL4 protein revealed predominantly phosphopeptide 1 for both kinases, indicating S699 phosphorylation (Fig. 12, HA-SRB10, HA-KIN28), although GAL4 was also 63 Results Figure 10. SRB 10 phosphorylates GAL4 at S699 in vivo. GAL4A683 protein expressed in WT (panel A) or srblO' (panel B) yeast was labeled with [32P]-orthophosphate in vivo (see section 2.3). Tryptic phosphopeptides were resolved by electrophoresis (horizontal dimension) and chromatography (vertical). A synthetic peptide corresponding to the predicted GAL4 S699-containing tryptic peptide (YVSPGSVGPSPVPLK, S699 underlined) was phosphorylated in vitro with HA-SRB10 (see section 2.3) and analyzed separately (panel C), or co-migrated with in vr'vo-labeled GAL4A683 peptides (panel D). 64 Results 65 Results phosphorylated at S837 by HA-KIN28 as indicated by the appearance of phosphopeptide 5 (Fig. 12, HA-KTN28). The identity of the numbered peptides was confirmed by co-migration with peptides generated from in vrvo-labeled GAL4A683 protein (not shown). I also examined phosphorylation of GAL4 in vitro by purified TFIIH, which contains KLN28 as the only kinase (Feaver et al., 1994b). In contrast to that observed with HA-tagged KIN28, GAL4 phosphorylated by TFIIH kinase in vitro only produced phosphopeptide 5, indicating S837 phosphorylation, and not phosphopeptide 1 (Fig. 12, TFIIH). 3.1.5 SRB10 specifically phosphorylates GAL4 at S699 in vitro To compare the relative abilities of immunopurified HA-SRB10 and HA-KIN28 to phosphorylate the S699 on GAL4, a synthetic peptide corresponding to the predicted S699-containing GAL4 tryptic peptide was used as substrate. For both HA-tagged kinases recovered from cells by immunoprecipation, a corresponding synthetic peptide containing an S699A substitution was phosphorylated approximately three-fold less efficiently than the wild type peptide, demonstrating that S699 was phosphorylated by both kinases in vitro (Fig. 13). Significantly, HA-SRB10 phosphorylated the wild type peptide approximately 50 fold more than did HA-KIN28 (Fig. 13), despite the fact that the kinases were recovered in approximately equivalent amounts (Fig. 14). Although this result does not rule out the possibility that HA-KIN28 is incorrectly postranslationally modified, combined with the fact that TFIIH phosphorylates GAL4 exclusively at S837 66 Results Figure 11. Immunopurified complexes containing HA-KTN28 and HA-SRB10 phosphorylate GAL4 in vitro. In vitro kinase reactions were performed with immunopurified HA-SRB10 (lanes 1-4) or HA-KTN28 (lanes 5-8) (see section 2.3). Reactions contained lOOng of WT GAL4 protein (lanes 2, 4, 6 and 8), lOOng of GAL80 protein (lanes 3 and 7), or no added substrate (lanes 1 and 5). Lanes 4 and 8 contain lOOng of WT GAL4 incubated in the presence of a control immunoprecipitate with anti-vimentin antibodies. 67 Results SRB10 KIN28 kDa 200-116-97 -66-45 AL4 ^GAL80 1 2 3 4 5 6 7 8 68 Results Figure 12. Immunopurified HA-KIN28, HA-SRB10, and purified TFIIH phosphorylate GAL4 at S699 and S837, S699, and S837 respectively. lOOng WT GAL4 protein was phosphorylated in vitro with HA-SRB10, HA-KIN28, or purified TFIIH and subjected to tryptic phosphopeptide analysis (see section 2.3). Tryptic phosphopeptides were resolved by electrophoresis (horizontal dimension) and chromatography (vertical). 69 Results SrblO Kin28 TFIIH 70 Results (Fig. 12), this result indicates that SRB10 and KIN28 likely have different specificities towards GAL4. This also suggests that GAL4 phosphorylation in vitro by immunopurified HA-KJN28 complexes might result from co-immunopurified SRB10 protein. To investigate this possibility, in vitro kinase reactions were performed with HA-KJN28 complexes from WT and srblO cells. I found that KIN28 recovered from srblO cells did not phosphorylate S699 (Fig. 15B), but instead phosphorylated S837 (phosphopeptide 5), and several additional uncharacterized sites (Fig. 15). In contrast, HA-KIN28 isolated from SRB10 (WT) cells phosphorylated peptide 1 almost exclusively (Fig. 15A). In combination with the preceeding data, these results demonstrate that SRB10 is capable of phosphorylating GAL4 S699 in vitro, whereas KIN28 is not. Moreover, these data suggest that both KIN28 and SRB10 are required for GAL4 S837 phosphorylation in vivo. The fact that immunopurified HA-KTN28 contained SRB10 activity suggested the possibility that immunopurifications of HA-KTN28 and HA-SRB10 might be recovering complexes (holoenzymes) containing both of the CDKs, plus any other transiently or stably associated protein kinases. Therefore, to determine whether SRB10 was directly capable of phosphorylating GAL4 S699 in vitro, recombinant SRB107 11 complexes were expressed in insect cells using baculoviruses and purified by affinity chromatography as described in section 2.3. In vitro kinase reactions performed using this complex demonstrate that purified WT SRB107 11 complexes are capable of phosphorylating GAL4 but not GAL80 (Fig. 16, lanes 2 and 3). In contrast, complexes containing the kinase inactive D290A mutant SRB10/ SRB11 were incapable of both efficient autophosphorylation and phosphorylation of recombinant GAL4 (Fig. 16, lane 1). 7 1 Results Figure 13. Phosphorylation of synthetic peptides with HA tagged protein kinases. HA-KIN28 (A) and HA-SRB10 (B) irnmunoprecipitates from figure 14 were used for in vitro kinase reactions with synthetic peptides as described in section 2.3 Reactions contained no peptide, lOug WT S699 peptide (YVSPGSVGPSPVPLK, S699 underlined), or lOug S699A mutant peptide (YVSPGAVGPSPVPLK, A699 underlined). Peptides were separated from the reaction and 32P-incorporation was determined (CPM) as described in section 2.3. 