Tissue specific demethylation is required for proper B-cell differentiation and function

Many studies have demonstrated that somatic cell differentiation during development is accompanied by extensive demethylation of specific sites relevant to each cell type. Although the mechanism of this process has not yet been elucidated, it is likely to involve the conversion of 5mC to 5hmC by Tet enzymes [Ziller, 2013 #5787]. In this paper, we show that a Tet2/Tet3 conditional knockout at early stages of B-cell development largely prevents lineage-specific programmed demethylation events. This lack of demethylation affects the expression of nearby B-cell lineage genes, probably by inhibiting enhancer activity, thus causing defects in B- cell differentiation. Our studies represent the first demonstration that tissue-specific demethylation may be necessary for proper development in vivo. DNA methylation profile of different developmental stages of B-cells isolated from wild-type or Tet2/Tet3 single and double knock out. The data was generated using the RRBS protocol followed by deep sequencing. RNAseq was performed on these samples as well.多项研究已证实，发育过程中的体细胞分化（somatic cell differentiation）会伴随与各细胞类型特异性相关的特定位点发生广泛的去甲基化。尽管该过程的具体机制尚未阐明，但推测其可能通过Tet酶（Tet enzymes）将5-甲基胞嘧啶（5-methylcytosine, 5mC）转化为5-羟甲基胞嘧啶（5-hydroxymethylcytosine, 5hmC）[Ziller, 2013 #5787]。本研究显示，在B细胞（B-cell）发育早期阶段实施Tet2/Tet3条件性敲除（conditional knockout），可显著阻断谱系特异性程序性去甲基化事件。这种去甲基化缺失会影响邻近B细胞谱系基因的表达，其机制大概率为抑制增强子（enhancer）活性，进而导致B细胞分化缺陷。本研究首次证实，组织特异性去甲基化可能对体内正常发育至关重要。本数据集涵盖从野生型（wild-type）或Tet2/Tet3单敲除、双敲除样本中分离的不同发育阶段B细胞的DNA甲基化谱（DNA methylation profile）。该数据集的数据通过简化代表性亚硫酸氢盐测序（Reduced Representation Bisulfite Sequencing, RRBS）方案结合深度测序生成，同时对上述样本开展了RNA测序（RNA sequencing, RNAseq）。