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Exploring the genetic interactome of a Saccharomyces cerevisiae separase mutant Ho, Krystina 2013

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EXPLORING THE GENETIC INTERACTOME OF A SACCHAROMYCES CEREVISIAE SEPARASE MUTANT by Krystina Ho  B.MSc., The University of Western Ontario, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Biochemistry and Molecular Biology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2013  © Krystina Ho, 2013  ii Abstract The budding yeast Saccharomyces cerevisiae is used as a model organism for understanding cellular dynamics such as cell cycle regulation.  Separase endopeptidases are essential to these events, being responsible for cleavage of cohesin – the molecular glue holding sister chromatids together.  To examine the function of separases in a systematic fashion, a temperature sensitive mutant of the yeast homologue, Esp1, was subjected to a high-throughput technique known as synthetic genetic array to identify genetic interactions. Examination of the list of alleles confirmed to cause a synthetic lethal or synthetic dosage lethal phenotype in the query esp1-1 mutant (“hits”) established the legitimacy of these screens, as many related to known Esp1 functions.  Surprisingly, categorization of these hits by biological process revealed an enrichment for genes involved in RNA metabolic processes.  Concurrent affinity immunoprecipitation of separase followed by mass spectrometry further showed a physical interaction between Esp1 and the integrase portion of Ty1 retrotransposons.  Evidence that this interaction was indicative of a role for separase in retrotransposition was attained when the esp1-1 mutant was found to have transposition defects.  Similar defects were also present in mutants of both the cohesin loader, SCC2, and the structural maintenance of chromosome, SMC3, genes.  Interestingly, cohesin loads at loci transcribed by RNA polymerase III, while hotspots of Ty1 integration are upstream of the same sites.  I propose that separase acts as a bridge to aid in targeting the Ty1 pre-integration complex to these hotspots.  The aforementioned categorization of esp1-1 genetic interactors also indicated that separase may somehow be involved in mRNA biogenesis.  As esp1-1 mutants have intact translational machinery, my data suggests that separase functions either  iii directly in transcription or, more likely, post-transcriptionally.  In all, screening for esp1-1 genetic interactions has revealed several new avenues for separase studies.  iv Preface The work presented in this dissertation comprises my graduate work from September 2007 to present.  I performed all the work presented under the supervision of Dr. Vivien Measday at the Wine Research Centre at the University of British Columbia, with the following noted exceptions.  To do the SGA screening and analyses described in Chapter 2, I worked primarily in the Loewen laboratory at the University of British Columbia through November and December 2008 with the help of a research associate, Dr. Barry Young. Under my supervision, an undergraduate student, Cynthia Xe, aided with the confirmation of esp1-1 SDL genetic interactions presented in Figure 2-4.  A graduate student in Dr. Charlie Boone’s laboratory at the University of Toronto, Anastasia Baryshnikova, compared the unconfirmed esp1-1 SL interactions to the Boone SGA database with the help of Dr. Michael Costanzo (Figure 2-6) and also looked at protein-protein interactions amongst the confirmed SL and SDL esp1-1 genetic interactors (Figure 4-4).  Nancy Fang in the Mayor laboratory at the University of British Columbia performed the affinity immunoprecipitation mass spectrometry data found in Figure 3-3, and Dr. Thibault Mayor the analysis in Table 3-1.  Dr. Lina Ma in the Measday laboratory undertook the Ty1 plasmid induction analysis (Figure 3- 7).  Finally, the Coller lab at Case Western University analyzed esp1-1 polysome formation (Figure 4-9).  A subset of the work presented in Chapter 2 and 3 is currently in the process of being written up for submission.  v Table of contents Abstract .................................................................................................................................... ii! Preface ..................................................................................................................................... iv! Table of contents ..................................................................................................................... v! List of tables............................................................................................................................ xi! List of figures ......................................................................................................................... xii! List of symbols and abbreviations ...................................................................................... xiv! Nomenclature ..................................................................................................................... xviii! Acknowledgements .............................................................................................................. xix! Dedication ............................................................................................................................. xxi! Chapter  1: Perspectives on separase – a general introduction .......................................... 1! 1.1! An overview of the eukaryotic cell cycle as it pertains to budding yeast .................... 1! 1.2! Regulating the cell cycle: a matter of checkpoints ...................................................... 2! 1.3! Cohesins: the molecular glue holding sister chromatids together ............................... 3! 1.3.1! The core cohesin complex and its architecture ..................................................... 3! 1.3.2! The cohesin cycle .................................................................................................. 5! 1.4! Bipolar attachment: one sister chromatid to each spindle pole body ........................... 5! 1.4.1! Microtubule-kinetochore interactions ................................................................... 5! 1.4.2! Sensing tension: the spindle assembly checkpoint ............................................... 7! 1.5! Cutting the ties that bind: the budding yeast separase, Esp1 ....................................... 8! 1.5.1! Securing Esp1 activity by Pds1 ............................................................................. 8! 1.5.2! The kleisin subunit Scc1 as a target for separase .................................................. 9! 1.5.3! Other known targets for Esp1 ............................................................................. 11!  vi 1.5.4! Using FEAR to exit mitosis ................................................................................ 12! 1.5.5! Separase activity ensures an efficient anaphase .................................................. 14! 1.6! Evolutionarily conserved separase – beyond budding yeast ...................................... 15! 1.6.1! Separases are present from yeast to humans ....................................................... 15! 1.6.2! Sister chromatid separation in vertebrates .......................................................... 17! 1.6.3! The importance of human separase: clinical implications .................................. 17! 1.7! Analyzing ESP1 function using a systematic, high-throughput screening method ... 18! Chapter  2: Examining the scope of genetic interactions for the esp1-1 temperature sensitive allele of separase .................................................................................................... 30! 2.1! Introduction ................................................................................................................ 30! 2.2! Results ........................................................................................................................ 33! 2.2.1! Creation of the esp1-1 allele for SGA analysis ................................................... 33! 2.2.2! Examination of the SGA esp1-1 allele ................................................................ 33! 2.2.3! SGA screening of esp1-1 negative genetic interactions ..................................... 35! 2.2.4! Analysis of confirmed negative esp1-1 genetic interactions .............................. 36! 2.2.5! Comparison of esp1-1 SL profile with other SGA analyses ............................... 37! 2.2.6! Screening of SDL hits for an Esp1 substrate ...................................................... 39! 2.2.7! Screening for suppressors of the esp1-1 temperature sensitivity ........................ 40! 2.3! Discussion .................................................................................................................. 42! 2.3.1! A dense negative genetic interaction network for a mutant of ESP1 ................. 42! 2.3.2! Genetic interactions of esp1-1 relate to essential Esp1 functions ....................... 44! 2.3.3! Negative genetic interactions of esp1-1 emphasize a role for Esp1 in maintaining spindle stability ............................................................................................................... 46!  vii 2.3.4! The genetic interaction network of esp1-1 suggests potential new roles for separase ........................................................................................................................... 46! 2.4! Materials and methods ............................................................................................... 47! 2.4.1! Yeast strains and growth ..................................................................................... 47! 2.4.2! Testing temperature sensitivity of the esp1-1 allele ........................................... 48! 2.4.3! Assaying for Esp1 substrates .............................................................................. 49! 2.4.4! Western blotting .................................................................................................. 49! 2.4.5! SGA screening .................................................................................................... 50! 2.4.6! Confirmation of genetic interactions .................................................................. 51! 2.4.7! Comparison of esp1-1 SL profile with Boone lab SL database .......................... 52! Chapter  3: The budding yeast separase, Esp1, regulates Ty1 retrotransposition ......... 77! 3.1! Introduction ................................................................................................................ 77! 3.2! Results ........................................................................................................................ 80! 3.2.1! esp1-1 genetic interactors are involved in the life cycle of Ty1 retrotransposons .............................................................................................................. 80! 3.2.2! Mass spectrometry reveals a physical interaction between Esp1 and Ty1 proteins ............................................................................................................................ 82! 3.2.3! esp1-1 has defects in Ty1 transposition .............................................................. 83! 3.2.4! esp1-1 does not fail to induce the pGAL1Ty1-H3mHIS3AI plasmid .................. 85! 3.2.5! Endogenous levels of TY1 mRNA are not decreased in esp1-1 .......................... 86! 3.2.6! Gag processing is intact in esp1-1 ...................................................................... 87! 3.2.7! Several cohesin mutants also show defects in Ty1 transposition ....................... 87! 3.3! Discussion .................................................................................................................. 88!  viii 3.3.1! Screening for genetic and physical interactions point to a potential role for Esp1 in Ty1 retrotransposition ................................................................................................. 89! 3.3.2! Assaying for Ty1 transposition in conditional alleles ......................................... 89! 3.3.3! Esp1 has a role in promoting the integration of Ty1 cDNA during retrotransposition ............................................................................................................ 90! 3.3.4! Esp1 may be responsible for targeting the PIC to hotspots of Ty1 integration .. 92!!A model for PIC recruitment to hotspots of transposition .............................. 92!!Evidence for an Esp1/cohesin mediated recruitment of PIC to hotpots of integration ................................................................................................................... 93!!Removal or translocation of cohesin may be necessary for successful integration ................................................................................................................... 94! 3.4! Materials and methods ............................................................................................... 96! 3.4.1! Yeast strains and growth conditions ................................................................... 96! 3.4.2! Affinity mass spectrometry ................................................................................. 97!!Affinity IP and mass spectrometry ................................................................. 97!!Analysis of mass spectrometry data ................................................................ 99! 3.4.3! Co-immunoprecipitation ..................................................................................... 99! 3.4.4! Western blotting ................................................................................................ 100! 3.4.5! Ty transposition assay ....................................................................................... 100! 3.4.6! Quantitative PCR analysis ................................................................................ 101!!RNA preparation ........................................................................................... 101!!Conversion to cDNA ..................................................................................... 101!!Real-time PCR .............................................................................................. 102!  ix Chapter  4: Examination of a role for the budding yeast separase, Esp1, in mRNA biogenesis ............................................................................................................................. 119! 4.1! Introduction .............................................................................................................. 119! 4.2! Results ...................................................................................................................... 122! 4.2.1! Protein-protein interactions amongst the confirmed esp1-1 SL and SDL hits are enriched for mRNA regulatory complexes ................................................................... 122! 4.2.2! Separase-mediated gene expression .................................................................. 125!!Analysis of an inducible heat shock gene, HSP30 ........................................ 125!!Analysis of the silenced mating cassette through HMRA2 ........................... 127! 4.2.3! Esp1 and decapping factors .............................................................................. 128! 4.2.4! Translational machinery is intact in an esp1-1 mutant ..................................... 129! 4.3! Discussion ................................................................................................................ 130! 4.3.1! A role for Esp1 prior to translation ................................................................... 130! 4.3.2! Sister chromatid cohesion and gene regulation: transcriptional implications for Esp1? ....................................................................................................................... 131! 4.3.3! Esp1 and the 7-methylguanosine cap ................................................................ 133! 4.3.4! Esp1 and nuclear export .................................................................................... 134! 4.3.5! A new landscape for Esp1 study ....................................................................... 134! 4.4! Materials and methods ............................................................................................. 135! 4.4.1! Yeast strains and growth ................................................................................... 135! 4.4.2! Spot assays ........................................................................................................ 136! 4.4.3! qPCR analysis ................................................................................................... 137! 4.4.4! Flow cytometry ................................................................................................. 137!  x 4.4.5! Co-immunoprecipitation ................................................................................... 138! 4.4.6! Western blotting ................................................................................................ 138! 4.4.7! Polysome assay ................................................................................................. 139! Chapter  5: Conclusions and future directions ................................................................ 151! 5.1! Summary and perspectives ...................................................................................... 151! 5.2! Future Studies .......................................................................................................... 154! 5.2.1! Saturation of separase genetic interactions ....................................................... 154! 5.2.2! Further defining the role of separase in Ty1-mediated integration ................... 155!!Testing the model .......................................................................................... 155!!Cohesin, Esp1 and the PIC ............................................................................ 156! 5.2.3! Esp1 and mRNA biogenesis ............................................................................. 158! 5.3! Final thoughts .......................................................................................................... 160! Bibliography ........................................................................................................................ 161!!   xi List of tables Table 2-1.  Classification of SL hits for esp1-1 ...................................................................... 53! Table 2-2.  Classification of SDL hits for esp1-1 ................................................................... 54! Table 2-3.  Fold enrichment of GO biological processes identified by analysis of esp1-1 SL and SDL interactions ............................................................................................................... 56! Table 2-4.  Comparison of esp1-1’s SL profile to the Boone database .................................. 58! Table 2-5.  SR hits for esp1-1 ................................................................................................. 62! Table 2-6.  Strains used in this chapter ................................................................................... 64! Table 3-1.  Affinity mass spectrometry peptides identified for Esp1 ................................... 103! Table 3-2.  Strains used in this chapter ................................................................................. 108! Table 4-1.  Strains used in this chapter ................................................................................. 140!   xii List of figures Figure 1-1.  The eukaryotic cell cycle .................................................................................... 19! Figure 1-2.  Cohesin establishment and maintenance ............................................................. 21! Figure 1-3.  A bioriented mitotic spindle in budding yeast .................................................... 22! Figure 1-4.  Kinetochore-MT interactions .............................................................................. 24! Figure 1-5.  The spindle assembly checkpoint ........................................................................ 25! Figure 1-6.  Scc1 cleavage by Esp1 ........................................................................................ 26! Figure 1-7.  FEAR and MEN pathways .................................................................................. 27! Figure 1-8.  Separase in mammalian cells .............................................................................. 28! Figure 1-9.  Separase as a oncogene ....................................................................................... 29! Figure 2-1.  Genetic interactions reveal functional relationships ........................................... 66! Figure 2-2.  Analysis of the SGA esp1-1 allele ...................................................................... 68! Figure 2-3.  Confirmation of SL hits ...................................................................................... 69! Figure 2-4. Confirmation of esp1-1 SDL hits ......................................................................... 71! Figure 2-5.  GO analysis of confirmed esp1-1 SL and SDL genetic interactions .................. 72! Figure 2-6.  Comparison of esp1-1’s SL profile ..................................................................... 73! Figure 2-7.  lrs4∆  is SL with esp1-1 ...................................................................................... 74! Figure 2-8.  Assaying for an esp1-1 substrate ......................................................................... 75! Figure 2-9.  Rescue of esp1-1 lethality by a overeexpression of a truncated Ty4 element .... 76! Figure 3-1.  Structure of the Ty family of elements ............................................................. 109! Figure 3-2.  Genes identified in esp1-1 SL and SDL screens have roles in the life cycle of Ty1 retrotranposons .............................................................................................................. 110! Figure 3-3.  Mass Spectrometry reveals a physical interaction between Esp1 and Ty1-IN . 112!  xiii Figure 3-4.  The defect in Ty1 transposition in an esp1-1 mutant is rescued by scc1-73 ..... 114! Figure 3-5.  Expression of IN from the pGAL1Ty1-H3mHISA1 plasmid ............................. 115! Figure 3-6.  esp1-1 strains are capable of Ty1 element expression and TyA-TyB polypeptide processing ............................................................................................................................. 116! Figure 3-7.  scc2-4 and smc3-1 strains have defects in transposition defects ....................... 117! Figure 3-8.  Model for Esp1-mediated insertion ................................................................... 118! Figure 4-1.  Overview of eukaryotic transcription ................................................................ 141! Figure 4-2.  Schematic representation of cap-dependent processes ..................................... 142! Figure 4-3.  mRNA decapping .............................................................................................. 143! Figure 4-4.  esp1-1 protein-protein interaction network ....................................................... 144! Figure 4-5.  esp1-1 is sensitive to changes in mRNA regulation .......................................... 145! Figure 4-6.  HSP30 mRNA levels are upregulated in the esp1-1 mutant in response to heat shock ..................................................................................................................................... 146! Figure 4-7.  HMRA2 transcript levels are upregulated in esp1-1 .......................................... 147! Figure 4-8.  Pat1 and Dhh1 do not physically interact with separase ................................... 149! Figure 4-9.  Polyribosome analysis of the esp1-1 mutant ..................................................... 150!   xiv List of symbols and abbreviations ∆  Deletion, null mutant 6AU  6 azauracil AI  Artificial intron APC  Anaphase promoting complex ATP  Adenosine triphosphate Bp  Base pair C-  Carboxy Cdc  Cell division cycle Cdk  Cyclin dependent kinase cDNA  Complementary DNA CHX  Cycloheximide C-Mad2 Closed isoform of Mad2 CTD  C-terminal domain DaMP  Decreased abundance by mRNA perturbation DMSO  Dimethylsulfoxide DNA  Deoxyribonucleic acid DTT  Dithiothreitol Esp1  Extra spindle poles 1 EDTA  Ethylenediaminetetraacetic acid Eif  Eukaryotic initiation factor FACS  Fluorescence activated cell sorting FEAR  Cdc14 early anaphase release  xv GO  Gene ontology GST  Glutathione s-transferase GTase  Guanosine triphosphate:mRNA guanylyltransferase HA  Hemagglutinin His  Histidine HU  Hydroxyurea hSecurin Human Securin hSeparase Human separase IB  Immunoblot IN  Integrase IP  Immunoprecipitation Log  Logarithmic Lrs4  Loss of rDNA silencing Mcd1  Mitotic chromosome determinant 1 MEN  Mitotic exit network mRNA  Messenger RNA MT  Microtubule N-  Amino NP-40  Nonidet P-40 Nz  Nocodazole O.D.  Optical density ORF  Open reading frame O-Mad2 Open isoform of Mad2  xvi Paf1C  Paf1 complex PCR  Polymerase chain reaction PIC  Pre-integration complex PP2A  Protein phosphatase 2A PR  Protease qPCR  Quantitative PCR rDNA  Ribosomal DNA RNA  Ribonucleic acid RNAPII RNA polymerase II RNAPIII RNA polymerase III RT  Reverse transcriptase RTPase RNA 5’-triphosphatase S. cerevisiae Saccharomyces cerevisiae SC  Synthetic complete Scc1  Sister chromatid cohesion 1 SDL  Synthetic dosage lethal SDR  Synthetic dosage rescue SDS  Synthetic dosage sick SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SGA  Synthetic genetic array SL   Synthetic lethal SPT  Suppressor of Ty Smc  Structural maintenance of chromosomes  xvii SPB  Spindle pole body SR  Synthetic rescue SS  Synthetic sick rDNA  Ribosomal DNA RENT  Regulator of nucleolar silencing and telophase RH  Ribonuclease H RT  Reverse transcriptase tRNA  Transfer RNA Ts  Temperature sensitive URA  Uracil WCL  Whole cell lysate WT  Wild type VLP  Virus like particle YPD  Yeast peptone dextrose  xviii Nomenclature Wild type alleles of S. cerevisiae are represented by capitalized and italicized letters (e.g. ESP1).  Mutant alleles are denoted by lower cased italics, indicating a deletion (e.g. lte1∆), point mutation (e.g. C1513A, esp1C1531A) or conditional allele (e.g. esp1-1).  Genes are expressed from their endogenous promoter unless another is indicated (e.g. GAL1, GAL1/10).  Gene products have their first letter capitalized.   Plasmids/vectors are indicated by a p and are italicized.   xix Acknowledgements They say it takes a village, and graduate training is no exception.  Without the support system I had around me, I can say with certainty that I wouldn’t have made it through.  First and foremost, I’d like to thank my family, especially since they’ve had to put up with being across the country for six years.  So, thank you a million times mom and dad who have always been there for me and have never been more than a phone call away.  Mikey and Kalia (and Dyson too!), thank you for giving me a place to crash without question every time I’m in the city.  Ama and Yeye, you taught me it could be done; Granny and Pepere, j’suis toujours ta p’tite poupoune. To all my friends back in Ontario, I miss all your faces and love seeing you whenever I’m back east.  To all my friends here in Vancouver, thank you for giving me a reason to get out of the lab on occasion. To everyone in the Measday lab, past and present, thank you being there for me through it all!  Nina, I don’t think I could’ve gotten anything done without your help.  Jenny, you always have my back, so thank you for always listening.  Lina, thank you for your help throughout the years, especially lately with all the work on my project.  Mike and Jay, my two wine aficionados, you guys always make the lab fun, and thank you for giving me an education in yeast that was outside the cell cycle.  Flo and Cynthia, thank you for all your hard work on my project.  Everyone else at the WRC, in the Van Vuuren and Lund labs, you guys make it a great place to work.  Maria, you are one of the most impressive women I have ever met.  Thank you for always coming in with a smile and making sure I’m fed and get home safely!  xx  To my committee, Dr. Chistopher Loewen, Dr Thibault Mayor and Dr. Ivan Sadowski, your advice over the years has been very much appreciated.  Vivien, thank you for this opportunity and all your guidance over the years.  Finally, thank you to all my funding sources over the years, specifically the University Graduate Fellowships, CIHR Banting and Best Doctoral Fellowship Award, and the Four Year Fellowship.  I wouldn’t have had the same opportunities throughout graduate school without them.  xxi Dedication “Wisdom is not a product of schooling but the life attempt to acquire it” ~Albert Einstein   1 Chapter  1: Perspectives on separase – a general introduction 1.1 An overview of the eukaryotic cell cycle as it pertains to budding yeast The eukaryotic cell cycle is typically defined in terms of chromosomal events, beginning with the replication of each chromosome during S phase.  This whole genome duplication is followed by M phase, which can be subdivided into two distinct categories: nuclear division (mitosis) and cell division (cytokinesis).   Throughout mitosis, duplicated chromosomes – referred to hereafter as sister chromatids – are paired until each is attached to an opposite microtubule organizing centre.  Once this bipolar attachment is achieved (“metaphase”), sister chromatids are permitted to separate and are pulled to opposite sides of the cell (“anaphase”).  There are also two designated “Gap” phases which allow for cell growth; G1 occurs before replication while G2 precedes progression into M phase (Morgan, 2007). Cell cycle dynamics are well established to be a product of a central biochemical oscillator governed by cyclin-cyclin dependent kinase (Cdk) complexes.  Indeed, greater than ten percent of yeast genes are cell cycle regulated, being transcribed at specific intervals during the process (Cho et al., 1998; Pramila et al., 2006; Spellman et al., 1998).  Cdks themselves are constitutively present throughout the cell cycle; rather, their oscillatory activity depends on binding various cyclins that are expressed at different stages.  G1 cyclin- Cdk complexes commit the cell to a new cell cycle (“Start”) during late G1 and activate S phase cyclin-Cdk complexes to initiate deoxyribonucleic acid (DNA) replication.  M phase cyclin-Cdk activity then results in the progression through mitosis, and subsequent cyclin destruction initiates mitotic exit (Morgan, 2007) (Figure 1-1A).  2 Our lab uses the budding yeast Saccharomyces cerevisiae (S. cerevisiae), as a model organism, taking advantage of the intrinsically simplistic nature of its cellular processes. The capacity of S. cerevisiae to propagate as a haploid at a rapid doubling rate (~two hours) allows for ease of genetic manipulation within the yeast genome.  