Redundant and specific roles of cohesin STAG subunits in chromatin looping and transcription control (Hi-C). Redundant and specific roles of cohesin STAG subunits in chromatin looping and transcription control (Hi-C)

Cohesin is a ring-shaped multiprotein complex that is crucial for 3D genome organization and transcriptional regulation during differentiation and development. It also confers sister chromatid cohesion and facilitates DNA damage repair. Besides its core subunits SMC3, SMC1A and RAD21, cohesin contains in somatic cells one of two orthologous STAG subunits, SA1 or SA2. How these variable subunits affect the function of the cohesin complex is still unclear. SA1- and SA2-cohesin were initially proposed to organize cohesion at telomeres and centromeres, respectively. Here, we uncover redundant and specific roles of SA1 and SA2 in gene regulation and chromatin looping using HCT116 cells with an auxin-inducible degron (AID) tag fused to either SA1 or SA2. Following rapid depletion of either subunit, we perform high resolution Hi-C, RNA-sequencing and sequential ChIP studies to show that SA1 and SA2 do not co-occupy individual binding sites and have distinct ways how they affect looping and gene expression. These findings are supported at the single cell level by single-molecule localizations via dSTORM super-resolution imaging. Since somatic and congenital mutations of the SA subunits are associated with cancer (SA2) and intellectual disability syndromes with congenital abnormalities (SA1 and SA2), we verified SA1-/SA2-dependencies using human neural stem cells, hence highlighting their importance for understanding particular disease contexts. Overall design: HiC experiments on HCT116 cells with an auxin-inducible degron (AID) tag fused to either the SA1 or SA2 cohesin complex subunit.

黏连蛋白（Cohesin）是一种环状多蛋白复合物，在细胞分化与发育过程中对三维基因组组织及转录调控发挥关键作用。该复合物同时介导姐妹染色单体黏连，并促进DNA损伤修复。除核心亚基SMC3、SMC1A及RAD21外，黏连蛋白在体细胞中还包含两种同源STAG亚基之一：SA1或SA2。目前这类可变亚基如何影响黏连蛋白复合物的功能仍不明确。最初研究认为，结合SA1与SA2的黏连蛋白分别在端粒与着丝粒处介导染色单体黏连。本研究借助分别融合了生长素诱导降解域（auxin-inducible degron, AID）标签的SA1或SA2的HCT116细胞，揭示了SA1与SA2在基因调控及染色质环形成中的冗余与特异性功能。在快速降解任一亚基后，我们通过高分辨率Hi-C、RNA测序及连续染色质免疫沉淀（sequential ChIP）实验，证实SA1与SA2不会共同结合至单个结合位点，且二者通过不同机制影响染色质环形成与基因表达。单分子定位超分辨率成像（dSTORM）在单细胞水平上的实验结果进一步验证了上述发现。鉴于SA亚基的体细胞突变与先天性突变分别与癌症（SA2）及伴先天性异常的智力障碍综合征（SA1与SA2）相关，我们通过人神经干细胞验证了SA1与SA2的功能依赖性，从而凸显了二者在特定疾病机制研究中的重要价值。实验设计：针对分别融合了生长素诱导降解域（AID）标签的SA1或SA2黏连蛋白亚基的HCT116细胞开展Hi-C实验。