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Core Promoter Function in Transcriptional Regulation of Constitutively Expressed Genes in Drosophila

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Core Promoter Function in Transcriptional Regulation of Constitutively Expressed Genes in Drosophila The Pennsylvania State University The Graduate School Eberly College of Science CORE PROMOTER FUNCTION IN TRANSCRIPTIONAL REGULATION OF CONSTITUTIVELY EXPRESSED GENES IN DROSOPHILA A Dissertation in Biochemistry and Molecular Biology by Douglas G Baumann © 2018 Douglas G Baumann Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2018 The dissertation of Douglas G Baumann was reviewed and approved* by the following: David S. Gilmour Professor of Biochemistry and Molecular Biology Dissertation Adviser Chair of Committee Lu Bai Assistant Professor of Biochemistry and Molecular Biology Joseph C. Reese Professor of Biochemistry and Molecular Biology Shaun Mahony Assistant Professor of Biochemistry and Molecular Biology Tae-Hee Lee Associate Professor of Chemistry Wendy Hanna-Rose Associate Professor of Biochemistry and Molecular Biology Interim Department Head, Biochemistry and Molecular Biology * Signatures are on file in the Graduate School. ii Abstract Regulation of gene expression is a key driver of organismal development and cellular differentiation. Misregulation of gene expression disrupts development and drives many disease states. While cells modulate gene expression through a variety of processes, transcription is thought to be the most critical and highly regulated process in gene regulation. Ultimately, all transcription regulatory signals converge at the core promoter. Here, I investigate the function of the conserved Drosophila core promoter element Motif 1. Most of our knowledge of transcription initiation comes from studies on promoters with a TATA box and Initiator. While these studies have yielded invaluable information, most promoters lack this combination of elements. Thus, our knowledge of the mechanisms that drive initiation from promoters with different core promoter elements is limited. In order to better understand alternative mechanisms of initiation, I investigated ribosomal protein (RP) gene transcription in Drosophila. I focused on RP genes for several reasons. First, Motif 1 is enriched at RP gene promoters in Drosophila. Second, TBP is not required for transcription of RP genes. Third, in all higher eukaryotes examined, the TCT motif replaces the Initiator sequence at RP gene transcription start sites. Finally, RP genes represent a well-defined gene-regulatory network and are thought to be coordinately expressed. For these reasons, the RP genes offer a compelling model system to study alternative initiation mechanisms and the coordinate transcription of gene networks. Here, I demonstrate that Motif 1 and M1BP are required for RP gene transcription. Additionally, I found that M1BP works at RP genes by recruiting the TBP-related factor 2 (TRF2) to RP gene promoters. My data shows for the first time that TRF2 is recruited to promoters by sequence-specific binding factors. This iii resolves a long-standing question in the field since TRF2 does not bind DNA in a sequence- specific manner. Furthermore, I show that the largest subunit of the general transcription factor TFIID associates with all RP gene promoters in vivo. Thus, while previous reports called into question TFIID’s role in initiation at TCT motif-containing RP genes, this finding further attests to TFIID’s key role in transcription. I also identify an M1BP interacting protein called GFZF and investigated its function. I show that GFZF is a glutathione S-transferase (GST) and that it is essential for transcription activation. These results provide the first example of a transcription factor with GST activity. GFZF has appeared in a number of genetic screens that implicate it in processes as disparate as hybrid inviability and positive regulation of RAS/MAPK signaling. Because little was known about GFZF’s molecular function, the authors of these reports explained and interpreted GFZF’s appearance in these screens in complex and unclear terms. My findings suggest GFZF’s involvement in these disparate cellular processes results from its association with over 1800 housekeeping gene promoters. Many of these housekeeping genes are directly involved in the processes that were being investigated. The knowledge of GFZF’s molecular and cellular function should guide future interpretations when GFZF appears in large-scale screens. Finally, while it remains unclear why a transcription factor possesses a functional GST domain, I discuss ways that a transcription factor with GST activity might function in gene regulation. This might include roles in the transcriptional response to stress and the maintenance of genome integrity. iv Table of Contents List of Figures ................................................................................................................................ ix List of Tables ................................................................................................................................ xii Abbreviations ............................................................................................................................... xiii Acknowledgments........................................................................................................................ xiv Chapter 1: Introduction ................................................................................................................... 1 Genome Accessibility ................................................................................................ 1 Transcription Activators and Mediator ...................................................................... 2 The General Transcription Machinery and Preinitiation Complex Formation .......... 5 Core Promoter Elements .......................................................................................... 13 The TBP-related Factors .......................................................................................... 18 Additional Core Promoter Sequence Elements........................................................ 19 Motif 1 and M1BP ................................................................................................... 20 GFZF and Glutathione S-transferases (GSTs) ......................................................... 21 Questions to Be Addressed ...................................................................................... 23 Chapter 2: M1BP recruits TRF2 to coordinately transcribe ribosomal protein genes .................. 26 Introduction ................................................................................................................... 27 Results ........................................................................................................................... 30 RP genes in Drosophila are coordinately transcribed at exceptionally high levels . 30 M1BP activates transcription of RP genes in cells .................................................. 31 v M1BP recruits TRF2 to the RP gene promoter ........................................................ 38 ChIP-exo provides evidence that M1BP recruits TRF2 to the majority of RP genes in vivo ..................................................................................................................... 42 ChIP-exo analysis detects TAF1 at RP gene promoters. ......................................... 47 Colocalization of M1BP and TRF2 is largely restricted to the RP genes ................ 51 RP gene promoters act as enhancers of other RP genes .......................................... 51 Discussion ..................................................................................................................... 55 Materials and Methods .................................................................................................. 60 RNAi Knockdown in S2R+ Cells Followed by Chromatin Immunoprecipitation .. 60 Western Blots ........................................................................................................... 61 Nuclear Extracts and Immunodepletion of M1BP ................................................... 61 In vitro Transcription Reactions and Primer Extension Assay ................................ 62 Expression and Purification of M1BP ..................................................................... 62 Synthesis of radiolabeled TRF2 ............................................................................... 63 Immobilized template pulldown experiments .......................................................... 64 Maltose-binding protein expression and pulldown experiments ............................. 64 ChIP-Exo.................................................................................................................. 65 Bioinformatics.......................................................................................................... 66 Peak Calling and ChIP-seq Analysis ....................................................................... 66 Antibodies ................................................................................................................ 67 vi Chapter 3: GFZF, a glutathione S-transferase protein implicated in cell cycle regulation and hybrid inviability, is a transcriptional co-activator ........................................................ 68 Abstract ......................................................................................................................... 69 Introduction ................................................................................................................... 70 Results and Discussion ................................................................................................
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