Resetting the Cancer Transcriptome with Epigenetic Therapy Public
Bell, Joshua S. (2017)
Published
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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