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. 2014 Dec;9(12):1588-95.
doi: 10.4161/15592294.2014.983379.

Genome-wide DNA methylation analysis in precursor B-cells

Md Almamun  1 , Benjamin T Levinson, Susan T Gater, Robert D Schnabel, Gerald L Arthur, J Wade Davis, Kristen H Taylor
Affiliations

Genome-wide DNA methylation analysis in precursor B-cells

Md Almamun et al. Epigenetics. 2014 Dec.

Abstract

DNA methylation is responsible for regulating gene expression and cellular differentiation and for maintaining genomic stability during normal human development. Furthermore, it plays a significant role in the regulation of hematopoiesis. In order to elucidate the influence of DNA methylation during B-cell development, genome-wide DNA methylation status of pro-B, pre-BI, pre-BII, and naïve-B-cells isolated from human umbilical cord blood was determined using the methylated CpG island recovery assay followed by next generation sequencing. On average, 182-200 million sequences were generated for each precursor B-cell subset in 10 biological replicates. An overall decrease in methylation was observed during the transition from pro-B to pre-BI, whereas no differential methylation was observed in the pre-BI to pre-BII transition or in the pre-BII to naïve B-cell transition. Most of the methylated regions were located within intergenic and intronic regions not present in a CpG island context. Putative novel enhancers were identified in these regions that were differentially methylated between pro-B and pre-BI cells. The genome-wide methylation profiles are publically available and may be used to gain a better understanding of the involvement of atypical DNA methylation in the pathogenesis of malignancies associated with precursor B-cells.

Keywords: CG dinucleotide; CLP, common lymphoid progenitor cells; CpGI, CpG island; DMRs, differentially methylated regions; DNA methylation; FDR, false discovery rate.; H3K27ac, histone H3 lysine 27 acetylation; H3K4me1, histone H3 lysine 4 monomethylation; HCB, human umbilical cord blood; HSCs, haematopoietic stem cells; MBDs, methyl CpG binding domains; MIRA-seq, methylated CpG island recovery assay (MIRA) followed by next generation sequencing; MeCP2, methyl CpG binding protein 2; Pre-B, precursor B-cell; CD; Pro-B, progenitor B-cell; ROIs, regions of interest; TFs, transcription factors; acute lymphoblastic leukemia; CpG; cluster of differentiation; ALL; enhancer; next-generation sequencing; precursor B-cell; umbilical cord blood.

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Figures

Figure 1.
Figure 1.
Average read and alignment statistics. Read and alignment statistics were averaged across all individuals for each precursor B-cell subset. The top of each bar represents the total number of sequencing reads (blue), the total number of mapped reads (dark gray), and the total number of unique reads (light gray).
Figure 2.
Figure 2.
Genomic distribution of methylation peaks in pro-B, pre-BI, pre-BII, and naïve-B-cells.
Figure 3.
Figure 3.
Percentages of methylation peaks associated with CpG islands, CpG shores, and CpG shelves.
Figure 4.
Figure 4.
Differentially methylated regions in the pro-B to pre-BI transition. Circos plots representing chromosomes 1-22. The X and Y chromosomes were excluded from analysis. A total of 4,210 loci gained methylation (hypermethylated in pre-BI) and 14,294 loci lost methylation (hypomethylated in pre-BI). Blue represents hypomethylated regions in pre-BI cells and yellow represents hypermethylated regions in pre-BI cells.
Figure 5.
Figure 5.
Genomic distribution of hypomethylated and hypermethylated loci during the pro-B to pre-BI transition.
Figure 6.
Figure 6.
Isolation of precursor B-cell subsets from human umbilical cord blood. Mononuclear cells were isolated using density gradient centrifugation to remove all non B-cells. B-cells were labeled with cell surface antibodies and sorted into 4 separate tubes. R4: pro-B-cells; R5: pre-BI cells; R6: pre-BII cells; R7: naïve B-cells.
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