72 Results A No Peptide S699 Peptide S699A Peptide o o o o o o o o o VO o o o oo CPM B No Peptide S699 Peptide S699A Peptide o o o o o o o o o o o © o o o co o o o o o CPM 73 Results HA-SRB10 HA-KIN28 1 2 3 Figure 14. Analysis of immunopurified HA-SRB10 and HA-KIN28 expressed in yeast. Extracts of W303 bearing a vector control (lane 1) or expressing HA-KIN28 (lane 2) or HA-SRB10 (lane 3) were prepared as described in section 2.3. 2mg of extracts were immunoprecipated with a-HA MAb (section 2.3), separated by SDS-PAGE and detected by western blot with a-HA MAb. 7 4 Results Figure 15. Tryptic phosphopeptide analysis of WT GAL4 treated with HA-KIN28 from SRB10 and srblO yeast strains. lOOng of WT GAL4 protein was phosphorylated in vitro with HA-KTN28 recovered from WT cells (SRB10), or HA-KIN28 recovered from cells bearing an srblO disruption (srblO) and subjected to tryptic phosphopeptide analysis (see section 2.3). Tryptic phosphopeptides were resolved by electrophoresis (horizontal dimension) and chromatography (vertical). 7 5 Results Results As with immunopurified SRB 10 complexes, tryptic phosphopeptide analysis demonstrated that phosphorylation occurs at S699 (Fig. 17). 3.1.6 GAL4 S699 is phosphorylated by SRBlO-containing RNA Pol II holoenzymes in vitro To determine whether R N A Pol II holoenzyme-associated SRB 10 is capable of phosphorylating GAL4 S699, purified holoenzymes were used for in vitro kinase reactions. R N A Pol II holoenzymes were purified3 by affinity chromatography with GST-TFIIS from either WT or srblO' yeast (Pan et al., 1997). Western blotting demonstrated that GST-TFIIS-purified holoenzyme complexes from WT yeast contained all the GTFs, including the largest R N A Polymerase II core subunit RPB1, TFIIB, TFIIE, TFIIH, SRB5 and SRB 10 (Fig. 18, and not shown). Many different proteins within the purified holoenzymes from both WT and srblO yeast were found to be phosphorylated 32 after incubation with y-[ P]-ATP in vitro confirming that R N A polll holoenzyme associated kinases have multiple targets in the holoenzyme (see section 1). G A L 4 was found to be phosphorylated by holoenzymes from both WT and srblO yeast, after recovery from the reactions by immunoprecipitation (Fig. 19, lanes 4 and 10). In contrast, GAL80 was not phosphorylated by either holoenzyme preparation (Fig. 19, lanes 6 and 12). Phosphopeptide mapping of WT GAL4 phosphorylated in vitro with WT holoenzymes produced all of the peptides normally observed with GAL4A683 in vivo (Fig. 20, panel A) plus an additional peptide (N) that must occur within 3 Holoenzymes were prepared by M. Kobor (Greenblatt Lab) from yeast extracts prepared and shipped on dry ice. Purified holoenzymes were shipped back at 4°C for use in kinase assays 77 Results Figure 16. Recombinant SRB107 SRB11 complexes specifically phosphorylate GAL4. In vitro kinase reactions were performed with lOOng of purified recombinant WT SRB107 SRB11 (lanes 2-4) or a kinase inactive mutant of SRB10 (D290A) (lane 1) as described in section 2.3. Reactions contained either 50ng of recombinant WT GAL4 (lanes 1 and 2), lOOng of recombinant GAL80 (lane 3) or no added substrate (lane 4). 78 Results - + + + SRB10 + - - - SRB10D290A + + - - GAL4 GAL80 1 2 3 4 79 Results Figure 17. Purified recombinant SRB 10/ SRB11 complexes phosphorylate GAL4 at S699 and S837. In vitro kinase reactions containing lOOng of recombinant SRB10/ SRB11 and 50ng of recombinant GAL4, were separated by SDS page and subjected to tryptic phosphopeptide analysis as described in section 2.3. Tryptic phosphopeptides were resolved by electrophoresis (horizontal dimension) and chromatography (vertical). 80 8 1 Results Figure 18. Affinity chromatography of RNA polll holoenzymes. Cell extracts prepared from WT (W303) and srblO (H617) yeast as described in section 2.3 were chromatographed on GST (as a control) or GST-yTFIIS (GST-IIS) columns. Bound proteins were eluted in IX SDS-PAGE sample loading buffer, separated by SDS-PAGE, and western blotted with the indicated antibodies (left). 8 2 83 Results the central portion of GAL4 (see Fig. 5). Significantly, I found that GAL4 phosphorylated by holoenzymes purified from srblO yeast was missing several phosphopeptides, including peptide 1 (Fig. 20, panel C), which co-migrates with the synthetic S699-containing tryptic peptide phosphorylated in vitro with recombinant SRB 10 (Fig. 20, panel B). These results are consistent with the notion that GAL4 S699 can be phosphorylated by RNA Pol II holoenzyme-associated SRB 10. 3.1.7 The requirement of SRB10 for GAL induction is epistatic to GAL4 S699 phosphorylation Several groups have noted that induction of transcription in response to galactose is impaired in strains lacking SRB10 (Balciunas and Ronne, 1995; Kuchin et al., 1995; Liao et al., 1995). I observed a similar effect in strains expressing WT GAL4, where GAL induction is approximately three-fold lower in srblO cells (Fig. 21). Significantly however, I also found that the GAL4 S699A mutation does not cause an additional defect in galactose-inducible transcription in srblO cells, as it does in WT cells (Fig. 21, and see Fig. 6). In fact, in the S288C strain background disruption oisrblO, or mutation of GAL4 S699 to alanine, produce approximately equivalent defects in GAL induction (Fig. 21). This finding demonstrates that SRB 10 and GAL4 S699 phosphorylation are genetically epistatic to one another, and represent an equivalent, rather than parallel, regulatory mechanism. S699 phosphorylation is required for GAL induction when GAL4's negative regulator GAL80 is present, but is unnecessary for transcriptional activation by 84 Results Figure 19. Cdk activity in purified RNA polll holoenzymes. In vitro kinase reactions were performed with immobilized GST control (odd lanes) or GST-yTFIIS affinity purified RNA polll holoenzymes (even lanes) from WT (lanes 1- 6) or srblO' (lanes 7-12) yeast as described in section 2.3. Reactions contained lOOng of WT GAL4 (lanes 3-4 and 9-10), lOOng of GAL80 (lanes 5-6 and 11-12), or no added substrate (lanes 1-2, 7-8). Substrates were recovered after the reaction by immunoprecipitation with aGAL4 (lanes 1-4, 7-10) or ccGAL80 (lanes 5-6, 11-12) antibodies, separated by SDS-PAGE and visualized by autoradiography. 