Resetting the Cancer Transcriptome with Epigenetic Therapy Open Access

Bell, Joshua S. (2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/pz50gx01d?locale=en
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Abstract

Abstract

DNA methylation is a key regulator of transcription in mammals, and aberrant methylation changes drive tumorigenesis. Epigenetic therapy inhibiting DNA methylation decreases the growth rate and invasive potential of solid tumor cells, and is a standard treatment for certain hematological cancers. However, many questions remain surrounding the exact mechanism of this epigenetic reprogramming, and the role of methylation in distinct genomic compartments. The human genome is a heavily methylated, CpG-depleted, dominantly heterochromatic terrain disrupted by CpG Islands (CGI), essential hypomethylated CpG dense regulatory elements, associated with most human promoters. CGI are characterized by high levels of transcriptional initiation, but transcription into the gene body is limited by promoter-proximal RNA Polymerase II (Pol II) pausing. Here, we document an additional Pol II pausing step at CGI boundaries known as distal pausing, and link the location and degree of pausing to GC-skew. Many 'orphan' CGI are also found far from any annotated transcript, and we establish most such CGI are highly active enhancers. We also examine the nascent transcriptome of cancer cells using Precision Run-on Sequencing (ProSeq) during epigenetic therapy to document that DNA hypomethylation results in pervasive changes including the down-regulation of oncogenes due to loss of gene body methylation, reactivation of genes silenced by promoter methylation, reactivation of enhancers hypermethylated in breast cancer, and induction of repetitive elements. Critically, many of these transcripts remain highly paused or are unstable and thus have evaded detection by RNA-Seq. Analyzing remethylation kinetics following therapy, we find that normal methylation returns quickly, while aberrant cancer-related hypermethylation is largely forgotten. These findings suggest new mechanisms by which epigenetic therapy reprograms cancer cells and asserts novel roles for DNA methylation in regulating transcription more broadly.

Table of Contents

Table of Contents

Chapter I: Introduction

Epigenetics.............................................................................................................5

DNA Methylation and CpG Islands.........................................................................6

Histone Modifications ….........................................................................................9

Crosstalk Between DNA Methylation and Histone Modifications …......................12

Transcription and Pol II Pausing …........................................................................13

Enhancers..............................................................................................................16

Epigenetic Aberrations in Cancer.......................................................................... 19

Epigenetic Therapy …............................................................................................20

Objectives.............................................................................................................. 22

Bibliography........................................................................................................... 23

Chapter II: GC-Skew Defines Distinct RNA Polymerase Pause Sites in CpG Island Promoters

Abstract ..................................................................................................................31

Introduction..............................................................................................................32

Results …................................................................................................................35

Discussion...............................................................................................................46

Methods...................................................................................................................50

Figures.....................................................................................................................56

References...............................................................................................................63

Supplement..............................................................................................................70

Chapter III: Factors Affects the Persistence of Drug-Induced Reprogramming of the Cancer Methylome

Abstract …................................................................................................................89

Introduction...............................................................................................................90

Results ….................................................................................................................92

Discussion.................................................................................................................101

Methods.....................................................................................................................106

References …............................................................................................................111

Figures …...................................................................................................................119

Chapter IV: Orphan CpG Islands Define a Class of Highly Active Enhancers

Abstract..........................................................................................................................140

Introduction.....................................................................................................................141

Results............................................................................................................................142

Discussion......................................................................................................................154

Methods..........................................................................................................................159

References......................................................................................................................165

Figures............................................................................................................................ 170

Chapter V: Therapeutic Inhibition of DNA Methylation Resets the Cancer Transcriptome

Abstract...........................................................................................................................193

Introduction......................................................................................................................194

Results.............................................................................................................................196

Discussion........................................................................................................................206

Methods …........................................................................................................................212

Figures …...........................................................................................................................214

References ….....................................................................................................................231

Chapter VI: Discussion

CpG Islands are Unique Chromatin Environments …........................................................234

CGI Conservation …..........................................................................................................237

DNA Methylation as a Master Regulator of Transcription.................................................. 240

Resetting the Cancer Epigenome....................................................................................... 244

Future Directions ….............................................................................................................245

Bibliography ….....................................................................................................................248

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