BRWD1 orchestrates chromatin topology by converting static to dynamic cohesin complexes (ChIP-Seq). BRWD1 orchestrates chromatin topology by converting static to dynamic cohesin complexes (ChIP-Seq)

Lymphocyte development consists of sequential and mutually exclusive cell states of proliferative selection and antigen receptor gene recombination. Transitions between each state require large, coordinated changes in epigenetic landscapes and transcriptional programs. How this occurs remains unclear. Herein, we demonstrate that in small pre-B cells, the lineage and stage-specific epigenetic reader BRWD1 reorders three-dimensional chromatin topology to affect transition between proliferative and gene recombination molecular programs. BRWD1 regulated the switch between poised and active enhancers interacting with promoters and coordinated this with Igk locus contraction. BRWD1 did so by converting chromatin-bound static cohesin to dynamic complexes competent to mediate long-range looping. Remarkably, ATP depletion recapitulated cohesin distributions observed in Brwd1-/- cells. Therefore, in small pre-B cells, cohesin conversion appears to be the main energetic mechanism dictating where dynamic looping occurs in the genome. Our findings provide a new mechanism of cohesin regulation and reveal how cohesin function can be regulated by lineage contextual mechanisms to facilitate specific cell fate transitions. Overall design: ChIP-seq of CTCF, H3K27Ac, H3M4me1, NIPBL, RAD21, SMC3, and WAPL in small pre-B cells isolated from mice, with WT, BRWD1KO genotypes , and WT small pre-B cells treated with oligomycin (ATPneg). Two biological replicates were collected for each ChIP sample. Input samples were collected in duplicate, or were used as previously released (GSM2753125).

淋巴细胞发育包含增殖选择与抗原受体基因重排这一系列依次发生且互斥的细胞状态。各状态间的转换，需要表观遗传景观与转录程序发生大规模且协同的变化，其具体调控机制至今尚未阐明。本研究证明，在小型前B细胞中，谱系与阶段特异性表观遗传读取器（epigenetic reader）BRWD1可重排三维染色质拓扑结构，进而调控增殖程序与基因重排程序间的转换。BRWD1可调控与启动子相互作用的poised增强子（poised enhancer）与活性增强子之间的转换，并协同调控免疫球蛋白κ轻链基因座（Igk locus）的收缩。该蛋白通过将结合于染色质的静态黏连蛋白（cohesin）转化为可介导长距离环化的动态复合物，实现上述调控功能。值得注意的是，ATP耗竭可重现Brwd1基因敲除（Brwd1-/-）细胞中观察到的黏连蛋白分布模式。因此，在小型前B细胞中，黏连蛋白的转化似乎是决定基因组内动态环化发生位点的主要能量依赖型机制。本研究发现揭示了黏连蛋白调控的全新机制，并阐明了谱系特异性背景机制如何调控黏连蛋白功能，以促进特定细胞命运的转换。

整体实验设计：对从小鼠体内分离的小型前B细胞进行染色质免疫共沉淀测序（ChIP-seq），检测靶标包括CTCF、H3K27Ac、H3M4me1、NIPBL、RAD21、SMC3及WAPL；实验分组涵盖野生型（wild type，WT）、BRWD1基因敲除（BRWD1 knockout，BRWD1KO）基因型细胞，以及经寡霉素（oligomycin）处理的ATP耗竭型（ATPneg）野生型小型前B细胞。每个ChIP样本均设置2次生物学重复。输入对照（Input）样本同样设置2次重复，或使用已公开的测序数据（GSM2753125）。