Moreover, the high degree of correlation of cell cycle regulatory mechanisms to higher eukaryotes makes S. cerevisiae an ideal model for studying cell cycle control.  S. cerevisiae, as its colloquial name suggests, divides by budding.  The daughter (“bud”) first makes its appearance at Start and continues to grow throughout mitosis.  After receiving a full set of chromosomes, cytokinesis occurs at the bud neck, separating the mother cell from its daughter (Forsburg and Nurse, 1991; Morgan, 2007).  These cell cycle dynamics are modulated by one central Cdk (Cdk1/Cdc28) in conjunction with both Clns (G1 cyclins) and Clbs (S and M cyclins) (Morgan, 2007) (Figure 1-1B).  1.2 Regulating the cell cycle: a matter of checkpoints The ultimate goal of mitotic cell division is for a cell to faithfully propagate its genetic material.  Although a simple notion in principle, the actual execution necessitates a complex series of events.  As a result, quality control mechanisms known as checkpoints continually monitor a cell’s progression through the cell cycle and serve to effect an appropriate response should an error occur.  Without checkpoints, mistakes may result in the inheritance of abnormal genomic content.  From a clinical perspective, this can lead to a variety of ailments, with genetic disorders (e.g. Down’s Syndrome) and cancer being predominant examples.  3 Generally speaking, cell cycle checkpoints are coordinated through complex molecular signaling cascades.  For example, the DNA replication checkpoint responds to the stalling of replication forks during DNA replication or to the depletion of nucleotide pools by the ribonucleotide reductase inhibitor hydroxyurea (HU).   This signaling cascade, which results in the phosphorylation of the kinase Rad53 by Mec1, prevents premature entry into mitosis while simultaneously restraining spindle elongation by inhibiting the microtubule- associated proteins Cin8 and Stu2 (Bachant et al., 2005; Krishnan et al., 2004; Ma et al., 2007).  A similar S-phase checkpoint can occur as a response to genotoxic stress; this so- called DNA damage checkpoint is also capable of inducing arrest in both the G1 and G2 phases.  Coordinated by either the aforementioned Mec1 or another similar kinase Tel1, the downstream effectors (Rad53 or Chk1) stall progression through the cell cycle and concurrently activate relevant DNA damage response proteins [reviewed in (Melo and Toczyski, 2002; Putnam et al., 2009)].  How these pathways inhibit mitotic entry will be the subject of further discussion of this chapter as the internal regulatory dynamics of sister chromatid segregation are explained.  1.3 Cohesins: the molecular glue holding sister chromatids together 1.3.1 The core cohesin complex and its architecture To tether sister chromatids together, a proteinaceous structure known as cohesin embraces the two strands of DNA.  The core cohesin complex consists of four members: two structural maintenance of chromosome (Smc1 and Smc3) subunits and two non Smc subunits (Scc1/Mcd1 and Scc3) [(Guacci et al., 1997; Michaelis et al., 1997), reviewed in (Nasmyth and Haering, 2009)]. The Smc proteins are folded to form a rod shaped structure: an  4 intramolecular coiled-coil separates a globular “hinge” domain from an adenosine triphosphate (ATP) binding head.  Together, the Smc subunits form a V-shaped heterodimer, with the hinge domain at one end and the ABC-like ATPases at the other (Haering et al., 2002).   The kleisin subunit Scc1/Mcd1 completes the canonical tripartite ring structure by acting as a bridge between the ATPase heads, forming a primary contact with Smc1 through its carboxy (C-) terminus while its amino (N-) terminus binds the Smc3 subunit (Gruber et al., 2003; Haering et al., 2004) (Figure 1-2A).   This bridge-like subunit was first identified as a mitotic chromosome determinant (Mcd1) in the Koshland laboratory, but for the purposes of this dissertation will be referred to by its other common name, sister chromatid cohesion 1 or Scc1 (Guacci et al., 1997).  Based on the architecture, the ring is thought to encircle both sister chromatids, however there remains the possibility that a dimer ring formation occurs instead (the “handcuff model”) (Huang et al., 2005; Nasmyth and Haering, 2009).  Binding of Scc1 to the Smc heterodimer also recruits other critical members for sister chromatid cohesion, including the other kleisin subunit Scc3 and a HEAT containing protein known as Pds5 (Gruber et al., 2003; Hartman et al., 2000; Mc Intyre et al., 2007; Panizza et al., 2000; Toth et al., 1999b).   Another protein, Wapl (Wpl1/Rad61 in yeast), complexes with Pds5 to inhibit cohesin loading (Sutani et al., 2009).  Other pertinent members of cohesin include those that are necessary for establishment of cohesin but not its maintenance, such as the Scc2/4 loading complex and the acetyl-transferase Eco1 (Ciosk et al., 2000; Toth et al., 1999b) (Figure 1-2A).  Cohesin is not only essential for sister chromatid tethering, but is also versatile, with roles in regulating gene expression, DNA recombination and repair and centromeric structure [reviewed in (Hagstrom and Meyer, 2003)].  Cohesin’s role in transcription will be touched on further in Chapter 4 of this dissertation.  5 1.3.2 The cohesin cycle Cohesin loading at centromeric DNA and discrete loci along chromosome arms during late G1/early S phase requires the Scc2/4 loading complex (Laloraya et al., 2000; Lengronne et al., 2004; Megee et al., 1999; Tanaka et al., 1999).  Though the exact mechanism is still unknown, loading is dependent on two criteria: (1) ATP hydrolysis by Smc1 and Smc3, and (2) opening of the hinge domain (Arumugam et al., 2003; Gruber et al., 2006).  Cohesin loading is initially counteracted by Wap1-Pds5 activity, allowing cohesin to release sister chromatids.  Thus, cohesin establishment is a dynamic process, with loading occurring through the hinge “entrance” gate and releasing through a kleisin-ATPase “exit” gate (Chan et al., 2012).   Eco1-mediated acetylation of the Smc3 subunit neutralizes Wap1- Pds5 activity and is thought to lock cohesin shut (Unal et al., 2008).  This post-translation modification persists until anaphase, when countered by the Hos1 deacetylase (Beckouet et al., 2010; Borges et al., 2010; Xiong et al., 2010) (Figure 1-2B).  Stable cohesin eventually translocates/slides away from sites of loading to the 3’ end of genes, coming to rest at intergenic regions throughout the genome, and remains present on the genome until the G2/M transition (Glynn et al., 2004; Lengronne et al., 2004).  1.4 Bipolar attachment: one sister chromatid to each spindle pole body 1.4.1 Microtubule-kinetochore interactions When a new round of cell division is triggered, not only are chromosomes duplicated, but the microtubule organizing centre (spindle pole body, SPB, in yeast) is as well.   Each SPB then migrates to opposite sides of the nuclear membrane, forming a dynamic array of microtubules (MTs) that is referred to as the mitotic spindle.  Long pole-pole MTs  6 interdigitate to structure the mitotic spindle while shorter MTs contact sister chromatids through the highly structured kinetochore complex that assembles on centromeric DNA [reviewed in: (Jaspersen and Winey, 2004; Westermann et al., 2007; Winey and O'Toole, 2001)] (Figure 1-3).  Kinetochore-MT interactions are primarily mediated by both the Dam1/DASH complex - a ten-membered protein structure encircling MTs - and the Ndc80 complex (Lampert et al., 2010; Tien et al., 2010).  The latter is an evolutionarily conserved four-membered complex, whose rod shaped structure comes from the Ndc80-Nuf2 and Spc24-Spc25 dimers forming two separate heads that connect through a coiled-coiled shaft (Cheeseman et al., 2006; Ciferri et al., 2005; Wei et al., 2005).  The Spc24-25 head interacts with the inner kinetochore, protruding the rod structure out and enabling the Ndc80-Nuf2 dimer to bind MTs through the Dam1/DASH complex (Cheeseman et al., 2006; DeLuca et al., 2006; Lampert et al., 2010; Tien et al., 2010) (Figure 1-4A). The kinetochores first contact the lateral surface of dynamically growing and shrinking MTs (“search and capture”), and subsequently start sliding towards the SPB from which the MT emanates, in a manner dependent on the Kar3 kinesin motor protein (Tanaka et al., 2005).  This lateral sliding is subject to frequent pausing, and is often converted to end- on pulling - whereby the kinetochore is attached to the MT plus end as it shrinks - in a process that requires the Dam1/DASH complex (Kitamura et al., 2007; Tanaka et al., 2007). Simply contacting each chromosome sister chromatid, however, does not ensure that sister chromatids are bioriented.  Bipolar – or amphitelic – attachments are indeed the goal; however, both sister chromatids can also contact the same SPB (syntelic attachment) or one sister chromatid can even fail to attach (monotelic attachment).  In higher eukaryotes, where more than one MT contacts each kinetochore, sister chromatids are also able to attach to both  7 poles (merotelic attachment) [reviewed in (Pinsky and Biggins, 2005; Tanaka, 2008)] (Figure 1-4B).  To rectify this situation, the Ipl1 Aurora kinase localizes to kinetochores and promotes the turnover of kinetochore-MT interactions by phosphorylating key players in kinetochore-MT interactions (i.e. the Dam1/DASH and Ndc80 complexes) until there is proper tension across the spindle (Akiyoshi et al., 2009; Cheeseman et al., 2002; Cheeseman et al., 2006; DeLuca et al., 2006) (Figure 1-4C).  1.4.2 Sensing tension: the spindle assembly checkpoint As turnover frees sister chromatids from inappropriate kinetochore-MT attachments, the lack of tension generated by these unattached kinetochores will stall cycle progression until all sister chromatids are bioriented.  The binding of hyperphosphorylated Mad1 to MT- free kinetochores propagates this signal.  Mad1 acts as the receptor, recruiting the closed form of Mad2 (C-Mad2) to kinetochores.  The open isoform of Mad2 (O-Mad2) subsequently binds to this docked Mad1/Mad2 dimer, undergoes a conformational change and is released back into the cytosol as C-Mad2 (De Antoni et al., 2005; Mariani et al., 2012; Nezi et al., 2006).  This cytosolic C-Mad2 is proposed to form a mitotic checkpoint complex, in cooperation with Mad3 and Bub3, in order to inactivate Cdc20, an anaphase promoting complex (APC) coactivator (Fraschini et al., 2001; Lau and Murray, 2012).  The mitotic checkpoint complex-Cdc20 interaction is further thought to act as a cytosolic amplification signal, recruiting and converting more Mad2 in order to fully inhibit Cdc20 (De Antoni et al., 2005) (Figure 1-5).  APCCdc20 activity is also targeted by the DNA replication checkpoint and the DNA damage response, as its downregulation is critical to preventing precocious sister chromatid separation and inappropriate mitotic entry (Clarke et al., 2003).  8 1.5 Cutting the ties that bind: the budding yeast separase, Esp1 In the late 1980s, Breck Byers’ laboratory discovered, through ethyl methanosulfonate mutagenesis, a temperature sensitive (ts) mutant that confers a multipolar phenotype, ultimately designating the wild type (WT) allele with the moniker extra spindle poles 1, or ESP1 (Baum et al., 1988).  Subsequent analysis of the esp1-1 mutation found that this allele is essential to successful nuclear division, culminating in the identification of Esp1 as the enzyme responsible for separating sister chromatids at the G2/M transition (McGrew et al., 1992; Uhlmann et al., 1999; Uhlmann et al., 2000).  A large protein at a molecular weight of 180kDa, Esp1 belongs to the evolutionarily conserved separin family of endopeptidases, whose members possess a C-terminal “separase” domain that has been predicted to adopt the fold of CD clan cysteine proteases (Aravind and Koonin, 2002; Ciosk et al., 1998; Uhlmann et al., 2000).  1.5.1 Securing Esp1 activity by Pds1 Temporal regulation of Esp1 involves the activity of Pds1, a 42kDa protein of the securin family that is capable of restricting Esp1’s proteolytic activity until the cell is ready to enter anaphase (Ciosk et al., 1998; Yamamoto et al., 1996a, b) (Figure 1-6).  Throughout the cell cycle, Esp1 levels are relatively stable, with a three-fold decrease in expression in G1 being the noted exception (Jensen et al., 2001).  In contrast, Pds1 expression is highly cell cycle regulated, with transcript levels peaking as a new round of DNA synthesis begins and protein levels reaching their maximal levels shortly thereafter during mitosis (Cohen-Fix and Koshland, 1999; Spellman et al., 1998).  An Esp1/Pds1 complex is established early on in the cell cycle, forming as soon as securin appears (Jensen et al., 2001).  Pds1 acts as a direct  9 inhibitor of separase proteolytic activity by contacting both the N- and C- termini of Esp1, disrupting interactions between the two ends that are critical for enzymatic function (Hornig et al., 2002).  In addition to inhibiting the catalytic activity of Esp1, phosphorylated securin also enables efficient nuclear targeting of Esp1 (Agarwal and Cohen-Fix, 2002; Jensen et al., 2001). Once proper bipolar assembly has been achieved, the APC - an E3 ubiquitin ligase – in conjunction with Cdc20, targets Pds1 for ubiquitin-mediated degradation (Cohen-Fix et al., 1996; Visintin et al., 1997).  Analysis of Pds1’s open reading frame (ORF) revealed a nine amino acid sequence (RLPLAAKDN) that bears similarity to cyclin destruction boxes and is necessary for the degradation of securin (Cohen-Fix et al., 1996; Yamamoto et al., 1996a).  Specificity of Pds1-ubiquitination is provided through the recognition of this motif by Cdc20 (Hilioti et al., 2001).  1.5.2 The kleisin subunit Scc1 as a target for separase At the turn of the century, separase was shown to promote the disassociation of the cohesin complex from chromosomes by targeting Scc1 for cleavage.  Amino-terminal sequence analysis revealed that Esp1-mediated Scc1 cleavage occurs between a pair of arginine residues at positions 268 and 269.  Interestingly, abrogation of this site continues to support Scc1 cleavage, albeit with a cleavage product that is now approximately 10 kDa larger.  Cleavage at this new site is immediately C-terminal to arginine 180.  Though R268 appears to be more readily cleaved, the presence of either site is sufficient for anaphase initiation.  However, mutation of both R180 and R268 to aspartic acid is lethal, as cell proliferation is prohibited by a failure to separate sister chromatids (Uhlmann et al., 1999).  10 After cleavage occurs, the C-terminal fragment is short lived; R269 as a newly formed N- terminal residue is destabilizing by the N-end rule and as such the fragment is subject to ubiquitin/proteasome dependent degradation.  Perhaps this degradation is necessary to circumvent the formation of new cohesin complexes until the next cell cycle, though this point remains conjecture (Rao et al., 2001; Varshavsky, 1996). Not long after these discoveries, in vitro cleavage assays confirmed that Esp1 itself is responsible for Scc1 cleavage, with mutation of a critical histidine residue at position 1505 or cysteine at 1531 sufficient to abolish Esp1 proteolytic activity (Uhlmann et al., 2000) (Figure 1-6).  Interestingly, only phosphorylated Scc1 is capable of being efficiently processed by Esp1, as phosphatase treated Scc1 is almost completely resistant to cleavage.  This post- translational modification is cell cycle regulated, with hyperphosphorylation of Scc1 occurring shortly before Scc1 is cleaved – in a manner dependent on the activity of the Polo/Cdc5 kinase (Alexandru et al., 2001; Uhlmann et al., 2000).  The finding that it is specifically chromatin-bound Scc1 that is targeted for phosphorylation events demonstrates that the cell has a securin independent means of regulating chromosome segregation (Hornig and Uhlmann, 2004).  Indeed, these overlapping regulatory mechanisms explain why pds1 mutants demonstrate WT kinetics with regards to sister chromatid separation (Alexandru et al., 1999; Uhlmann et al., 2000).  Two serine phosphorylations (S175 and S263) are essential for Scc1 regulation in the absence of pds1 (Alexandru et al., 2001) (Figure 1-6).  Events regulating the phosphorylation of Scc1 will be discussed further in section 1.5.5.     11 1.5.3 Other known targets for Esp1  Cohesin cleavage by Esp1 is not limited to mitosis; around the same time that separase’s interaction with Scc1 was discovered, Esp1 was also found to cleave Rec8 – a meiotic specific cohesin protein.  The similarity between the two interactions is striking. Rec8 cleavage also occurs at two separate locations along the polypeptide, both of which are C-terminal to arginine residues (R431 and R453).  Mutation of either site alone is not sufficient to prevent cleavage, but the combination proves to be unable to support fragmentation of the protein.  The resultant carboxy fragment of cleavage is then subject to the N-end rule pathway for degradation (Buonomo et al., 2000).  Also like Scc1, Esp1 recognition of Rec8 is dependent on the cohesin protein’s hyperphosphorylation (Ishiguro et al., 2010; Katis et al., 2010) Alignment of both cohesin cleavage sites allowed Sullivan et al to identify a third target for separase cleavage: the kinetochore associated protein Slk19 (Sullivan et al., 2001; Zeng et al., 1999).  Phosphorylated Slk19 is cleaved at the G2/M transition at only one arginine residue (R77) - though unlike both Scc1 and Rec8, the carboxy cleavage fragment is both stable and persistent throughout the next cell cycle.  This is because the amino terminus of the newly formed fragment is predicted to carry a stabilizing serine residue (Sullivan et al., 2001; Varshavsky, 1996).  Sullivan et al also determined a degenerate core consensus sequence for Esp1: (DE)XXR/X, where X is any amino acid and / denotes the cleavage site. Other than an invariant arginine and an acidic residue in the P4 position, not much specificity is rendered by this motif.  Indeed, they were unable to find any new substrates by screening ORFs within the yeast genome with this sequence, limiting the currently known Esp1 substrates to the aforementioned three proteins. (Sullivan et al., 2004).  12 1.5.4 Using FEAR to exit mitosis Like the G2/M transition, exiting from mitosis is a highly regulated process, requiring a switch from a state of high to low Clb-Cdk1 activity.  This inhibition of mitotic cyclins occurs by two known mechanisms, the first of which is APC-mediated degradation.  The APC not only facilitates the G2/M transition, but also cooperates with a different co-activator known as Cdh1/Hct1 to stimulate Clb degradation at the end of mitosis (Schwab et al., 1997; Visintin et al., 1997).  Clb-Cdk1 activity is also repressed through binding of the Cdk1 inhibitor Sic1 (Mendenhall, 1993; Schwob et al., 1994).  If either of these two pathways is defective, the other is sufficient to trigger the exit from mitosis (Schwab et al., 1997; Visintin et al., 1997). Both mechanisms of Clb destruction, however, hinge on the activity of the Cdc14 phosphatase (Visintin et al., 1998).  Cdc14 serves to reverse inhibitory phosphorylation on Cdh1, while its mechanism of action on Sic1 is twofold (Jaspersen et al., 1999; Visintin et al., 1998).  (1) The SIC1 transcriptional regulator Swi5 is inhibited by Cdk1 kinase activity, being relegated to the cytoplasm until dephosphorylated by Cdc14 (Knapp et al., 1996; Moll et al., 1991; Toyn et al., 1997; Visintin et al., 1998).  (2) Sic1 itself is dephosphorylated, preventing recognition by the SCFCdc4 ubiquitin-protein ligase complex (Verma et al., 1997a; Verma et al., 1997b; Visintin et al., 1998).  Cdc14 is sequestered in the nucleolus until the Cdc5/Polo kinase releases the phosphatase from its inhibitor, Cfi1/Net1 (Visintin et al., 1999; Visintin et al., 2003).  The release of Cdc14 is promoted through two separate pathways, one of which is stimulated by separase [reviewed in (Bardin and Amon, 2001; D'Amours and Amon, 2004)].  13 Initially, Cdc14 release from the nucleolus was thought to be subject to only one regulatory series of events: the mitotic exit network (MEN) (Shou et al., 1999; Visintin et al., 1999) (Figure 1-7).  MEN promotes Cdc14 release through a signaling cascade that initiates with the Tem1 GTPase.  The migration of one SPB into the daughter cell during mitosis brings inhibited Tem1 into contact with its putative guanine exchange factor Lte1 (Bardin et al., 2000; Pereira et al., 2000).  Active Tem1 then stimulates the Cdc15 kinase to phosphorylate the Dbf2-Mob1 downstream effectors (Asakawa et al., 2001; Mah et al., 2001).  Disruption of MEN causes cells to arrest in telophase with Cdc14 still sequestered in the nucleolus (Shou et al., 1999; Visintin et al., 1999).  However, examination of individual MEN mutants as they progress through the cell cycle revealed a transient release of Cdc14 in early anaphase, a pathway that came to be aptly termed Cdc14 early anaphase release (FEAR) (Stegmeier et al., 2002) (Figure 1-7). Cdc14 release through the FEAR pathway is contingent on both the activity of Esp1 and Slk19.  Interestingly, a cleavage-resistant Slk19 is able to stimulate FEAR, and a strain carrying a mutation of the critical cysteine residue in separase, esp1C1531A, is able to rescue the mitotic exit defects of esp1-1 (Stegmeier et al., 2002; Sullivan and Uhlmann, 2003). These observations suggest that the role for Esp1 in the FEAR pathway is not dependent on its proteolytic activity.  Instead, separase is thought to cooperate with Zds1 and Zds2 to inhibit the protein phosphatase 2A (PP2A) subunit Cdc55 (Queralt and Uhlmann, 2008b). Downregulation of PP2ACdc55  stimulates Net1 phosphorylation, allowing Cdc14 to escape the nucleolus (Queralt et al., 2006) (Figure 1-7).  Though the FEAR pathway has yet to be fully understood, Spo12 and Bns1 are also positive regulators while other negative factors  14 include Pds1 and the nucleolar protein Fob1 (Cohen-Fix and Koshland, 1999; Stegmeier et al., 2004; Stegmeier et al., 2002; Tinker-Kulberg and Morgan, 1999). So why do cells exhibit two distinct waves of Cdc14 release?  The first (FEAR) is transient, causing Cdc14 to be rapidly resequestered into the nucleolus until late anaphase, where the more prolonged release occurs (MEN) (Stegmeier et al., 2002).  One explanation is that Cdc14 acts as a feedback mechanism, with FEAR required for timely activation of MEN – a reasoning confirmed when it was found that dephosphorylation by Cdc14 stimulates Cdc15 activity (Stegmeier et al., 2002).   The biphasic Cdc14 release is also thought to mediate oscillations in CDK substrate degradation; S-phase Cdk substrates are subject to FEAR-induced dephosphorylation and mitotic Cdk substrates to MEN-induced dephosphorylation (Jin et al., 2008).  1.5.5 Separase activity ensures an efficient anaphase The antagonist relationship between Esp1 and PP2ACdc55 is mutual, with the overexpression of Cdc55 capable of preventing cohesin cleavage.  PP2ACdc55 activity is thought to be downstream of the cohesin protector shugoshin (Sgo1 in yeast), which senses a lack of MT tension thereby initiating events to prevent premature Scc1 degradation (Clift et al., 2009).  Presumably, PP2ACdc55 promotes the dephosphorylation of chromatin bound Scc1, preventing its recognition by Esp1 (Yaakov et al., 2012).  This is similar to meiotic events, where Rec8 is dephosphorylated by PP2A, though with a different catalytic subunit, Rtf1 (Kitajima et al., 2006; Riedel et al., 2006).  During nuclear division the inhibition of PP2ACdc55 by separase can therefore be thought of as having a twofold effect.  At the G2/M transition, active separase targets chromatin bound Scc1 as well as inhibits PP2ACdc55.  This  15 inhibition not only stimulates the release of Cdc14 but also leads to an increase in Scc1 phosphorylation, resulting in a rapid and efficient cleavage of all cohesin while simultaneously initiating mitotic events that will cause mitotic exit (Yaakov et al., 2012). Esp1 activity also contributes to anaphase spindle dynamics.  Though delocalized throughout G1, Esp1 quickly accumulates in the nucleus upon interacting with Pds1 and is eventually found exclusively at SPBs and the spindle midzone as the cell progresses through anaphase (Ciosk et al., 1998; Hornig et al., 2002; Jensen et al., 2001).  Not only is Pds1 needed for nuclear accumulation of Esp1 as described, but securin is also crucial for the spindle localization of separase.  An explanation for this localization phenotype was provided when esp1tsscc1∆ double mutants were found to exhibit short spindles – suggesting that Esp1 is necessary for anaphase spindle elongation in a manner distinct from its role in sister chromatid segregation (Jensen et al., 2001).   Additionally, Esp1 cleavage of Slk19 is necessary for the stabilization of MT spindles, which follows with evidence for a role for FEAR in the stabilization of MT dynamics at the onset of anaphase (Higuchi and Uhlmann, 2005; Sullivan et al., 2001).  1.6 Evolutionarily conserved separase – beyond budding yeast 1.6.1 Separases are present from yeast to humans In Schizzasaccharomyces pombe, CUT1 - a gene that causes unequal distribution of DNA in a phenotype that is classified as cell ultimately torn – was found to share significant homology to Esp1 (Hirano et al., 1986; Uzawa et al., 1990).  Similarly, separase endopeptidases have been identified in many eukaryotes, with Aspergillus (bimB), Caenorhabditis elegans (Sep-1), Drosophila melanogaster (SSE), Xenopus and humans  16 being prime examples (Jager et al., 2001; May et al., 1992; Siomos et al., 2001; Zou et al., 1999).  Among the various separases, homology at the level of the primary amino acid sequence is most notable in the C-terminal domain.  That is not to say that the amino termini of these endopeptidases are dissimilar, as computational analysis showed that they adopt similar tertiary alpha-alpha superhelix folds (Jager et al., 2004).  Nor is it accurate to assume their unimportance; indeed, in S. cerevisiae, the amino region of Esp1 was found to be required for its activity (Hornig et al., 2002).  As well, in Drosophila melanogaster, SSE was thought to be a uniquely small separase, until it was discovered that SSE associates not only with the securin protein Pimples (PIM) but also with the Three Rows Protein (THR).  The latter is now known to correspond to the N-terminal domains in other separase homologs (Leismann et al., 2000). The structure of human separase (hSeparase) was solved using electron microscopy (Viadiu et al., 2005). Interestingly, hSeparase also separates the N- and C- termini of the protein by undergoing an autocatalytic cleavage prior to anaphase (Waizenegger et al., 2002; Waizenegger et al., 2000).  However, though this proteolysis of hSeparase itself is not essential for Scc1 cleavage, it is necessary for both timely entry into M phase and correct assembly of the bipolar spindle (Papi et al., 2005; Waizenegger et al., 2002).  The exact mechanistic purpose behind hSeparase autocleavage was discovered when it was observed that autocleavage disrupts hSeparase binding to PP2A (Gorr et al., 2005; Holland et al., 2007; Wardlaw, 2010).  Closer to anaphase initiation, human securin (hSecurin) is degraded and hSeparase auto-cleaves, releasing PP2A and allowing a second inhibitor, Cdk1-cyclin B1, to bind hSeparase until anaphase onset (Wardlaw, 2010) (Figure 1-8A).  This has led to a model that hSecurin restricts hSeparase activity only throughout early mitosis.  17 1.6.2 Sister chromatid separation in vertebrates Though separase is commonly found in both yeast and higher eukaryotes, in vertebrates, cohesin removal is distinctly more complex than in budding yeast.  During prophase, the majority of cohesin is removed, specifically from chromosome arms (Losada et al., 1998).  However, this disassociation does not occur through cohesin cleavage, but rather the phosphorylation of the cohesin subunit SA2 – a mitotic orthologue of Scc3 present in higher eukaryotes - by Plk1 and Aurora-B in a process that also involves the Sororin and Wap1 proteins (Gimenez-Abian et al., 2004; Hauf et al., 2005; Sumara et al., 2002). Phosphorylated Sororin, a cohesin protein for which an orthologue has not yet been identified in budding yeast, initiates the removal of arm cohesin by recruiting Plk1 to phosphorylate SA2 (Zhang et al., 2011).  Plk1 further phosphorylates Sororin itself, leading to its displacement by Wapl, which then facilitates the removal of cohesin complexes from sister chromatid arms (Gandhi et al., 2006; Kueng et al., 2006; Nishiyama et al., 2010; Zhang et al., 2011).  SA2 at centromeric bound cohesin is protected from phosphorylation by Shugoshin, leaving centromeric cohesin to be removed by separase-mediated cleavage of Scc1 at the metaphase to anaphase transition (Hauf et al., 2001; Kitajima et al., 2006; McGuinness et al., 2005) (Figure 1-8B).  1.6.3 The importance of human separase: clinical implications The oncogenic potential of hSeparase was first suggested when small interfering ribonucleic acid (RNA) knockdown of separase in human cells resulted in polyploidy and when separase-deficiency in mice had similar genetic anomalies (Kumada et al., 2006; Waizenegger et al., 2002; Wirth et al., 2006).  Further evidence came from aneuploidy  18 caused by the overexpression of separase in p53-null mice mammary epithelial cells exposed to hormone (Pati et al., 2004).  Direct testing showed that induction of separase expression in transplanted mammary cells results in tumour development in mice within a month (Zhang et al., 2008).  Further analysis by immunofluorescence microscopy of tumour specimens revealed that hSeparase is overexpressed in osteosarcomas as well as in breast and prostate cancers (Meyer et al., 2009) (Figure 1-9).  All this data supports the theory that hSeparase is an oncogene, with its overexpression also associated with a higher recurrence rate, metastasis and a lower five year survival rate (Meyer et al., 2009).  1.7 Analyzing ESP1 function using a systematic, high-throughput screening method The clinical importance of hSeparase has underlined the need for a thorough understanding of separase activity.  In higher eukaryotes, separases have also been shown to have roles outside cohesin cleavage, being implicated in such processes as centrosome duplication in humans, cell polarity in Arabidopsis thaliana and epithelial re-organization in Drosophila melanogaster (Pandey et al., 2005; Tsou et al., 2009; Yang et al., 2011).   In S. cerevisiae, the relationship of Esp1 with Slk19 has revealed a role for separase in mitotic exit and spindle stability.  The goal of this dissertation is to examine Esp1 function using a systematic, high-throughput screening method in order to discover whether any other roles could be ascribed to separase in budding yeast.  My investigation into the genetic interactome of a mutant of separase, esp1-1, has revealed involvement of Esp1 in the transposition of Ty elements as well as potentially in messenger RNA (mRNA) biogenesis.   19                                 Figure 1-1.  