TET-mediated Hydroxymethylation in Reprogramming to Induced Pluripotency Open Access

Wang, Tao (2013)

Permanent URL: https://etd.library.emory.edu/concern/etds/6d56zx00s?locale=en


5-Hydroxymethylcytosine (5hmC) is a newly discovered modified form of cytosine that has been suspected to be an important epigenetic modification in stem cells and during neurodevelopment. Here, we report the roles of 5-hmC during reprogramming as well as neurodevelopment. Mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem cells (ESCs). Human ES cells contain 5hmC, which is generated through the oxidation of 5-methylcytosine by the TET enzyme family. Here we show that 5hmC levels increase significantly during reprogramming to human iPSCs mainly owing to TET1 activation, and this hydroxymethylation change is critical for optimal epigenetic reprogramming, but does not compromise primed pluripotency. Compared with hES cells, we find iPS cells tend to form large-scale (100 kb-1.3 Mb) aberrant reprogramming hotspots in subtelomeric regions, most of which display incomplete hydroxymethylation on CG sites. Strikingly, these 5hmC aberrant hotspots largely coincide (~80%) with aberrant iPS-ES non-CG methylation regions. Our results suggest that TET1-mediated 5hmC modification could contribute the epigenetic variation of iPSCs and iPSC-hESC differences.

Table of Contents

Table of Contents

Chapter 1: Toward pluripotency by reprogramming: looking back, looking forward

Introduction 1

Abstract 2

Introduction 3

Reprogramming factors 5

Effect of stoichiometry 8

DNA methylation and de-methylation 10

Small molecule-mediated reprogramming 12

iPS and ES cells differences 14

Elite, stochastic, and deterministic models 16

MicroRNAs in somatic reprogramming 17

Disease modeling 19

Concluding remarks 22

References 25

Chapter 2: Subtelomeric hotspots of aberrant 5-hydroxymethylcytosine-mediated epigenetic modifications during reprogramming to pluripotency

Abstract 34

Introduction 35

Results 37

TET1-mediated hydroxymethylation plays a critical role during reprogramming to pluripotency in human cells 37

5hmC epigenomic landscape during reprogramming 38

5hmC is bi-directionally correlated with DNA methylation changes and associated with pluripotency related gene networks 39

Sequence preferences of 5hmC modification during reprogramming 41

Aberrant 5hmC reprogramming hotspots cluster in telomere-proximal regions 41

Concordance of large-scale 5hmC hotspots and iPS-ES non-CG DMRs 43

Base-resolution 5hmC analyses reveal large-scale hotspots are mainly caused by aberrant CG hydroxymethylation 44

Discussion 47

Methods 51

References 99

Chapter 3: Genome-wide DNA hydroxymethylation changes are associated with neurodevelopmental genes in the developing human cerebellum

Abstract 106

Introduction 107

Results 110

Dynamics of DNA hydroxymethylation and its genomic features in human

cerebellum 110

5-hmC genomic features during cerebellum development 111

The fetus-specific DhMRs displays pluripotent epigenetic memories 112

DhMRs are associated with genes involved in neurodevelopmental disorders 113

Discussion 117

Material and Methods 122

References 141

Chapter 4: Discussion and future directions

Potential distinct roles between TET1 and TET2 147

DNA methylation and demethylation 149

Future direction for hydroxymethylation studies 150

References 153

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