Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College London, Du Cane Road, London W12 0NN, UK.
Nature. 2011 Aug 10;476(7361):467-71. doi: 10.1038/nature10312.
Cohesin enables post-replicative DNA repair and chromosome segregation by holding sister chromatids together from the time of DNA replication in S phase until mitosis. There is growing evidence that cohesin also forms long-range chromosomal cis-interactions and may regulate gene expression in association with CTCF, mediator or tissue-specific transcription factors. Human cohesinopathies such as Cornelia de Lange syndrome are thought to result from impaired non-canonical cohesin functions, but a clear distinction between the cell-division-related and cell-division-independent functions of cohesion--as exemplified in Drosophila--has not been demonstrated in vertebrate systems. To address this, here we deleted the cohesin locus Rad21 in mouse thymocytes at a time in development when these cells stop cycling and rearrange their T-cell receptor (TCR) α locus (Tcra). Rad21-deficient thymocytes had a normal lifespan and retained the ability to differentiate, albeit with reduced efficiency. Loss of Rad21 led to defective chromatin architecture at the Tcra locus, where cohesion-binding sites flank the TEA promoter and the Eα enhancer, and demarcate Tcra from interspersed Tcrd elements and neighbouring housekeeping genes. Cohesin was required for long-range promoter-enhancer interactions, Tcra transcription, H3K4me3 histone modifications that recruit the recombination machinery and Tcra rearrangement. Provision of pre-rearranged TCR transgenes largely rescued thymocyte differentiation, demonstrating that among thousands of potential target genes across the genome, defective Tcra rearrangement was limiting for the differentiation of cohesin-deficient thymocytes. These findings firmly establish a cell-division-independent role for cohesin in Tcra locus rearrangement and provide a comprehensive account of the mechanisms by which cohesin enables cellular differentiation in a well-characterized mammalian system.
黏合蛋白通过在 S 期 DNA 复制后到有丝分裂期间将姐妹染色单体保持在一起，从而实现复制后 DNA 修复和染色体分离。越来越多的证据表明，黏合蛋白还形成长距离染色体顺式相互作用，并可能与 CTCF、中介体或组织特异性转录因子一起调节基因表达。人类黏合蛋白病，如 Cornelia de Lange 综合征，被认为是由于非规范黏合蛋白功能受损所致，但在脊椎动物系统中，尚未明确区分黏合蛋白与细胞分裂相关和与细胞分裂无关的功能——这在果蝇中得到了例证。为了解决这个问题，我们在胸腺细胞发育的特定时间，即在这些细胞停止循环并重新排列其 T 细胞受体 (TCR)α 基因座（Tcra）时，在发育中的小鼠胸腺细胞中删除了黏合蛋白基因 Rad21。Rad21 缺陷型胸腺细胞具有正常的寿命，并保留了分化的能力，尽管效率降低。Rad21 的缺失导致 TCRa 基因座的染色质结构缺陷，其中黏合蛋白结合位点位于 TEA 启动子和 Eα 增强子的侧翼，并将 TCRa 与分散的 Tcrd 元件和相邻的管家基因区分开来。黏合蛋白对于长距离启动子-增强子相互作用、Tcra 转录、募集重组机制的 H3K4me3 组蛋白修饰以及 TCRa 重排是必需的。预先排列的 TCR 转基因的提供在很大程度上挽救了胸腺细胞分化，表明在基因组中的数千个潜在靶基因中，Tcra 重排的缺陷限制了黏合蛋白缺陷型胸腺细胞的分化。这些发现牢固确立了黏合蛋白在 TCRa 基因座重排中独立于细胞分裂的作用，并提供了一个全面的解释，说明黏合蛋白如何在一个特征明确的哺乳动物系统中使细胞分化。