The eukaryotic cell cycle (A) Oscillations in CDK-cyclin complexes govern cell division, as cells progress through the cell cycle.  Cited from (Morgan, 2007). Reprinted with permission © Oxford University Press. (B) S. cerevisiae divides by budding.  The daughter cell forms as a bud at “Start”, eventually pinching off the mother at cytokinesis.  G1 (The “Clns”) and S and M phase cyclins (“Clbs”) interact with one major Cdk – Cdk1/Cdc28.  Cited from (Bloom and Cross, 2007).  Reprinted with permission © Nature Publishing Group.  A. B.  20                            A. B.  21 Figure 1-2.  Cohesin establishment and maintenance (A) Cohesin forms a tripartite ring consisting of two Smc subunits and Scc1 (kleisin) bridge. Another kleisin subunit, Scc3, is thought to be a member of the core cohesin complex as well, recruited by Scc1.  All known cohesin/cohesin-related proteins for yeasts, Drosophila melanogaster and humans are indicated. Cited from (Haering et al., 2002; Nasmyth, 2011). Reprinted with permission © Elsevier and Nature Publishing Group.  (B) The establishment of sister chromatid cohesion begins with a dynamic process as chromatids are loaded through the entry hinge gate by Scc2/4 and released by Wapl/Pds5.  Eco1 acetylation of the Smc3 subunit locks cohesin onto sister chromatids, inhibiting precocious separation of the replicates. Cited from (Chan et al., 2012).  Reprinted with permission © Elsevier.   22                      Figure 1-3.  A bioriented mitotic spindle in budding yeast Pole-pole (interpolar) MTs emanate from spindle pole bodies, structuring the mitotic spindle. Shorter MTs contact sister chromatids through a complex known as the kinetochore in such a way that each sister chromatid binds to an opposing spindle pole body.  + refers to the plus end of the microtubule, the minus end is at the spindle pole body.  Cited from (Morgan, 2007).  Reprinted with permission © Oxford University Press.    23                                               A. B. C. C en tro m er ic  D N A Microtubule Ndc80 complex Dam1/DASH complex  24 Figure 1-4.  Kinetochore-MT interactions (A) Kinetochore-MT interactions are dependent on the ringed Dam1/DASH complex and the rod-shaped Ndc80 complex.  Cited from (Bloom and Joglekar, 2010).  Reprinted with permission © Nature Publishing Group. (B) MTs can contact sister chromatids in the following way: (1) the preferred bioriented manner (amphitelic attachments) (2) only one sister chromatid attached (monotelic)  (3) sister chromatids attach to the same pole (syntelic) (4) unequal distribution of attachments (merotelic).  (C) The Ipl1Aurora kinase corrects kinetochore-MT attachments by phosphorylating key proteins (e.g. Dam1/DASH and Ndc80 complexes).  Cited from (Tanaka, 2008).  Reprinted with permission from © Springer.   25                       Figure 1-5.  The spindle assembly checkpoint Unattached kinetochores signal the recruitment of the Mad1 receptor.  Mad1 binds C-Mad2 to form a heterodimer, which is recognized by O-Mad2.  O-Mad2 comes to the relevant kinetochore, and is released back into the cytosol as C-Mad2.  This newly formed C-Mad2 functions within the mitotic checkpoint complex (MCC) to inactivate Cdc20.  Cited from (Mariani et al., 2012).  Reprinted with permission © Elsevier.   26            Figure 1-6.  Scc1 cleavage by Esp1 Phosphorylated, chromatin bound Scc1 is targeted for cleavage upon Esp1 release from its inhibitor, Pds1.  Cited from (Alexandru et al., 2001).  Reprinted with permission © Elsevier.   27                        Figure 1-7.  FEAR and MEN pathways Throughout the FEAR pathway, Esp1 in cooperation with Zds1/2 inactivates PP2ACdc55, thereby allowing Cdc5 to phosphorylate Net1 and stimulate the release of Cdc14 from the nucleolus.  A more prolonged release of Cdc14 occurs later in the cell cycle, through Lte1 stimulation of the Tem1 GTPase activity.  Downstream phosphorylation of Cdc15 and then Dbf2-Mob1 stimulates Net1 release of Cdc14.  Cited from (Queralt and Uhlmann, 2008a). Reprinted with permission © Elsevier.   28                                 Figure 1-8.  Separase in mammalian cells (A) The dual inhibition of hSeparase.  hSecurin inhibits hSeparase during early mitosis. Closer to anaphase initiation, PP2A binding is inhibited, allowing cyclin B1-Cdk to inhibit hSeparase until anaphase onset.  Cited from (Wardlaw, 2010).  Reprinted with permission © Oxford University Press.  (B) Cohesin is removed in prophase from the chromosomal arms by Plk1 phosphorylation of the SA2 subunit.  Centromeric chromatin is protected, and is only removed at the metaphase-anaphase transition by separase cleavage of Scc1/Rad21.  Cited from (Gutierrez-Caballero et al., 2012).  Reprinted with permission © Elsevier.  A. B.  29                         Figure 1-9.  Separase as a oncogene Separase is overexpressed in prostate cancers.  Separase is in red while 4’,4-diamidino-2- phenylindole staining (blue) indicates DNA.  Cited from (Meyer et al., 2009).  Reprinted with permission © American Association for Cancer Research.   30 Chapter  2: Examining the scope of genetic interactions for the esp1-1 temperature sensitive allele of separase 2.1 Introduction An inherent advantage afforded by the use of S. cerevisiae as a model organism is the ease of genetic manipulation.  In a worldwide collaborative effort, budding yeast was the first fully sequenced eukaryotic genome (Goffeau et al., 1996).  Since then, numerous large scale yeast collections have been created, including ones that have systematically deleted each gene (the yeast deletion mutant collection) and epitope tagged each gene either endogenously [with green fluorescent protein, tandem affinity purification, V5 HIS6, or hemagglutinin (HA) tags] or on an overexpression plasmid [with a glutathione s-tranferase (GST) tag] (Ghaemmaghami et al., 2003; Giaever et al., 2002; Huh et al., 2003; Kumar et al., 2002; Sopko et al., 2006).  These collections have allowed high-throughput analyses of protein- protein and genetic interactions as well as protein localization studies and represent a significant advance of yeast systems biology.  In 2001, a technique known as synthetic genetic array (SGA) was developed to examine – on a more global scale - a genetic relationship where the combination of two mutants demonstrate a fitness defect more pronounced than either mutant on its own: a synthetic lethal (SL) interaction.  With the use of the yeast knockout collection, the seminal SGA paper performed SL analyses with eight null mutants as query strains and found 291 interactions amongst 204 genes (Tong et al., 2001). The notion of genetic interactions was first introduced by William Bateson, who presented the idea that simple Mendelian genetics could be distorted through gene interaction (Bateson and Mendel, 1909).  He used the term “epistasis” to describe the phenotypic effect whereby one gene masks the allelic effects at another locus.  A more statistical approach was  31 then conducted by R.A. Fisher, who explored the idea of genetic interactions as deviations from what one would expect by the combination of alleles (Fisher, 1919) [reviewed in (Boone et al., 2007; Phillips, 1998)].  In order to assess these genetic interactions systematically, SGA methodology was developed as a series of replica pinning steps that allows for the selection of recombinant haploid double mutants - after a query mutant is mated to the entire deletion mutant collection and the resultant diploids are sporulated. Double mutants that have smaller than expected colony sizes are considered to be synthetic sick (SS) or SL (Tong et al., 2001).  Similarly, synthetic dosage lethal (SDL) interactions occur when overexpression of a gene is detrimental to a mutant strain but not a WT strain. The SGA technique has been adapted to enable high-throughput analyses of SDL interactions using either the galactose inducible pGAL1/10-GST-ORFX overexpression collection in a single deletion mutant or a small set of pGAL overexpression plasmids in the deletion mutant collection (Measday et al., 2005; Sopko et al., 2006). The purpose of creating a complex genetic interaction network is primarily for the identification of functional relationships, as physical interaction data to date does not carry significant overlap with genetic data (Tong et al., 2004).  Though the meaning behind a particular genetic relationship is not always obvious, interpretations of the mechanistic basis can often be applied - in particular for SL interactions.  For example, for SL relationships between nonessential null mutants, “between pathway” interactions can occur when the genes function in separate pathways that both contribute to an essential biological function. Therefore, while the interference with only one pathway is buffered, destruction of both pathways can result in cell lethality (Guarente, 1993; Kelley and Ideker, 2005) (Figure 2-1A). With essential genes, whereby conditional alleles are used to examine genetic relationships,  32 “within” pathway interactions can occur instead.  Here, the mutation of one protein can disrupt the interaction of a protein complex, though not enough to impinge on cell viability. However, by mutating another member of the same complex, the interaction may be sufficiently weakened as to disturb the essential pathway, resulting in cell death (Guarente, 1993; Kelley and Ideker, 2005) (Figure 2-1B).  In addition, SDL interactions identified in the pho85∆ mutant has served as a proof of principle that disrupting the function of an enzyme can be lethal in combination with the overexpression of a substrate (Measday and Hieter, 2002; Sopko et al., 2006) (Figure 2-1C).  By understanding the genetic interaction network of a gene, not only can known functional relationships be identified, but novel ones as well.  For example, in the first SGA screens, the profile of bbc1∆ SL interactions was enriched for genes involved in actin regulation.  Follow up experiments demonstrated that Bbc1, a previously uncharacterized protein, localized to corticol actin patches and bound proteins that control the assembly of these patches (Tong et al., 2001). To attain a more comprehensive understanding of the cellular role for separase, I assembled a genetic interaction network for ESP1.   As Esp1 is an essential protein, a conditional allele of ESP1, esp1-1, was subjected to SGA analysis.  Not only did I examine negative (SL and SDL) genetic interactions, I also looked for potential suppressors of the temperature sensitivity of esp1-1.  The analysis of the spectrum of esp1-1 genetic interactions is explored in this chapter while follow up examination of new roles for separase is the subject of subsequent chapters.     33 2.2 Results 2.2.1 Creation of the esp1-1 allele for SGA analysis  To uncover potential new roles for Esp1 using systematic genetic interaction studies, I subjected the prototypical ESP1 ts mutant, esp1-1, to SGA analysis (Baum et al., 1988).  To that end, the entire ~5000 base pair (bp) ORF of esp1-1 was sequenced to determine the location of the relevant mutation(s).  Fortuitously, I identified a single point mutation at bp 4211 (CT), resulting in conversion of proline 1404 to leucine (Figure 2.2A). The proximity of this mutation to histidine 1505 and cysteine 1531, the canonical proteolytic residues in Esp1, implicated a physical disruption of the catalytic dyad as a possible reason for the conditional phenotype of esp1-1 (Uhlmann et al., 2000).  The placement of this mutation allowed me to amplify the C-terminus of esp1-1 (including bp 4211) with polymerase chain reaction (PCR), and then use that fragment to integrate the mutation into the SGA starting strain as described (Tong et al., 2001).  2.2.2 Examination of the SGA esp1-1 allele  Several quality control experiments were undertaken to ensure that the esp1-1 allele functioned as expected in the SGA strain background.  As a first step, esp1-1 was confirmed to be a ts conditional allele by analyzing its growth on rich media using a serial dilution spot assay at various temperatures: 25˚C, 30˚C, 33˚C and 35˚C (Figure 2-2B).  esp1-1 growth was slightly impeded at 30˚C as compared to control, negligible at 33˚C and non-existent at 35˚C. The nature of the temperature sensitivity of esp1-1 in the SGA strain background could thus be defined as a non-restrictive/permissive temperature of 25˚C, a semi-permissive temperature of 30˚C, and a restrictive/non-permissive temperature of 35˚C.  The esp1-1  34 growth phenotype in the SGA strain background is consistent with the severity of temperature sensitivity demonstrated by both an esp1-1 mutant in another strain background and broadly to other conditional alleles of Esp1 (Baskerville et al., 2008).  The length of time at the restrictive temperature that was necessary to adversely affect esp1-1 viability in the SGA background was also examined in detail. WT and esp1-1 cells were first grown to mid-logarithmic (log) phase at 25˚C then shifted to 35˚C for a total of four hours, with samples taken every two hours.  Samples were immediately serially diluted and spotted onto yeast peptone dextrose (YPD), with cell viability determined by growth that occurred after three days of incubation at 25˚C (Figure 2-2C).  In the span of approximately one doubling (two hours), cell viability was noticeably affected in the esp1-1 strain.  Cell lethality was exacerbated further at four hours, suggesting the SGA esp1-1 allele cannot recover well when subjected to a non-permissive temperature for even a few doublings.  Finally, as the conditional phenotype of esp1-1 was expected to correspond with a disruption in the proteolytic activity of separase, a known Esp1 substrate (Slk19) was examined to ensure proteolytic processing was indeed affected in the SGA esp1-1 allele.  WT and esp1-1, both carrying a pGAL1/10-GST-SLK19 vector, were grown in raffinose at 35˚C for one hour in order to reduce esp1-1 activity.  Overexpression of the N-terminally tagged GST-Slk19 was then induced by switching of the carbon source from raffinose to galactose and growing at the restrictive temperature for another hour.  Western blot analysis revealed a band that was reliably present when Slk19 was overexpressed in WT cells but not esp1-1 cells (Figure 2-2D).  The migration of this band was consistent with the expected molecular weight of the N-terminal fragment produced upon cleavage of Slk19 (Sullivan et al., 2001). Clearly, cleavage of Slk19 is impeded in the SGA esp1-1 strain, and the integrated mutation  35 affected separase proteolytic activity.  Taken together, the sequence validation of the point mutation and the analysis of both temperature sensitivity and proteolytic function confirmed that the esp1-1 allele behaved as expected in the SGA background.  2.2.3 SGA screening of esp1-1 negative genetic interactions  After careful analysis of the allele, the esp1-1 query strain was mated to the ~4700 nonessential deletion mutant collection (geneX∆)  and the ~5300 strains from the pGAL1/10- GST-ORFX overexpression collection to identify SL and SDL interactions, respectively (Giaever et al., 2002; Sopko et al., 2006).  A total of 114 SL and 248 SDL interactions (“hits”) were identified in this manner, and ranked according to the severity of the phenotype. SL genetic interactions were scored by comparing the colony size of the double mutant to geneX∆ on its own using a quantitative scoring program (Colony Scorer) (Tong et al., 2004). Similarly, SDL genetic interactions were scored by comparing colony growth of esp1-1 strains expressing the pGAL1/10-GST-ORFX  plasmid to the viability of overexpressing ORFX on its own.  The top 60 SL and top 100 SDL hits (e.g. the ones that were most lethal to esp1-1 viability) were confirmed through rigorous testing.  For SL interactions, heterozygous diploids were independently sporulated, and the growth phenotype of individual haploids analyzed by tetrad dissection (Figure 2-3).  Expected esp1-1 geneX∆ haploids that were consistently unable to produce viable haploid progeny were considered to be SL; double mutants that reproducibly formed smaller colonies than either single mutant on its own were identified as SS.   SDL interactions were confirmed by extracting the pGAL1/10-GST-ORFX plasmid from the colony, retransforming each plasmid into WT and esp1-1 cells, performing a serial dilution spot assay to compare growth on galactose versus viability on dextrose, and  36 finally sequence validation of ORFX (Figure 2-4).  Genes that caused a growth defect in esp1-1 but not WT when overexpressed on galactose were scored as SDL or synthetic dosage sick (SDS).  The final list of confirmed hits consisted of 15 null mutants and 44 overexpressed proteins that were shown to negatively impact the growth of esp1-1 mutants (Tables 2-1 and 2-2).  This corresponded to a confirmation rate of 50-60% for both screens.  Two of the esp1- 1 SL interactors - hsc82∆ and lte1∆ - have been previously described.  The former terminal phenotype is perhaps indicative of the Hsp90 type chaperone protein, Hsc82, being involved in the promotion of Esp1 function while the latter is due to defects in promoting both pathways controlling mitotic exit (FEAR and MEN) (D'Aquino et al., 2005; Sarin et al., 2004; Stegmeier et al., 2002).  I also identified Slk19 in the esp1-1 SDL screen which supports the notion that increased dosage of a substrate in the presence of its defective enzyme can be detrimental to cells (Measday and Hieter, 2002; Sopko et al., 2006; Sullivan et al., 2001).  2.2.4 Analysis of confirmed negative esp1-1 genetic interactions  Confirmed SL and SDL genes were classified according to Gene Ontology (GO) Biological Process using Yeast-GO SLIM analysis and examined for those that exhibited enrichment above genome frequency (Figure 2-5, Table 2-3).   As expected, the cell cycle (8 genes out of 59 total, or 13.6% frequency) was an enriched biological process, exhibiting an almost three-fold enrichment over the 4.7% genome frequency for this category (Figure 2-5, Table 2-3).  Cell cycle genes identified included the aforementioned lte1∆ and SLK19, as well as two MT (kar3∆ and CIK1) and one actin (MYO2) motor protein.  We also identified a  37 2.6-fold enrichment for genes involved in DNA repair which is consistent with a recently described role for Esp1 in promoting dissociation of cohesin during DNA double-strand break repair (McAleenan et al., 2013) (Figure 2-5, Table 2-3).  Other biological processes of note were: protein folding (3/59 hits, correlating to a 3.64 fold enrichment), nuclear transport (also 3/59 hits, correlating to a 1.96 fold enrichment) and carbohydrate metabolic processes (4/59 hits; 1.59 fold enrichment) (Figure 2-5, Table 2-3).  Most notably, a number of RNA metabolic processes were enriched, including: RNA catabolism, regulation of translation and RNA polymerase II (RNAPII) transcription (Figure 2-5, Table 2-3).  Several of the RNA catabolic genes encode P-body proteins  (lsm1∆, lsm7∆, PAT1, DHH1, SCD6) while RNAPII transcription genes encode members of the Paf1 complex (rtf1∆, leo1∆, cdc73∆) (Parker and Sheth, 2007; Rosonina and Manley, 2005).  The enrichment of these categories was unexpected and suggested that Esp1 may be involved in a pathway that is dependent on a number of these proteins.  2.2.5 Comparison of esp1-1 SL profile with other SGA analyses  As previously mentioned, SL relationships with mutations in essential genes, such as esp1-1, may imply that genes function in parallel pathways that contribute to a common, essential biological function or that they function in the same essential pathway [reviewed in (Boone et al., 2007)].  It has been demonstrated that genes that function in the same pathway may have similar genetic interaction profiles, e.g. a significant number of SL hits in common (Tong et al., 2004).  To determine which genes had a similar SL interaction profile to esp1-1, I collaborated with Dr. Charlie Boone’s lab at the University of Toronto.  Anastasia  38 Baryshnikova and Dr. Michael Constanzo compared the entire unconfirmed SL profile of esp1-1 to the database of SGA analyses compiled by the Boone lab (Costanzo et al., 2010).  The SL profiles of over 200 query mutants were identified as sharing a significant correlation with esp1-1 (Pearson coefficient above 0.2, data not shown), with the top 63 alleles subjected to further scrutiny (Figure 2-6, Table 2-4).  As expected, several alleles identified corresponded to genes with known roles in cell cycle regulation, including ts mutants of cell division cycle (cdc) genes (cdc4-3, cdc7-4, cdc7-1, cdc4-2, cdc37-1, cdc28- 13, cdc23-1) and the MT motor CIK1 (cik1∆).  Crucially, several query mutants identified (cdc20-3, slk19∆, spo12∆) encode proteins directly involved in the same functional pathways as Esp1.  As well, though not among the top 63, a conditional mutation of securin shared similarity with the SL profile of esp1-1 (Pearson coefficient 0.203, pds1-128, data not shown).  The pathways associated with these query mutants include the activation of Esp1 through APCCdc20  and the FEAR pathway (Stegmeier et al., 2002; Visintin et al., 1997). Further, mutants of the cohesin loader (scc2-4) and cohesin components themselves (smc1- 259) showed significant correlation (Pearson coefficient above 0.2) with the SL profile of esp1-1 (Figure 2-6, Table 2-4, data not shown). Direct evidence of shared functional relationships between Esp1 and genes with similar SL interactions thus underscored the robustness and strength of the SL screen in particular. Interestingly, the query allele with the highest correlation to esp1-1’s SL profile was lrs4∆ (Pearson coefficient 0.402, Figure 2-6, Table 2-4).  Lrs4 is a protein involved in ribosomal DNA (rDNA) silencing (loss of rDNA silencing) (Smith et al., 1999).  Along with Csm1, deletion of which also results in an overlapping SL profile with esp1-1, Lrs4 has been shown to interact with the regulator of nucleolar silencing and telophase exit (RENT)  39 complex to suppress unequal crossing over at the rDNA (Huang et al., 2006).  The RENT complex, which includes Cdc14 and Cfi1/Net1, is responsible for restricting Cdc14 to the nucleolus until release is triggered by the FEAR pathway or MEN (Shou et al., 1999; Stegmeier et al., 2002; Visintin et al., 1999).  Once released from the nucleolus, Cdc14 is not only responsible for promoting mitotic exit, but also the segregation of rDNA (D'Amours et al., 2004; Visintin et al., 1998). To further investigate the potential for a relationship between Esp1 and Lrs4, I looked for genetic interactions between esp1-1 and lrs4∆ and found them to be SL (Figure 2-7). Whether this genetic interaction is due to Esp1’s role in the FEAR pathway or, more excitingly, a direct role for Esp1 in either rDNA silencing or segregation remains to be investigated.  2.2.6 Screening of SDL hits for an Esp1 substrate  Sullivan et al described a consensus sequence for separase cleavage but were unable to identify any novel Esp1 substrates out of 63 candidate spindle associated proteins (Sullivan and Uhlmann, 2003).  However, the confirmation of Slk19 as an SDL hit for esp1-1 lent credence to the idea that an SDL screen has the potential to identify substrates of a protease, and provided a new list of potential Esp1 targets to test.  As all three known substrates of Esp1 are cleaved, a phenomenon visible by western blot analysis, I used the proteolytic processing of potential substrates to my advantage.  In developing a screen that would allow me to identify Esp1 substrates in a relatively high throughput manner, I built on the approach used to confirm the proteolytic functionality of esp1-1 in the SGA background (see: section 2.2.2).  The pGAL1/10-GST-ORFX vectors of interest were first overexpressed  40 individually at 25˚C in WT cells as a preliminary screen.  Only proteins encoded by genes identified in the esp1-1 SDL screen that showed clear evidence of lower migrating bands by subsequent immunoblot analysis were further investigated (Figure 2-8A).   From 44 initial proteins, 36 displayed evidence of potential cleavage products.  For these 36 proteins, as with the pGAL1/10-GST-SLK19 vector, the plasmid of interest was induced at a non-permissive temperature in both WT and esp1-1 strains.  Samples were then examined by western blot analysis for differences in protein mobility (Figure 2-8B).  Apart from Slk19, none of the proteins tested showed notable variation between WT and esp1-1 when blots were probed for GST.  Thus, at present, none of the SDL hits are obvious substrates for Esp1 though I cannot rule out the possibility that cleavage occurs at such a specific point of the cell cycle as to preclude being observed by such a high throughput analysis.  2.2.7 Screening for suppressors of the esp1-1 temperature sensitivity  The conditional nature of the esp1-1 allele provides the opportunity to investigate not only genetic interactions that result in lethality, but also those that serve to improve the cell viability of esp1-1 (epistatic relationships).  To that end, the esp1-1 strains that were mated to both the deletion mutant and pGAL1/10-GST-ORFX overexpression collections were also grown at 35˚C after the final selective pinning steps to look for synthetic rescue (SR) and synthetic dosage rescue (SDR).  25 SR interactions were identified where esp1-1 geneX∆ double mutants grew but esp1-1 alone could not (Table 2-5).  Though the genetic interactions were not confirmed, GO analysis demonstrated that like SL/SDL interactions, the SR relationships of esp1-1 were enriched for genes involved in RNA metabolic processes, with the transcription from RNAPII promoter (hap4∆, htz1∆, rpb4∆, rsc1∆, spt3∆, yap7∆) and  41 RNA modification (isu2∆, rit1∆) categories showing a 3.3 and 3.2 fold enrichment above genome frequency, respectively.  Only one gene, kel1∆, has a role in cell cycle regulation, though, interestingly, Kel1 activity inhibits MEN.  Kel1, a kelch domain containing protein, functions in a complex with Kel2 to repress mitotic exit through interaction with the MEN effectors Lte1 and Tem1 (Hofken and Schiebel, 2002; Nelson and Cooper, 2007; Philips and Herskowitz, 1998; Seshan et al., 2002).  The downregulation of the MEN through deletion of LTE1 has already been shown to negatively impact the growth of esp1-1 (D'Aquino et al., 2005) (Figure 2-5).  Therefore, stimulation of MEN (by deletion of KEL1) may rescue the growth of esp1-1. When the pGAL1/10-GST-ORFX overexpression collection was screened for high- copy suppressors of esp1-1, only one protein was able to consistently suppress the ts phenotype – a truncated form of a Ty4 element containing just the reverse transcriptase and ribonuclease H domains (hearafter referred to as pGAL1/10-GST-TY4-RT/RH).  The rescue was confirmed by spot assay on both dextrose (control) and galactose media (to overexpress the protein) (Figure 2-9).  Ty4 is a member of the long terminal repeat retrotranposons, of which there are 5 families (Ty1-5) in S. cerevisiae.  Though Ty elements do not have an infectious stage, they have similar life cycles to retroviruses.  They are composed of both TyA, which forms the virus like particle, and the TyB polyprotein, which contains the protease, integrase, RT and RH components [reviewed in (Lesage and Todeschini, 2005)]. Currently, it is unclear why this particular truncated Ty element is capable of rescuing the ts nature of esp1-1 as neither full-length elements nor individual proteins alone were able to consistently rescue esp1-1 (data not shown).   42 2.3 Discussion  In this chapter, I examine the scope of genetic interactions of the esp1-1 allele as a means to better understand the complexity of Esp1 function.  The conditional nature of the esp1-1 strain afforded me the opportunity to examine both negative genetic interactions (SL and SDL) as well as epistatic relationships in a high-throughput manner.  The identification of known esp1-1 SL interactors (hsc82∆ and lte1∆) and a known Esp1 substrate (SLK19) in the screens served as initial confirmation of the veracity of these screens.  The esp1-1 genetic interaction network formed by these screens reflected known Esp1 functions, while also isolating pathways of interest for further study.  2.3.1 A dense negative genetic interaction network for a mutant of ESP1 As an essential allele with a large ORF, technical barriers had prevented the performance of high-throughput, systematic screening of ESP1 until now.  Low-throughput genetic analysis of the esp1-1 strain identified SL interactions with cdc14(1-374), hsc82∆, lte1∆ and pds1∆, and an SDL interaction with AMN1 (Ciosk et al., 1998; D'Aquino et al., 2005; Rahal and Amon, 2008; Sarin et al., 2004; Stegmeier et al., 2002; Wang et al., 2003). However, by performing large scale screening, I was able to gain a better picture of the scope of esp1-1 genetic interactions. Essential synthetic genetic networks have been shown to have an interaction density five times that of non-essential networks (Davierwala et al., 2005).   SGA screening of esp1- 1 isolated a large number of negative genetic interactions - 114 and 248 (unconfirmed) SL and SDL hits respectively.  Though not all SL interactions were confirmed, the number of hits identified would indeed be more consistent with essential alleles, as nonessential genes  43 only average 34 SL interactions (Tong et al., 2004).  The number of SDL hits for esp1-1 also appears to be substantial; recent SGA analysis of the spectrum of kinase mutants showed that the highest number of SDL hits identified was 156 and 113 for cla4∆ and slt2∆ respectively (Sharifpoor et al., 2012).  As well, the depth of the SL and SDL screens in particular demonstrated the complexity and importance of ESP1, reflecting that the esp1-1 conditional allele impinges not only on the regulation of the metaphase to anaphase transition but also the exit from mitosis (Stegmeier et al., 2002; Uhlmann et al., 1999; Uhlmann et al., 2000).  I did not find any direct overlap between genes identified as SL and SDL, consistent with previous findings from kinetochore SL and SDL screening (Measday et al., 2005).  Though these two genetic screens probe different genetic relationships in general, GO analysis has shown consistency in relation to the various functions enriched from these hits. Inclusion of epistatic interactions in the analysis was an opportunity to further saturate the scale of the screen.  Although bub2∆ and kin4∆ rescue the lethality of an esp1-1 lte1∆ double mutant and the dosage imbalance observed upon overexpression of ESP1 is rescued by simultaneous overexpression of either CLB2, CDH1 or PDS1, the rescue of the esp1-1 ts phenotype alone has not been examined on this level (D'Aquino et al., 2005; Kaizu et al., 2010; Stegmeier et al., 2002).    While screening for epistatic relationships contributed to the overall picture of esp1-1 genetic interaction, the breadth of information provided was lower than that of lethal interactions, as only one dosage suppressor was identified.  Esp1’s importance to cell viability promotes the susceptibility of esp1-1 to various perturbations, leading to a dense negative genetic interaction network.    