RNA polymerase II is necessary for spatial chromatin reorganization following exit from mitosis [HiC]. RNA polymerase II is necessary for spatial chromatin reorganization following exit from mitosis [HiC]

Mammalian chromosomes are three-dimensional entities shaped by converging and opposing forces acting on chromatin. Mitotic cell division induces drastic chromatin condensation, but following reentry into the G1 cell cycle phase, condensed chromosomes unwind to re-establish interphase organization. Here, we use a cell line allowing auxin-mediated degradation of RNA polymerase II to test its role in this transition. In situ Hi-C and super-resolution imaging showed that RNAPII is required for compartment and loop formation following mitosis. RNAPs also often counteract loop extrusion and, in their absence, longer and more prominent loops arise. Evidence from chromatin fractionation and in silico modeling attribute these effects to RNAPII-mediated cohesin loading at active promoters upon G1 reentry. Our findings reconcile the role of RNAPII in gene expression with that in chromatin architecture. Overall design: HiC sequencing in DLD1 cells

哺乳动物染色体是受染色质上相向与相背作用力共同塑造的三维实体。有丝分裂会引发染色质发生剧烈浓缩，但当细胞重新进入细胞周期G1期后，浓缩的染色体解旋，重建间期染色质组织形式。本研究使用一种可实现生长素介导降解RNA聚合酶II（RNA polymerase II）的细胞系，探究其在该染色体重构过程中的作用。原位Hi-C与超分辨率成像实验结果表明，RNA聚合酶II（RNAPII）对于有丝分裂后染色质区室与环结构的形成不可或缺。RNA聚合酶还通常会拮抗环挤出过程；当RNA聚合酶缺失时，细胞内会形成更长且更显著的染色质环。染色质分级分离实验与计算机模拟建模的结果表明，上述现象源于细胞重新进入G1期时，RNA聚合酶II介导的黏连蛋白在活性启动子区域的加载过程。本研究结果将RNA聚合酶II在基因表达中的功能与其在染色质构象调控中的功能进行了统一阐释。实验整体设计：在DLD1细胞系中开展Hi-C测序实验。