8 5 Results WT kDa 205-112-87-56-GAL4 GAL80 « i- H h 00 00 00 00 00 C/) oo 0 = 0 = 0 = 0 ( 0 srblOr GAL4 GAL80 0 = h 00 00 0 = -*GAL4 ^GAL80 1 2 3 4 5 6 7 8 9 10 11 12 86 Results Figure 20. SRB 10 is required for phosphorylation of GAL4 at S699 by purified RNA pol II holoenzymes. Tryptic phosphopeptide analysis of WT GAL4 protein phosphorylated in vitro with purified immobilized RNA polll holoenzymes from WT (panels A and B) or srblO (panel C) yeast as described in section 2.3. Reactions contained immobilized holoenzymes and lOOng of recombinant GAL4. GAL4 was recovered after the reaction by immunoprecipitation with aGAL4 antibodies, separated by SDS-PAGE and treated to tryptic phosphopeptide analysis as described in section 2.3. Tryptic phosphopeptides were resolved by electrophoresis (horizontal dimension) and chromatography (vertical). WT GAL4 phosphopeptides produced by phosphorylation with WT holoenzymes were co-migrated with GAL4 S699 synthetic peptide (YVSPGSVGPSPVPLK, S699 underlined) phosphorylated in vitro with lOOng of purified recombinant SRB 10/ SRB11 as described in section 2.3 (panel B). 8 7 Results 1 \ N 61 3 1 * A 1 1 5 4 4 0 ? % 7 N 6 3 t B 5 4 o 8 2 % 7 N Is •.>*%•' ' • •'. t^y w™"'" " L • . 1 C • * 88 Results 6OO-1 | _ _ 1 | | | _ 1 | 1 WT srblOT gal8(T ga!80Srb10' Figure 21. The GAL4 S699A mutation is genetically epistatic to SRB10. GALl-LacZ expression was measured following 2 hrs induction with 2.0% galactose in the yeast strains YJMH4 (WT), YJMH5 (srblO), YJMH8 (go/50"), YJMH9 {gal80Tsrbl(r), bearing a control vector (-), or expressing WT GAL4 (WT), or GAL4 S699A (A699) from plasmids pRS315, pJR006, or pJR007 respectively. Results are the average of three independent determinations. Error bars represent standard deviation. 89 Results GAL4 in gal80 cells (Sadowski et al., 1996). If the negative effect of srblO disruptions on GAL transcription were caused by loss of S699 phosphorylation, one would expect that srblO disruptions should not have an effect on GAL transcription in strains lacking GAL80. Consistent with this prediction, I found that disruption of srblO does not affect GALl-LacZ expression in gal80 cells (Fig. 18, compare gal80~, and gal80~ srblO'). This result supports the hypothesis that the effect of SRB10 on GAL transcription is mediated by the GAL4 S699 phosphorylation. Combined with the fact that SRB10 is required for phosphorylation of S699 in vivo, and that HA-SRB10 phosphorylates GAL4 S699 in vitro, these results demonstrate that GAL4 is controlled by an SRBlO-mediated phosphorylation. 3.2 S699 phosphorylation regulates the affinity of GAL4 for GAL80 in vivo Although early models of induction postulated that GAL80 dissociated from GAL4, this model has been challenged by a number of laboratories (see introduction). When expressed in combination with a GAL4 mutant that was impaired for transcriptional activation, but which retains its ability to interact with GAL80, GAL80-VP16 was shown to cause transcriptional activation in both inducing and non-inducing conditions (Leuther and Johnston, 1992). This result led to the proposal that galactose induction may not require dissociation of GAL80 from GAL4. This idea was supported by experiments which demonstrated that GAL4 purified from galactose-induced yeast was associated with GAL80 (Parthun and Jaehning, 1992). More recently, experiments in vitro have demonstrated a terniary complex containing GAL4, GAL80 and GAL3 which is capable of activating transcription (Piatt and Reece, 1998). Taken together, 90 Results these results support a model for induction in which GAL3-galactose causes a conformational change in the interaction between GAL4 and GAL80 that allows GAL4 to activate transcription without dissociation of GAL80. This appears to be the current prevailing view. Several other observations complicate interpretation of these experiments. It was previously shown that GAL4 phosphorylation requires transcriptional activation (Sadowski, 1991). Results presented here are consistent with these observations. Furthermore phosphorylation at S699 is required for full induction in WT GAL80 strains, but is not required for activation of transcription in gal80~ yeast (Fig. 21, and Sadowski et al., 1996). These results suggest that the SRBlO-mediated phosphorylation on GAL4 at S699 must control induction by regulating GAL4-GAL80 interaction. Unfortunately, the design of the experiments mentioned above did not pay consideration to the role of phosphorylation. The two-hybrid experiment with the GAL80-VP16 fusion (Leuther. and Johnston, 1992), used a GAL4 mutant that was impaired for transcriptional activation, which by previous analysis would prevent phosphorylation (Sadowski, 1991). The co-purification of GAL4 and GAL80 was performed with yeast bearing a disruption of REG1, which relieves URS-mediated glucose repression, and grown in both glucose and galactose (Parthun and Jaehning, 1992). I have observed that glucose inhibits the kinase activity of HA-SRB10, but not HA-KIN28 (data not shown). Thus, it is likely that the GAL4 in the purification experiments was phosphorylated at S837, but not S699 (Parthun and Jaehning, 1992). Finally, the in vitro induction experiments demonstrating the GAL4/ GAL80/ GAL3 complex was performed with a truncated GAL4 derivative bearing only the DBD fused to AR2 and therefore did not possess the region where most 9 1 Results of the phosphorylations occur (Piatt and Reece, 1998). Considering these factors, one could argue that it has not been resolved whether full activation of the GAL genes in vivo requires dissociation or simply a conformational change between GAL4 and GAL80. To address this issue, and to further characterize the function of the S699 phosphorylation on GAL4,1 devised a novel two-hybrid strategy that would allow use of WT GAL4 as a bait protein. 