44 2.3.2 Genetic interactions of esp1-1 relate to essential Esp1 functions Esp1’s function in the FEAR pathway was reflected in esp1-1 SL (lte1∆) and SDL (SLK19) interactions as well as by the correlation of esp1-1’s SL profile with spo12∆ and slk19∆ mutants.  However, because of the technical limitations of the screens performed, the direct identification of hits specifically involved in sister chromatid segregation was not anticipated.  Screens using the deletion collection specifically probed non-essential alleles, which excluded a number of cell cycle genes (such as those encoding the core cohesin components) from the analysis (Giaever et al., 2002).  In addition, though pds1∆ esp1-1 double mutants are SL when independently tested, pds1∆ mutants grow very poorly, and as such was a member of the 18.2% of genes omitted from the nonessential deletion collection (Giaever et al., 2002; Yamamoto et al., 1996a, b).  For this reason, the pds1∆esp1-1 interaction was not recovered from the esp1-1 SL screen presented here.  Similar caveats arise from screening of the pGAL1/10-GST-ORFX collection, as many cell cycle genes (SCC1 and PDS1 included) are lethal even when overexpressed in WT cells (Sopko et al., 2006). The inherent limitations of the SGA screening method must also be acknowledged. For example, the SGA SL screening methods would exclude loci that are genetically linked to the ESP1 ORF as well as those null mutants that are prone to sporulation and germination defects.  As a result, bona fide esp1-1 SL interactions may be missed, which was reflected by finding that lrs4∆esp1-1 double mutants are SL through independent means (Figure 2-7).  In addition, as demonstrated by the ~50% confirmation rate for both the SL and SDL screens, SGA analyses can also produce false positives.  Many explanations may account for this, including the potential to spontaneously acquire secondary mutations throughout the process.  45 Note that the haploid selectable marker for the esp1-1 SGA strain used in the screen was the Schizzasaccharomyces pombe HIS5 gene under the control of the STE2 promoter and not the original MFA1pr-HIS3 SGA reporter in order to alleviate the false positive rates that have been known to occur with the latter recombinant allele due to leaky expression/HIS3 recombination (Daniel et al., 2006; Tong and Boone, 2007).  In spite of these aforementioned restrictions, the impact of the well characterized role for Esp1 on sister chromatid cohesion was demonstrated by the overlap between the SL interaction profiles of esp1-1 and cdc20-3 or cohesin mutants (scc2-4, smc1-259) (Figure 2-6, data not shown).  Cdc20, in conjunction with the APC, effectively enables the activation of Esp1, therefore null mutations exacerbating the conditional phenotype of cdc20-3 are likely to cause lethality in an esp1-1 mutant as well.  However, an explanation for the similarity between the SL interaction profiles of esp1-1 and cohesin mutants is not immediately apparent, as these mutants share opposing phenotypes with respect to sister chromatid segregation.  For example, cohesin loading in S phase requires Scc2, and in the scc2-4 mutant, cohesin association is weak and premature sister chromatid segregation occurs (Ciosk et al., 2000; Lengronne et al., 2004; Toth et al., 1999a).  In contrast, the opposite occurs in esp1-1 cells as sister chromatid segregation is impeded due to a failure to cleave Scc1 (Uhlmann et al., 1999).  Similarly, Smc1 mutants demonstrate defects in the tethering of sister chromatids (Toth et al., 1999a).  However, as both separase and cohesin function in the maintenance of temporal control of sister chromatid segregation, further interference with this regulation may be similarly lethal in conditional mutants of both despite the difference in phenotype.   46 2.3.3  Negative genetic interactions of esp1-1 emphasize a role for Esp1 in maintaining spindle stability When the confirmed SL and SDL genetic interactions were examined for enriched GO biological processes, I was particularly intrigued to find several spindle motor (Myo2 and Kar3) and motor-related proteins (Cik1) present in the expected cell cycle category.  All serve to regulate spindle dynamics.  Myo2 mediates proper spindle orientation throughout the cell cycle while a Kar3-Cik1 heterodimer promotes poleward transport of cargoes such as kinetochores along MTs (Barrett et al., 2000; Beach et al., 2000; Sproul et al., 2005; Tanaka et al., 2005; Yin et al., 2000).  Esp1 localizes to and promotes stability of the elongating anaphase spindle; therefore, perturbation of spindle mechanics in an esp1-1 strain, which has spindle defects, is expected to be detrimental to esp1-1 cell viability (Jensen et al., 2001; Sullivan et al., 2001).  The importance of proper spindle dynamics in esp1-1 viability at the permissive temperature was further exemplified through the similarity between the SL profiles of cik1∆ and esp1-1 mutants.  2.3.4 The genetic interaction network of esp1-1 suggests potential new roles for separase  The ability of the genetic interaction network of esp1-1 to reflect not only the well- studied roles of Esp1 (sister chromatid cohesion, FEAR) but also less characterized ones (spindle dynamics) reinforces the potential for identification of novel roles for Esp1.  Though no new novel substrates for Esp1 were found, I identified several functional pathways that merit further study.  Both lrs4∆ and csm1∆ were shown to have SL profiles similar to esp1-1. Though this similarity may be predicated on the failure of the esp1-1 mutant to properly  47 release Cdc14 from the nucleolus in anaphase, thus possibly causing defects in rDNA segregation, I cannot exclude the possibility that Esp1 plays a direct role in the maintenance and separation of rDNA.  However, the most striking finding from this study was the enrichment of RNA metabolic genes as genetic interactors of esp1-1.  This opens two avenues for further investigation.  The most direct possibility is that Esp1 plays some sort of role in the regulation of mRNA as SL and SDL hits specifically identified members of the Paf1C as well as P-body components.   A role for Esp1 in the regulation of mRNA is further explored in Chapter 4.  Another possibility is that Esp1 functions in another pathway whose regulation is dependent on these genes – a prospect that became increasingly likely when a truncated Ty element was identified as a dosage suppressor of esp1-1.  Interestingly, many esp1-1 SL and SDL hits have an effect on the regulation of Ty elements in budding yeast (see: Chapter 3 for details) and a possible role for Esp1 in the regulation of Ty transposition is the subject of the next chapter.  2.4 Materials and methods 2.4.1 Yeast strains and growth  Strains used for these analyses are indicated in Tables 2-6.  Strains were grown in either YPD or synthetic complete (SC) media lacking uracil (SC-URA) and were incubated at 25˚C unless otherwise indicated.  Standard protocols for yeast culture and transformation were followed (Guthrie and Fink, 2004).  LRS4 was deleted using a PCR-based, homologous recombination method for S. cerevisiae (Longtine et al., 1998).  48 To create the esp1-1::NAT mutation in the SGA starting strain, a C-terminal fragment was amplified from esp1-1 (W303 strain background, gift from Yanchang Wang) (Baum et al., 1988).  This fragment included the P1404L (bp4211 C→T) mutation, approximately 200 bp downstream of the stop codon and an additional 25 bp overlapping the TEF promoter in p4339 (Tong et al., 2001).  Concurrently, the NATMX4 cassette from p4339 was also amplified to include 45 bp immediately downstream of the ESP1 ORF.   Both amplicons were transformed simultaneously into Y7092. Successful double recombination was confirmed in three ways: (1) growth on YPD media containing clonNAT (2) temperature sensitivity at 37˚C (3) sequencing of the esp1-1::NAT mutation (Macrogen).  2.4.2 Testing temperature sensitivity of the esp1-1 allele WT and esp1-1 strains were grown to mid-log phase (optical density, O.D.600 = 0.5- 1.0) in YPD at which point cells were diluted to an O.D.600 of 0.1.  Subsequently, cells were serially diluted four times by a factor of 1:5 in YPD.  4µL of each dilution was spotted onto YPD and plates grown at 25˚C, 30˚C, 33˚C or 35˚C for three days. WT and esp1-1::NAT cells grown to mid log phase were also diluted in prewarmed YPD and allowed to grow for four hours at 35˚C, with time points taken at 0, 2 and 4 hours. For each time point, samples were diluted to an O.D.600 of 0.1 and serially diluted as described above.  Dilutions were spotted onto YPD and plates allowed to incubate at 25˚C for three days.     49 2.4.3 Assaying for Esp1 substrates  To assay strictly for potential cleavage products, WT cells containing the pGAL1/10- GST-ORFX plasmid were grown overnight in SC-URA + 2% raffinose + 0.1% dextrose. Cells were then diluted in SC-URA + 2% raffinose and grown to log phase.  Cells were pelleted, washed and resuspended in SC-URA + 2% galactose, and allowed to grow for one hour before samples for western blot analysis were collected.  To examine whether ORFX encoded a substrate for Esp1, WT and esp1-1::NAT cells containing pGAL-GST-ORFX were grown to log phase in SC-URA + 2% raffinose and transferred to 35˚C for one hour.  Cells were then pelleted, washed and resuspended in prewarmed SC-URA + 2% galactose.  Cells were then placed back at 35˚C for another hour at which point samples were taken for western blotting.  2.4.4 Western blotting 10-25mL of cells were harvested, washed with ice cold H20 and resuspended in lysis buffer [50mM Tris-HCl pH 7.5, 10% Nonidet P-40 (NP-40), 250mM NaCl, 0.5mM ethylenediaminetetraacetic acid (EDTA), 1mM dithiothreitol (DTT) and protease inhibitors] at 4˚C.  Glass beads were added, and the suspension vortexed at 4˚C in two minutes cycles, with two minutes on ice in between, until > 90% lysis was achieved.  Lysates were centrifuged at 14 000 x g for ten minutes, and supernatant was kept as the whole cell lysate (WCL).  Protein concentration was quantified using a NanoDrop® ND-1000.  40 µg of WT and 80µg of esp1-1::NAT were run using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and transferred onto nitrocellulose membranes by wet transfer. Membranes were immunoblotted with anti-GST HRP Conjugate (1:3000, GE Healthcare).  50 2.4.5 SGA screening SGA screens have been previously described in detail, and were all performed in Dr. Christopher Loewen’s lab at the University of British Columbia under the supervision of a research associate, Dr. Barry Young (Tong and Boone, 2006; Tong et al., 2001; Tong and Boone, 2007).  Using a Singer® RoToR HDA robot, esp1-1::NAT was mated to both the haploid deletion collection of nonessential alleles (BY4741, MATa, KanMX, a gift from C. Boone) and the 2 micron pGAL1/10-GST-ORFX overexpression array (Open Biosystems) (Sopko et al., 2006; Tong et al., 2001).  For SL and SDL screens respectively, either double mutants (esp1-1::NAT geneX∆) or esp1-1::NAT carrying pGAL1/10-GST-ORFX were obtained through sporulation of heterozygous diploids with at least three colonies of each genotype being compared.  A series of replica pinning steps to various selective medias isolated recombinant MATa cells of interest (esp1-1 in combination with geneX∆ or pGAL1/10-GST-ORFX for the SL or SDL screens, respectively.  See (Tong and Boone, 2007) for full details on selection criteria.  On the final selective media, MATa recombinants were allowed to grow at 25˚C or 35˚C, for negative interactions or suppressors respectively.  Note that for SGA screens involving the pGAL-GST-ORFX collection, the final selective media was supplemented with 2% galactose as a carbon source instead of dextrose in order to induce overexpression of ORFX.  After incubation, plates were scanned, colonies detected using Colony Imager, and colony size quantified using Colony Scorer (Tong et al., 2004). The ratio of colony size to control determined a genetic interaction.  Only ORFs demonstrating growth below (for SL and SDL) or above (for SR and SDR) the cutoff score for all three replicates were considered to be genetic interactions (“hits”).  SL interactions (colony size that was <0.19 that of control) and SDL interactions (colony size growing <0.3  51 of control) were chosen for further testing, narrowing each list to 43 and 74 different ORFs, respectively.  All SDR hits (colony size >1.5 above background growth) were tested.  2.4.6 Confirmation of genetic interactions SL hits were confirmed by sporulation of the heterozygous diploid of interest. Tetrads were then dissected and germinants analyzed.  To confirm the identity of geneX∆, PCR analysis of heterozygous diploids was performed using the general KanB primer as well as each gene’s specific A confirmation primer (Giaever et al., 2002).  esp1-1::URA3 lrs4∆::kanMX6 heterozygous diploids were also analyzed in this manner to confirm SL. Plasmids carrying the 74 genes identified as SDL with esp1-1::NAT were rescued from yeast and retransformed into both WT and esp1-1::NAT strains.  Transformants were then grown overnight to log phase (O.D.600,= 0.5-1.0) in SC-URA + 2% raffinose + 0.1% dextrose media.  Strains were then diluted to an O.D.600 of 0.1, and subsequently serially diluted four times by a factor of 1:5, all in SC-URA + 2% raffinose.  Dilutions were then spotted onto either SC-URA + 2% dextrose or SC-URA + 2% galactose plates, and incubated for four days at the appropriate temperature (25˚C or 35˚C for SDL or SDR hits respectively).  The identity of ORFX was confirmed by sequencing (Operon) using both a GST forward primer (OVM487 5’ TGCAGGGCTGGCAAG 3’, sequence from C. Loewen) and reverse primer specific to the vector (Sopko et al., 2006).  SDR hits were analyzed similarly to SDL hits, though SC-URA plates were grown at 35˚C.  esp1-1::NAT cells containing pGAL1/10-GST (empty vector) was used as a control.    52 2.4.7 Comparison of esp1-1 SL profile with Boone lab SL database  An esp1-1 genetic interaction profile was generated from the entire (unconfirmed) SL SGA dataset by averaging the values associated with the same ORF.  A Pearson correlation coefficient between the esp1-1 profile and every screen in the published SGA dataset was generated (Costanzo et al., 2010).   This was computed in two ways: (1) correlating across all pairs (both positive and negative interactions), and (2) correlating across negative genetic interactions only.  By standard Precision-Recall analysis, the correlation strictly across negative (SL) interactions provided the most meaningful set of correlations.    53 Table 2-1.  Classification of SL hits for esp1-1 Gene name Systematic name SL/ SS Descriptiona APQ12 YIL040W SL Protein required for nuclear envelope morphology, nuclear pore complex localization, mRNA export from the nucleus BTS1 YPL069C SS Geranylgeranyl diphosphate synthase CDC73 YLR418C SS Component of the Paf1p complex HSC82 YMR186W SL Cytoplasmic chaperone of the Hsp90 family HSL1 YKL101W SS Nim1p-related protein kinase that regulates the morphogenesis and septin checkpoints KAR3 YPR141C SL Minus-end-directed microtubule motor that functions in mitosis and meiosis LEO1 YOR123C SL Component of the Paf1p complex LSM1 YJL124C SL Lsm (Like Sm) protein; forms heteroheptameric complex (with Lsm2p- Lsm7p) involved in degradation of cytoplasmic mRNAs LSM7 YNL147W SL Lsm (Like Sm) protein; part of heteroheptameric complexes (Lsm2p-7p and either Lsm1p or 8p) LTE1 YAL024C SL Protein required for asymmetric localization of Bfa1p at daughter-directed spindle pole bodies and for mitotic exit at low temperatures MMS22 YLR320W SL Subunit of an E3 ubiquitin ligase complex involved in replication repair NUP60 YAR002W SL FG-nucleoporin component of central core of the nuclear pore complex (NPC) RPS27B YHR021C SL Protein component of the small (40S) ribosomal subunit RTF1 YGL244W SL Component of the Paf1p complex YDJ1 YNL064C SL Type I HSP40 co-chaperone aadapted from Saccharomyces genome database (SGD –  54 Table 2-2.  Classification of SDL hits for esp1-1 Gene name Systematic name SDL/ SDS Descriptiona ACO1 YLR304C SDL Aconitase, required for the tricarboxylic acid (TCA) cycle ADE6 YGR061C SDL Formylglycinamidine-ribonucleotide (FGAM)-synthetase CDC6 YJL194W SDS Essential ATP-binding protein required for DNA replication CIK1 YMR198W SDL Kinesin-associated protein required for both karyogamy and mitotic spindle organization COP1 YDL145C SDS Alpha subunit of COPI vesicle coatomer complex CYM1 YDR430C SDS Lysine-specific metalloprotease of the mitochondrial intermembrane space DHH1 YDL160C SDL Cytoplasmic DExD/H-box helicase, stimulates mRNA decapping DOT1 YDR440W SDS Nucleosomal histone H3-Lys79 methylase DTR1 YBR180W SDS Putative dityrosine transporter of the major facilitator superfamily EBS1 YDR206W SDL Protein involved in inhibition of translation and nonsense-mediated decay ECM10 YEL030W SDL Heat shock protein of the Hsp70 family EFT2 YDR385W SDL Elongation factor 2 (EF-2); catalyzes ribosomal translocation during protein synthesis GAR1 YHR089C SDL Protein component of the H/ACA snoRNP pseudouridylase complex GSP2 YOR185C SDL GTP binding protein (mammalian Ranp homolog) GZF3 YJL110C SDS GATA zinc finger protein HRP1 YOL123W SDL Subunit of cleavage factor I MNN4 YKL201C SDL Putative positive regulator of mannosylphosphate transferase Mnn6p MYO2 YOR326W SDS Type V myosin motor involved in actin-based transport of cargos PAN2 YGL094C SDL Essential subunit of the Pan2p-Pan3p poly(A)-ribonuclease complex PAT1 YCR077C SDL Deadenylation-dependent mRNA-decapping factor PDR1 YGL013C SDS Transcription factor that regulates the pleiotropic drug response PEX19 YDL065C SDS Chaperone and import receptor for newly-synthesized class I PMPs PEX8 YGR077C SDS Intraperoxisomal organizer of the peroxisomal import machinery PRD1 YCL057W SDL Zinc metalloendopeptidase RAD27 YKL113C SDS 5' to 3' exonuclease, 5' flap endonuclease  55 Table 2-2.  Classification of SDL hits for esp1-1 (continued) Gene name Systematic name SDL/ SDS Descriptiona RAD53 YPL153C SDL Protein kinase, required for cell-cycle arrest in response to DNA damage REG1 YDR028C SDS Regulatory subunit of type 1 protein phosphatase Glc7p RSC30 YHR056C SDL Component of the RSC chromatin remodeling complex SCD6 YPR129W SDL Repressor of translation initiation SGS1 YMR190C SDS Nucleolar DNA helicase of the RecQ family SIP4 YJL089W SDL C6 zinc cluster transcriptional activator SLK19 YOR195W SDL Kinetochore-associated protein required for normal segregation of chromosomes in meiosis and mitosis SPT10 YJL127C SDS Putative histone acetylase with a role in transcriptional silencing SPT23 YKL020C SDS ER membrane protein involved in regulation of OLE1 transcription STE4 YOR212W SDS G protein beta subunit, forms a dimer with Ste18p to activate the mating signaling pathway TOF2 YKR010C SDL Protein required for rDNA silencing and mitotic rDNA condensation; stimulates Cdc14p phosphatase activity and biphasic release to promote rDNA repeat segregation; required for condensin recruitment to the replication fork barrier site; TPN1 YGL186C SDS Plasma membrane pyridoxine (vitamin B6) transporter TPS3 YMR261C SDS Regulatory subunit of trehalose-6-phosphate synthase/phosphatase TRM732 YMR259C SDS Interacts with Trm7p for 2'-O-methylation of C32 of substrate tRNAs UBP12 YJL197W SDL Ubiquitin-specific protease; cleaves ubiquitin from ubiquitinated proteins YDR374C YDR374C SDS Unknown YIR007W YIR007W SDL Putative glycosidase YPK1 YKL126W SDS Serine/threonine protein kinase YPR174C YPR174C SDL Unknown aadapted from Saccharomyces genome database (SGD-   56 Table 2-3.  Fold enrichment of GO biological processes identified by analysis of esp1-1 SL and SDL interactions GO term Frequency Genome Frequency Gene(s) Fold Enrichment RNA catabolic process 5 of 59 genes,  8.5% 108 of 6334 genes, 1.7% PAT1, DHH1, EBS1, LSM1, LSM7 5 protein folding 3 of 59 genes, 5.1% 89 of 6334 genes, 1.4% ECM10, HSC82, YDJ1 3.64 mitotic cell cycle 8 of 59 genes, 13.6% 300 of 6334 genes, 4.7% LTE1, CDC6, HSL1, TOF2, CIK1, SLK19, MYO2, KAR3 2.89 DNA repair 6 of 59 genes, 10.1% 239 of 6334 genes, 3.8% PAN2, RAD27, MMS22, SGS1, RAD53, DOT1 2.66 regulation of translation 2 of 59 genes, 3.4% 90 of 6334 genes, 1.4% EFT2, SCD6 2.42 nuclear transport 3 of 59 genes, 5.1% 164 of 6334 genes, 2.6% APQ12, NUP60, GSP2 1.96 transcription from RNA polymerase II promoter 8 of 59 genes, 13.6% 453 of 6334 genes, 7.2% PDR1, RTF1, RSC30, GZF3, SPT10, SPT23, CDC73, LEO1 1.89 carbohydrate metabolic process 4 of 59 genes, 6.8% 268 of 6334 genes, 4.2% MNN4, TPS3, REG1, SIP4 1.61 cellular respiration 1 of 59 genes, 1.7% 90 of 6334 genes, 1.4% ACO1 1.21 ribosomal small subunit biogenesis 1 of 59 genes, 1.7% 126 of 6334 genes, 2% RPS27B 0.85 protein targeting 2 of 59 genes, 3.4% 262 of 6334 genes, 4.1% PEX19, PEX8 0.83 protein phosphorylation 1 of 59 genes, 1.7% 176 of 6334 genes, 2.8% YPK1 0.61 Golgi vesicle transport 1 of 59 genes, 1.7% 187 of 6334 genes, 3% COP1 0.57 mRNA processing 1 of 59 genes, 1.7% 192 of 6334 genes, 3.0% HRP1 0.57 nucleobase-containing small molecule metabolic process 1 of 59 genes, 1.7% 187 of 6334 genes, 3% ADE6 0.57 proteolysis involved in cellular protein catabolic process 1 of 59 genes, 1.7% 190 of 6334 genes, 3% CYM1 0.57 signaling 1 of 59 genes, 1.7% 228 of 6334 genes, 3.6% STE4 0.5   57 Table 2-3.  Fold enrichment of GO biological processes identified by analysis of esp1-1 SL and SDL interactions (continued)  GO term Frequency Genome Frequency Gene(s) Fold Enrichment transmembrane transport 1 of 59 genes, 1.7% 220 of 6334 genes, 3.5% DTR1 0.49 biological process unknown 5 of 59 genes, 8.5% 1188 of 6334 genes, 18.8% YDR374C, YIR007W, UBP12, TRM732, YPR174C 0.45 lipid metabolic process 1 of 59 genes, 1.7% 295 of 6334 genes, 4.7% BTS1 0.36 rRNA processing 1 of 59 genes, 1.7% 296 of 6334 genes, 4.7% GAR1 0.36 other 2 of 59 genes, 3.4%  PRD1, TPN1     58 Table 2-4.  Comparison of esp1-1’s SL profile to the Boone database SGA query ORFa SGA query gene name Pearson correlation to esp1-1 screen Descriptionb YDR439W LRS4 0.402 Nucleolar protein that forms a complex with Csm1p, and then Mam1p at kinetochores during meiosis I to mediate accurate homolog segregation; required for condensin recruitment to the replication fork barrier site and rDNA repeat segregation YFL009W_tsq334 cdc4-3 0.347 F-box protein required for G1/S and G2/M transition YJR140C HIR3 0.333 Subunit of the HIR complex; a nucleosome assembly complex involved in regulation of histone gene transcription YMR078C CTF18 0.333 Subunit of a complex with Ctf8p that shares some subunits with Replication Factor C and is required for sister chromatid cohesion YNL199C GCR2 0.332 Transcriptional activator of genes involved in glycolysis YCR086W CSM1 0.33 Nucleolar protein that forms a complex with Lrs4p and Mam1p; see LRS4 YPR070W MED1 0.319 Subunit of the RNA polymerase II mediator complex YNL127W FAR11 0.318 Protein involved in recovery from cell cycle arrest in response to pheromone, in a Far1p-independent pathway YAR002W NUP60 0.31 FG-nucleoporin component of central core of the nuclear pore complex (NPC) YLR438C- A_DAmP LSM3_DAmP 0.308 Lsm (Like Sm) protein; part of heteroheptameric complexes (Lsm2p-7p and either Lsm1p or 8p) YGL116W_tsq368 cdc20-3 0.305 Activator of anaphase-promoting complex/cyclosome (APC/C) YGL061C_tsq239 duo1-2 0.303 Essential subunit of the Dam1 complex YIL084C SDS3 0.301 Component of the Rpd3p/Sin3p deacetylase complex YDR435C PPM1 0.298 Carboxyl methyltransferase, methylates the C terminus of the protein phosphatase 2A catalytic subunit YBR111W-A SUS1 0.294 Component of both the SAGA histone acetylase and TREX-2 complexes YBR026C ETR1 0.293 2-enoyl thioester reductase  59 Table 2-4.  Comparison of esp1-1’s SL profile to the Boone database (continued) SGA query ORFa SGA query gene name Pearson correlation to esp1-1 screen Descriptionb YLR320W MMS22 0.293 Subunit of an E3 ubiquitin ligase complex involved in replication repair YBR058C UBP14 0.292 Ubiquitin-specific protease that specifically disassembles unanchored ubiquitin chains YOR259C_tsq260 rpt4-150 0.292 One of six ATPases of the 19S regulatory particle of the 26S proteasome involved in degradation of ubiquitinated substrates YDL017W_tsq131 cdc7-4 0.291 DDK (Dbf4-dependent kinase) catalytic subunit required for origin firing and replication fork progression YKL193C_tsq572 sds22-5 0.29 Regulatory subunit of the type 1 protein phosphatase (PP1) Glc7p YER035W EDC2 0.289 RNA-binding protein that directly activates mRNA decapping YOR123C LEO1 0.287 Component of the Paf1 complex YFR004W_tsq534 rpn11-14 0.286 Metalloprotease subunit of the 19S regulatory particle of the 26S proteasome lid YDL017W_tsq880 cdc7-1 0.285 See above: cdc7-4 allele YBR087W_tsq887 rfc5-1 0.284 Subunit of heteropentameric Replication factor C (RF-C) YFR052W_tsq405 rpn12-1 0.283 Subunit of the 19S regulatory particle of the 26S proteasome lid YJR097W JJJ3 0.282 Protein of unknown function YDL028C_tsq169 mps1-417 0.281 Dual-specificity kinase; required for spindle pole body (SPB) duplication and spindle checkpoint function YER112W_DAmP LSM4_DAmP 0.281 Lsm (Like Sm) protein; part of heteroheptameric complexes (Lsm2p-7p and either Lsm1p or 8p) YGL044C_tsq652 rna15-58 0.28 Component of the cleavage and polyadenylation factor I (CF I) YHR167W THP2 0.28 Subunit of the THO and TREX complexes YHR039C-A VMA10 0.279 Subunit G of the eight-subunit V1 peripheral membrane domain of the vacuolar H+-ATPase (V-ATPase) YDR180W_tsq69 scc2-4 0.273 Subunit of cohesin loading factor (Scc2p-Scc4p)   60 Table 2-4.  Comparison of esp1-1’s SL profile to the Boone database (continued) SGA query ORFa SGA query gene name Pearson correlation to esp1-1 screen Descriptionb YDR207C UME6 0.273 Key transcriptional regulator of early meiotic genes YHR152W SPO12 0.273 Nucleolar protein of unknown function, positive regulator of mitotic exit YMR198W CIK1 0.271 Kinesin-associated protein required for both karyogamy and mitotic spindle organization YPR101W SNT309 0.27 Member of the NineTeen Complex (NTC) YDR363W-A SEM1 0.268 Component of lid subcomplex of the 26S proteasome regulatory subunit YER093C_tsq658 tsc11-1 0.267 Subunit of TORC2 (Tor2p-Lst8p-Avo1-Avo2-Tsc11p-Bit61p) YHR191C CTF8 0.267 Subunit of a complex with Ctf18p that shares some subunits with Replication Factor C and is required for sister chromatid cohesion YBL058W SHP1 0.266 UBX (ubiquitin regulatory X) domain-containing protein that regulates Glc7p phosphatase activity and interacts with Cdc48p YBL034C_tsq822 stu1-12 0.263 Component of the mitotic spindle that binds to interpolar microtubules via its association with beta-tubulin YFL009W_tsq415 cdc4-2 0.263 F-box protein required for G1/S and G2/M transition YGL173C KEM1 0.263 Evolutionarily-conserved 5’-3’ exonuclease component of cytoplasmic processing (P) bodies involved in mRNA decay YOL012C HTZ1 0.262 Histone variant H2AZ YDR168W_tsq317 cdc37-1 0.261 Essential Hsp90p co-chaperone YPR119W CLB2 0.261 B-type cyclin involved in cell cycle progression YLR410W VIP1 0.26 Inositol hexakisphosphate (IP6) and inositol heptakisphosphate (IP7) kinase YFR002W_DAmP NIC96_DamP 0.259 Linker nucleoporin component of the nuclear pore complex (NPC) YLR272C_tsq432 ycs4-1 0.259 Subunit of the 60ondensing complex YNL136W EAF7 0.259 Subunit of the NuA4 histone acetyltransferase complex   61 Table 2-4.  Comparison of esp1-1’s SL profile to the Boone database (continued) SGA query ORFa SGA query gene name Pearson correlation to esp1-1 screen Descriptionb YOR083W WHI5 0.258 Repressor of G1 transcription that binds to SCB binding factor (SBF) at SCB target promoters in early G1 YBR160W_tsq175 cdc28-13 0.257 Catalytic subunit of the main cell cycle cyclin-dependent kinase (CDK) YGR270W YTA7 0.257 Protein that localizes to chromatin and has a role in regulation of histone gene expression YOR027W STI1 0.257 Hsp90 cochaperone YDR225W HTA1 0.256 Histone H2A, core histone protein required for chromatin assembly and chromosome function YMR235C_tsq172 rna1-S116F 0.255 GTPase activating protein (GAP) for Gsp1p, involved in nuclear transport YHR083W_tsq493 sam35-2 0.254 Essential component of the sorting and assembly machinery (SAM complex or TOB complex) of the mitochondrial outer membrane YBR193C_tsq741 med8-51 0.252 Subunit of the RNA polymerase II mediator complex YHR019C_DAmP DED81_DAmP 0.252 Cytosolic asparaginyl-tRNA synthetase YHR166C_tsq89 cdc23-1 0.251 Subunit of the Anaphase-Promoting Complex/Cyclosome (APC/C) YOR195W SLK19 0.251 Kinetochore-associated protein required for normal segregation of chromosomes in meiosis and mitosis; component of the FEAR regulatory network a(Costanzo et al., 2010) badapted from Saccharomyces genome database (SGD-    62 Table 2-5.  SR hits for esp1-1 Gene Name Systematic Name Ratio above background Standard Deviation p-value Descriptiona GYP6 YJL044C 2.884241667 ± 0.810697 0.017235041 GTPase-activating protein (GAP) for Ypt6p MUQ1 YGR007W 2.621442 ± 0.528578 0.003016929 Ethanolamine-phosphate cytidylyltransferase SPT3 YDR392W 2.351997333 ± 0.714284 0.048982443 Subunit of the SAGA and SAGA-like transcriptional regulatory complexes YER004W YER004W 2.248904 ± 0.468281 0.010612262 Protein of unknown function CIS1 YDR022C 1.846820667 ± 0.250445 0.003441325 Autophagy-specific protein required for autophagosome formation TOM5 YPR133W-A 1.746660667 ± 0.085004 5.8463E-12 Component of the TOM (translocase of outer membrane) complex YGL152C YGL152C 1.735899 ± 0.255116 0.013270446 Dubious open reading frame unlikely to encode a protein APM1 YPL259C 1.633821 ± 0.128945 0.000160931 Mu1-like medium subunit of the clathrin-associated protein complex (AP-1) ISU2 YOR226C 1.629425 ± 0.074595 3.66134E-10 Protein required for synthesis of iron-sulfur proteins YJR030C YJR030C 1.579406 ± 0.051141 5.95591E-16 Putative protein of unknown function YAP7 YOL028C 1.913483333 ± 0.552570 0.089231678 Putative basic leucine zipper (bZIP) transcription factor RCE1 YMR274C 1.900415333 ± 0.668476 0.137272246 Type II CAAX prenyl protease RIT1 YMR283C 2.287966667 ± 0.794785 0.079768792 2'-O-ribosyl phosphate transferase ROD1 YOR018W 2.023172333 ± 0.939081 0.181802133 Membrane protein, binds the ubiquitin ligase Rsp5p KEL1 YHR158C 1.723950333 ± 0.339991 0.051623979 Protein required for proper cell fusion and cell morphology; functions negatively regulate mitotic exit HTZ1 YOL012C 2.111911333 ± 0.664830 0.078275137 Histone variant H2AZ YOR139C YOR139C 3.343506667 ± 1.494872 0.072976629 Dubious open reading frame unlikely to encode a protein YKL075C YKL075C 2.371866333 ± 0.774513 0.060358616 Putative protein of unknown function YIA6 YIL006W 2.022037667 ± 0.672625 0.102624894 Mitochondrial NAD+ transporter RSC1 YGR056W 1.978989667 ± 0.667040 0.112601965 Component of the RSC chromatin remodeling complex HAP4 YKL109W 2.064200333 ± 0.725156 0.108766631 Subunit of the heme-activated, glucose-repressed Hap2p/3p/4p/5p CCAAT-binding complex  63 Table 2-5.  SR hits for esp1-1 (continued) Gene Name Systematic Name Ratio above background Standard Deviation p-value Descriptiona RPB4 YJL140W 2.680497333 ± 1.138821 0.092358777 RNA polymerase II subunit B32 MPD2 YOL088C 1.837100333 ± 0.631344 0.145339002 Member of the protein disulfide isomerase (PDI) family YGL235W YGL235W 2.197648 ± 0.770046 0.091015434 Putative protein of unknown function YJR146W YJR146W 2.430699333 ± 1.127255 0.131702287 Protein of unknown function aadapted from Saccharomyces genome database (SGD-  64 Table 2-6.  