The Pennsylvania State University The Graduate School Department of Biochemistry and Molecular Biology GLOBAL REGULATION OF GENE EXPRESSION IN SACCHAROMYCES CEREVISIAE VIA TATA BINDING PROTEIN REGULATORY FACTORS A Thesis in Biochemistry, Microbiology, and Molecular Biology by Kathryn L. Huisinga Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2005 The thesis of Kathryn L. Huisinga was reviewed and approved* by the following: B. Franklin Pugh Professor of Biochemistry and Molecular Biology Thesis Advisor Chair of Committee Joseph C. Reese Associate Professor of Biochemistry and Molecular Biology Ross C. Hardison T. Ming Chu Professor of Biochemistry and Molecular Biology Naomi S. Altman Associate Professor of Statistics Robert A. Schlegal Professor of Biochemistry and Molecular Biology Head of the Department of Biochemistry and Molecular Biology *Signatures are on file in the Graduate School ABSTRACT The TATA Binding Protein (TBP) is a key component of gene regulation. It binds to the promoter region of eukaryotic genes and facilitates assembly of the transcription initiation machinery, including RNA Polymerase II. Many proteins interact with TBP to both positively and negatively regulate gene expression. My thesis utilized genome-wide expression profiling in Saccharomyces cerevisiae to define the target genes of, and relationships between, the factors that regulate transcription via TBP. I found the SAGA and TFIID co-activator complexes, both of which can deliver TBP to promoters, make overlapping contributions to the expression of nearly all yeast genes. The SAGA complex functions predominantly at ~10% of the genome, targeting genes that contain TATA boxes and are up regulated upon an environmental stress response. TFIID functions primarily at TATA-less genes, including genes down-regulated upon stress, while playing a predominant role in activation at the remaining 90% of the genome. The SAGA-dominated genes tend to be coordinately down regulated by a variety of additional transcriptional regulators. They include Mot1 and the NC2 complex both of which down-regulate transcription primarily at TATA-containing genes, in a manner that often counteracts the positive role of SAGA. Additionally, Mot1 and NC2 target many of the same genes, whose down-regulation generally requires the combined action of both factors. Mutations that disrupt repression of transcription via TBP dimerization increase expression of very lowly expressed genes, generally located in subtelomeric regions that are also repressed by chromatin-associated mechanisms. TBP interactions with DNA are iii most critical at highly transcribed genes, and the TAF1 N-terminal domain plays a minimal role by itself in gene regulation but is partially redundant with other regulatory factors. In total, this work has defined a highly intertwined, gene-specific, network of transcription regulators, which stimulate and inhibit gene expression via interactions with the TATA Binding Protein. iv TABLE OF CONTENTS LIST OF FIGURES.........................................................................................................x LIST OF TABLES ........................................................................................................xii ACKNOWLEDGEMENTS..........................................................................................xiii 1 Introduction: An Overview of the Regulation of Gene Expression .............................1 1.1 Genes and Gene Expression................................................................................1 1.2 Regulation of RNA Polymerase II transcription ..................................................2 1.3 TATA Binding Protein’s Role in Pol II transcription.........................................10 1.3.1 Identification of TFIID in yeast and higher eukaryotes ...............................10 1.3.2 TBP Structure and Interaction with DNA ...................................................12 1.3.3 Role of yeast TAFs in transcription ............................................................13 1.3.4 Regulators of TBP......................................................................................14 1.4 A Genome-wide approach to investigating transcription regulation...................15 1.4.1 Genome-wide Technology .........................................................................15 1.4.2 Benefits and Limitations of a Genome-wide approach................................16 1.5 References ........................................................................................................18 2 Interplay of TBP Inhibitors in Global Transcriptional Control .................................26 2.1 Summary ..........................................................................................................26 2.2 Introduction ......................................................................................................27 2.3 Results..............................................................................................................33 2.3.1 Genome-wide Effects of ΔTAND and TBP Mutations................................33 v 2.3.2 Distinct Gene Expression Groups Reveal Combinatorial Interactions of TBP Regulators .............................................................................................................39 2.3.3 Repressive Subtelomeric Regions are Intrinsically Accessible to the General Transcription Machinery........................................................................................47 2.4 Discussion ........................................................................................................49 2.4.1 The Yeast Genome is Negatively Regulated in Part by a Variety of TBP Inhibitors ...............................................................................................................49 2.4.2 NC2 Attenuates Highly Active Genes ........................................................51 2.4.3 Multiple Inhibitory Interactions Along TBP’s Concave Surface Provide Redundant Mechanisms for Preventing Unregulated Transcription ........................53 2.4.4 The Repressive Subtelomeric Environment is Accessible to the General Transcription Machinery........................................................................................55 2.5 Experimental Procedures ..................................................................................57 2.6 References ........................................................................................................65 3 A genome-wide housekeeping role for TFIID and a highly regulated stress- related role for SAGA in Saccharomyces cerevisiae ...............................................72 3.1 Summary ..........................................................................................................72 3.2 Introduction ......................................................................................................73 3.3 Results..............................................................................................................75 3.3.1 Determination of transcriptional effects via expression microarray analysis75 3.3.2 TFIID and SAGA each contribute to the expression of nearly all genes......79 3.3.3 TFIID dominates at ~90% of all genes, while SAGA dominates at ~10%...84 vi 3.3.4 Stress-induced genes tend to be SAGA-dominated while stress-repressed genes tend to be TFIID-dominated.........................................................................88 3.3.5 Genes having highly acetylated histone H4 tails tend to be TFIID-dominated 93 3.3.6 TAFs make a greater positive contribution at TFIID-dominated genes than at SAGA-dominated genes ........................................................................................96 3.3.7 Bdf1 and histone H4 tails are linked to TFIID regulation............................97 3.3.8 SAGA-dominated genes are coordinately regulated....................................99 3.4 Discussion ......................................................................................................101 3.4.1 TFIID and SAGA contribute to the expression of essentially all genes .....101 3.4.2 Shared TAFs are more important for TFIID than for SAGA.....................102 3.4.3 Histone H4 tail acetylation, Bdf1 binding, and TFIID function are linked.102 3.4.4 SAGA-dominated genes reveal a highly regulated stress-response pathway 103 3.5 Materials and methods ....................................................................................105 3.6 References ......................................................................................................108 4 Coordination of gene expression in Saccharomyces cerevisiae through positive and negative regulation of the TATA-Binding Protein............................. 117 4.1 Summary ........................................................................................................117 4.2 Introduction ....................................................................................................118 4.3 Results............................................................................................................126 4.3.1 Design of the study...................................................................................126 vii 4.3.2 Perturbing different combinations of interactions (nodes) in the TBP regulatory network have distinct effects on cell growth........................................131 4.3.3 A portion of the yeast genome is subject