Dynamic changes in chromatin accessibility and nucleosome positioning in the control of antigen receptor rearrangement

Developing lymphocytes diversify their antigen receptor (AgR) loci by V(D)J recombination. Here, we use a low-input version of the MACC (MNase Accessibility) assay to define the chromatin accessibility landscape of primary cells during lymphoid development tracking the changes as different AgR loci become primed for recombination. Distinct chromatin structures define unique features of IgH, Igk and TCRα loci. In particular, we find locus-specific temporal changes in accessibility both across megabase-long AgR loci and locally at the recombination signal sequences (RSSs). These changes appear to be regulated independently. We further identify local dynamic rearrangements of individual nucleosomes at RSSs during B-cell commitment. As predicted by the original “accessibility” hypothesis, these changes correlate with AgR lymphoid lineage and developmental-stage specificity. We suggest that local and global changes in chromatin openness, in concert with nucleosome occupancy and histone modifications, represent additional regulatory layers for governing the temporal order of AgR recombination. MNase-seq profiles of hematopoietic stem cells (HSCs); lymphoid-primed multipotent progenitors (LMPPs); common lymphoid “A” type precursors (ALPs or CLPs); B cell–biased lymphoid progenitors (BLPs); B-cell progenitors (pro-B); large B-cell precursors (Lpre-B); and small B-cell precursors (small pre-B). Lung epithelium cells (LungEp) were used as nonlymphoid and recombinationally inactive cells. Wild-type C57BL/6N mice (4-6 weeks old) were used to isolate the above cell populations.

正在发育的淋巴细胞通过V(D)J重组对抗原受体（antigen receptor, AgR）基因座实现序列多样化。本研究采用微球菌核酸酶可及性检测（MNase Accessibility, MACC）的低起始量版本，解析淋巴细胞发育过程中原代细胞的染色质可及性图谱，并追踪不同AgR基因座被重组激活时的动态变化。独特的染色质结构赋予免疫球蛋白重链（IgH）、免疫球蛋白κ轻链（Igk）以及T细胞受体α链（TCRα）基因座以独特的特征。具体而言，本研究发现，无论是在兆碱基级别的AgR基因座整体区域，还是在重组信号序列（RSSs）的局部区域，均存在基因座特异性的可及性时序变化。此类变化似乎受到独立调控。本研究还进一步发现，在B细胞定型过程中，RSS位点处的单个核小体发生了局部动态重排。正如原始‘可及性假说’所预测的那样，此类变化与AgR的淋巴系谱系特异性及发育阶段特异性密切相关。本研究提出，染色质开放状态的局部与全局变化，联合核小体占位与组蛋白修饰，共同构成了调控AgR重组时序的额外调控层级。本数据集包含造血干细胞（HSCs）、淋巴定向多能祖细胞（LMPPs）、共同淋巴样“A型”前体细胞（ALPs或CLPs）、B细胞偏向性淋巴样祖细胞（BLPs）、B细胞前体（pro-B）、大B细胞前体（Lpre-B）以及小B细胞前体（small pre-B）的微球菌核酸酶测序（MNase-seq）图谱。本研究以肺上皮细胞（LungEp）作为非淋巴系且无重组活性的对照细胞。所有细胞群体均通过4~6周龄的野生型C57BL/6N小鼠分离得到。