Strains used in this chapter Strain Genotype Source Y7092  Mat α can1∆::STE2pr-Sp_his5 lyp1∆ his3∆1 leu2∆0 ura3∆0 met15∆0 (Tong and Boone, 2006) YKH64/65 Mat α can1∆::STE2pr-Sp_his5 lyp1∆ his3∆1 leu2∆0 ura3∆0 met15∆0  esp1-1::NATMX4 This study YKH60 Mat a ura3-52 lys2-801_amber ade2-101 trp1-Δ63 his3- Δ200 leu2-Δ1 esp1-1::URA3 This study YKH519 Mat α ura3-52 lys2-801_amber ade2-101 trp1-Δ63 his3- Δ200 leu2-Δ1 lrs4∆::KanMX6 This study    65                                A. B.  66              Figure 2-1.  Genetic interactions reveal functional relationships (A and B) Proteins functioning in parallel pathways (A1, A2, A3 in pathway A; B1, B2, B3 in pathway B) contributing to an essential biological function can have “between pathway” SL interactions (denoted by lines), as impinging on both pathways simultaneously (through gene mutation) is lethal to the cell.  Essential proteins (C1, C2) functioning within the same pathway can also be SL, as mutating two genes encoding proteins that function within the same complex can result in lethality whereas mutating each individually would not.  Cited from (Boone et al., 2007).  Reprinted with permission © Nature Publishing Group. (C) Overexpressing a substrate in a strain carrying a mutation in the substrate’s enzyme can lead to SDL relationships - especially if the enzyme-substrate interaction (such as cleavage) is critical for cell function    Mutant Substrate Viable Enzyme Viable+ Overexpressed Substrate + Overexpressed Substrate Mutant + Dead C.  67                                 68 Figure 2-2.  Analysis of the SGA esp1-1 allele (A) Sequence analysis of the esp1-1 allele (Query) revealed a point mutation at bp 4211 (highlighted by the black box) when compared to the ESP1 ORF (Sbjct).  This caused a proline to leucine mutation within the catalytic domain at residue 1404.  To illustrate the placement of this mutation in context, the esp1-1 ORF is shown as a rectangle, with the C- terminal proteolytic “separase” domain represented as a gradient filled box and the leucine (L) point mutation at residue 1404 highlighted.  The locations of the critical catalytic histidine and cysteine residues (amino acid 1505 and 1531 respectively) are also indicated. (B) esp1-1 temperature sensitivity analysis by spot dilution assay on YPD at 25˚C, 30˚C, 33˚C and 35˚C.  (C) Cell viability was assessed by spot dilution assay for cells grown for 0, 2 and 4 hours at 35˚C.  (D) pGAL1/10-GST-SLK19 was overexpressed at 35˚C for one hour in WT and esp1-1 strains and immunoblotted against GST.  A band corresponding to the Slk19 cleavage product is denoted by *.  Molecular weight bands are indicated on the left.  A line between lanes indicates that, although samples were run on the same gel, they were not run side by side.     69                    Figure 2-3.  Confirmation of SL hits SL hits were confirmed using tetrad analysis.  Two representative examples are shown here. (A) hsc82Δ is known to be SL with esp1-1 and was identified in the SGA screen.  (B) kar3Δ is a novel SL hit identified from the SGA screen.  PD - parental ditype; NPD - non parental ditype; T – tetratype  PD NPD T KVFǻ esp1-1 PD NPD T NDUǻ esp1-1 A. B.  70                    Dextrose Galactose Gal-GST-ACO1 Gal-GST-ADE6 Gal-GST-CDC6 Gal-GST-CIK1 Gal-GST-COP1 Gal-GST-CYM1 Gal-GST-DHH1 Gal-GST-DOT1 Gal-GST-DTR1 Gal-GST-EBS1 Gal-GST-ECM10 Gal-GST-EFT2 Dextrose Galactose Gal-GST-GAR1 Gal-GST-GSP2 Gal-GST-GZF3 Gal-GST-HRP1 Gal-GST-MNN4 Gal-GST-MYO2 Gal-GST-PAN2 Gal-GST-PAT1 Gal-GST-PDR1 Gal-GST-PEX19 Gal-GST-PEX8 Gal-GST-PRD1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1  71                       Figure 2-4. Confirmation of esp1-1 SDL hits Plasmids of interest were transformed into both WT and esp1-1 strains. Logarithmically growing cells were serially diluted and plated onto dextrose (control) or galactose containing media  Dextrose Galactose Dextrose Galactose Gal-GST-RAD27 Gal-GST-RAD53 Gal-GST-REG1 Gal-GST-RSC30 Gal-GST-SCD6 Gal-GST-SGS1 Gal-GST-SLK19 Gal-GST-SIP4 Gal-GST-SPT10 Gal-GST-SPT23 Gal-GST-STE4 Gal-GST-TOF2 Gal-GST-TPN1 Gal-GST-TPS3 Gal-GST-UBP12 Gal-GST-YDR374C Gal-GST-YIR007W Gal-GST-YMR259C Gal-GST-YPK1 Gal-GST-YPR174C Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1 Wild Type esp1-1  72                     Figure 2-5.  GO analysis of confirmed esp1-1 SL and SDL genetic interactions Genes identified in the esp1-1 SL and SDL screens were categorized according to biological process using Saccharomyces Genome Database GO Slim Mapper.   Enriched GO categories as well as unknown genes are highlighted; genes belonging to biological processes that were not enriched are presented as “Other”.  SL hits are represented by italics; SDL hits are indicated with uppercase letters.   Protein Folding Cell Cycle Unknown Carbohydrate Metabolic Process Other RNA Catabolic Process DNA Repair Nuclear Transport Transcription from an RNAPII promoter Regulation of Translation  73                   Figure 2-6.  Comparison of esp1-1’s SL profile esp1-1’s SL hits were compared to the SL hits of other mutants that have been screened by Dr. Charlie Boone’s lab (Costanzo et al., 2010).  Shown on the X axis are strains that shared a significant correlation with the esp1-1 SL profile, with Pearson coefficient on the Y-axis.  Gene name alone indicates a deletion mutant.  Analysis was performed by Anastasia Baryshnikova and Michael Costanzo.   74       Figure 2-7.  lrs4∆  is SL with esp1-1 Tetrad analysis from lrs4∆ esp1-1 heterozygotes.  PD= parental ditype, NPD=non parental ditype, T=tetratype.    OUV¨ esp1-1 PD NPD T  75                      Figure 2-8.  Assaying for an esp1-1 substrate (A) pGAL1/10-GST-ORFX plasmids were overexpressed in WT cells and immunoblotted for the GST fusions. GST-PRD1 is shown as a representative candidate that did not show cleavage products while GST-ECM10 is an example of one that was tested in both WT and esp1-1 strains. (B) pGAL1/10-GST-ORFX was overexpressed in both WT and esp1-1 at a non-permissive temperature (35˚C) for one hour.  Banding patterns of GST-immunoblots were compared between the two strains.  GST-SLK19, a known Esp1 substrate, is shown as a proof of principle for the assay.  A band corresponding to the Slk19 cleavage product is denoted by *.  Molecular weight bands are indicated on the left.  A line between lanes indicates that, although samples were run on the same gel, they were not run side by side. 70 G ST -S LK 19 70 G ST -P R D 1 G ST -E C M 1070 A. 70 GST-RAD27 W ild  Ty pe esp 1-1 B. * GST-SLK19 W ild  Ty pe esp 1-1 70 100 55 35 100 55 35 100 100 55 55 35 35 100 55 35  76                 Figure 2-9.  Rescue of esp1-1 lethality by a overeexpression of a truncated Ty4 element An esp1-1 strain carrying either the pGAL1/10-GST (EV) or pGAL1/10-TY4-RT/RH were serially diluted and spotted onto media carrying 2% glucose (control) or 2% galactose (to overexpress the plasmid).  Plates were then allowed to incubate at either 25˚C or 32˚C.  Glucose Galactose Glucose Galactose EV EV Û& Û& Ty4- RT/RH Ty4- RT/RH  77 Chapter  3: The budding yeast separase, Esp1, regulates Ty1 retrotransposition 3.1 Introduction  Retroviruses such as Human Immunodeficiency Virus and Human T-cell Lymphotrophic Virus-1 are characterized by their capacity to self-propagate via an RNA intermediate (Barre-Sinoussi et al., 1983; Gallo, 1985; Poiesz et al., 1980; Poiesz et al., 1981).  Similarly, eukaryotic retroelements encode the necessary factors for RNA-mediated mobility, though they lack an extracellular infectious stage (Boeke et al., 1985; Boeke and Sandmeyer, 1991).  In S. cerevisiae, there are five families of retrotransposons known broadly as Ty elements (Ty1-5) (Figure 3-1).  Most belong to the copia (Pseudoviridae) class of Ty elements (Ty1,2,4,5), but Ty3 is a member of the gypsy (Metaviridae) family [reviewed in: (Beauregard et al., 2008; Lesage and Todeschini, 2005)] (Figure 3-1).  Ty retrotransposons are approximately 5.5-6 kb in size and are comprised of both a TYA and TYB sequence - analogous to retroviral gag and pol respectively - that are flanked by long terminal repeats.  The TYA gene product is the structural protein that forms the virus like particles (VLPs), while TYB encodes a polyprotein, which is processed into the factors necessary for transposon mobility, namely a protease (PR), integrase (IN) and reverse transcriptase (RT) (Beauregard et al., 2008; Boeke and Sandmeyer, 1991; Lesage and Todeschini, 2005).  The TYA-TYB mRNA transcript produces two proteins corresponding to either TyA alone or a TyA-TyB fusion product whose formation is dictated by a translational frameshifting event (Belcourt and Farabaugh, 1990; Clare and Farabaugh, 1985; Mellor et al., 1985; Wilson et al., 1986).  Both the TyA and TyA-TyB fusion product are subsequently processed by the PR into the mature forms of their individual elements (VLPs, IN, RT, PR)  78 which are then capable of supporting successful Ty retrotransposition (Garfinkel et al., 1991; Merkulov et al., 1996; Youngren et al., 1988a).  Together, Ty elements represent 3.1% of the S. cerevisiae genome, with the highest proportion of elements located on chromosome III.  The Ty1 and Ty2 families of retrotransposons are by far the most abundant, with 32 and 13 full-length sequences in the genome respectively; by contrast, Ty3-5 have a total of five full-length elements.  Amid a 62% amino acid identity, Ty1 and Ty2 elements are considered highly similar, with distinguishing characteristics between the two families arising primarily from within the TYA sequence and an extremely variable region of TYB that corresponds to the carboxy terminus of IN (Kim et al., 1998).  Curcio et al monitored spontaneous Ty insertions into a promotorless HIS3 gene and found that 95% of events corresponded to Ty1 elements, while the rest were Ty2 (Curcio et al., 1990).  Based on the number of sequences found in the genome for each family, the efficiency of Ty1 transposition relative to that of Ty2 was unexpected.  However, the rate was instead found to correlate with the comparative levels of each transcript in the cell, with the cell containing 20-fold more Ty1 than Ty2 RNA.  Together, Ty1 and Ty2 are thought to account for 0.1%-0.8% of total RNA in the cell, or as much as half the polyadenylated RNA (Curcio et al., 1990; Elder et al., 1981).  Despite the abundance of transcripts, endogenous Ty transposition remains a relatively rare event, occurring at an approximate rate of 10-5-10-7 elements/generation (Curcio and Garfinkel, 1991).  The transposition events stimulated by Ty elements can influence genome organization, with increases in Ty transposition and subsequent homologous recombination capable of “scrambling the yeast genome” (Fink et al., 1986).  For example, RNA-mediated  79 recombination resulting from TY1 element expression has specifically been reported as a factor in intron loss and pseudogene formation in S. cerevisiae (Derr and Strathern, 1993). Thus, to mitigate the chance of insertional events that would negatively affect cell viability, Ty elements are targeted to specific areas of the genome to avoid disrupting promoters or ORFs (Boeke and Devine, 1998).  Both Ty1 and Ty3 elements favour insertion into sequences upstream of RNA polymerase III (RNAPIII) transcribed genes with Ty1 inserting within ~1kb upstream and Ty3 inserting 2bp upstream of the RNAPIII gene.  Ty5 is targeted to transcriptionally silent areas of the genome such as the mating loci and telomeres (Chalker and Sandmeyer, 1992; Devine and Boeke, 1996; Zou et al., 1996).  The TFIIIB and TFIIIC RNAPIII transcription factor subunits have been found to mediate Ty3 specificity while Sir3 and Sir4 are necessary for targeting Ty5 (Kirchner et al., 1995; Zhu et al., 1999).  To better understand the Ty1 retrotransposition cycle, several genome-wide studies have been undertaken to identify genes capable of influencing the rate of Ty1 retrotransposition.  These investigations included systematic screening of the yeast deletion mutant collection to identify genes that inhibit and promote Ty1 transposition, or using transposition-mediated mutagenesis to identify loss of function mutations which cause upregulation of Ty1 transposition (Griffith et al., 2003; Nyswaner et al., 2008; Scholes et al., 2001).  Other studies used classical mutagenesis techniques to uncover genes that influence individual stages of the Ty1 retrotransposition cycle.  For example, a screen for mutants able to restore expression of genes whose 5’ region had been interrupted by a Ty sequence identified suppressor of Ty (SPT) genes that regulate transcription of the TY1 element (Winston, 1992; Yamaguchi et al., 2001); a rare transfer RNA (tRNA) gene was found to modulate translational frameshifting (Kawakami et al., 1993; Xu and Boeke, 1990); mutation  80 of FUS3 caused destabilization of Ty1 VLPs (Conte et al., 1998); mutations in PMR1 dramatically decreased the rate of reverse transcription (Bolton et al., 2002; Yarrington et al., 2007); and, multiple genes have a role in complementary DNA (cDNA) conversion/stability (Lee et al., 1998; Liebman and Newnam, 1993; Rattray et al., 2000).  Genes that influence chromatin structure have also been shown to influence Ty1 target site distribution (Bachman et al., 2005; Gelbart et al., 2005; Mou et al., 2006).  However, the method by which TY1 cDNA is recruited to hotpots of chromosomal integration has not yet been fully elucidated.  Genetic interaction data of the esp1-1 budding yeast separase mutant suggested Esp1 might be involved in Ty retrotransposition (Chapter 2 of this dissertation).  In this chapter, I present physical interaction data and analysis of Ty1 retrotransposition in esp1-1 and cohesin mutants, which suggest that Esp1 and cohesin have a previously unanticipated role in mediating transposition by interacting with Ty1 integrase.  3.2 Results 3.2.1 esp1-1 genetic interactors are involved in the life cycle of Ty1 retrotransposons  The retrotransposition cycle of Ty1 elements can be described in the following steps (A) transcription of the TY1 element by RNAPII (B) nuclear export of the TY1 mRNA (C) translation of TY1 mRNA (D) assembly and maturation of the VLP (E) reverse transcription of the TY1 mRNA to cDNA (F) import into the nucleus and (G) integration of the TY1 cDNA into the genome (Beauregard et al., 2008).  A closer examination of the host S. cerevisiae genes required for individual steps of the Ty1 life cycle revealed that many of the esp1-1 genetic interactors involved in RNA/DNA metabolism either promote or impede the Ty1 life cycle (Figure 3-2).  Genome-  81 wide studies have implicated the P-body proteins Pat1, Dhh1 and Lsm1-7 in Ty1 retrotransposition as enhancers of the formation of retrotransposition-competent Ty1 VLPs (Beckham and Parker, 2008; Checkley et al., 2010; Griffith et al., 2003).  Pat1 and Dhh1 were identified in the esp1-1 SDL screen whereas lsm1∆ and lsm7∆ were identified in the esp1-1 SL screen (Figure 2-5, Tables 2-1 and 2-2).  The RecQ helicase Sgs1, which is SDS when overexpressed in esp1-1 cells, is likely involved in the suppression of TY1 cDNA recombination.  Strains lacking SGS1 transpose heterogeneous Ty1 multimers/tandem arrays, which effectively increase the number of TY1 cDNAs per transposition event (Bryk et al., 2001; Nyswaner et al., 2008) (Figure 2-5, Table 2-1).  The Fen-1 nuclease Rad27, which is also SDS when overexpressed in esp1-1 cells, is thought to degrade TY1 cDNA to prevent the formation of Ty1 multimers (Nyswaner et al., 2008; Sundararajan et al., 2003) (Figure 2-5, Table 2-2).  Further, members of the Paf1C restrict Ty1 transposition, possibly post- transcriptionally or by limiting the amount of favourable hotspots for integration (Nyswaner et al., 2008).  Deletion mutants of three members of the Paf1C - cdc73∆, leo1∆ and rtf1∆ - are SL when combined with esp1-1 (Figure 2-5, Table 2-1).  Finally, two esp1-1 SDL hits are SPT genes (Figure 2-5, Table 2-2).  Either mutation (SPT10) or multicopy expression (SPT23) of these loci confers suppression of Ty1 insertion mutations (Burkett and Garfinkel, 1994; Fassler and Winston, 1988).  Clearly, several genetic interactors of esp1-1 have important roles in promoting or impeding successful Ty1 transposition.     82 3.2.2 Mass spectrometry reveals a physical interaction between Esp1 and Ty1 proteins  To corroborate the genetic interaction data, and better understand its functional significance, a search for physical interactors of Esp1 was also undertaken.   Nancy Fang, a Ph.D. student in Dr. Thibault Mayor’s laboratory at the University of British Columbia, performed an immunoprecipitation (IP) of 13Myc-tagged Esp1 and an untagged strain (mock IP).  The total number of peptides IP’d from the Esp1-13Myc strain was then compared to the mock, and we found 152 proteins that co-purify with Esp1 but were not enriched in mock experiments (Table 3-1).  Among the physical interactors identified was the known separase inhibitor, Pds1 (Ciosk et al., 1998).  Other proteins known to associate with Esp1 (Scc1, Slk19, Cdc55 and Zds1 as examples) may have been missed due to the transient nature or cell cycle specificity of these interactions (Queralt et al., 2006; Queralt and Uhlmann, 2008b; Sullivan et al., 2001; Sullivan and Uhlmann, 2003; Uhlmann et al., 2000).  In addition to the Esp1 co-purifying proteins, a more than five-fold increase of spectral counts for Ty1 peptides corresponding to Ty1-IN and Ty1-RT was observed (Figure 3-3A).  In comparison, peptides from the Ty1 coat protein and protease were readily identified in both the mock and Esp1 IPs, suggesting these Ty1 proteins may not specifically interact with Esp1.  I confirmed the physical interaction of Esp1 with Ty1 proteins by independent co-IP experiments.  The VLP can be detected at endogenous levels.  However, using current methods, the TyB polypeptides cannot and must be overexpressed (Curcio and Garfinkel, 1992).  Therefore, I induced pGTy1-H3, a Ty1 expression plasmid under control of the GAL1 promoter, in galactose media in both an untagged control strain and the Esp1-13Myc strain. Esp1-13Myc was purified, immunoblot analysis was performed and probed for the presence  83 of VLP, IN and RT (Figure 3-3B).   I found that IN was specifically present in the Esp1- 13Myc IP but not in the purification from the untagged strain.  VLP was also identified in the Esp1-13Myc IP, however high background levels of VLP were detected in the untagged strain IP suggesting that this interaction was not specific.  A very faint band corresponding to the RT in the Esp1-13Myc purification was also detected but the level was so low that it was difficult to determine that the RT was not present in the untagged control strain.  Both the mass spectrometry and co-IP data suggested that Esp1 specifically interacts with Ty1 IN and possibly– to a lesser extent – the RT.  Esp1 interacts with securin immediately after PDS1 expression in S phase until Pds1 is degraded in metaphase, whereby Esp1 is released to cleave the Scc1 cohesin (Jensen et al., 2001; Uhlmann et al., 1999; Uhlmann et al., 2000).  I was therefore interested in testing whether Pds1 and Scc1 could also physically interact with Ty1 polypeptides.  13Myc-tagged Pds1 and Scc1 were IP’d individually from yeast lysates and immunoblot analysis performed.  Similar to the analysis with Esp1, I found that Pds1-13Myc and Scc1-13Myc specifically co-purified with Ty1-IN, and a very faint band was detected when I probed the IPs with anti-RT antibodies (Figure 3-3C).  The VLP immunoblot had high levels of background in the untagged control strain, suggesting that this interaction is nonspecific. These results demonstrated that the interaction of Esp1 with Ty1 proteins may occur when Esp1 is in a complex with Pds1 and associated with Scc1.  3.2.3 esp1-1 has defects in Ty1 transposition  The interaction of esp1-1 with a large number of genes involved in Ty1 transposition along with mass spectrometry data suggested that Esp1 itself might have a role in Ty1  84 transposition. I employed a Ty1 transposition assay whereby Ty1 is overexpressed on a plasmid (pGAL1Ty1-H3mHIS3AI) carrying the HIS3 gene with an artificial intron (AI) effectively interrupting its expression.  After induction of Ty1 expression on galactose media, the intron is removed from the Ty1-H3mHIS3A1 mRNA intermediate, and successful cDNA integration renders the strain HIS3+(Curcio and Garfinkel, 1991) (Figure 3-4A).  To determine whether Esp1 has a role in Ty1 transposition, pGAL1Ty1-H3mHISA1 was transformed into WT, esp1-1 and spt10∆ strains.  SPT10, which was identified in the esp1-1 SDL screen, is required for efficient Ty1 transposition and was used as a negative control (Griffith et al., 2003).  After allowing strains to grow on dextrose plates, cells were replica plated to galactose media to induce transposition for two days.  To simultaneously impinge on Esp1 function in an esp1-1 mutant, strains were first grown at a permissive temperature (25˚C) overnight before shifting to a semi-restrictive temperature of 30˚C for the remainder of the 48 hours.  I chose these conditions as opposed to incubating immediately at 30˚C because increasing temperature results in a decrease in transposition efficiency (Lawler et al., 2002; Paquin and Williamson, 1984) (data not shown).  Transposition at 25˚C was also used as a comparative control.  After induction, strains were tested for successful transposition by plating on media lacking histidine (His).  At the permissive temperature of 25˚C, esp1-1 was able to induce transposition, as shown by colony growth on SC-His plates (Figure 3-4B).  However, when shifted to a semi-permissive temperature of 30˚C, only a few colonies from the esp1-1 strain were able to grow on SC-His media compared to a lawn of colonies in the WT strain.  The esp1-1 strain grew on SC media demonstrating that the strain is still viable at 30˚C (Figure 3- 3B).  The transposition defect of the esp1-1 mutant was also more severe than that of the  85 spt10∆ mutant at 30˚C (Figure 3-4B).  Therefore, Esp1 is required for efficient transposition of a Ty1 element.  Because Ty1 IN and VLP interacted with Pds1 and Scc1 in addition to Esp1, I tested if Ty1 transposition was affected in strains carrying mutations in the corresponding genes. I found that both pds1-128 and scc1-73 mutants were able to grow on SC-His media after induction of Ty1 transposition suggesting that, unlike Esp1, Pds1 and Scc1 are not required for Ty1 transposition at 30˚C (Figure 3-4C).   Since Esp1 is required to cleave Scc1 in anaphase, I also wondered if the Ty1 transposition defect in the esp1-1 mutant was due to the prolonged presence of cohesin.  Therefore, an esp1-1 scc1-73 double mutant strain was created and I found that the esp1-1 transposition defect was rescued in the double mutant (Figure 3-4D).  3.2.4 esp1-1 does not fail to induce the pGAL1Ty1-H3mHIS3AI plasmid  There are many reasons why a mutant of separase could affect the rate of Ty1 transposition.  The first possibility investigated was that the failure of esp1-1 to transpose the Ty1-HIS3+ element could be explained by an inability to properly induce the pGAL1Ty1- H3mHIS3AI plasmid.  Thus, IN levels were assessed by Dr. Lina Ma (a research associate in the Measday laboratory) for WT and esp1-1 strains carrying pGAL1Ty1-H3mHIS3AI that had been grown in galactose media for a total of 24 hours.  As endogenous levels of IN cannot be detected by current immunoblotting techniques, only strains that had successfully overexpressed pGAL1Ty1-H3mHIS3AI and processed the TyB polypeptide would have a band corresponding to IN when probed with the anti-IN 8B11 antibody (Curcio and Garfinkel, 1992; Eichinger and Boeke, 1990).  Therefore, induction of the vector was  86 successful in an esp1-1 mutant, as a visible IN band was observed by western blotting (Figure 3-5).  Lina also tested scc1-73 and the esp1-1 scc1-73 double mutants, and found that they too had the ability to induce the pGAL1Ty1-H3mHIS3AI plasmid.  Evidently, the HIS3 auxotrophy observed in esp1-1 is not due to a failure to overexpress the TY1 element.  3.2.5 Endogenous levels of TY1 mRNA are not decreased in esp1-1  As a number of esp1-1 SL and SDL genetic interactors are genes involved in RNA metabolism, I was curious to determine whether endogenous TY1 mRNA levels were affected in strains defective for separase activity.  Thus, mRNA levels of TY1 were measured using quantitative PCR (qPCR).  Though transposition efficiency decreases at higher temperatures, Lawler et al showed that this temperature sensitivity was due to a deficiency in the proteolysis of the Ty1 Pol polyprotein and not because of transcriptional or translational effects (Lawler et al., 2002; Paquin and Williamson, 1984).   I was therefore able to assay expression levels of TY1 for WT and esp1-1 at both a permissive (25˚C) as well as a non- permissive (35˚C) temperature.   TY1 mRNA expression levels were modestly (two-fold) elevated in an esp1-1 mutant as compared to control at the permissive temperature.  After a subsequent shift to 35˚C for two hours, both WT and esp1-1 TY1 mRNA levels were equivalently increased (one to two-fold) compared to mRNA levels at 25˚C (Figure 3-6A). There did not appear to be an observable temperature effect on TY1 expression levels that is specific to esp1-1.  The increase in TY1 mRNA levels in esp1-1 is interesting and may be due to a role for Esp1 in RNA metabolism (see: Chapter 4).  This qPCR analysis suggests that a decrease in Ty1 mRNA levels is not likely the cause for Ty1 transposition defects in the esp1-1 mutant.  87 3.2.6 Gag processing is intact in esp1-1  The protease known to be responsible for proteolytic processing of TyA-TyB is encoded within the element itself.  However, since Esp1 is also a protease, I was curious to determine whether Ty1 processing was affected in an esp1-1 mutant.  To that end, endogenous levels of precursor (p49) and mature (p45) gag were examined in both a WT and esp1-1 strain by immunoblot.  Gag production and processing are critical for both VLP maturation and reverse transcription and are thus considered a sensitive indicator of PR function (Adams et al., 1987; Checkley et al., 2010; Youngren et al., 1988b).  Thus, I subjected both WT and esp1-1 to a semi-permissive temperature of 30˚C for six hours. At neither 25˚C nor 30˚C did esp1-1 accumulate greater amounts of precursor (p49) than WT, and both have a greater level of p45 when compared to p49 (Figure 3-6B).  This is contrast to mutants defective in PR function such as P body mutants (dhh1∆, kem1∆, lsm1∆ and pat1∆) which showed ratios of p45/49 of ~0.8 (Checkley et al., 2010).   As the protease deficiency of esp1-1 did not appear to impinge on proteolytic processing of the TyA-TyB polypeptide, proteolysis of TyA-TyB is not likely to be the cause of the esp1-1 transposition deficiency. This data is consistent with the fact that proteolytic sites within the TyA-TyB polypeptide do not match recognition motifs for separase, suggesting that Esp1 activity is unlikely to be involved in these events (Merkulov et al., 2001; Merkulov et al., 1996; Sullivan et al., 2004).  3.2.7 Several cohesin mutants also show defects in Ty1 transposition  The failure of esp1-1 to successfully undergo Ty1 transposition could be a separase- specific phenotype or perhaps indicative of a more general consequence of failure to properly regulate sister chromatid segregation.  To distinguish between these two possibilities, I also  88 looked at Ty1 transposition in conditional alleles of the cohesin loader Scc2 (scc2-4) and a structural component of cohesin, Smc3 (smc3-1) (Figure 3-7).  Using the same Ty1 transposition assay described in 3.2.3, pGAL1Ty1-H3mHIS3AI was overexpressed in both scc2-4 and smc3-1 and assayed for the ability to become HIS3+.  Both scc2-4 and smc3-1 were able to grow on media lacking histidine at 25˚C, but continued to be auxotrophic for HIS3 when shifted to a semi-restrictive temperature.  As scc2-4 and smc3-1 both display premature sister chromatid segregation phenotypes, their failure to undergo Ty1 transposition would imply that cohesin-loading and a competent cohesin ring hinge domain are critical for retrotransposition to occur.  3.3 Discussion  In assessing the genetic interactome of esp1-1 (see: Chapter 2), I was surprised to find that although genes with a cell cycle function were uncovered in the esp1-1 SL and SDL screens, a number of genes identified in the screens have roles in other biological processes. Indeed, using mass spectrometry analysis, we made the unexpected discovery that Esp1 interacts with peptides encoded by the Ty1 retrotransposon.  I confirmed that Esp1, its inhibitor Pds1 and its substrate Scc1 all interacted with the Ty1-IN.  Moreover, esp1-1 mutants were unable to carry out Ty1 transposition and this defect was partially rescued by scc1-73.     89 3.3.1 Screening for genetic and physical interactions point to a potential role for Esp1 in Ty1 retrotransposition The esp1-1 genetic screens revealed that RNA metabolic processes are critical to esp1-1 viability (Figure 2-5).   As well, a prominent number of genes identified in our esp1-1 SL and SDL screens have a role in the life cycle of Ty1 retroelements (Figure 3-2).  The potential significance of this result was underscored when mass spectrometry data and subsequent co-IP confirmation studies demonstrated that Esp1 physically interacted with the Ty1-IN protein (Figure 3-3).  Note that in order to discount the possibility that these interactions are a result of the Myc epitope, an alternative Esp1 tag could be tested for co-IP with Ty1-IN, or an unrelated protein C-terminally tagged with Myc. I do not yet know if defects in Ty1 transposition are responsible for the cell lethality upon overexpression of genes such as Pat1 and Dhh1 in the esp1-1 mutant.  The esp1-1 SDL screen was performed at 25˚C where Ty1 transposition still occurs.  The SDL phenotype could be due to mistargeting of Ty1 elements into essential genes or in their regulatory regions.  Alternatively, errors in Ty1 element insertion may affect chromatin structure, which in combination with the defect in sister chromatid separation, is lethal.  Instead, Esp1 may have a novel role in regulating transcription and/or translation, which is discussed further in Chapter 4.  3.3.2 Assaying for Ty1 transposition in conditional alleles The Ty1-H3mHIS3AI element has provided researchers with an efficient mechanism of quantifying Ty1 transposition – a tool that has proven useful in both large-scale and low- throughput studies.  However, because of the temperature sensitive nature of  90 retrotransposition, essential genes have not to my knowledge been previously tested to determine the effect their disruption may have on the rate of retrotransposition.  In these studies, I used a modification of the conventional plasmid based assay that allowed sufficient transposition to occur in WT cells at a higher temperature while providing a mechanism for testing conditional alleles.  Using this technique, esp1-1 was shown to have defects in retrotransposition (Figure 3-4B).  