3.2.1 The Repressed Transactivator Assay All transcriptional activators contain two basic elements, a DNA-binding domain, which allows the protein to bind to specific sequences upstream of the genes they regulate; and activation domains, which recruit GTFs to promoters leading to initiation of transcription (Brent and Ptashne, 1985). The yeast two-hybrid system utilizes the fact that these functional elements can work when brought together as fusions or hybrids with separate proteins that form a strong interaction (Fields and Song, 1989). In the standard yeast two-hybrid assay, a protein of interest is fused to a well-characterized DNA binding domain to create the "bait" hybrid protein. Coexpression of a protein fused to a strong activation domain allows detection of an interaction by the physical tethering of the activator domain to the promoter via interaction with the bait hybrid protein causing expression of a reporter gene. However, the standard two hybrid assay is not suitable for assaying interactions with transactivating "bait" proteins since they cause constitutive expression of the reporter gene. 92 Results Figure 22. Rationale for the Repressed Transactivtor Two-Hybrid System. Panel A, Representation of the GAL1 gene in glucose-grown cells, indicating the binding sites for GAL4 (UAS), and MIG1 (URS). MIG1 recruits the general repressors SSN6 and TUP1. The N-terminal repression domain (RD) of TUP 1 is indicated in black. Panel B, Activation of a GAL1-URA3 reporter gene by a GAL4 DNA-binding domain (DBD)-activator fusion protein (Activator Bait, X) in ura3~ yeast gives a URA+ phenotype, and causes sensitivity to 5-Fluoro-orotic acid (5-FOA). Panel C, Interaction between a TUP1 RD fusion (TUP1-RD prey, Y) with the Activator Bait causes repression of transcription, resulting in 5-FOA resistance. 93 Results Results Eukaryotic cells also contain another class of proteins that regulate gene expression by inhibiting expression of nearby genes (Herschbach et al., 1994; Kadosh and Struhl, 1997; Keleher et al., 1992; Kirov et al., 1996; Madden et al., 1993; Margolin et al., 1994; Moosmann et al., 1996; Ogbourne and Antalis, 1998). One such protein is the general repressor TUP1 (Keleher et al., 1992). TUPl is a 713 amino acid protein implicated in the repression of a diverse set of genes involved in adaptation to wide variety of environmental conditions including carbon source, cell type, DNA damaging agents, and oxidiative stress (Keleher et al, 1988; Nehlin et al., 1991; Zitomer and Lowry, 1992). TUPl is unable to bind DNA on its own but is recruited to the promoter by partners which bind to DNA and/or are modified in response to specific signals (Huang et al., 1998; Komachi et al., 1994; Park et al., 1999; Treitel and Carlson, 1995; Tzamarias and Struhl, 1995). Repression by TUPl appears to involve two separate mechanisms, direct inhibition of the RNA pol II holoenzyme (Kuchin and Carlson, 1998) and establishment of repressive chromatin structure (Cooper et al., 1994). Both mechanisms function through the N-terminal domain of TUPl and actively repress when artificially tethered to a promoter by a heterologous DNA binding domain (Tzamarias and Struhl, 1994). The system I developed to assay GAL4/ GAL80 interaction in vivo exploits this ability to repress transcription from an activated promoter (Tzamarias and Struhl, 1994). I call this system the Repressed Transactivator Assay (RTA) which is similar to the standard two-hybrid assay, but in which the "bait" is a transcriptional activator. The activator bait is challenged with protein fragments fused to the N terminus of the general repressor TUPl. If an interaction occurs between the activator bait and the TUPl fusion 95 Results prey the reporter gene should be repressed (Figure 22). By the use of a counterselectable marker such as URA3 (Boeke et al., 1987), interactions can be scored by their ability to allow growth in the presence of a toxic substrate for the reporter enzyme, which in the case of URA3 is 5-FOA. 3.2.2 The N terminus of TUP1 represses transcription as a fusion in a two hybrid system. To investigate the possibility that TUP 1 could repress transcription in the context of a two hybrid assay, I examined the well-defined interaction between GAL4 and its inhibitor protein GAL80. GAL4 (Fig 23, Bait) over-expressed on its own from the ADH1 promoter in a strain bearing a GAL1-URA3 reporter gene causes activation of transcription and renders the yeast sensitive to 0.1% 5-FOA (compare Fig 23, A and B). As expected, expression of GAL80 from the ADH1 promoter (Gal80 Prey, Fig. 23) in the same strain causes some inhibition of GAL4, allowing infrequent formation of colonies on 5-FOA. Expression of the N-terminus of TUP 1 on its own has no effect on activation by GAL4 (Fig 23, GAL4/ Tupl), but in contrast, expression of a protein consisting of the TUP 1 N-terminal 200 residues fused to GAL80 (Fig. 23, GAL4/ Tupl-Gal80) causes significantly more growth on 5-FOA than does expression of GAL80 on its own (compare Fig. 23 B, GAL4/ Gal80 to GAL4/ Tupl-Gal80). I observed this same result whether the transformed yeast were grown on glucose (not shown), or galactose-containing plates. These results demonstrate that the repression domain of TUP 1 can function as a fusion to the N-terminus of GAL80. Furthermore, the fact that TUP1-96 Results GAL80 represses transcription of a GAL4-dependent reporter gene under both inducing and non-inducing conditions, supports the view that the presence of galactose does not cause dissociation of GAL4 from GAL80 (see below). 