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Yyou\ the Saccharomyces Cerevisiae Paf1 Transcription Elongation The Saccharomyces cerevisiae Paf1 transcription elongation complex is connected to chromatin modification through the multifunctional Rtf1 subunit and the inositol polyphosphate signaling pathway by Marcie Helene Warner B.S., Duquesne University, 2000 Submitted to the Graduate Faculty of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2008 YYou\ UNIVERSITY OF PITTSBURGH SCHOOL OF ARTS AND SCIENCES This dissertation was presented by Marcie Helene Warner It was defended on October 10, 2008 and approved by Deborah Chapman, PhD, Associate Professor, Dept. of Biological Sciences Sanford Leuba, PhD, Assistant Professor, Dept. of Cell Biology and Physiology William Saunders, PhD, Associate Professor, Dept. of Biological Sciences Anthony Schwacha, PhD, Assistant Professor, Dept. of Biological Sciences Dissertation Advisor: Karen Arndt, PhD, Associate Professor, Dept. of Biological Sciences ii Copyright © by Marcie Helene Warner 2008 iii The Saccharomyces cerevisiae Paf1 transcription elongation complex is connected to chromatin modification through the multifunctional Rtf1 subunit and the inositol polyphosphate signaling pathway Marcie Helene Warner, PhD University of Pittsburgh, 2008 Transcription in eukaryotes takes place in the context of a repressive chromatin template. Access to the DNA is facilitated by histone modifying enzymes and ATP-dependent chromatin remodeling complexes, which modify chromatin structure. The activities of chromatin modifying proteins are often coordinated by nonenzymatic accessory factors that interact with actively transcribing RNA Polymerase II (Pol II). One such factor is the Saccharomyces cerevisiae Paf1 transcription elongation complex. This complex, which is minimally composed of Paf1, Ctr9, Rtf1, Cdc73, and Leo1, physically interacts with Pol II and localizes to the coding regions of active genes. The Rtf1 subunit of the Paf1 complex performs several cotranscriptional functions: it facilitates recruitment of the chromatin remodeling enzyme Chd1, promotes covalent modification of specific lysine residues in histones H2B and H3, and mediates association of other Paf1 complex subunits. Using a collection of internal deletion mutations that remove 20 to 50 amino acid segments across the length of Rtf1, I demonstrated that Rtf1's known functions are mediated by nonoverlapping regions, implying that the multiple functions of this protein are not completely interrelated. Deletion of the regions of Rtf1 that are required for promoting histone modification or its association with active genes resulted in the strongest transcription-related phenotypes, which suggested that promoting cotranscriptional histone modification is a critical means by which Rtf1 exerts its effects on transcription. Detailed analysis of the region of Rtf1 iv required for histone modification determined that it is sufficient to promote Rtf1-dependent histone modifications and that this function is dependent on several highly conserved residues. Additionally, a screen for factors that become essential in the absence of Rtf1 uncovered mutations in the first two enzymes of the inositol polyphosphate (IP) signaling pathway: Plc1 and Arg82. The IP signaling pathway has been linked to the function of several chromatin remodeling complexes. I uncovered strong genetic interactions between Arg82, Paf1, and mutations in the SWI/SNF and INO80 chromatin remodeling complexes and demonstrated that the expression of several target genes was strongly impaired by mutations in these factors. Together, these data suggest that transcription elongation, IP signaling, and chromatin remodeling cooperate to coordinate proper gene expression. v TABLE OF CONTENTS PREFACE ................................................................................................................................. XVI 1.0 INTRODUCTION ........................................................................................................ 1 1.1 THE EUKARYOTIC GENOME IS PACKAGED INTO CHROMATIN .... 2 1.2 CHROMATIN STRUCTURE IS DYNAMICALLY REGULATED BY SEVERAL MECHANISMS ................................................................................................ 5 1.2.1 Nucleosome disassembly and the incorporation of histone variants .......... 5 1.2.2 Histone modification........................................................................................ 7 1.2.2.1 Histone Acetylation ............................................................................. 11 1.2.2.2 Histone Ubiquitylation ........................................................................ 14 1.2.2.3 Histone Methylation ............................................................................ 18 1.2.3 ATP-Dependent Chromatin Remodeling .................................................... 25 1.2.3.1 SWI/SNF .............................................................................................. 29 1.2.3.2 RSC ....................................................................................................... 30 1.2.3.3 ISW1 ..................................................................................................... 31 1.2.3.4 ISW2 ..................................................................................................... 32 1.2.3.5 CHD1 .................................................................................................... 33 1.2.3.6 INO80 ................................................................................................... 33 1.2.3.7 SWR1 .................................................................................................... 34 vi 1.3 TELOMERIC SILENCING IS CONTROLLED BY GLOBAL PATTERNS OF HISTONE MODIFICATION ..................................................................................... 35 1.4 TRANSCRIPTION BY RNA POLYMERASE II IS HIGHLY REGULATED BY ACCESSORY PROTEINS ............................................................... 37 1.4.1 Promoter Binding and Transcription Initiation ......................................... 44 1.4.1.1 Assembly of the Preinitiation Complex ............................................. 45 1.4.1.2 Transcriptional activators and coactivators ..................................... 49 1.4.2 Transcription Elongation .............................................................................. 51 1.4.2.1 Paf1 complex ........................................................................................ 51 1.4.2.2 Spt4/Spt5 complex ............................................................................... 61 1.4.2.3 TFIIS .................................................................................................... 62 1.4.2.4 yFACT .................................................................................................. 63 1.4.3 3’ End Formation and Transcription Termination at Protein Coding Genes ………………………………………………………………………………. 64 1.4.4 mRNA export ................................................................................................. 68 1.5 THE INOSITOL POLYPHOSPHATE SIGNALING PATHWAY AFFECTS TRANSCRIPTION ......................................................................................... 70 1.5.1 Arg82 is required for normal expression of arginine metabolic genes ..... 73 1.5.2 IP6 is necessary for mRNA export................................................................ 74 1.5.3 Chromatin remodeling is affected by inositol polyphosphates .................. 74 1.6 THESIS AIMS ................................................................................................... 76 2.0 RTF1 IS COMPOSED OF DISCRETE FUNCTIONAL REGIONS ................... 77 2.1 INTRODUCTION ............................................................................................. 77 vii 2.2 MATERIALS AND METHODS ...................................................................... 78 2.2.1 Media and yeast strains ................................................................................. 78 2.2.2 Plasmid construction ..................................................................................... 79 2.2.3 Yeast growth assays ....................................................................................... 80 2.2.4 Sequence Alignment ...................................................................................... 80 2.2.5 Immunoblotting analyses .............................................................................. 81 2.2.6 Analysis of histone H2B K123 monoubiquitylation ................................... 82 2.2.7 Chromatin immunoprecipitation assays ..................................................... 82 2.3 RESULTS ........................................................................................................... 86 2.3.1 Conserved regions of Rtf1 direct normal transcription ............................. 86 2.3.2 A region near the amino-terminus of Rtf1 mediates physical interaction with Chd1 .................................................................................................................... 92 2.3.3 Conserved regions of Rtf1 are required for Rtf1-dependent histone modifications and telomeric silencing ...................................................................... 96 2.3.4 Association of Rtf1 with active ORFs requires a conserved central region ………………………………………………………………………………. 99 2.3.5 Rtf1 interacts with other Paf1 complex components through its carboxy- terminus ...................................................................................................................
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