I do not believe the transposition defect in esp1-1 was a consequence of temperature sensitivity, as other conditional mutants (pds1-128, scc1-73) did not display similar decreases in Ty1 transposition (Figure 3-4C).  Further evidence to this effect was demonstrated by the ability of scc1-73 to restore Ty1 retrotransposition in esp1-1 without substantial rescue of the temperature sensitivity (Figure 3-4D; data not shown). Thus, a modified Ty1-H3mHIS3AI assay using esp1-1 served as a proof of principle for a screen able to monitor Ty1 transposition in conditional alleles, while also suggesting Esp1 has a role in the stimulation of Ty1 retrotransposition events.  That said, it does remain possible that reversion of the his3∆1 auxotrophy could be accounted for by gene conversion rather than transposition, and it is this process that is affected in the mutant strains tested.  To investigate this possibility, a Southern blot analysis could be used to determine the frequency of the his3∆1 gene conversion, or the assay could be performed in a strain with the HIS3 allele completely deleted.  3.3.3 Esp1 has a role in promoting the integration of Ty1 cDNA during retrotransposition  Though Ty1 integration occurs through IN-mediated mechanisms, TY1 cDNA is also capable of inserting itself through homologous recombination – a process that is dependent  91 on the RAD52 DNA recombination/repair gene (Melamed et al., 1992; Resnick, 1975; Sharon et al., 1994).  I have been unable to determine whether Esp1 contributes to recombination mediated integration, as esp1-1 rad52∆ haploid double mutants were unable to germinate - an SL relationship that is likely a consequence of an increase in DNA damage induced by esp1-1 lethality in combination with defects in DNA repair (Mendoza et al., 2009; Paques and Haber, 1999; Symington, 2002) (data not shown).  However, further investigation into the relationship of Esp1 and IN indicated that an involvement for Esp1 in homologous recombination events is unlikely. A few aspects of the data suggest that Esp1 may more specifically have a role in TY1 cDNA integration into the genome and not in another part of the Ty1 life cycle.  First, endogenous TY1 mRNA levels were not substantially decreased in an esp1-1 mutant; indeed, TY1 mRNA levels may in fact be slightly elevated (Figure 3-5A).  Second, the ratio of endogenous Ty1 gag mature and precursor protein levels were not noticeably different in an esp1-1 strain, demonstrating that Esp1 did not affect proteolytic processing of the Ty1 polypeptide (Figure 3-5B).  Third, our lab has also examined the TY1 cDNA profile of both WT and esp1-1 strains by Southern blot (data not shown), and we did not observe any change in the levels of TY1 cDNA in separase mutants, suggesting that Esp1 is unlikely to affect transposition at the level of reverse transcription.  This assay also indicates that the esp1-1 transposition defect is not due to a failure to splice out the AI, as there was no detectable size difference between the WT and esp1-1 TY1 cDNA bands (data not shown).    Finally, Esp1 directly interacted with Ty1-IN - implicating Esp1 in targeting of TY1 cDNA into the genome (Figure 3-3B).  92 Following reverse transcription, TY1 cDNA and IN are transported into the nucleus as components of a pre-integration complex (PIC) that has not been well defined, though it is known that a nuclear localization sequence within Ty1-IN mediates its entry into the nucleus (Kenna et al., 1998; Moore et al., 1998).  The process of integration suggests two possible mechanisms of action for Esp1, which are not necessarily mutually exclusive:  (1) Esp1 is required for effective PIC entry into the nucleus, and (2) Esp1 is responsible for targeting of the PIC to hotspots of Ty1 integration.  Esp1 is initially expressed in the cytoplasm until S phase where Pds1 is expressed and the Esp1-Pds1 complex is transported into the nucleus (Jensen et al., 2001).  In strains lacking Pds1, Esp1 no longer localizes to the nucleus suggesting that the nuclear transport machinery recognizes the Esp1-Pds1 complex, and not Esp1 alone (Jensen et al., 2001).  Though I have not ruled out the possibility that the Esp1- Pds1 complex functions to promote entry of the PIC into the nucleus, successful Ty1 transposition in a pds1-128 strain would suggest that this is not the case.  pds1-128 strains have been shown to decrease the efficiency of the nuclear import of Esp1, thus if the function of Esp1 in mediating Ty1 transposition was strictly to aid nuclear import, I would expect pds1-128 strains to exhibit a similar transposition defect (Jensen et al., 2001).  3.3.4 Esp1 may be responsible for targeting the PIC to hotspots of Ty1 integration A model for PIC recruitment to hotspots of transposition In S phase, cohesin loads on chromosomes at sites defined by the Scc2/4 cohesin loading complex after which it moves to sites of convergent transcription (Ciosk et al., 2000; Lengronne et al., 2004).  The sites of cohesin loading correlate with tRNA sites and other genes transcribed by RNAPIII (D'Ambrosio et al., 2008).  Notably, Ty1 elements  93 preferentially integrate upstream of genes transcribed by RNAPIII, such as tRNA genes, in a loosely defined window ranging from about 80 to several hundred base pairs (Devine and Boeke, 1996; Hani and Feldmann, 1998; Ji et al., 1993; Kim et al., 1998).  This hallmark specificity of target integration events is by design to avert catastrophic insertion events – e.g. interruption of an essential genes (Boeke and Devine, 1998).  I therefore propose a model whereby an Esp1-Pds1-Scc1-IN complex may form shortly after cohesin has loaded during S phase, thereby targeting the PIC to preferred sites of integration.  I further speculate that upon targeting, cohesin must subsequently move away from sites of insertion or be removed altogether for IN-mediated Ty1 integration to occur (Figure 3-8). Evidence for an Esp1/cohesin mediated recruitment of PIC to hotpots of integration Evidence for an Esp1-Pds1-Scc1-IN complex formation for recruitment came from co-IP experiments demonstrating that each are capable of physically interacting with Ty1 IN (Figure 3-3).  However, as both pds1-128 and scc1-73 mutants did not show a transposition defect, it appears that the Esp1-IN interaction is critical for recruitment of the PIC complex (Figure 3-4).  Ty1 transposition defects in a conditional mutant of the Scc2 loader then further suggested that the timing of a cohesin-Esp1-PIC complex formation might be limited to S phase (Figure 3-7).  S phase is the perfect window of opportunity for successful integration, as chromosomes are in a decondensed state, providing the integration machinery with unimpeded access to the genome. Although the failure of smc3-1 mutants to undergo successful transposition is also consistent with this model, mutation of the Scc1 kleisin subunit would at first glance appear  94 to contradict it (Figure 3-4, 3-7).  However, the scc1-73 mutant supporting Ty1 transposition may still be consistent with the model that cohesin-Esp1 recruits the PIC.  Although scc1-73 interferes with the interaction of Scc1 with the Smc1/3 heterodimer, the possibility exists that scc1-73 mutants attempt loading of the Smc1/3 cohesin proteins, which would then fail to securely entrap sister chromatids due to the absence of a functional Scc1 (Haering et al., 2004).  If this is the case, Esp1 may be able to recruit the PIC by interacting with either the cohesin loader or Smc1/3 heterodimer – a physical interaction that I have not yet tested. Thus, the successful Ty1 transposition in the scc1-73 mutant does not necessarily preclude recruitment of the PIC to hotspots of integration by an Esp1-cohesin complex.  In addition, the scc1-73 mutant, also known as mcd1-1, is known to have sharp change in viability at its restrictive temperature.  Increasing the temperature to 30˚C may have been enough to rescue the Ty1 transposition defect of the esp1-1 allele, but not enough to impinge on a potential role for Scc1 in transposition [(Guacci et al., 1997) B. Lavoie, personal communication]. Further investigation into the phenotype of the scc1-73/mcd1-1 allele at 30˚C and perhaps using an integrated Ty1-mHIS3AI element to quantify the rate of transposition for this mutant may resolve some of these questions. Removal or translocation of cohesin may be necessary for successful integration  The impact that regulation of chromatin structure has on IN-mediated Ty1 integration was demonstrated when a member of the yeast ISW1 remodeling complex, Isw2, was shown to be recruited to tRNA gene sites by RNAPIII transcription.  Mutants of ISW2 were subsequently shown to disrupt the prototypical 80bp periodicity of Ty1 target sites  95 though actual recruitment to the tRNA gene target sites was not affected (Bachman et al., 2005; Gelbart et al., 2005).  Interestingly, human ISW1 complexes have also been associated with the loading of cohesin proteins (Hakimi et al., 2002).  The impact of nucleosome positioning on Ty1 element integration events reflects the importance of local chromatin structure on integration events.  Thus, just as cohesin translocation is necessary to allow the transcriptional machinery access to transcribed genes, once recruited, Ty1 IN may also require that chromatin be unhindered by cohesin for successful integration to occur (Lengronne et al., 2004).  This step would either likely occur through cohesin translocation or its local removal. scc2-4 mutants are known to support initial loading events, though cohesin is then unable to move from sites of loading (Lengronne et al., 2004).  Thus, the transposition defects are likely not a result of failure to recruit the Esp1-PIC complex to hotspots of transposition, but likely because cohesin fails to move from them.  Loading of cohesin onto DNA is thought to occur by ATP hydrolysis of the Smc head domains which disassociates them from the Scc1 kleisin, creating a gateway for DNA to pass through (Arumugam et al., 2003).  Likewise, ATP hydrolysis is necessary for translocation of cohesin from its initial loading sites (Hu et al., 2011).  It would therefore be of interest to determine whether ATP hydrolysis is necessary for Ty1 transposition. The rescue of the esp1-1 transposition defect by scc1-73 suggests that disruption of cohesin from sister chromatids is capable of rescuing the transposition defect of esp1-1. Whether cohesin is capable of translocation in an esp1-1 mutant has still yet to be determined.  Another possibility is that cohesin needs to be locally removed for IN to gain access to chromatin – a process that would be dependent on the protease activity of Esp1.  96 Studies in both fission and budding yeast have shown that separase is required for the local removal of cohesin during DNA double-strand break repair, therefore precedence exists for non-mitotic separase activity (McAleenan et al., 2013; Nagao et al., 2004).  The loading of cohesin at tRNA genes and the interaction of Ty1-IN with Esp1-Pds1 and Scc1 suggest a means for how Ty1 elements propagate themselves without damage to the host genome. Ty1-IN may hitch a ride with the cohesin loading machinery to insert Ty1-cDNA into regions of active transcription at the same time that cohesin is loaded onto chromatin.  3.4 Materials and methods 3.4.1 Yeast strains and growth conditions  All strains for this chapter are listed in Table 3-2.  Strains were grown in either rich (YPD) or minimal (SC) media and were incubated at 25˚C unless otherwise indicated. Standard protocols for yeast culture and transformation were followed (Guthrie and Fink, 2004).  Strains were C-terminally tagged with 13Myc using a PCR-based homologous recombination method (Longtine et al., 1998).  To assess endogenous expression of TY1, WT and esp1-1 were grown to log phase in YPD, spun down and resuspended in prewarmed YPD before being shifted to 35˚C for two hours.  2mL of cells were harvested for qPCR analysis.  Endogenous gag processing was also examined in WT and esp1-1; cells were grown to log phase and shifted to 30˚C for six hours. 10-25mL of cells was pelleted for western blot analysis (western blot sample preparation, see: section 2.4.4).  To ensure proper expression of the pGAL-Ty1H3-mHIS3AI plasmid in WT, esp1-1::NAT, scc1-73::KanMX6 and the esp1-1::NAT scc1-73::KanMX6 double mutant, strains transformed with the vector were grown in SC-URA + 2% raffinose + 0.1%  97 dextrose overnight to log phase.  Switching of carbon sources to 2% galactose induced expression of the plasmid, and samples for western blot analysis were taken after 24 hours (western blot sample preparation, see: section 2.4.4).  3.4.2 Affinity mass spectrometry Affinity IP and mass spectrometry Esp1 was IP’d using a one-step Myc-tag approach based on Field et al (Field et al., 1988).  Briefly, 1L each of WT and ESP1-13Myc::KanMX6 strains were grown to an O.D.600 of ~0.9.  Cells were lysed in 100mM Tris-HCl pH 7.5, 150mM NaCl, 0.1% Tween 20, 1% NP-40, 10% glycerol, 5mM EDTA, 1mM DTT, 1mM phenylmethylsulfonyl fluoride, 10mM chloroacetamide and a 1x protease inhibitor cocktail by glass bead beating. Lysates were cleared by centrifugation at 10,000 x g, 4˚C for 15 minutes. IPs were performed using a 9E10 anti-Myc antibody cross-linked to Sepharose (MMS-150P, Covance). 400µl beads (bed volume) were mixed with each lysate for four hours at 4˚C and washed five times in 100mM Tris-HCl pH 7.5, 500mM NaCl, 0.05% Tween 20, 0.5% NP-40, 10% glycerol, 5mM EDTA and 1mM DTT. Proteins were eluted twice with 200µL of SDS loading sample buffer without reducing agent prior to TCA precipitation. Proteins were then separated by SDS- PAGE followed by in-gel digestion for mass spectrometry analysis. The protein gel was first incubated in fixation buffer (5% acetic acid, 47.5% methanol) with coomassie Blue G for 30 minutes following by a four hour wash in H2O. Gel pieces from each sample (Esp1-IP and control) were divided into five fractions: three fractions above 55 kDa; one fraction bellow 55 kDa without immunoglobulins; and one fraction containing the immunoglobulin G heavy chain and light chains (55kDa and 25kDa bands). In-gel trypsin digestion was based on  98 Shevchenko et al. with minor changes (Shevchenko et al., 2000). Digestion buffer contained 50mM ammonium bicarbonate, and gel pieces were dehydrated using ethanol. Gel pieces were incubated at 56˚C for 45 minutes in 10mM DTT followed by a 30-minute incubation in 55mM chloroacetamide at room temperature. Peptides were extracted once with 0.5% acetic acid, twice with 30% acetonitrile, 0.5% acetic acid and twice with 100% acetonitrile. Dried peptide-extraction samples were resuspended in 0.5% acetic acid solution and acetified to a pH below 2 using 100% acetic acid. Peptide samples were then purified by solid phase extraction on C-18 stage tips (Ishihama et al., 2006; Rappsilber et al., 2003; Rappsilber et al., 2007). Purified peptides were analyzed using a linear-trapping quadrupole - Orbitrap mass spectrometer (LTQ-Orbitrap Velos; ThermoFisher Scientific) coupled to an Agilent 1200 Series high-performance liquid chromatography using a nanospray ionization source that included a 100-µm-inner diameter fused silica trap column packed with 5 µm-diameter Aqua C-18 beads (Phenomenex,, 50-µm-inner diameter fused silica fritted analytical column packed with 3 µm-diameter Reprosil-Pur C-18-AQ beads (Dr. Maisch, and a 20-µm-inner diameter fused silica gold coated spray tip. Fractions from each sample were run at a 60-minute high-performance liquid chromatography gradient from 0.5% acetic acid to 0.5% acetic acid and 26% acetonitrile). The LTQ-Orbitrap was set to acquire a full-range scan at 60,000 resolution from 350 to 1600 Th in the Orbitrap and to simultaneously fragment the top ten peptide ions in each cycle in the LTQ (minimum intensity 1000 counts).  Singly charged ions were excluded and parent ions were then excluded from MS/MS for the next 30 sec. The Orbitrap was continuously recalibrated using lock-mass function (Olsen et al., 2005).  99 Analysis of mass spectrometry data Centroided fragment peak lists were processed with Proteome Discoverer v. 1.2 (ThermoFisher Scientific) followed by a Mascot analysis (2.3.0, Matrix Science) against a Saccharomyces cerevisiae protein database (; 05012010, 6147 protein sequences) with the following parameters: peptide mass accuracy 10 parts per million; fragment mass accuracy 0.6 Da; trypsin enzyme specificity; fixed modifications – carbamidomethyl; variable modifications - methionine oxidation, lysine acetylation, serine/threonine/tyrosine phosphorylation; ESI-TRAP fragment characteristics.  Only those peptides with IonScores exceeding the individually calculated 99% confidence limit (false positive discovery rate <=1%) were considered as accurately identified.  3.4.3 Co-immunoprecipitation  WT as well as epitope-tagged ESP1-13Myc::KanMX6, PDS1-13Myc::KanMX6 and SCC1-13Myc::HIS3MX6 strains were transformed with pGAL-Ty1-H3 (pJEF724, generous gift from Jef Boeke) and were grown overnight to log phase in SC-URA + 2% raffinose + 0.1% dextrose.  Cells were then diluted down to an O.D.600  <0.08, resuspended in SC-URA + 2% galactose to induce Ty1 expression for 24 hours.  Cells were harvested, washed with ice cold H20 and resuspended in lysis buffer (50mM Tris-HCl pH 7.5, 10%NP-40, 350mM NaCl, 0.5mM EDTA, 1mM DTT and protease inhibitors) at 4˚C.  Glass beads were added, and the suspension vortexed two minutes on, two minutes off at 4˚C until > 90% lysis was achieved.  Lysates were centrifuged at 14 000 x g for ten minutes, and supernatant was kept as the WCL.  Protein concentration was quantified using NanoDrop 1000.   Equivalent amounts of protein were incubated with 20µL of a 1:1 Covance® 9E10 affinity matrix slurry  100 at 4˚C for 2.5 hours.  Beads were then washed four times with wash buffer (lysis buffer without protease inhibitors), and finally resuspended in 40µL 2X SDS loading buffer containing 200mM β mercaptoethanol.  Equal concentration of WCL, and equal amounts of IP were loaded for western blot analysis  3.4.4 Western blotting Samples were run on 8% or 10% SDS PAGE gels and transferred to polyvinylidene difluoride.  Membranes were probed with anti-c-Myc (1:5000, 9E10, Roche), anti-IN [1:1000, 8B11, J. Boeke (Eichinger and Boeke, 1990)],  anti-VLP [1:10 000, J. Boeke (Youngren et al., 1988a)], or anti-RT (1:1000, J.Boeke).  Blots were imaged with a ChemiDoc™ MP (Biorad).  3.4.5 Ty transposition assay  The pGAL1-Ty1H3-mHIS3AI plasmid was generously provided by Jef Boeke (Curcio and Garfinkel, 1991).  Strains were transformed with pGAL1-TY1H3-mHIS3AI and first grown on SC-URA + 2% dextrose plates.  After three days of growth, plates were then replicated to SC-URA + 2% galactose and grown for two days at either 25˚C (permissive temperature for esp1-1::NAT and scc1-73::KanMX6 strains) or 25˚C for 12-16 hours and then shifted to 30˚C (semi-restrictive temperature for esp1-1::NAT and scc1-73::KanMX6 strains).   Subsequently, plates were replicated to YPD for one day and then SC and SC- histidine media for two days.    101 3.4.6 Quantitative PCR analysis RNA preparation  Samples were thawed on ice and subjected to a modified RNA extraction protocol to extract total RNA (Rneasy Mini Kit, Qiagen).  100µL of buffer RLT with β-mercaptoethanol (10µL/mL) was added to pellets along with equivalent amount of glass beads.  After vortexing samples twice for 45 seconds (4˚C), with five minutes on ice between cycles, an additional 260µL of buffer RLT + β-mercaptoethanol was added and samples mixed by inversion.  Samples were then spun at 14 000 x g (4˚C) for two minutes.  The resulting supernatant was mixed with 350µL of 70% ethanol and immediately placed on provided columns.  Columns were then washed once with 700µL RWI buffer and twice with 500µL buffer RPE.  For each wash, columns were spun at 10 000 x g for 15 seconds with the exception of the last wash, which was spun for two minutes.  Residual ethanol was carefully pipetted off the ring of the column and the column switched to a fresh RNAse-free eppendorf.  40µL of diethylpyrocarbonate treated ddH2O was added and column spun at 10 000 x g for one minute.  1µL of each RNA sample was run on a 1% agarose gel as quality control, and then concentration determined by NanoDrop ND 1000. Conversion to cDNA  1µg of total RNA was used to generate cDNA in a 20µL reaction volume using the SuperScript® VILO™ kit (Invitrogen).  The reverse transcription reaction was performed using a themocycler (RoboCycler Gradient 96, Stratagene).  Samples were incubated at 25˚C for 10 minutes, 42˚C for 60 minutes, and finally 85˚C for five minutes.   102 Real-time PCR Real-time PCR was performed in triplicate for each sample in a 96 Well Optical Reaction Plate with Barcode (Applied Biosystems) with a reaction volume of 25µL for each replicate [14.65µL ddH2O, 6.25µL SYBR® Green Master Mix (Applied Biosystems), 0.05 µL each of 100mM forward and reverse primer, 4µL of 1/100 cDNA]. A standard two hour comparative PCR analysis was performed using a 7500 Real Time PCR System (Applied Bioystems).   Primers were designed against YML054W as a representative TY1 element using Primer Express v. 2.0 (Applied Biosystem) (forward primer, OVM760, 5’ ACCCACAGCAGTGCATGATG3’, and reverse primer, OVM761, 5’ TGGCCCAGGTGGAAAGTACA 3’).  TAF10 was used as an internal control [forward primer, OVM695, 5’ GGCGTGCAGCAGATTTCAC 3’, and reverse primer, OVM696, 5’ TGAGCCCGTATTCAGCAACA 3’] (Teste et al., 2009).   Relative transcript values and error bars (upper/lower limits) were quantified as described (Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR, Applied Biosystems, view at:    103 Table 3-1.  Affinity mass spectrometry peptides identified for Esp1 ORF Name Protein Score Peptides Identified Sequence Coverage (%) Peptides Identified in mock YNR016C ACC1    1020 46 17 10 YGL195W GCN1    799 35 10.7 0 YMR229C RRP5    392 32 13.9 6 YGR098C ESP1    546 29 14.3 0 YPL160W CDC60   738 29 23.5 6 YJL034W KAR2    608 29 32.4 7 YBR079C RPG1    451 22 21.6 5 YOR335C ALA1    522 21 20.9 2 YBL076C ILS1    571 21 17.1 5 YLR153C ACS2    318 20 16 5 YDR127W ARO1    313 19 11.4 4 YER036C ARB1    773 18 22 3 YGL206C CHC1    296 18 8.7 4 YPL226W NEW1    316 17 15.7 0 YDR037W KRS1    314 17 18.8 3 YHR020W YHR020W 349 16 28.6 0 YMR012W CLU1    370 16 13 0 YDR113C PDS1    359 16 32.4 0 YOR204W DED1    319 16 21.9 2 YIL018W RPL2B   381 16 35.8 3 YGL173C KEM1    272 15 8.3 0 YDL171C GLT1    308 15 5.8 3 YGR264C MES1    261 15 14.6 3 YKL210W UBA1    208 14 12 3 YOR153W PDR5    277 13 8.5 0 YLR342W FKS1    354 12 8.1 0 YER091C MET6    175 12 16.7 0 YGL137W SEC27   253 12 10.7 2 YPL131W RPL5    194 12 28.3 2 YGR285C ZUO1    231 12 17.1 2 YOR341W RPA190  189 11 5.9 0 YDL145C COP1    111 11 10 0 YOR168W GLN4    163 11 13.3 0 YDR238C SEC26   215 11 11.6 0 YOR361C PRT1    151 10 13 0 YHR027C RPN1    160 10 9.9 2 YGL207W SPT16   200 10 11 2  104 Table 3-1.  Affinity mass spectrometry peptides identified for Esp1 (continued) ORF Name Protein Score Peptides Identified Sequence Coverage (%) Peptides Identified in mock YIL041W GVP36   208 10 30.1 2 YMR108W ILV2    164 10 13.5 2 YHR047C AAP1    166 9 8.9 0 YGR061C ADE6    150 9 8 0 YGL234W ADE5,7  176 9 11.3 2 YEL046C GLY1    185 9 22.5 2 YBR196C PGI1    125 8 18.6 0 YDR234W LYS4    271 8 16.7 0 YDR341C YDR341C 81 8 11.9 0 YKR001C VPS1    117 8 13.1 0 YNL104C LEU4    74 8 11 0 YNL287W SEC21   215 8 9.4 0 YHR183W GND1    137 8 16.4 2 YGR162W TIF4631 233 7 10.2 0 YIL075C RPN2    75 7 5.6 0 YBR084W MIS1    157 7 8.3 0 YDL084W SUB2    135 7 14.3 0 YER086W ILV1    88 7 15.6 0 YFR009W GCN20   104 7 9.8 0 YEL031W SPF1    135 6 3.7 0 YLR106C MDN1    178 6 1.5 0 YDL143W CCT4    174 6 12.5 0 YER025W GCD11   135 6 13.3 0 YGL150C INO80   51 6 1.1 0 YGL210W YPT32   62 6 33.8 0 YGR185C TYS1    76 6 12.7 0 YMR080C NAM7    41 6 3.1 0 YOR046C DBP5    133 6 9.5 0 YGL120C PRP43   160 6 9 0 YGR175C ERG1    136 6 9.7 0 YLR100W ERG27   31 6 12.4 0 YPR181C SEC23   168 6 5.6 0 YGR218W CRM1    80 5 4.4 0 YKL145W RPT1    151 5 9.2 0 YNL132W KRE33   114 5 4 0 YBR143C SUP45   150 5 8.7 0 YCL018W LEU2    31 5 15.1 0  105 Table 3-1.  Affinity mass spectrometry peptides identified for Esp1 (continued) ORF Name Protein Score Peptides Identified Sequence Coverage (%) Peptides Identified in mock YCL040W GLK1    62 5 6.6 0 YCR009C RVS161  55 5 18.1 0 YDL140C RPO21   58 5 2.3 0 YER031C YPT31   51 5 24.7 0 YER062C HOR2    122 5 10 0 YFR010W UBP6    62 5 13.4 0 YGR116W SPT6    68 5 2.4 0 YKR095W MLP1    84 5 2.3 0 YLR058C SHM2    100 5 10.4 0 YLR447C VMA6    95 5 11 0 YBR080C SEC18   59 4 4.6 0 YDR382W RPP2B   195 4 60.9 0 YMR241W YHM2    245 4 7 0 YFR002W NIC96   51 4 4.3 0 YBL022C PIM1    122 4 3.8 0 YBR145W ADH5    75 4 2.3 0 YDL147W RPN5    70 4 8.5 0 YDR101C ARX1    55 4 5.1 0 YDR170C SEC7    68 4 1.8 0 YER070W RNR1    109 4 5.2 0 YER125W RSP5    51 4 6.8 0 YFL004W VTC2    61 4 5.8 0 YHR137W ARO9    53 4 6.2 0 YHR179W OYE2    75 4 10.5 0 YIR006C PAN1    57 4 2.8 0 YJL050W MTR4    93 4 3.9 0 YJR064W CCT5    92 4 6.9 0 YKL009W MRT4    82 4 20.3 0 YKL104C GFA1    97 4 7.3 0 YLR303W MET17   116 4 9.9 0 YLR384C IKI3    48 4 3 0 YMR038C CCS1    59 4 9.2 0 YMR125W STO1    78 4 4.9 0 YOR108W LEU9    74 4 6 0 YBL039C URA7    159 4 6.6 0 YBR039W ATP3    113 4 15.4 0 YBR221C PDB1    58 4 13.4 0  106 Table 3-1.  Affinity mass spectrometry peptides identified for Esp1 (continued) ORF Name Protein Score Peptides Identified Sequence Coverage (%) Peptides Identified in mock YDR211W GCD6    82 4 4.5 0 YDR406W PDR15   81 4 1.9 0 YER178W PDA1    44 4 7.4 0 YPR041W TIF5    68 4 9.1 0 YDR381W YRA1    99 3 14.2 0 YKL029C MAE1    140 3 7 0 YKL120W OAC1    74 3 10.8 0 YML123C PHO84   31 3 6.6 0 YPR019W MCM4    25 3 1.7 0 YCR057C PWP2    34 3 2.7 0 YCR084C TUP1    109 3 4.6 0 YDL100C GET3    36 3 7.9 0 YDR129C SAC6    187 3 5.8 0 YDR158W HOM2    63 3 5.8 0 YDR353W TRR1    144 3 15.7 0 YER003C PMI40   27 3 9.1 0 YER052C HOM3    41 3 6.6 0 YGL026C TRP5    98 3 5.9 0 YGR229C SMI1    69 3 5.9 0 YGR256W GND2    79 3 6.7 0 YHR098C SFB3    105 3 3.9 0 YIL109C SEC24   57 3 3.8 0 YJL033W HCA4    27 3 3.4 0 YKL126W YPK1    90 3 5.7 0 YKL216W URA1    33 3 4.8 0 YLL018C DPS1    53 3 6.5 0 YLR196W PWP1    73 3 4.5 0 YNL163C RIA1    31 3 2.4 0 YNL220W ADE12   66 3 5.3 0 YNL231C PDR16   26 3 9.4 0 YNL241C ZWF1    100 3 7.5 0 YNL307C MCK1    48 3 5.9 0 YOR151C RPB2    53 3 2.9 0 YOR165W SEY1    51 3 3.5 0 YPL043W NOP4    58 3 5 0 YPL058C PDR12   66 3 1.5 0 YPL190C NAB3    28 3 3.5 0  107 Table 3-1.  Affinity mass spectrometry peptides identified for Esp1 (continued) ORF Name Protein Score Peptides Identified Sequence Coverage (%) Peptides Identified in mock YPL242C IQG1    19 3 0.7 0 YDL058W USO1    53 3 1.5 0 YIL043C CBR1    34 3 9.9 0 YPL119C DBP1    0 3 2.9 0    108 Table 3-2.  Strains used in this chapter Strain Genotype Source Y7092  Mat α can1∆::STE2pr-Sp_his5 lyp1∆ his3∆1 leu2∆0 ura3∆0 met15∆0 (Tong and Boone, 2006) YKH64/65 Mat α can1∆::STE2pr-Sp_his5 lyp1∆ his3∆1 leu2∆0 ura3∆0 met15∆0  esp1-1::NATMX4 This study TS collection MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ ura3Δ0 pds1- 128::KanMX6 (Li et al., 2011) TS collection MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ ura3Δ0 scc1- 73::KanMX6 (Li et al., 2011) TS collection MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ ura3Δ0 scc2- 4::KanMX6 (Li et al., 2011) TS collection MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ ura3Δ0 smc3- 1::KanMX6 (Li et al., 2011) YKH612 MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ ura3Δ0 scc1- 73::KanMX6 esp1-1::NATMX6 This study YKH186 Mat a ura3-52 lys2-801_amber ade2-101 trp1-Δ63 his3- Δ200 leu2-Δ1 ESP1-13Myc::KanMX6 This study YM8 Mat a ura3-52 lys2-801_amber ade2-101 trp1-Δ63 his3- Δ200 leu2-Δ1 PDS1-13Myc::KanMX6 This study YKH407 Mat a ura3-52 lys2-801_amber ade2-101 trp1-Δ63 his3- Δ200 leu2-Δ1 SCC1-13Myc::His3MX6 This study    109                  Figure 3-1.  Structure of the Ty family of elements Cited from (Lesage and Todeschini, 2005).  Reprinted with permission © S Karger AG.   110                            Figure 3-2.  Genes identified in esp1-1 SL and SDL screens have roles in the life cycle of Ty1 retrotranposons The retrotransposition cycle of Ty1 elements can be described in the following steps: (A) transcription of the element by RNAPII (B) nuclear export (C) translation of retrotransposon mRNA (D) assembly and maturation of the VLP (E) reverse transcription of the Ty1 element to cDNA (F) import to the nucleus and (G) integration of the cDNA into the genome.  Host proteins that stimulate Ty1 retrotransposition are in green, host proteins that inhibit Ty1 retrotransposition are in red. Genes identified in the esp1-1 SDL screen are marked with filled stars while those identified in the SL screen are marked with outlined stars. Image adapted from (Beauregard et al., 2008). TY1 AAAA AAAA A A A Pat1 Dhh1 Lsm1-7 P Body Nucleus Cytoplasm Rad27 Paf1 Rtf1 Leo1 Sgs1 A B C D E F G esp1-1 SDL hits esp1-1 SL hitsAAAA TY1 mRNA Pat1/Dhh1 Lsm1-7 VLP TY1 cDNA  111        Esp1 IP 4 2 1 3 4 1 3 4 1 1 1 1 2 1 2 1 2 1 1 2 1 2 15 7 13 6 Esp1 IP Mock 2 1 1 1 2 0 1 3 0 0 0 0 0 0 0 0 0 0 0 1 0 0 7 4 0 1 Mock Pe pt id e D IL SV D Y TD IM K  EV H TN Q D PL D V SA SK  LN N N G IH IN N K  VA C Q LI M R  V TN II D R  Y V R PP PM LT SP N D FP N W V K  A H N V ST SN N SP ST D N D SI SK  ST TE PI Q LN N K  AV SP TD ST PP ST H TE D SK R  EV D PN IS ES N IL PS K  LD Q FN Y D A LT FD ED LN R  M LA H A N A Q TI R  N Q FQ A SV LV IQ M D R  SA PS Y FI SF TD ET TK  SD G TV LA PI V K  TL LD D C R  TV D TT N Y V IL Q G K  TV PQ IS D Q ET EK  EV N Q LL K  LG M EN SL TE K  LN V PL N PK  Y D EA IT Y N K  G ag /c oa t p ro te in  Pr ot ea se  In te gr as e R ev  T ra ns cr ip ta se /R N A se  H  A. B. Wild Type Esp1-13Myc WCL IP WCL IP IB: (descending) Myc, IN, VLP and RT IP: Myc Wi ld Ty pe Pd s1- 13 My c WCL IP WCL IP Sc c1 -13 My c WCL IP C. 55 55 130 100 Esp1-13Myc IN VLP RT IB: (descending) Myc, IN, VLP and RT IP: Myc Pds1-13Myc IN VLP RT Scc1-13Myc  112 Figure 3-3.  Mass Spectrometry reveals a physical interaction between Esp1 and Ty1-IN (A) Ty1 peptides identified in Esp1-13Myc mass spectrometry versus untagged (mock) strain are color coded as follows:  coat protein (blue), PR (yellow), IN (orange) and RT/RNAse H (green).  (B) and (C) Immunoblot (IB) of anti-Myc IPs carried out from untagged WT, Esp1- 13Myc Pds1-13Myc, Scc1-13Myc WCLs.  Expression of a Ty1 element (pGAL1-Ty1-H3) was induced in all strains for 24 hours prior to cell lysis.  Blots were probed with anti-Myc, anti-IN, anti-VLP and anti-RT antibodies.  Where possible, molecular weights are indicated to the left of blots.   113  B. W ild  T yp e es p1 -1 VS W ¨ SC SC - His SC SC - His Û& Shift C. SC SC - His SC SC - His pd s1 -1 28 sc c1 -7 3 Û& Shift A.  114          Figure 3-4.  The defect in Ty1 transposition in an esp1-1 mutant is rescued by scc1-73 (A) Design of the pGAL1Ty1-H3mHIS2AI.  Cited from (Curcio and Garfinkel, 1991). Reprinted with permission © Joan Curcio and David Garfinkel. (B-D) WT, esp1-1, spt10∆, pds1-128, scc1-73 and esp1-1 scc1-73 strains carrying pGAL1Ty1-H3mHIS3A1 were patched onto SC-URA + 2% galactose plates for two days, then replica plated to SC (control) or SC- His to test for successful transposition. Strains were tested at both 25˚C and a temperature shift (25˚C overnight during initial induction, and shifted to 30˚C for the rest of the assay).   