3.2.3 SRB 10 is required for repression by the N terminus of TUPl TUPl causes repression of diverse sets of genes by interacting with different DNA-binding proteins (DeRisi et al., 1997). In the case of glucose-repressed genes, TUPl's function was characterized genetically by identification of mutations (suppressors of sucrose non-fermentors, ssri) that relieved constitutive repression from a URSGLU.(Carlson et al., 1984). Some of the SSN genes encode components of the RNA-polll mediator subcomplex, for example, SSN3 and 8 are identical to the cdk/ cyclin pair SRB10ISRB11 (Song et al., 1996). SRB10 kinase activity has been shown to be required for at least part of the repressive effect of the TUPl repression domain in vivo, thus demonstrating that one function of TUPl may involve modulation of RNA Pol II holoenzyme function (Kuchin and Carlson, 1998). To determine whether the N-terminus of TUPl inhibits transcription by its normal repressive mechanism when fused to GAL80,1 examined whether SRB 10 was required for repression of GAL4-dependent reporter gene expression by TUP1-GAL80. For these experiments I used a GALl-LacZ reporter gene. Consistent with the results of Figure 23,1 found that expression of the TUP1-GAL80 fusion caused significant inhibition of transcriptional activation by GAL4 in WT SRB 10 yeast (Fig. 24, WT/ Tupl-Gal80). Expression of GAL80 alone in this strain caused only slight inhibition of GAL4, and TUPl expressed on its own had no effect (Fig. 24, WT/ GAL80, and WT/ Tupl). In contrast to these results, I found that in srblO yeast TUP1-GAL80 was incapable of inhibiting activation of GALl-LacZ reporter 97 Results A Bait Prey 0 % 5 - F O A GAL4 Vector GAL4 Gal80 GAL4 Tup1 GAL4 Tup1-Gal80 B Bait Prey GAL4 Vector GAL4 Gal80 GAL4 Tup1 GAL4 Tup1-Gal80 0.1% 5 - F O A Figure 23. TUP1-GAL80 represses GAL transcription under inducing conditions. Yeast strain yJMHl was co-transformed with plasmid pMA210 expressing WT GAL4 (Bait), and: pJMH106, expressing GAL80 (line 2); pJMH105, expressing TUP1RD (line 3); pJMH107, expressing TUP1-GAL80 (line 4); or YephalAlac (line 1). Equivalent numbers of cells were spotted, in 10 fold serial dilutions (from left to right), onto selective media containing galactose and raffinose without 5-FOA (GAL/ RAF), or with 0.1% 5-FOA (GAL/ RAF/ 5-FOA). 98 Results gene expression (Fig. 24, srblOl TUP1-GAL80). This result supports the conclusion that the TUPl repression domain fused to GAL80 causes repression through its normal function, rather than by simply exaggerating an inhibitory effect of GAL80. 3.2.4 Repression of GAL transcription by TUP1-GAL80 requires the C-terminal 30 residues of GAL4. Deletion analysis has identified two regions of GAL4 that are capable of causing activation of transcription in vivo when fused individually to the DNA binding domain, known as activating region 1 and activating region 2 (ARI, AR2)(Ma and Ptashne, 1987b). The C-terminal AR2 also overlaps the primary region of GAL4 required for interaction with GAL80 (Ma and Ptashne, 1987a). Deletion of the C-terminal 30 residues of GAL4 produces a constitutive activator that is incapable of efficient interaction with GAL80. To determine whether the RTA system could be used to map a site of protein-protein interaction on a transactivator protein, I determined whether repression of GAL-reporter gene expression by TUP1-GAL80 requires the C-terminal 30 residues of GAL4. For this purpose I assayed the ability of TUP1-GAL80 to repress transcription activated by a series of GAL4 deletion/ fusion derivatives (Fig 25). I found that only GAL4-derivatives bearing the C-terminal 30 residues allowed survival of yeast on 5-FOA when co-expressed with TUP1-GAL80. Thus, yeast expressing GAL4( 1-848) in combination with TUP1-GAL80 could not grow on 5-FOA indicating that loss of the C-terminal 30 residues prevents repression by the fusion. Similarly, a deletion derivative bearing both ARI and AR2 but lacking the central region (GAL4 (1-238+768-881)) was inhibited by TUP1-GAL80, but a comparable derivative lacking the C-terminal 30 residues was not (GAL4 (1-238+768-848)). Derivatives bearing the GAL4 DNA-binding domain and only ARI, or fused to the artificial activating domain B17 were also found to be insensitive to 9 9 Results Figure 24. Repression of GAL transcription by TUP1-GAL80 requires SRB 10. Yeast strains W303:131 (SRB10, black bars), and H617:131 (srbl0, open bars) were transformed a vector control (YephalAlac) (Vector) or plasmids expressing GAL80 (pJMH106), TUPl(pJMH105), or TUPl-GAL80(pJMH107). Yeast were grown to mid-log phase in selective medium containing galactose, and expression of the GALl-LacZ reporter gene was assayed by measuring p-galactosidase activity (see section 2.2). Results are the average of three independent determinations. Error bars indicate standard deviations. 100 Results Vecto 1 Vector Tup1 GALsmmmmmmm H H 1 T U P I :GALQmmmmmmm 50 100 150 (3-galactosidase 200 1 0 1 Results inhibition by TUP1-GAL80 (Fig. 25, GAL4(l-238), GAL4-B17). Therefore, despite the fact that all of the GAL4 derivatives shown in Fig. 25 are capable of efficient transcriptional activation (Fig. 25C), only those bearing the C-terminal 30 residues are inhibited by TUP1-GAL80. These results indicate that repression by TUP1-GAL80 requires the normal site of interaction between GAL4 and GAL80, and suggest that the RTA two-hybrid system could be used to map interactions between an activator protein and its targets. 