Epigenetic regulation of B cell differentiation Pubblico
Barwick, Benjamin Gabriel (2016)
Abstract
Appropriate regulation of B cell differentiation is essential to humoral immunity and controlled through transcription factors that dictate B cell fate and function. Transcription factor control of gene expression is restrained by the epigenetic accessibility of target loci, a process that is mediated, in part, by DNA methylation at CpG dinucleotides. Here, DNA methylation changes during plasma cell differentiation were studied in mice three days post challenge with LPS. As compared to naïve B cells, plasma cells were found to change DNA methylation state at 10% of CpG loci, 99.7% of which were demethylation events. Losses in DNA methylation tended to be distal to transcription start sites but overlapped enhancers of transcription. Chemical inhibition of DNA methylation augmented plasma cell commitment in a cell division-dependent manner, suggesting DNA methylation losses must occur prior to plasma cell differentiation. Analysis of in vivo differentiating B cells separated by the number of cellular divisions the B cell had undergone revealed that highly divided cells increased mRNA levels fivefold as compared to undivided B cells. This was coupled to a hierarchy of DNA hypomethylation events at specific transcription factor motifs in a cell division dependent manner. Conversely, accumulation of DNA methylation was less apparent and mice that lack Dnmt3a and Dnmt3b enzymes, which deposit de novo DNA methylation, had phenotypically normal B cell development and similar plasma cell responses to LPS over three days. However, upon extended immunologic challenge, Dnmt3ab-deficient mice accumulated more antigen-specific B cells and exhibited a fivefold increase in splenic and bone marrow plasma cells. Molecular analysis revealed that Dnmt3ab-deficient bone marrow plasma cells failed to repress gene expression at several hundred genes to the same levels as Dnmt3ab-sufficient mice. This was coupled with a failure of Dnmt3ab-deficient germinal center B cells and plasma cells to gain and/or maintain DNA methylation at several thousand loci that were clustered in enhancers of genes that function in B cell activation and homing. These data provide mechanistic insight into cell-division coupled transcriptional and epigenetic reprogramming and suggest that while DNA hypomethylation events reflect the recent cis-regulatory history of plasma cells, extended responses are controlled by de novo DNA methylation that function to help maintain plasma cell homeostasis. 
Table of Contents
Chapter 1 : General Introduction 1
A brief history of B cells and antibodies 1
B cells in health and disease 3
Hematopoiesis and B cell development 3
B cell subset function 6
The cellular and molecular programming of B cell activation 7
Transcriptional regulation of B cell differentiation 11
The conception of epigenetics 14
Mechanisms of DNA methylation reprogramming 15
Histone modifications and control of gene expression 19
The "Epigenetic Code" 20
Epigenetic programming in hematopoiesis 23
Epigenetic programming in B cell development and differentiation
25
Chapter 2 : Plasma cell differentiation is coupled to
division-dependent DNA hypomethylation and gene regulation 30
Abstract 30
Introduction 30
LPS mediated B-cell differentiation is coupled to unique
transcriptional states 32
Plasma cells undergo targeted DNA hypomethylation 34
Demethylated regions overlap enhancers and transcription factor
motifs 38
Inhibition of DNA methylation facilitates plasma cell
differentiation 40
DNA demethylation and gene expression are coupled to
activation-induced cell division 42
Cell division is marked by dynamic gene regulation and progressive
DNA hypomethylation. 46
Discussion 56
Acknowledgements 59
Funding 59
Author Contributions 60
Competing Financial Interests 60
Supplemental Figures 60
Methods 65
Accession codes 65
Mice and LPS challenge 65
Cell Isolation and Flow Cytometric Analysis 66
Ex Vivo Differentiation and 5-aza-Cytidine Treatment 67
Quantitative real-time PCR (qRT-PCR) Analysis 67
Microarray Analysis 68
RNA-seq Analysis 69
Mapping and Quantification of RNA-seq Data 69
Bioinformatic Analyses of Expression Data 70
DNA Methylation Assays 71
DNA Methylation Bioinformatic Analyses 73
Correlation of Gene Expression and DNA Methylation 74
Meta-Analysis 74
Chapter 3 : De novo DNA methylation limits B cell proliferation and
differentiation 76
Abstract 76
Introduction 77
B cell development proceeds normally in the absence of the de novo
DNA methyltransferases 78
Dnmt3a and Dnmt3b control germinal center B cell expansion and
plasma cell differentiation 81
Dnmt3a and Dnmt3b control repression of proliferation and homing
genes. 85
De novo DNA methylation at genes silenced in B cell differentiation
87
Dnmt3a and Dnmt3b repress enhancer accessibility in B cell
differentiation 91
Discussion 94
Chapter 4 : Discussion 96
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