D. SC SC - His SC SC - His es p1 -1 sc c1 -7 3 Û& Shift  115           Figure 3-5.  Expression of IN from the pGAL1Ty1-H3mHISA1 plasmid The pGAL1Ty1-H3mHISA1 plasmid was induced for 24 hours in galactose media in WT, esp1-1, scc1-73, esp1-1 scc1-73 strains.  Immunoblot probed with anti-IN; a non-specific band identified by anti-IN was used to ensure equal loading.  Molecular weights are indicated on left.  Analysis performed by Lina Ma.  IN Non-specific band Wi ld Ty pe esp 1-1 scc 1-7 3 esp 1-1  scc 1-7 3 100 70 27  116                              Figure 3-6.  esp1-1 strains are capable of Ty1 element expression and TyA-TyB polypeptide processing (A) Relative transcript levels of TY1 mRNA in the esp1-1 mutant compared to WT quantified by qPCR.  qPCR was performed in triplicate on samples taken at 25˚C and after a two hour incubation at 35˚C.   Samples are displayed on the X-axis while relative transcript level compared to WT Nz treatment is shown on the Y-axis.  Upper and lower limits for the three technical replicates are also displayed accordingly as error bars.  TAF10 was used as an internal control.  (B) Endogenous gag processing in WT versus the esp1-1 mutant at 25˚C and after 6 hours incubation at 30˚C.  p49 = unprocessed; p45 = processed.  spt3∆ serves as a negative control for gag expression  WT esp1-1  Û&  Û&  Û&  Û& VS W ¨ p49 p45 A. Wild Type - Û& Wild Type - Û& esp1-1 - Û& esp1-1 - Û& R el at iv e Tr an sc ri pt  L ev el s Sample B.  117                    Figure 3-7.  scc2-4 and smc3-1 strains have defects in transposition defects WT, scc2-4, smc3-1 and spt10∆ strains carrying pGAL1Ty1-H3mHIS3A1 were induced to undergo transposition on galactose media and tested for successful transposition indicated by a HIS3+ phenotype.  Strains were tested at both 25˚C and a temperature shift (25˚C overnight during initial induction, and shifted to 30˚C for the rest of the assay).  sc c2 -4 sm c3 -1 W ild  T yp e SC SC - His SC SC - His VS W ¨ Û& Shift  118                                     Figure 3-8.  Model for Esp1-mediated insertion As cohesin is loaded, Esp1 targets the PIC to hotspots of integration.  After targeting, cohesin slides or is removed to allow the integration of the cDNA.     119 Chapter  4: Examination of a role for the budding yeast separase, Esp1, in mRNA biogenesis 4.1 Introduction   Eukaryotic transcription is a result of the activity of the multimeric RNAPII, an enzyme conserved from yeast to humans [reviewed in (Young, 1991)].  The regulation of gene expression rests on the presence of DNA elements – upstream activation or repressive sequences.  Recognition of these elements is what drives transcriptional regulation by promoting (through transcriptional activators) or blocking (through transcriptional repressors) the recruitment of RNAPII.  Basal transcription is initiated through minimalist transcriptional architecture via assembly of the pre-initiation complex on the core promoter element.  In S. cerevisiae, assembly of this complex is dependent on co-activators, such as SAGA, TFIID and mediator, which recognize transcriptional activators and modulate RNAPII recruitment [reviewed in (Hahn and Young, 2011)] (Figure 4-1).  RNAPII is a distinctive RNA polymerase, because it possesses an extra C-terminal domain (CTD) on its largest subunit, Rpb1.  This CTD consists of multiple YSPTSPS heptamer repeats, which are highly phosphorylated throughout transcriptional events.  During assembly of the pre-initiation complex, the mediator complex functions as a bridge between upstream activators and RNAPII, recruiting the polymerase to the promoter.  In doing so, the CTD kinase activity of the transcription factor TFIIH is stimulated, phosphorylating serine 5 of the heptamer repeat.  This modification is present for the first few hundred nucleotides of elongation, and has been linked to mRNA capping events.  As serine 5 phosphorylation levels drop, serine 2 kinases are recruited to induce phosphorylation of the second serine residue, which in turn stimulates transcriptional elongation [reviewed in (Buratowski, 2009)]  120 (Figure 4-1).  In order to escape the nucleus to be translated, pre-mRNAs must first be capped, spliced and polyadenylated to form mature transcripts.  Capping is a three-step enzymatic process that adds a 7-methyl guanosine moiety to the 5’ of the transcript.  In the first step, an RNA 5’-triphosphatase (RTPase) removes the γ phosphate from the 5’ end of the mRNA and a guanosine triphosphate:mRNA guanylyltransferase (GTase) then transfers a guanine monophosphate to the newly exposed diphosphate.  This guanosine cap is subsequently methylated to produce the final cap (m7GpppN where N is the first nucleotide) [reviewed in: (Topisirovic et al., 2011)].  In budding yeast, the capping enzyme is a heterodimeric complex, with RTPase and GTase subunits encoded by CET1 and CEG1, respectively (Itoh et al., 1987; Shibagaki et al., 1992; Tsukamoto et al., 1997).   The importance of the cap is considerable. Not only is it necessary for efficient translation, but it also serves to protect transcripts from exonucleolytic degradation, as well as promoting the transcription, polyadenylation, splicing and nuclear export of the mRNA.  These processes are mediated by two known cap-binding proteins, one of which is found in the nucleus (nuclear cap binding complex) while the other is present in the cytoplasm (eIF4E).  The heterodimeric nuclear cap binding complex binds to mRNA transcripts as they are capped, and is critical for nuclear export and splicing while eIF4E binding is needed for cap-dependent recruitment of the ribosome (Topisirovic et al., 2011) (Figure 4-2).  Mature mRNA transcripts can subsequently be regulated by mRNA decay, which generally begins in budding yeast with shortening of the polyadenylated tail by the Pan2/Pan3 or Ccr4-NOT deadenylases (Boeck et al., 1996; Brown et al., 1996; Tucker et al., 2001).  The translational repressors Dhh1 and Pat1 then mediate assembly of the decapping  121 complex, recruiting other stimulatory factors such as the Lsm1-7 complex to initiate the activity of the decapping enzymes Dcp1/2 (Figure 4-3) [reviewed in (Ling et al., 2011)]. Upon removal of the cap, the transcript is decayed through Xrn1 5’ to 3’ exonucleolytic activity (Stevens, 1980) [for mRNA turnover review, see: (Meyer et al., 2004)].  This turnover became a point of interest in cellular regulation when it was found that eukaryotic cells have cytoplasmic ribonucleolytic complexes, such as P-bodies and stress granules, which are thought to be sites of translationally repressed mRNAs/transcript storage.  These transient structures are actively assembled on free mRNA transcripts and determine the cellular fate of these transcripts – decay, storage or ultimately translation [reviewed in (Kedersha and Anderson, 2009)].  The exact mechanisms by which events within cytoplasmic P-bodies are decided still remain a point of investigation.  Mature transcripts can also be carefully regulated throughout translation - indeed, 40% of the proteome variability cannot be accounted for by changes at the transcript level (Brockmann et al., 2007).  The events of translation can be succinctly described in four major stages: initiation, elongation, termination and recycling.  The small 40S subunit of the ribosome is first recruited to the transcript by general initiation factors (eukaryotic initiation factors, eIFs).  Scanning of the transcript then occurs until the start codon is identified, at which point an initiation tRNA is brought in, eIFs disassociate and the large 60S ribosomal subunit is recruited to form a functional 80S ribosome. Throughout elongation, the following cycle occurs: (1) binding of the relevant tRNA to the aminoacyl site of the ribosome (2) establishment of a peptide bond with the growing polypeptide in the peptidyl site, and finally (3) translocation of the ribosome so that a new tRNA can enter the aminoacyl site, the newly elongated peptidyl-tRNA moves to the peptidyl site and the newly deacylated tRNA transfers  122 to the exit site.  Synthesis of the polypeptide chain progresses until a stop codon is reached and termination is initiated [reviewed in (Marintchev and Wagner, 2004)].  The enrichment of genes involved in RNA metabolic processes in the esp1-1 SGA screen (see: Chapter 2) motivated an investigation of a potential role for the budding yeast separase in mRNA biogenesis – a notion that was further evidenced by closer examination of protein-protein interactions among the SL and SDL hits.  Interestingly, a role for separase in mRNA biogenesis has already been suggested when the overexpression of ESP1 was found to rescue the temperature sensitivity of capping enzyme mutant, ceg1-ts (Schwer and Shuman, 1996).  Follow up studies in this chapter on transcription and translation efficacy in esp1-1 mutants highlight mRNA regulation as a potential new area of Esp1 study.  4.2 Results 4.2.1 Protein-protein interactions amongst the confirmed esp1-1 SL and SDL hits are enriched for mRNA regulatory complexes   In the second chapter of this dissertation, I confirmed the phenotype of 15 SL and 44 SDL relationships for the esp1-1 mutant (see: section 2.2.3).  Surprisingly, GO analysis of these hits demonstrated that negative esp1-1 genetic interactions were enriched for genes involved in RNA metabolic processes (Figure 2-5).  To obtain a better understanding of the nature of this relationship, Anastasia Baryshnikova (Boone Lab) isolated the protein-protein interactions that exist strictly amongst the confirmed SL and SDL hits (Figure 4-4). Interestingly, two complexes involved in mRNA biogenesis were identified in this manner. Several genes whose products form the Paf1C were isolated in the esp1-1 SL screen: CDC73, LEO1, and RTF1 (Chang et al., 1999; Mueller and Jaehning, 2002; Wade et al.,  123 1996).  Paf1C is a transcriptional elongation complex that is distinct from mediator and directly affects the expression of only a specific subset of genes (Betz et al., 2002; Chang et al., 1999; Penheiter et al., 2005; Shi et al., 1997; Shi et al., 1996; Zhang et al., 2009).  This complex has also been implicated in posttranscriptional events as well, with mutants of the Paf1C being defective in pre-mRNA 3’ end processing (Penheiter et al., 2005; Sheldon et al., 2005).  Notably, deletion of any member of the Paf1C caused cell lethality in combination with the esp1-1 mutant, emphasizing the importance of Paf1C-mediated events in the survival of a conditional mutant of separase at permissive temperature. In addition to the enrichment of the Paf1C, factors involved in the decapping step of mRNA degradation were specifically identified in esp1-1 SL and SDL screens.  The overexpression of the stimulatory mRNA decapping factors PAT1 and DHH1 triggered cell lethality in esp1-1, as did the deletion of the Lsm genes, LSM1 and LSM7 (Bonnerot et al., 2000; Coller et al., 2001; Fischer and Weis, 2002; Sweet et al., 2012; Wyers et al., 2000) (Figure 4-4).  Lsm proteins in budding yeast are able to form two distinct heteroheptameric ring complexes; Lsm2-8 functions in mRNA splicing while Lsm1-7 stimulates decapping (Bouveret et al., 2000; Tharun et al., 2000).  The Dcp1-Dcp2 decapping enzyme itself was not identified in the screens; however, both Dcp1 and Dcp2 are essential genes which are not present in the haploid yeast deletion mutant collection (Dunckley and Parker, 1999; Giaever et al., 2002; LaGrandeur and Parker, 1998).  Though there was no consistency in whether it was the loss of or gain of function of these decapping factors that caused lethality in esp1-1, the regulation of mRNA decay appears to be consequential in maintaining cell viability.   124 Drugs affecting transcription and translation induce a growth phenotype in esp1-1  To determine if mRNA regulation is of critical importance to the esp1-1 strain, I monitored the effect of disrupting the transcriptional or translational machinery. I assessed the growth of the esp1-1 mutant in the presence of two different drugs at both permissive and semi-restrictive temperatures: 6-azauracil (6AU) and cycloheximide (CHX). 6AU inhibits inosine-5’-monophosphate dehydrogenase leading to a reduction in intracellular guanosine triphosphate pools and stalling of RNAPII (Exinger and Lacroute, 1992).  Meanwhile, CHX blocks translation by interfering with the translocation step of elongation (Schneider-Poetsch et al., 2010).  As a pyrimidine analogue, the action of 6AU requires that media where the drug is present be depleted of uracil  - meaning that strains tested for growth on 6AU must be capable of expressing the URA3 gene.  Therefore, the auxotrophic WT and esp1-1 mutant were first transformed with the centromeric URA3 pRS316 plasmid, and grown to log phase before being serially diluted and spotted onto SC-URA media supplemented with either dimethylsulfoxide (DMSO) or 100µg/mL 6AU (Sikorski and Hieter, 1989).   After five days of growth at a permissive temperature (25˚C), esp1-1 mutants were clearly sensitive to the presence of 6AU when compared to both WT and DMSO controls (Figure 4-5A).  Using a similar approach, WT and esp1-1 cells, without the pRS316 vector, were spotted onto YPD alone or YPD containing a subinhibitory amount of CHX (0.025µg/mL) (Figure 4-5B).  After three days of growth at 33˚C, CHX mildly rescued the esp1-1 ts phenotype.  Note that WT and esp1-1 cells here are both in the YPH499 background, where the esp1-1 restrictive temperature is slightly higher, allowing for some growth at 33˚C (data not shown).  125 The drug sensitivity data suggests that disruption of either transcription or translation has the opposite effects on esp1-1 growth.  Perhaps the sensitivity of esp1-1 to 6AU but the rescue by CHX reflects a role for Esp1 prior to translation – either in transcription or the post-transcriptional regulation of mRNA transcripts.  If this were the case, stalling transcriptional machinery would be detrimental to esp1-1; however, by using modest amounts of CHX - which would slow but not completely stall translational elongation - defects in transcription/post-transcriptional regulation may be more easily tolerated resulting in the increase of the esp1-1 restrictive temperature.  4.2.2 Separase-mediated gene expression Analysis of an inducible heat shock gene, HSP30  As discussed in Chapter 3, cohesin is translocated after loading at site of RNAPIII transcription to areas of convergent transcription. Lengronne et al found that alterations in the transcriptional program can influence cohesin positioning along chromosomes.  For example, upon activation of the heat shock gene HSP30, cohesin that initially covered most of the HSP30 ORF was quickly shifted downstream to the neighbouring MAK32/PET18 locus (Lengronne et al., 2004).  Similarly, a gene that is actively repressed during heat shock, SR09, was free of cohesin until a shift to high temperature resulted in the relocation of cohesin to the SR09 ORF (Gasch et al., 2000; Lengronne et al., 2004).  I therefore sought to investigate the possibility that separase is involved in the promotion of cohesin translocation, thereby influencing transcriptional events. To determine whether this is the case, the transcript level of HSP30 and SRO9 were evaluated in both a WT and esp1-1 mutant by qPCR (Figure 4-6).  WT and esp1-1 strains  126 were first treated with nocodazole (Nz) at 30˚C for two hours before being shifted to 37˚C for 15 minutes, again in media containing Nz.   The reason for the initial treatment was twofold. First, by arresting cells in metaphase using Nz, the sister chromatid segregation defect in the esp1-1 mutant at high temperature would not factor into the analysis when cells were subjected to heat shock.  Any difference in the growth rate between the two strains at restrictive temperatures would also be negated.  Second, a two hour incubation at 30˚C prior to heat shock would serve to suppress the activity of esp1-1 – an effect that would likely not be achieved within a 15-minute heat shock window.  Samples for real-time PCR analysis were taken both before and after heat shock. Prior to heat shock, the transcript level of both HSP30 and SR09 was slightly increased in an esp1-1 strain compared to WT (approximately 1.2-1.3 fold).  After a shift to 37˚C, HSP30 levels in WT cells rose fourfold as expected while SR09 transcripts showed a threefold decrease.  In contrast, esp1-1 mutants exhibited a stronger induction of HSP30 (sixfold) while SRO9 levels were comparable to WT.  A gene that did not undergo a change in cohesin pattern during heat shock, YCR016W, was also included and I found that although the amount of YCR016W mRNA decreased when shifted to 37˚C, there was no notable difference in transcript levels between WT and esp1-1 (Lengronne et al., 2004).  It is likely that in the event of a defect in heat shock-mediated cohesin relocation, HSP30 transcript levels would not rise as expected and amount of SR09 mRNA would either stay the same or decrease, depending on whether cohesin translocation is critical for SR09 repression.  As esp1-1 was able to induce HSP30 upon heat shock, separase does not appear critical for cohesin translocation; or, if indeed it is, there seems to be buffering mechanisms allowing the mutant to overcome this defect.  A possible explanation, since esp1-1 HSP30 levels appear to  127 increase even more readily than in WT, is that separase somehow contributes to the repression of transcription. Analysis of the silenced mating cassette through HMRA2  If Esp1 indeed does have a role in repressing transcription, it may also affect silencing.  In budding yeast, hallmark sites of silenced chromatin include the HM mating loci, telomeres and ribosomal RNA sites [reviewed in (Rusche et al., 2003)].  The HML and HMR loci, which each contain a cryptic copy of either MATa and MATα respectively, allow for interconversion of mating type in homothallic strains through the action of the site- specific HO endonuclease (Kostriken et al., 1983; Nickoloff et al., 1986; Strathern et al., 1982).  Silencing at HM mating loci is mediated by Sir proteins, but as it is a canonical heterochromatic site, I wondered whether Esp1 also had an effect on the silencing of the HM locus [reviewed in (Hickman et al., 2011)].  Thus, I examined the transcript levels of the HMRA2 allele by qPCR.  WT and esp1-1 cells were shifted to 35˚C for two hours in rich media, and samples were taken for real-time PCR analysis (Figure 4-7).  In comparison to WT, esp1-1 cells seemed to show a slight rise in HMRA2 mRNA at a permissive temperature (~1.4 fold), which grew more substantial upon shifting to a restrictive temperature.  This apparent increase may be consistent with a potential role for Esp1 in maintaining silenced chromatin.  However, as a similar phenomenon was not seen at ribosomal RNA loci (data not shown), the role of Esp1 in transcriptional regulation is likely more complex than initially anticipated.    128 4.2.3 Esp1 and decapping factors  Aside from transcriptional control, Esp1 may also function in the posttranscriptional regulation of mRNA transcripts.  The identification of mRNA decapping factors (PAT1, DHH1, lsm1∆, lsm7∆) highlighted the fact that Esp1 may function in mRNA biogenesis to regulate these factors (Figure 4-8).  As PAT1 and DHH1 overexpression negatively affected the viability of esp1-1, their gene products are the most obvious potential Esp1 targets since substrate overexpression is capable of causing an SDL phenotype in a relevant enzyme mutant (Measday and Hieter, 2002; Sopko et al., 2006).  I have already tested pGAL1/10- GST-PAT1 and pGAL1/10-GST-DHH1 as candidate substrates (see: section 2.2.6), and neither was shown to be an obvious Esp1 target.  However, in order to rule out that either was a substrate when under the influence of their native promoter or that their cleavage could be cell cycle specific, I subjected both epitope-tagged Pat1-HA and Dhh1-HA strains to various drug treatments that would result in cell cycle arrest at G1 (mating pheromone), S (HU) and metaphase (Nz).   After two hours of treatment, samples for each strain were taken for immunoblotting.  Neither Pat1 nor Dhh1 showed any difference in banding pattern at any of the stages of the cell cycle tested when compared to their log phase control or the pGAL1/10-GST-ORFX experiments (Figure 4-8A).  Subsequent analysis of esp1-1 cells grown at a restrictive temperature for a total of four hours confirmed that, for Pat1, no band visible by western analysis was due to Esp1 cleavage in logarithmically growing cells (Figure 4-8B).  As protease independent functions have also been ascribed to Esp1, even though Pat and Dhh1 were not demonstrably cleaved, I still wondered whether a physical interaction existed between Esp1 and either Pat1 or Dhh1(Sullivan and Uhlmann, 2003).  Esp1-13Myc  129 was IP’d from strains grown to log phase that also contained either Pat1 or Dhh1 epitope tagged with HA.   As neither Pat1-HA nor Dhh1-HA was visible above background in the IP fractions when samples were immunoblotted and probed for HA, no physical interaction between Esp1 and Pat1/Dhh1 was confirmed (Figure 4-8C).  It does, however, remain a possibility that a transient interaction occurs that was not detected by the attempted methods. Follow up work with IPs of cells that are arrested at specific stages of the cell cycle - or else treated with CHX to stimulate mRNA decapping - could help illuminate whether this remains a possibility.  4.2.4 Translational machinery is intact in an esp1-1 mutant  The identification of a genes in the esp1-1 SL and SDL screens that function in translation, such as elongation factor 2 which catalyzes ribosomal translocation during protein synthesis, prompted me to test if separase has a role in maintaining functional translational machinery (Perentesis et al., 1992).  I collaborated with Dr. Jeff Coller’s laboratory at Case Western University to analyze polysome formation in WT and esp1-1 strains.  After growing cells to log phase and then shifting them to 35˚C for two hours to suppress esp1-1 activity, CHX was added and cells were harvested to make extracts that were run on sucrose gradients to separate free 40S and 60S ribosomal subunits from polysomes. No increase in individual subunit peaks (40S, 60S) was observed in esp1-1, nor was any decrease in the amount of polysomes noted.  Therefore, mutants of esp1-1 appear to be capable of forming functional polysomes even at high temperatures, indicating that there are no defects in the translation of mRNA transcripts in this mutant.   130 4.3 Discussion  Screening for SL and SDL genetic interactions with the budding yeast separase mutant, esp1-1, as the query identified genes involved in RNA and DNA metabolism. Further examination of the protein-protein interactions amongst these hits has revealed a potential role for Esp1 in mRNA regulation.  Subsequent studies of gene expression, post- transcriptional regulation and translational control have illuminated several avenues through which Esp1 may exert influence on mRNA biogenesis.  4.3.1 A role for Esp1 prior to translation  The hallmark function of Esp1 is the cleavage of a specific subset of proteolytic targets – namely the Scc1 or Rec8 kleisin subunits of cohesin and the kinetochore associated Slk19 protein (Buonomo et al., 2000; Sullivan et al., 2001; Uhlmann et al., 1999; Uhlmann et al., 2000).  However, the examination of esp1-1 negative genetic interactions – most specifically, the enrichment of the Paf1C and mRNA decapping complex - intimated that separase-mediated regulation might not only occur post-translationally, but at the transcript level as well (Figure 4-1). Additionally, several pieces of evidence suggested that this potential role for Esp1 in mRNA regulation would likely occur prior to translation.  Firstly, drugs that inhibit translation would likely further exacerbate a mutant’s translational defects - but instead, the addition of CHX rescued the growth of esp1-1 (Figure 4-2).  Secondly, the translational machinery was intact in an esp1-1 mutant (Figure 4-9).  This data is consistent with the notion that separase functions directly in transcription or as a regulator of post-transcriptional modifications.  131 4.3.2 Sister chromatid cohesion and gene regulation: transcriptional implications for Esp1?  Studies have found that cohesin association patterns in the S. cerevisiae genome are influenced by the local transcriptional status (Bausch et al., 2007; Lengronne et al., 2004). Moreover, more recently, Skibbens et al have shown that cohesin inactivation in late G1 can cause significant transcriptional changes for 29 unique loci (Skibbens et al., 2010).  This direct influence of sister chromatid cohesion on transcription is consistent with other species. In Schizosaccharomyces pombe, for example, the Rad21 kleisin subunit promotes G2-specific transcription termination between convergent genes (Gullerova and Proudfoot, 2008). Nipped B and Stromalin, orthologues of Scc2 and Scc3 respectively, regulate the long-range activation of the cut gene in Drosophila melanogaster (Rollins et al., 2004).  And, crucially, differential gene expression patterns were observed in patients with Cornelia de Lange Syndrome – a clinical manifestation of mutations within the NIPLB cohesin regulator or the SMC1A cohesin structural component (Liu et al., 2009).  To my knowledge, separase, though involved in the regulation of sister chromatid cohesion, has not previously been associated with transcriptional regulation.  Yet, the enrichment of Paf1C mutants in esp1-1 SL interactions suggests that Esp1 might influence gene expression.  In considering how Esp1 may exert such a control, two avenues of possible explanations emerge, neither of which has been negated by these studies: (1) Esp1 directly activates/represses gene expression, or (2) Esp1 exerts transcriptional control through its influence on sister chromatid cohesion.  Notably, it was strictly the loss of function mutations in Paf1C genes that negatively impacted the growth of the esp1-1 mutant at a permissive temperature (Figure 4-1).  132 Interestingly, although associated with RNAPII at all transcriptionally active yeast loci, mutants of the Paf1C only affect a small subset of genes (~200) – a substantial number of which are involved in cell wall biosynthesis, RNAPII transcription and ribosomal RNA processing (Penheiter et al., 2005; Shi et al., 1997).  Therefore, it is reasonable to suggest that, like the Paf1C complex, separase may influence the expression of a specific number of genes, whose transcription would then be misregulated in esp1-1 mutants.  Such defects, in combination with the transcriptional irregularities introduced by Paf1C mutants, could then be lethal.  The observed increase in mRNA transcript level of the HSP30 and the HMRA2 loci in esp1-1 would suggest that this is a possibility (Figures 4-3, 4-4).  Note that the qPCR analysis was performed in triplicate on technical replicates, and would need to be repeated with biological replicates to determine whether this increase is in fact statistically significant. In addition, global microarray analysis would be necessary to state with certainty that Esp1 in fact regulates transcription.  The mechanism by which Esp1 may directly influence gene expression is also unclear at this point.  Testing of the HSP30 locus also implied that any transcriptional influence Esp1 may have is unlikely due to a failure in cohesin translocation (Figure 4-3).  However, this does not prohibit the possibility that Esp1’s influence on cohesin impacts gene expression in another manner.  Indeed, the noted increase in HMRA2 expression in separase mutants could implicate a role for Esp1 in maintaining silenced chromatin (Figure 4-4). Although the SL of Paf1C mutants with esp1-1 suggested that separase may have a role in transcription, the alternative prospect that ESP1 expression itself is subject to Paf1C regulation must also be given due consideration.  However, as ESP1 – or any obvious factors that may influence Esp1 activity such as PDS1, CDC20 and core cohesin components – were  133 not among the some 200 genes that exhibited changes in transcription upon deletion of critical Paf1C components, this explanation for the noted SL did not appear likely (Penheiter et al., 2005).  4.3.3 Esp1 and the 7-methylguanosine cap In comparison to the Paf1C, the genetic relationship between mRNA decapping factors and esp1-1 was not straightforward.  It was the loss of function of the Lsm proteins, but contrastingly the overexpression of the Pat1 and Dhh1 stimulatory decapping factors, that was the cause of lethality in an esp1-1 mutant (Figure 4-4).  Interestingly, a relationship between Esp1 and the 5’ 7-methylguanosine cap of mature transcripts was already established when the overexpression of ESP1 was found to be capable of rescuing the temperature sensitivity of a mutant of the budding yeast capping enzyme guanylyltransferase (CEG1) (Schwer and Shuman, 1996).  Specifically, it was the amino terminus of Esp1 – which lacks the catalytic domain – that proved sufficient for suppressing ceg1-ts growth defects.  Schwer and Shuman speculated to the existence of a physical interaction between Ceg1 and Esp1, thereby stabilizing the capping enzyme against degradation (Schwer and Shuman, 1996).  Another explanation, however, could be that Esp1 itself is directly involved in mRNA capping.  Indeed, Schwer and Shuman noted segments of homology between the N-terminal domain of Esp1 and the D12 subunit of the vaccinia virus capping enzyme (Schwer and Shuman, 1996).   Whether Ceg1 proves to be unstable in separase mutants or esp1-1 has defects in the capping reaction itself, both explanations could indeed explain how the misregulation of decapping factors impinges on esp1-1 viability.  Further, Paf1C mutants cause an increase in transcripts targeted for nonsense mediated decay due to defects in pre-  134 mRNA 3’ end processing (Penheiter et al., 2005).  In combination with capping mutants, the pervasive lack of stable mature transcripts could negatively impact cellular fitness.  As a caveat, however, it is notable that ceg1-ts mutants result in a decline in mRNA levels by pulse-chase experiments, which is contrary to what was found for the HSP30 and HMRA2 loci (Schwer et al., 1998).  Again, a genome wide analysis of esp1-1’s transcriptional profile at a restrictive temperature would be key in examining this further.  4.3.4 Esp1 and nuclear export Separase could also affect the rate of decapping – an explanation that could also account for the suppression of ceg1-ts defects.  A disruption of these activities in an esp1-1 mutant would also be exacerbated by the misregulation of decapping factors (Figure 4-1).  I could not find evidence for a direct physical relationship between Esp1 and either Pat1 or Dhh1, making a direct role in decapping unlikely (Figure 4-5).  So how else might Esp1 affect the rate of decapping?  The answer might again lie in esp1-1 SGA screening hits (Figure 2-5, Tables 2-1, 2-2).  