Epigenetic programming during B cell maturation. (a) Top, FACS sorting markers used to isolate the analyzed B cell subsets after selection of CD19+ cells. Bottom, the frequency of IGHV3 mutations in each subpopulation. (b) Top, TWGBS summary comparing naive B cells and high-maturity memory B cells. Bottom, methylation heat maps for the top 5,000 most variable windows. (c) Enrichment of differentially methylated windows among chromatin states, defined using the 15-state ChromHMM model20 (hypermethylated, >20% change; hypomethylated, >40% change), in the comparison of naive B cells and high-maturity memory B cells. Fold enrichment was calculated independently in 19 lymphoblastoid B cell lines (error bars, s.e.m.). TSS, transcriptional start site. (d) Bubble scatterplot of 223 transcription factor motifs displaying the prevalence and fold enrichment of each motif in hypomethylated windows (>40% change). Bubble size corresponds to the P value. (e) Bubble scatterplot of 78 transcription factor ChIP-seq peaks (determined in GM12878 cells) in hypomethylated windows (>40% change). Bubbles are colored according to the cognate motif in d. (f) Examples of loci differentially methylated in normal B cells at single-CpG resolution. Regions of hypermethylation (pink) within the gene body of MSI2 (left) and hypomethylation (blue) upstream of NOTCH1 (right) are shown. Chromatin states (colors correspond to those used in c) and transcription factor binding site peaks from GM12878 cells are indicated. (g) Average methylation level of hypomethylated windows (>20% change) containing high-confidence transcription factor binding sites (TFBSs) and hypermethylated windows (>20% change) overlapping regions of transcriptional elongation in each B cell subtype. (h) Left, DNA methylation phylogenetic tree diagram of blood cell development of common hematopoietic cell types. Each branch tip represents a single sample. Right, diagram of B cell maturation constructed using high-confidence methylation (450K) at transcription factor binding sites. HSCs, hematopoietic stem cells; NK cells, natural killer cells; CC, centrocytes; CB, centroblasts. (i) Scatterplots displaying the change in methylation of 450K probes in naive B cells after in vitro stimulation and 5 d in culture (y axis) versus the difference in methylation between naive B cells and high-maturity memory B cells (x axis). Naive B cells were stimulated with CD40L (left) or with CD40L and antibody to IgM (αIgM; right). (j) Bubble scatterplot of transcription factor motifs overlapping hypomethylated CpGs following CD40L stimulation and 5 d in culture.
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