3.2.5 S699 inhibits the GAL4 / GAL80 interaction in vivo Having established that the RTA system can detect specific interactions between GAL80 and GAL4 in vivo, I sought to determine whether the phosphorylation at serine 699 affects this interaction. In order to ensure any differences in growth were solely a reflection of the state of GAL80's interaction with GAL4 and not simply a result of activation difference, I overexpressed WT GAL4 and S699A GAL4 from the ADH1 promoter on a high copy plasmid. Under these conditions both WT GAL4 and S699A activate transcription equally well (Fig. 26A). When yeast co-expressing TUP1-GAL80 with WT GAL4 or GAL4 S699A were plated on 5-FOA containing galactose I found that cells expressing GAL4 S699A grew more efficiently than did cells expressing WT GAL4. This result suggests that GAL4 S699A is more strongly repressed by TUP1-GAL80, indicating a stronger interaction than with WT GAL4. Furthermore, this result suggests that interaction between GAL4 and GAL80 may be destabilized by phosphorylation at S699. 102 Results Figure 25. Repression of GAL transcription by TUP1-GAL80 requires the GAL4 C-terminal 30 amino acids. Panel A. Diagrams of various GAL4 deletions Panel B. Yeast strain yJMHl was co-transformed with pJMH107 expressing TUP1-GAL80 and plasmids expressing the GAL4 deletion/ fusion derivatives indicated in Panel A. Cells were spotted, in 10-fold serial dilutions (from left to right) on selective plates containing galactose and raffinose without 5-FOA (Panel A, GAL/RAF), or with 0.1% 5-FOA (Panel B, GAL/RAF/ 5-FOA). Panel C. Yeast co-transformed with GAL4 derivatives (indicated in Panel A) and pJMH105 expressing TUP1 were spotted as above on minimal selective plates lacking uracil (URA7 GAL/ RAF) to assay activation of the GAL1-URA3 reporter. 103 Results A G A L 4 Bait DBD AR1 AR2 238 147 £38 238 IB17 881 B C T U P 1 - G A L 8 0 Prey T U P 1 Vector G A L / R A F / U R A 7 G A L / R A F 5 - F O A G A L / R A F 848 768 881 768 848 1 0 0 t % ••# si dp w i • W^F f^iF "il^ yP «jr» I w WW 1 © § * 1 0 4 Results Figure 26. S699 functions by destabilizing the GAL4/ GAL80 interaction. (A) YT6:171 transformed with pMA210 (WT GAL4) or pMA210A699 (GAL4S699A) and either empty vector (Vector) or pJMH106 (GAL80) were grown to mid-log phase in selective medium containing galactose, and expression of the GALl-LacZ reporter gene was assayed by measuring |3-galactosidase activity (Section 2.2). Results are the average of three independent determinations. Error bars indicate standard deviations. For B and C , yJMHl was transformed with pJMH107 (Tupl-Gal80) and either pMA210 (WT GAL4) or pMA210A699 (GAL4S699A). Cells were spotted, in 10-fold serial dilutions (from left to right) on SD his",trp" containing 2% GAL/ RAF (B) or SD his, trp" containing 2% GAL/ RAF supplemented with 0.1 % 5-FOA (C). 1 0 5 A Results O 0) WT GAL4 GAL4 S699A oo < CD WT GAL4 GAL4 S699A B 1000 2000 p-galactosidase 3000 Bait Prey GAL4 S699A Tup1-Gal80 WT GAL4 Tup1-Gal80 c Bait Prey GAL4 S699A Tup1-Gal80 WT GAL4 Tup1-Gal80 106 Discussion 4. DISCUSSION GAL4 was one of the first transcription factors demonstrated to be phosphorylated when it was initially shown that phosphorylation correlated with its activation function (Mylin et al, 1989; Mylin et al., 1990). However, the significance of these phosphorylations for gene regulation became enigmatic when it was realized that they likely occurred as a consequence of, rather than a contributing factor for, GAL4s transcriptional activation function (Sadowski et al., 1991). The data presented in this thesis demonstrate that mutations to RNA Pol II holoenzyme components prevent GAL4 phosphorylation at one or more sites in vivo, and that the mediator-associated cdk SRB 10 is required for a specific subset of GAL4s phosphorylations. Furthermore, GAL4 is specifically phosphorylated at S699 by recombinant SRB 10 in vitro, whereas GAL4 S837 can be phosphorylated in vitro by KIN28/ TFIIH. GAL4 is also phosphorylated by purified RNA Pol II holoenzymes in vitro at all of the sites normally observed in vivo. These results demonstrate that most, if not all, of GAL4s phosphorylations occur during transcriptional activation by the holoenzyme-associated cdks. Of the multiple phosphorylations on GAL4, only that at S699 appears to be necessary for efficient induction of the GAL genes. SRB10 is also required for GAL induction, and srblO disruptions are epistatic to the GAL4 S699A mutation. These results demonstrate a direct regulatory role for phosphorylation of GAL4 by SRB 10. 4.1 F u n c t i o n o f t h e R N A P o l y m e r a s e I I h o l o e n z y m e - a s s o c i a t e d c d k s The C-terminal domain (CTD) of the largest core subunit of RNA Pol II becomes phosphorylated during transition from the preinitiation to elongating complex (OBrien et 107 Discussion al., 1994). Three different protein kinases in yeast have been implicated in CTD phosphorylation, including the two holoenzyme-associated cdks KIN28 and SRB10, and a related enzyme encoded by CTK1 (Liao et al., 1995; Sterner et al., 1995; Valay et al, 1995). Much work characterizing the function of these kinases in transcriptional regulation has focused on their role in CTD phosphorylation, which in many analyses is considered to be overlapping and at least partially redundant (Lee and Lis, 1998). The results presented in this thesis demonstrate that GAL4 is a substrate for both SRB10 and KTN28, but each of these kinases phosphorylate separate specific sites, suggesting that each of these cyclin-dependent protein kinases have separate preferred substrates in vivo. GAL4 is the first physiologically relevant substrate demonstrated in vivo for yeast SRB10; substrates have not yet been identified for its mammalian counterpart CDK8. However, considering the archetypal position of GAL4 I expect that many different yeast and mammalian