APQ12, a gene that when deleted was found to be SL with esp1-1, has known defects in mRNA export (Baker et al., 2004).  This genetic relationship, along with the identification of a nucleoporin (nup60∆) as SL with esp1-1 and a Ran GTPase (GSP2) as SDL, could reflect a role for separase in the export of mature transcripts from the nucleus (Kadowaki et al., 1993; Rout et al., 2000) (Figure 2-5, Tables 2-1, 2-2).  4.3.5  A new landscape for Esp1 study  Investigating into the esp1-1 genetic interactome (see: Chapter 2) has ascribed a new area of Esp1 function in mRNA regulation that has not been previously explored.  The initial  135 studies described in this chapter have yet to elucidate the exact mechanism by which separase may influence the transcriptional landscape in S. cerevisiae.  Microarray studies which could examine gene expression across the genome in an esp1-1 mutant may help illuminate whether the transcriptional landscape itself is regulated by separase or instead Esp1 acts post- transcriptionally.  Subsequent investigations into the rate of mRNA capping/decapping and mRNA export and decay in esp1-1 would serve to facilitate further understanding of the exact nature of this new Esp1 function.  The studies presented in this chapter are a prelude to an exciting new area of investigation into separase function.  4.4 Materials and methods 4.4.1 Yeast strains and growth  Strains used for this study are indicated in Tables 4-1.   Strains were grown in either rich (YPD) or minimal (SC) media and were incubated at 25˚C unless otherwise indicated. Standard protocols for yeast culture and transformation were followed (Guthrie and Fink, 2004).  PAT1 and DHH1 were C-terminally tagged with 3HA::HIS3MX6 using a PCR-based method homologous recombination method for S. cerevisiae (Longtine et al., 1998).  For analysis of heat shock genes, WT and esp1-1::URA3 cells were grown to log phase, then treated for two hours with Nz (15µg/mL, Sigma-Aldrich) at 30˚C.  Cells were pelleted, resuspended in prewarmed YPD media, and incubated at 37˚C for 15 minutes. Samples were taken for fluorescence activated cell sorting (FACS, see: flow cytometry) and qPCR analysis before and after heat shock.  HMRA2 mRNA transcript levels were monitored in WT and esp1-1::NAT strains grown to log phase in YPD and then shifted to 35˚C for two hours.  Samples for qPCR were taken before and after temperature shift.  136  For observing Pat1 and Dhh1 banding patterns, WT strains carrying PAT1- HA::HIS3MX6 or DHH-:HA-HIS3MX6 alleles, generously provided by Roy Parker, were grown to log phase, and split into four, separate 10mL fractions.  One fraction was left untreated, while alpha factor (5µg/mL), HU (0.2M) or Nz (15µg/mL) was added to the other three fractions.  Samples were taken for FACS analysis, and the remainder of the fraction was harvested for immunoblotting. WT and esp1-1 were also grown to log phase in 50 mL of YPD.  Cells were then spun down and resuspended in prewarmed YPD and placed at 35˚C. Samples for immunoblotting were taken before and after four hours of temperature shift. Western blot sample preparation was as described in section 2.4.4, and 40µg of protein was run for each sample.  4.4.2 Spot assays WT and esp1-1::URA3 strains carrying the pRS316 vector were grown to mid-log phase (O.D.600 = 0.5-1.0) in SC-URA at which point cells were diluted to an O.D.600 of 0.1. Subsequently, cells were serially diluted four times by a factor of 1:5 in YPD.  4µL of each dilution was spotted onto SC-URA containing either DMSO or 100µg/mL 6AU (stock prepared in DMSO).  Plates were grown for a total of five days. The same strains, without pRS316, were also grown in YPD, serially diluted as described above, and 4µL of each dilution spotted on YPD and YPD plates supplemented with 0.025 µg/mL of CHX (stock prepared in ddH2O).  Plates were grown at 33˚C for three days.    137 4.4.3 qPCR analysis  RNA preparation and cDNA conversion for qPCR is described in and Real-time PCR was done in triplicate and analyzed as described in  For heat shock analysis, three genes of interest were investigated: HSP30 (primers: forward, OVM732, 5’ GCG GCTCCTGAAACAAAAGA 3’; reverse, OVM733, 5’ GGGTTCGTGGATTGCAG TCT), SR09 (primers: forward: OVM734, 5’ GAAGCCGCTACCGTCAATGT 3’; reverse, OVM735, 5’ TTGGCTTCACCGGTAACGTT 3’) and YCR016W (primers: forward, OVM736: 5’  HMRA2 was tested for the mating tester locus (forward primer, OVM754, 5’   CTT GGA CGA AAT CCC CTC AGT 3’; reverse, OVM755, 5’ TTC CAC ATA GTC GTT CCA TTG C 3’) GGCGGATGGGTTGGTATTTA 3’ ; reverse primer, OVM 755, 5’ GGGTTCTCGATGTTCTTTGCA 3’).  TAF10 was used as an internal control.  Primers were designed using Primer Express (Applied Biosystems).  4.4.4 Flow cytometry  For FACS analysis, 500µL-1mL of cells were harvested and resuspended in 1mL of 70% EtOH and kept overnight at 4˚C.  To process the samples, cells were pelleted, resuspended in 200µL of 1mg/mL RNAseA in 0.2M Tris-HCl pH7.5 and incubated overnight at 37˚C.  The next morning, 5µL of Proteinase K (20mg/mL) was added to the cells and incubated for an hour at 50˚C.    Cells were then pelleted, resuspended in 400µL of 0.2M Tris-HCl pH 7.5 and sonicated with a Branson sonifier for 20 seconds at 20% amplitude.  Samples were then diluted with 600µL of 1mg/mL propidium iodide diluted in 0.2M TrisHCl pH 7.5.  Samples were kept in the dark and allowed to stain for at least 30 minutes at room temperature.  Flow cytometry was performed by a BD FACScan (BD  138 Biosciences) with BD Cell Quest Pro software and analyzed using FlowJo (Version 10.0.4). Data was gated for 1N populations and greater, with N> 20 000.  4.4.5 Co-immunoprecipitation  WT and ESP1-13Myc::kanMX6 strains alone or in combination with PAT1- HA::HIS3MX6 or DHH1-HA::HIS3MX6 were grown in 50 mL of YPD.  Cells were harvested and lysed similarly to western blot samples (see: section 2.4.4).  Beads were prepared by washing, per sample, 15µL of immunoglobulin G coupled Dynabeads® (Invitrogen) three times with 1mL of 5mg/mL bovine serum albumin dissolved in phosphate buffered saline . Beads were then resuspended in the wash buffer to create a 30µL 1:1 bead slurry, and incubated with 1µL anti-c-Myc antibody (9E10, Roche) overnight at 4˚C.   For each strain tested, this 1:1 Myc-coupled bead slurry was added to 4mg of WCL and incubated overnight at 4˚C. After IP, beads were washed a total of 4 times with 1mL of 1X PBS/BSA and resuspended in 40µL 2x SDS loading buffer.  40µg of WCL and 5µL of IP were run for western blot analysis  4.4.6 Western blotting  All samples were run on a 10% SDS PAGE gels and transferred to a nitrocellulose membrane.  Blots were probed with either anti-Myc (1:5000, 9E10, Roche), anti-HA (1:500, 12CA5, Roche ) or anti-Pat1 antibodies [1:5000, generous gift from Roy Parker (Nissan et al., 2010)]    139 4.4.7 Polysome assay  Polysome analysis was then performed as described (Brengues et al., 2005).  200mL of WT and esp1-1::URA3 cells were grown to mid-log phase in synthetic media.  Cells were then pelleted, resuspended in prewarmed media and incubated at 35˚C for two hours.  After harvesting, CHX (212µL of 53µg/mL stock) was added and cells harvested.  Polysome extracts were then made and run on sucrose gradients to monitor polysome/ribosomal subunit distribution at an absorbance of 255 nm.  The polysome assay was done in the lab of Jeff Coller.   140 Table 4-1.  Strains used in this chapter Strain Genotype Source Y7092  Mat α can1∆::STE2pr-Sp_his5 lyp1∆ his3∆1 leu2∆0 ura3∆0 met15∆0 (Tong and Boone, 2006) YKH64/65 Mat α can1∆::STE2pr-Sp_his5 lyp1∆ his3∆1 leu2∆0 ura3∆0 met15∆0  esp1-1::NATMX4 This study YVM1761 Mat a ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1 P. Hieter YKH60 Mat a ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1 esp1-1::URA3 This study YKH186 Mat a ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1 ESP1-13myc::kanMX6 This study YJC751 Mat a ura3 leu2 his3 met15 PAT1-3HA::HIS3MX6 Parker lab YJC753 Mat a ura3 leu2 his3 met15 DHH1-3HA::HIS3MX6 Parker lab YKH299 Mat a S288Ca ESP1-13Myc::KanMX6 PAT1- 3HA::HIS3MX6 This study YKH305 Mat a S288Ca ESP1-13Myc::KanMX6 DHH1- 3HA::HIS3MX6 This study Deletion collection MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ ura3Δ0 pat1∆ (Giaever et al., 2002) aa combination of YPH499/YVM1761 (ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1) and yJC151 (ura3 leu2 his3 met15, Parker lab) thus the exact genotype is unknown   141                                Figure 4-1.  Overview of eukaryotic transcription (1-2) Transcription activation mediates assembly of the pre-initiation complex on the core promoter.  (3) Serine 5 phosphorylation of the Rpb1 CTD initiates transcriptional elongation. (4-5) Subsequent phosphorylation of Serine 2 stimulates processive elongation of RNAPII. (6-7) Termination signals cause diassociation and recycling of the RNAPII complex.  Cited from (Aygun and Svejstrup, 2010). Reprinted with permission © Elsevier.   142                     Figure 4-2.  Schematic representation of cap-dependent processes The 5’ mRNA cap binds to both nuclear (nuclear cap binding protein, denoted as nCBC here) and cytoplasmic (eIF4E) proteins to mediate important post-transcriptional events.  CE = capping enzyme, MT= methyltransferase, NMD = nonsense mediated decay, SMD = Staufen mediated decay.  Cited from (Topisirovic et al., 2011).  Reprinted with permission © John Wiley and Sons.    143                                  Figure 4-3.  mRNA decapping The translational repressors Pat1 and Dhh1 recruit the assembly of additional factors to mediate the removal of the 5’ cap by the Dcp1/2 decapping enzyme.  Cited from (Ling et al., 2011).  Reprinted with permission © John Wiley Sons.     144                        Figure 4-4.  esp1-1 protein-protein interaction network Protein-protein interactions among esp1-1 SL (red circles) and SDL (blue circles) genetic interactions.  Analysis was performed by Anastasia Baryshnikova.  Synthetic lethality hit Synthetic dosage lethality hit Protein-protein interaction Query gene mRNA decapping Paf1 complex  145                      Figure 4-5.  esp1-1 is sensitive to changes in mRNA regulation A serial dilution of esp1-1 and WT cells was spotted onto (A) YPD containing either DMSO or 100 µg/mL 6AU and incubated at the esp1-1 permissive temperature of 25˚C  (B) YPD or YPD containing 0.025 µg/mL CHX at the esp1-1 semi-permissive temperature of 33˚C.  Cycloheximide B. W ild Ty pe es p1 -1 DMSO 6-azauracil A. YPD W ild Ty pe es p1 -1  146                       Figure 4-6.  HSP30 mRNA levels are upregulated in the esp1-1 mutant in response to heat shock WT and esp1-1 cells were grown for 2 hours in Nz at 30˚C, and then subjected to a 15 minute heat shock.  qPCR analysis of the HSP30, SRO9 and YCR116W genes was performed in triplicate on cDNA from both strains before and after heat shock.  FACS analysis confirmed the metaphase cell cycle arrest induced by Nz treatment.   Samples are displayed on the X- axis while relative transcript level compared to WT Nz treatment is shown on the Y-axis. Upper and lower limits for the three technical replicates are also displayed accordingly as error bars.  TAF10 was used as an internal control.  0 1 2 3 4 5 6 7 !"#$%&'()%*+,-% ).(/0/%*+,-% !"#$%&'()%*1.-% ).(/0/%*1.-% HSP30 SR09 YCR016W Wild Type - Nz Wild Type - HS esp1-1- HSesp1-1- Nz 1N 2N N =2 0 00 00 Wild Type esp1-1 Samples R el at iv e Tr an sc ri pt  L ev el s  147                           Figure 4-7.  HMRA2 transcript levels are upregulated in esp1-1 WT and esp1-1 cells were grown at a restrictive temperature of 35˚C for two hours.  qPCR analysis of the HMRA2 locus was performed in triplicate on cDNA samples from before and after cells were shifted to the higher temperature.   Samples are displayed on the X-axis while relative transcript level compared to the WT 25˚C control is shown on the Y-axis.  TAF10 was used as an internal control.  Upper and lower limits for the three technical replicates are also displayed accordingly as error bars.  The location of the HMRA2 locus on chromosome III in relation to surrounding genes is also shown (, with the arrow denoting direction of transcription.  0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 !"#$%&'()%*+% ),(-.-%*+% !/%0+% ),(-.-%0+%Wild Type Û& Wild Type Û& esp1-1 Û& esp1-1 Û& Samples R el at iv e Tr an sc ri pt  L ev el s  148                                             /RJĮ)+81] /RJĮ)+81] 3DW+$ 'KK+$ Log Į) +8 1] Log Į) +8 1] 3DW+$ 'KK+$ 1 1 1      Wi ld Ty pe esp 1-1 SD W¨ Wi ld Ty pe esp 1-1 SD W¨ Û& Û& ,%Į3DW : &/ I P : &/ I P : &/ I P : &/ I P : &/ I P : &/ I P : &/ I P : &/ I P ,%Į0\F ,%Į+$ Wi ld Ty pe (V S 0 \F 'K K + $ Do ub le Wi ld Ty pe (V S 0 \F 3D W +$ Do ub le A. B. C.   55        55 ,%Į+$ ,30\F ,30\F  149 Figure 4-8.  Pat1 and Dhh1 do not physically interact with separase (A) Pat1-HA and Dhh1-3A full length and truncated protein migration in yeast lysates isolated from log phase, α-factor treated, HU treated and Nz treated cells.  Blots probed with anti-HA (B) Pat1 protein fragments in WT versus esp1-1 strains that were shifted from a 25˚C permissive temperature to a restrictive 35˚C temperature for four hours.  Blots were probed with anti-Pat1 antibody.  A line between lanes indicates that, though samples were run on the same gel, they were not run side by side. (C) Anti-Myc IP from WT, Esp1-13Myc, Pat1-HA, Dhh1-HA, Pat1-HA Esp1-13Myc and Dhh1-3HA Esp1-13Myc strains. Immunoblot analysis was performed and probed with anti-Myc to detect presence of Esp1, and anti-HA to detect presence of Pat1 or Dhh1 in the IPs.  Note: the ~55 kDa band present in all IPs (WT, Esp1-13Myc, Dhh1-HA and Double) shown in the HA immunoblot on the right corresponds to IgG.  Molecular weights for all immunoblots are indicated on the left.   150                    Figure 4-9.  Polyribosome analysis of the esp1-1 mutant Polyribosome formation was analyzed in WT versus the esp1-1 mutant at a non-permissive temperature of 35˚C.  The level of absorbance at 255nm is indicated by peak height, with density of fractions examined increasing from left to right.  Individual peaks corresponded to ribonucleoproteins (RNPs), the small (40S) and large (60S) ribosomal subunits, the fully functional 80S ribosome as well as polyribosomes. This analysis was performed in Jeff Coller’s lab.  Wild Type esp1-1 RNP 40S 60S 80S Polyribosome RNP 40S 60S 80S Polyribosome  151 Chapter  5: Conclusions and future directions 5.1 Summary and perspectives  Separin endopeptidases (separases) have been a marked area of research as they were discovered to be responsible for sister chromatid separation throughout the late 1990s to early 2000s (Uhlmann et al., 1999; Uhlmann et al., 2000).  Moreover, recent investigation into the clinical implications of separase activity has revealed a potential oncogenic role for hSeparase, with hSeparase overexpression common to many tumour profiles (Meyer et al., 2009).  hSeparase’s securin inhibitor, pituitary tumour transforming gene (PTTG), has also become a well-established and characterized oncogene, even serving as a marker for malignancy (Fong et al., 2012; Solbach et al., 2004).  As such, a thorough understanding of separase function using model organisms as a basis could prove important for future clinical research.  Indeed, the sister chromatid separation role of separase was first defined in budding yeast, predominantly through ground-breaking work from the Nasmyth laboratory while at the Research Institute of Molecular Pathology in Vienna, Austria (Uhlmann et al., 1999; Uhlmann et al., 2000).  Since then, research on Esp1 has uncovered further roles for the budding yeast separase in both spindle stability and mitotic exit (Jensen et al., 2001; Stegmeier et al., 2002; Sullivan et al., 2001).  Mitotic exit delays are also present in zebrafish mutants of separase, again emphasizing the relevance of model organism separase studies to higher eukaryotes (Shepard et al., 2007). Presently, there are three known separase targets in budding yeast: one mitotic (Scc1) and one meiotic (Rec8) cohesin protein as well as the kinetochore-associated Slk19 protein (Buonomo et al., 2000; Sullivan et al., 2001; Uhlmann et al., 1999; Uhlmann et al., 2000). However, to my knowledge, Esp1 function has not been examined before on a global level.  152 The goal of this dissertation was therefore to provide a genome-wide perspective of Esp1 that could generate data suggestive of a novel separase function in S. cerevisiae.   Taking advantage of the vast number of yeast genome collections, I collaborated with the Loewen laboratory here at the University of British Columbia to perform an array based robotic technique known as SGA, with the canonical esp1-1 separase mutant as a query (Tong et al., 2001).  Using this SGA analysis to probe both the yeast deletion collection as well as the 2 micron pGAL1/10-GST-ORFX overexpression collection for genetic interactions, I uncovered and confirmed 15 null mutants (geneX∆) and 44 overexpressed ORFs (pGAL1/10-GST- ORFX) capable of inducing lethality in the esp1-1 strain (Giaever et al., 2002; Sopko et al., 2006) (Figure 2-3 to 2-5, Tables 2-1 and 2-2).  Both known SL hits - hsc82∆ and lte1∆ - and an Esp1 substrate (SLK19) were identified, lending credence to the veracity of these screens (Sarin et al., 2004; Stegmeier et al., 2002; Sullivan et al., 2001).  Moreover, further examination of the genetic interactions using two approaches – GO analysis and comparison of the esp1-1 SL profile to the extensive database of query mutant SL profiles maintained by the Boone laboratory – further legitimized both screens, with both analyses identifying known Esp1 activities (Figures 2-5 and 2-6).  The observations presented in Chapter 2 of this dissertation therefore further support the evidence for already characterized roles for separase, and more excitingly, propose new avenues of separase study to pursue.  Two of these possibilities are presented in Chapter 3 and Chapter 4, and were followed up through work performed by Dr. Lina Ma and myself in the Measday laboratory as well as through collaborations with the Mayor laboratory here at the University of British Columbia and the Coller laboratory at Case Western University.  153 The most surprising finding from the SGA screens was the high enrichment of genes involved in RNA metabolic processes (Figure 2-5).  Many of the proteins identified within these functional pathways had known involvement in transposition events (Figure 3-2). Affinity purification of Esp1 followed by mass spectrometry performed by Nancy Fang in the Mayor laboratory uncovered a potential physical interaction between Esp1 and yeast Ty elements.  I confirmed that Esp1 interacts specifically with the IN portion of Ty1 elements by co-IP analysis (Figure 3-3).  Using a pGAL1-Ty1-H3mHIS3AI assay, I found that esp1-1, smc3-1 and scc2-4 mutants have transposition defects, and that the esp1-1 defect in Ty1 transposition is rescued by the scc1-73 conditional allele (Figure 3-4 and 3-7).  Interestingly, cohesin is first loaded at RNAPIII-transcribed genes while Ty1 integration is targeted upstream of genes transcribed by the same polymerase (Ciosk et al., 2000; D'Ambrosio et al., 2008; Devine and Boeke, 1996; Ji et al., 1993; Lengronne et al., 2004).  Taken together, these results suggested that Esp1 may somehow be involved in targeting of the PIC complex specifically to preferred hotspots of integration in the genome shortly after cohesin is loaded. Physical interactions amongst SL and SDL hits also highlighted an enrichment for the Paf1C and mRNA decapping proteins (Figure 4-4).  With a noticeable response of esp1-1 growth to drugs known to inhibit transcription/translation, I postulate that Esp1 may in fact function in mRNA biogenesis (Figure 4-5).  I would further speculate that this role is prior to translation, as separase mutants have intact translational machinery (Figure 4-9).  Analysis of expression levels of key genes in an esp1-1 mutant are consistent with the fact that separase may play a direct role in transcription (Figure 4-6 and 4-7).  However, other SL/SDL hits - and a prior study demonstrating a relationship between Esp1 and a subunit of mRNA capping enzyme - make a role in posttranscriptional modifications or nuclear export more likely  154 (Schwer and Shuman, 1996).  Both transposition and mRNA biogenesis are novel areas of study for Esp1 activity, and quite outside the realm of what is currently know about separase function.  In this way, these observations could prove significant to the already exciting field of separase study.  5.2 Future Studies 5.2.1 Saturation of separase genetic interactions The SGA screens presented in Chapter 2 have greatly increased the genetic interactome of esp1-1 – indeed, few SL and SDL interactions have previously been uncovered (see: for a full summary of known Esp1 physical and genetic interactions).  However, many cell cycle genes are essential and as such were not subject to SL screening.  There are several avenues to studying the ~1000 essential genes as conditional alleles, including promoter shutoff strains and heat-activated degron alleles (Dohmen and Varshavsky, 2005; Kanemaki et al., 2003).  There are also collections of hypomorphic alleles, e.g. the decreased abundance by mRNA perturbation (DAmP) collection (Breslow et al., 2008; Schuldiner et al., 2005). However, more recently, the Boone laboratory at the University of Toronto and the Hieter laboratory at the University of British Columbia has collated over 700 conditional ORFs under the control of their native promoters and linked to a kanMX marker for ease of SGA screening (Ben-Aroya et al., 2008; Li et al., 2011).  Performing another SGA analysis screen with this ts collection would allow us to amass another set of esp1-1 genetic relationships.  It may also be of interest to probe the non-essential deletion mutant array, the ts collection and the pGAL1/10-GST-ORFX collection using a different conditional mutant of esp1 as a query  155 strain, specifically one that interferes with a different aspect of separase function.  For example, the esp1-N5 mutant appears to have mutations confined to the first 500 bp of the ESP1 ORF and would be a good candidate as these analyses could serve to uncover further information about the as yet uncharacterized N-terminal domain of separase (Baskerville et al., 2008).  5.2.2 Further defining the role of separase in Ty1-mediated integration Testing the model  The model put forth in Chapter 3 suggests that the PIC is targeted in an Esp1 dependent manner shortly after cohesin is loaded, and subsequent cohesin translocation - perhaps even opening of the ring - is necessary to fulfill the integration (Figure 3-8).  To more directly test that loading is necessary to integration, Smc proteins with defects in their ATPase domains (smc1E1158Q and smc3E1155Q) can be transformed with pGAL-Ty1- H3mHIS3AI and tested for efficiency of Ty1 integration as described (see: section 3.4.4) (Hu et al., 2011).  If cohesin loading/ATPase activity is in fact necessary for Ty1 transposition, the aforementioned mutants should reflect that Ty1 transposition is impeded and remain auxotrophic for HIS3 throughout the assay. I have also confirmed that transposition defects in esp1-1 are rescued by the simultaneous mutation of the cohesin protein scc1-73, perhaps suggesting that the cohesin ring must be opened to alleviate Ty1 integration errors caused by the lack of separase activity (Figure 3-4).  To determine conclusively that separase-mediated integration is dependent on Esp1 proteolytic activity, a protease-deficient esp1 strain (esp1C1531A) can be examined (Uhlmann et al., 2000).  As the mutation on its own is lethal, esp1C1531A is expressed under  156 inducible control through a GAL1 promoter at the TRP1 locus.  Two strains should therefore be created - GAL1-ESP1 esp1-1 as a control and GAL1-esp1C1531A esp1-1 – and transformed with the pGAL1-Ty1-H3mHIS3AI vector.  Ty1 transposition assays will be performed as described in 3.4.4 and assessed for transposition efficiency by their ability to become HIS3+. Note that placing these strains on galactose media will allow the simultaneous expression of the TY1 element as well as the WT/protease deficient ESP1 allele.  WT ESP1 should consistently rescue the transposition defect of esp1-1, while the esp1C1531A allele would not be expected to if the proteolytic activity of separase is required for Ty1 transposition. Furthermore, under the conditions tested, the scc1-73 mutant itself did not appear to have transposition defects (Figure 3-4); however, the does not preclude the possibility that targeted Scc1 cleavage may in fact be necessary.  After examining whether separase proteolytic activity is necessary for Ty1 transposition, it would also be of interest to test a non-cleavable version of SCC1 (SCC1R180DR268D) using the pGAL1-Ty1-H3mHIS3AI assay (Uhlmann et al., 1999).  If Esp1 protease activity and Scc1 cleavage were necessary for Ty1 integration, it would support the notion that not only is translocation of the ring necessary for Ty1 integration necessary, but opening of the ring itself. Cohesin, Esp1 and the PIC The next step would then be to fully understand all the components of the cohesin complex that are required for Ty1 transposition by using the pGAL1-Ty1-H3mHIS3AI to test smc1, scc3, scc4, pds5 and wpl1 mutants.  Our lab has access to the extensive collection of ts mutants (Li et al., 2011).  Some ts mutants (pds1-128, scc1-73) have already been tested and do not display transposition defects (Figure 3-4).  Our methodology for examining ts mutants  157 for Ty1 transposition efficiency through the pGAL1-Ty1-H3mHIS3AI system, however, has the caveat that all ts mutants have different restrictive temperatures.  Thus, an alternative possibility would be to use the aforementioned DAmP collection. 842 essential yeast genes – including ESP1, PDS1, and all the major cohesin proteins - have been collected, with their 3’ untranslated region disrupted by an antibiotic resistance cassette (Breslow et al., 2008).  As a result of this disruption, the mRNA transcript levels for these genes are reduced two to tenfold.  Strains of interest can quite easily be obtained through Thermo Scientific and transformed with pGAL1-Ty1-H3mHIS3AI, affording us the ability to have consistency across strains and perform the assay at 25˚C as well (see: section 3.4.4).  Transposition efficiency can again be measured by a HIS3+ phenotype to determine which cohesin proteins are necessary for Ty1 tranposition.  Subsequently, all cohesin proteins of interest can be endogenously epitope tagged and examined for an interaction with the PIC through the co-IP approaches used for Esp1-13myc, Pds1-13myc and Scc1-13myc (see: section 3.4.3).  As I suspect that Esp1 is the critical bridge between the PIC and cohesin, it would be of interest to also examine these interactions in an esp1-1 strain that has been incubated at a restrictive temperature to repress Esp1 activity. Finally, to test whether integration patterns of Esp1 and cohesin proteins mediate correct Ty1 insertion events, Dr. Lina Ma in our laboratory is currently examining de novo transposition events that occur at the preferred SUF16 locus on chromosome III [see for details: (Nyswaner et al., 2008)].  WT, esp1-1 and cohesin mutant strains carrying the pGAL1-Ty1-H3mHIS3A1 plasmid are induced in triplicate in liquid galactose media for 24 hours first at the permissive temperature of 25˚C.  She will then also optimize a temperature for this assay that will allow us to inactivate Esp1 or cohesin but maintain some level of  158 transposition. Genomic DNA will be prepared and PCR analysis will be performed using one primer within the SNR33 gene and one primer in TY1.  TY1 insertion events are expected as a ladder of PCR products ~1000bp to 1700bp. If esp1-1 or cohesin mutants have defects in targeting Ty1 either no PCR products will be detected, or a change in the pattern of Ty1 integration events (e.g. changes in PCR banding patterns) will be noticeable.  5.2.3 Esp1 and mRNA biogenesis  As mentioned in discussing the observations presented in Chapter 4, the first approach to investigating Esp1’s role in mRNA biogenesis would be to determine definitively the manner in which Esp1 facilitates mRNA events – either a direct involvement in transcription or a post-transcriptional function.  Global examination of the impact of the esp1-1 mutation on transcriptional events would involve a collaboration with Dr. Zongli Luo in the Van Vuuren laboratory at the University of British Columbia.  WT and esp1-1 cells will be grown to log phase in rich media and shifted to the restrictive temperature of 35˚C. Total mRNA will be prepared from each strain using an RNeasy Mini Kit (Qiagen) and hybridized to GeneChIP Yeast Genome 2.0 (Affymetrix) S. cerevisiae microarrays, which are comprised of 5,744 probe sets for 5841 yeast genes.   Transcriptional events in esp1-1 mutants will be compared and contrasted to WT to determine the extent of transcriptional changes that exist when separase activity is repressed.  Further, our lab could examine whether RNAPII recruitment is altered in separase mutants in collaboration with the Howe and Kobor laboratories at the University of British Columbia.  WT and esp1-1 cells shifted to a nonpermissive temperature will be treated with 1% formaldehyde prior to chromatin extraction and shearing.  After cross link reversal, DNA that contacts polymerase will be  159 purified using antibodies against RNAPII and then amplified, labeled with biotin, and finally hybridized to GeneChip 1.0R (Affymetrix) S. cerevisiae microarrays, which are comprised of over 3.2 million probes covering the complete genome (Schulze et al., 2009).   Defects in polymerase recruitment in esp1-1 mutants would again be suggestive of a direct role in transcriptional stimulation.  As also indicated in Chapter 4, I feel it is more likely that Esp1 has a role in posttranscriptional events.  Overexpression of ESP1 has already been shown to alleviate the ts defects of the mRNA capping enzyme ceg1 mutant.  To examine the relationship between Esp1 and Ceg1 more in depth, I propose adding a C-terminal 3HA epitope tag to CEG1 and mating it to ESP1-13myc::KanMX6.  The resultant strain can then be used to determine whether Esp1 and Ceg1 co-IP, with a protocol similar to one described in 4.4.3.  Further, we can examine the amount of capped versus uncapped pre-mRNA in the esp1-1 for specific genes of interest by preparing total RNA from both WT and esp1-1 cells grown at the restrictive temperature and then using anti-m7G antibodies to IP capped mRNA.  Uncapped (supernatant) versus capped (IP’d) mRNA can then be examined using Northern analysis (He et al., 2008). The finding that esp1-1 SL/SDL hits enriched for nuclear export factors as well suggest that separase may have a role in nuclear export.   Our laboratory can perform a fairly conventional in situ hybridization of polyadenlyated RNA.  WT and esp1-1 cells will be grown at a restrictive temperature for two hours.  These cells will then be fixed with formaldehyde, adhered to slides and hybridized with labeled oligo (dT).  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