B-cell Division RNA-seq [RNA-Seq]

B cells provide humoral immunity by differentiating into antibody secreting plasma cells. Differentiation is dependent upon division and transcriptional changes, with commitment to B cell lineages associated with epigenetic changes. Analysis of early transcriptional and epigenetic events in B cell differentiation revealed that plasmablasts and plasma cells undergo dynamic changes in gene expression and a progressive DNA hypomethylation targeted to at least 10% of genes/loci. Of the differentially methylated loci, more than 99.7% were demethylated during differentiation and these clustered in cis-regulatory features such as enhancers and transcription factor binding sites. Changes in gene expression and DNA methylation coincided with each other at specific divisions during differentiation and inhibition of DNA methylation resulted in augmented plasma cell commitment in a division-dependent manner. These data identify a major epigenetic reprogramming event during early B cell differentiation coupled division and provide an approach to modulating humoral immune responses. Overall design: Splenic B cells (B220+ CD43-) from naïve C57/BL6J mice were labeled with CFSE or CTV and transferred into uMT mice and allowed to rest overnight prior to challenge with LPS. Three days post challenge adoptively transferred B cells representing distinct divisions were sorted out for molecular analysis. These divisions are labelled Div0, Div1, Div3, Div5, Div8- and Div8+. Division 8- refers to cells that divided at least 8 times but were CD138-, whereas Division 8+ refers to cells that divided at least 8 times but were CD138+. Cells were subjected to RNA-seq and Reduced Representation Bisulfite Sequencing.

B细胞通过分化为抗体分泌型浆细胞（antibody secreting plasma cells）介导体液免疫。该分化过程依赖于细胞分裂与转录组改变，而B细胞谱系定型与表观遗传调控变化密切相关。对B细胞分化早期转录与表观遗传事件的分析显示，浆母细胞（plasmablasts）与浆细胞会发生基因表达的动态变化，以及针对至少10%的基因/基因座的渐进式DNA低甲基化。在差异甲基化基因座中，超过99.7%会在分化过程中发生去甲基化，且这些位点富集于顺式调控元件（如增强子（enhancers）与转录因子结合位点（transcription factor binding sites））。分化过程中，特定细胞分裂阶段的基因表达变化与DNA甲基化变化呈现同步性；而DNA甲基化抑制会以细胞分裂依赖的方式增强浆细胞定型。本研究数据揭示了早期B细胞分化过程中伴随细胞分裂的关键表观遗传重编程事件，并为调控体液免疫应答提供了潜在策略。总体实验设计：取自未致敏（naïve）C57/BL6J小鼠的脾脏B细胞（B220+ CD43-）经CFSE或CTV标记后，移植至uMT小鼠体内，于脂多糖（LPS，lipopolysaccharide）刺激前静置过夜。刺激后第3天，对代表不同细胞分裂阶段的过继转移B细胞进行分选，用于分子层面分析。这些细胞分裂阶段被标记为Div0、Div1、Div3、Div5、Div8-与Div8+。Div8-指至少完成8次细胞分裂但CD138-的细胞，而Div8+指至少完成8次细胞分裂且CD138+的细胞。所有分选得到的细胞均进行RNA测序（RNA-seq）与简化代表性亚硫酸氢盐测序（Reduced Representation Bisulfite Sequencing, RRBS）。