transcription factors are regulated by similar mechanisms through the action of the holoenzyme-associated cdks. TFIIK, the kinase component of TFIIH, is known to be capable of phosphorylating several GTFs, including TBP, TFIIE, and TFIIF (RAP74 subunit) in addition to the CTD, as well as several transactivator proteins (Lu et al., 1997; Ohkuma and Roeder, 1994; Rochette-Egly et al., 1997). The functional significance of most of these phosphorylations has not been determined. CTD phosphorylation is required for entry into an elongation mode (O'Brien et al., 1994), and couples RNA processing to transcription (McCracken et al., 1997a; McCracken et al., 1997b). Direct interaction between yeast transcription factors and TFIIH has not yet been demonstrated, although I show here that TFIIH phosphorylates GAL4 at S837, a site known to be phosphorylated 108 Discussion in vivo (Sadowski et al., 1996). KTN28 is essential for cell viability (Valay et al., 1995) and is required for transcription of genes which are also dependent upon the mediator components SRB4 and SRB6 (Valay et al., 1995). Given the highly conserved mechanisms of transcriptional regulation in eukaryotes, combined with recent evidence of RARa and p53 phosphorylation by TFIIH (Lu et al., 1997; Rochette-Egly et al., 1997) it seems likely that a subset of yeast transactivators may also be regulated by TFIIH. Indeed, it is a possibility that the S837 phosphorylation may play a regulatory role for a GAL4 function that has yet to be characterized. It has been suggested that SRB 10 causes transcriptional repression by phosphorylating the CTD prior to preinitiation complex formation (Hengartner et al., 1998). However, I do not favor this model as a specific regulatory mechanism because it does not explain how SRB 10 can function both in activation and repression (see above), how it can function as an activator when tethered to DNA in the presence of excess SRB11 (Carlson, 1997), nor why some genes are completely unaffected by SRB 10 mutations (see above). Instead, I propose that SRB 10 regulates specific transcription factors bound to upstream sites during their interaction with the GTF complexes. 4.2 Regulation of RNA Polymerase holoenzyme cdk function Several recent observations implicate the RNA polymerase holoenzyme as a downstream target for physiological signal transduction mechanisms. CTD phosphorylation appears to be regulated during entry into stationary phase (Patturajan et al., 1998), in response to heat shock in yeast (Cooper et al., 1997; Cooper et al., 1999) 109 Discussion Figure 27. GAL4 activity is regulated by two independent signals. In non-inducing conditions (A), GAL4 activity is inhibited by the negative regulator GAL80. Upon galactose addition (B), GAL3-galactose interacts with GAL80 to cause a transient conformational alteration that allows GAL4 to activate transcription. During interaction with the RNA Pol II holoenzyme (C), GAL4 is phosphorylated at S699 by SRB10; this phosphorylation stabilizes the active GAL4-GAL80 conformation induced by GAL3-galactose. SRBlO's ability to phosphorylate GAL4 is regulated by independent environmental signals, thus modulating GAL induction to levels appropriate for the cellular environment. 1 1 0 Discussion 1 1 1 Discussion and mammalian cells (Dubois et al., 1997), and by stress activated MAP kinase pathways (Venetianer et al., 1995). A mouse mediator complex was also shown to be associated with a nuclear MAP kinase (Jiang et al, 1998). Finally, the SRBlO-associated cyclin C (SRB11) has been shown to be degraded in response to environmental signals including heat, peroxide stress, and growth on non-fermental carbon sources (Cooper et al., 1997; Cooper et al., 1999). Degradation of SRB11 during growth on non-fermentable carbon sources is not suprising in light of the fact that in gal80 cells, where the GAL genes are activated constitutively, GAL4 is hypophosphorylated in cells growing in non-fermentable carbon, but becomes rapidly hyperphosphorylated in response to fermentable sugars (Sadowski et al., 1996). These observations have broad implications for eukaryotic transcriptional regulation because environmental signals that influence holoenzyme cdk activity are also likely to modulate transcription of specific genes through phosphorylation of their cognate transcription factors. Consistent with this hypothesis, GAL gene induction is impaired under conditions of nutrient limitation and heat stress (Rohde, 1999). Therefore, I propose that a critical function of the RNA polymerase holoenzyme cdks involves modulation of specific transactivators in response to general physiological signals to coordinate inducible transcription with the cellular environment (see Fig. 27). 4.3 Regulation of GAL4 activity by S699 phosphorylation The results presented using the RTA system demonstrate that GAL4 and GAL80 are capable of interaction under inducing conditions (Figs. 23, 24 and 25). This is 112 Discussion consistent with the previous reports demonstrating constitutive association of GAL4 and GAL80 (see above). However, interaction of GAL80 with WT GAL4 is impaired in comparison to GAL4 bearing a S699A mutation as measured using the RTA system (Fig. 26). Taken together, these results suggest that induction of GAL4 involves two distinct states, one involving the formation of a GAL4/ GAL80/ GAL3 complex, capable of weak transactivation, the other involving dissociation of the GAL807 GAL3 complex allowing for frill activation by GAL4. In vivo, these two states would be in equilibrium depending on the conditions encountered. Phosphorylation at GAL4 S699 would stabilize the dissociated state, shifting the equilibrium and allowing for full activation. Induction by GAL4 S699A would therefore be reduced, its equilibrium would favor the GAL4/ GAL807 GAL3 complex. Thus, I propose that GAL3-galactose binds to the GAL4/ GAL80 complex allowing recruitment of the RNA polll holoenzyme. If conditions are right (see above), phosphorylation of GAL4 occurs, the GAL807 GAL3 complex dissociates, and full activation ensues (Fig. 27). 