Enhancing Transcription by Release of RNAPII from the Promoter-Proximal Pause Pubblico

Abstract

Promoter-Proximal Pause
Transcription regulation by promoter-proximal pausing is the rate limiting step for the
expression of a large number of genes. The steps necessary for release of RNA polymerase II
from this pause are not well characterized. Although covalent histone modifications have been
implicated in this process, their role in this regulatory step is not well understood. This study
elucidates some of the histone modifying enzymes and the modifications they perform in order
to characterize the link between histone phosphorylation and acetylation. 14-3-3 is identified as
a critical protein necessary for mediating the connection between histone phosphorylation and
acetylation that we show is necessary for release of RNA polymerase into transcriptional
elongation. Transcription factors bind through sequence specific interactions that ultimately
mediate transcription activation to the promoter region of the genes they regulate. In the large
percentage of genes that have paused RNA polymerase II at the 5' end of the gene, the
activating signal should mediate the release of the polymerase from a paused state into
transcriptional elongation. Histone modifications also play a role in regulation of transcription at
enhancers and we identify histone phosphorylation
14-3-3 recruitment and acetylation
of histones
regulate suggesting an interaction
between these two sequences. This interaction is confirmed by chromatin conformation capture
assays and shown to be dependent on JIL-1, 14-3-3 and CBP. Lastly, in order to investigate
these steps as a genome-wide phenomenon during transcription activation across we
demonstrate that histone phosphoacetylation of both H3K9acS10ph and H3K27acS28ph occur
at both enhancers and promoters.

Enhancing Transcription
Promoter-
Proximal Pause

M.S., Georgia State University, 2001
B.S., Bowling Green State University, 1998
Advisor: Victor G. Corces, Ph.D.
A dissertation submitted to the Faculty of the
James T. Laney School of Graduate Studies of Emory University
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
Graduate Division of Biological and Biomedical Sciences
Genetics and Molecular Biology
April, 2012

Table of Contents

TABLE OF CONTENTS Chapter 1 : Introduction 1

Regulation of transcription by promoter-proximal pausing 2

Histone code regulation of transcription 3

JIL-1 phosphorylates H3S10 upon transcriptional activation

during interphase 4

14-3-3 is able to bind phosphorylated histones 5

Drosophila as a model for transcriptional regulation 5

P-TEFb mediates the release of RNAP II from the promoter-

proximal pause 6

Enhancers regulate transcription 8

Scope of the Dissertation 9

Chapter 2: 14-3-3 Binding to H3S10ph Mediates Histone Crosstalk in

Drosophila 14

Abstract 15

Introduction 16 Results 17 Discussion 25 Materials and methods 27

Chapter 3: Genome-wide Phosphoacetylation of Histone H3 at

Drosophila Enhancers and Promoters 54

Abstract 55 Introduction 56 Results 57 Discussion 66 Materials and Methods 70 Chapter 4: Discussion 99

Regulation of promoter-proximal pausing 100

Enahancer regulated transcription 102

Enhancer-promoter contacts are required for activation 103

JIL-1 is present at enhancers prior to activation 104

Fs(1)h characterization in Drosophila 105

References 109 LIST OF TABLES

Table 3-1 Double stranded RNA Primers for gene knock down 95

Table 3-2 3C Primers 97

LIST OF FIGURES

Figure 1-1 Levels of H3S10ph and 14-3-3 in interphase cells are low

compared to those present in mitotic chromosomes. 11

Figure 1-2 Promoter-proximal pausing of RNAP II is a rate limiting set for a large

percentage of genes 12

Figure 2-1 JIL-1 is recruited to the promoter of the hsp70 gene after heat-shock 32

Figure 2-2 14-3-3 proteins are required for transcription elongation of most

Drosophila genes 34

Figure 2-3 H3S10ph and 14-3-3 overlap at the majority of sites with

active transcription 36

Figure 2-4 Binding of 14-3-3 to chromosomes during transcription is dependent

on JIL-1 kinase. 38

Figure 2-5 14-3-3 proteins interact with the elongation protein Elp3 and are

required for transcription elongation of most Drosophila genes 40

Figure 2-6 Elp3 co-localizes with active chromatin and is dependent on 14-3-3

for recruitment to the chromosomes 42

Figure 2-7 Elp3 recruitment to chromatin is dependent on JIL-1 and 14-3-3 44

Figure 2-8 JIL-1, 14-3-3, and Elp3 are required for H3 acetylation 46

Figure 2-9 Histone H3K9ac, a modification of active transcription carried out

by Elp3, is dependent on JIL-1, 14-3-3, and Elp3 48

Figure 2-10 H4K16 acetylation is dependent on JIL-1 but not 14-3-3 in females 50

Figure 2-11 H4K16 acetylation is dependent on JIL-1 but not 14-3-3 in males 52

Figure 3-1 JIL-1 kinase is necessary for H3S28 phosphorylation at actively

transcribed regions of polytene chromosomes 73

Figure 3-2 JIL-1 phosphorylation of H3S10 and H3S28 at promoters and

enhancers is necessary for transcriptional activation of ecdysone

responsive genes 75

Figure 3-3 JIL-1 is required for phosphorylation at enhancers and promoters

upon ecdysone transcriptional activation 77

Figure 3-4 Either of the phosphorylation marks pulls down equal amounts of

acetylated histone 79

Figure 3-5 CBP acetylation and 14-3-3 recruitment at promoters and enhancers is

necessary for transcription activation of ecdysone-responsive genes 81

Figure 3-6 Knockdown of CBP or 14-3-3 does not affect H3S28ph levels 83

Figure 3-7 14-3-3 recruitment and H3K27 acetylation are dependent on JIL-1

kinase at enhancers and promoters 85

Figure 3-8 JIL-1, 14-3-3, and CBP are required for enhancer-promoter interactions 87

Figure 3-9 JIL-1 and histone phosphoacetylation at promoters correlate with

transcript levels genome-wide 89

Figure 3-10 Distribution of JIL-1 with respect to genes 91

Figure 3-11 JIL-1, H3K9acS10ph and H3K27acS28ph are present at enhancers 93

Figure 4-1 Phosphoacetylation at enhancers and promoters is necessary for

contact 107

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Molecular
Parola chiave	Histone modification Enhancer Transcription
Committee Chair / Thesis Advisor	Corces, Victor, Emory University
Committee Members	Lucchesi, John, Emory University Kelly, William G, Emory University Reines, Daniel, Emory University Boss, Jeremy, Emory University

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