4.4 Significance of GAL4 S699 phosphorylation for GAL gene regulation For the yeast GAL genes, I propose that galactose, through the action of GAL3, relieves the repressive effect of GAL80 on GAL4 activity (Fig. 27B), while SRB10 modulates the level of induction by phosphorylating GAL4 (see Fig. 27C). The RNA polymerase holoenzyme-associated cdks, as targets for general signaling mechanisms (Fig. 27C), are ideally positioned to regulate the activity of gene-specific transactivators to levels that are appropriate for the cells physiological environment. Under conditions of stress or nutrient limitation, induction of specific genes can be limited by inhibiting 113 Discussion activity of the appropriate cdk, whereas ideal growth conditions can be signaled to gene-specific activators by their phosphorylation during interaction with the holoenzyme. Future studies should investigate the nature of the signaling mechanisms that control GAL4 activity through SRB10, and I would expect these to overlap signal transduction pathways that regulate cell stress and general nutrient responses. 4.5 Repressed Transactivator Assay The yeast two-hybrid (Song and Fields, 1989) and interaction-trap (Golemis, 1994) systems have provided simple and reliable genetic strategies for identification and characterization of interactions between proteins. These systems were developed consequent to the understanding that eukaryotic trancriptional activators have separable DNA-binding (DBD) and transcriptional activation domains (AD) that readily function when fused to heterologous proteins (Brent and Ptashne, 1985). Interaction of an AD "prey" fusion with a DBD "bait" fusion protein produces a functional transactivator complex that can activate reporter genes bearing upstream c/s-elements for the DBD. One limitation of these strategies is that they are unable to analyze the function of most naturally occuring transcriptional regulatory proteins, unless regions which cause transcriptional activation are deleted from the fusion. For many transcription factors, critical interactions with regulatory components are often mediated by the same regions which contact general intiatiation components and thereby cause transcriptional activation (for example see Jakobsen and Pelham, 1991; Murai et al., 1998; Thut et al., 1997; Wang et al., 1997). Furthermore transcriptional regulatory proteins by their very nature make 114 Discussion the best candidates for study by the two hybrid system since they are the natural occupants of promoters. In addition to the benefits of investigating the interactions with transcriptional activators, the RTA system is ideally suited to investigate interactions with the approximately 25% of protein fragments which activate transcription when artificially tethered to a promoter (Ma and Ptashne, 1987; Ruden, 1992). The RTA system therefore provides important addition to the arsenal of the molecular biologist. 4.6 Future Directions There are two clear directions for future studies of the role of RNA polll holoenzyme phosphorylation of GAL4. The first is to investigate the precise role of phosphorylation at S699. The in vivo results presented in this thesis suggest that phosphorylation at S699 regulates association of GAL80 with GAL4; however, in vitro studies should be performed to confirm or refute this possibility. Unfortunately, GAL4 is extremely difficult to study in vitro due to its tendency to form insoluble complexes when present at concentrations amiable to such studies. In light of this, other in vivo techniques could be employed to measure differences in affinity related to phosphorylation of S699. One such technique, chromatin immunoprecipitation (CHIP)(Aparicio, 1997), in combination with real time PCR analysis could be exploited to examine the effect of phosphorylation on the interaction between GAL4 and GAL80. CHIP involves the immunoprecipation of proteins crosslinked to DNA and is ideally suited to studies involving transcription factors. Using aGAL80 and/ or ocGAL3 115 Discussion antibodies to immunoprecipate GAL3/ GAL807 GAL4 complexes crosslinked to DNA, followed by real time PCR analysis would allow for the measurement of any differences in complex stability dependent on S699. The second direction involves the elucidation of the signalling pathway(s) which control SRB10. To this end, a genetic screen has been designed to identify mutants in this pathway which are epistatic to S699 (Rohde, 1999). In addition, recent work by Nelson et al. have demonstrated that SRB10 is capable of phosphorylating components involved in the mating pheromone response pathway of S. cerevisiae (Nelson, 1999). This is not surprising given the nature of the signals involved in regulation of mating, and further emphases the global nature of control by holoenzyme associated kinases. 4.7 Conclusion In summary, the work described in this thesis demonstrates that both holoenzyme associated cdks are capable of phosphorylating GAL4; SRB10 was shown to phosphorylate GAL4 at S699 while TFIIK was shown phosphorylate S837. Futhermore, using a novel two hybrid based assay, phosphorylation by the SRB10 cdk was shown to affect the interaction between GAL4 and its negative regulator GAL80. These results are significant for the study of transcription regulation in that they suggest a mechanism for the regulation of transcription in response to global stimuli such as carbon limitation or environmental stress. 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