Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for CTCF [SMF]. Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for CTCF [SMF]

Catalytic activity of the ISWI family of remodelers is critical for nucleosomal organization and transcription factor binding, including the insulator protein CTCF. To define which subcomplex mediate these diverse functions we phenotyped a panel of isogenic mouse stem cell lines each lacking one of six ISWI accessory subunits. Individual deletions of either CERF, RSF1, ACF, WICH or NoRC subcomplexes cause only moderate effects on the chromatin landscape, while removal of the NURF specific subunit BPTF leads to drastic reduction in chromatin accessibility and Snf2h ATPase localization around CTCF sites. While this reduces distances to the adjacent nucleosomes it only modestly impacts CTCF binding itself. In absence of accessibility bound CTCF is nevertheless impaired in function resulting in lower occupancy of cohesin and cohesin-loading factors, and reduced insulation at these sites, highlighting the need of NURF-mediated remodeling for open chromatin and proper CTCF function. Our comprehensive analysis reveals a specific role for NURF in mediating Snf2h localization and chromatin opening at bound CTCF sites showing that local accessibility is critical for cohesin binding and insulator function. Overall design: Amplicon-based Single Molecule Footprinting (SMF) in mouse Embryonic Stem Cells, in wild-type and Bptf knock-out (triplicates).

ISWI家族染色质重塑酶的催化活性对于核小体组织以及包括绝缘子蛋白CTCF在内的转录因子结合过程至关重要。为明确哪一种亚复合物介导了上述多样的生物学功能，我们对一组同基因小鼠干细胞系开展了表型分析，每株细胞系分别缺失6种ISWI辅助亚基中的一种。单独缺失CERF、RSF1、ACF、WICH或NoRC亚复合物仅会对染色质景观产生轻度影响；而缺失NURF特异性亚基BPTF，则会显著降低CTCF结合位点周围的染色质开放性与Snf2h ATP酶的定位水平。尽管该缺失会缩短相邻核小体之间的间距，但仅对CTCF自身的结合产生轻微影响。在染色质开放性受损的情况下，结合状态的CTCF仍会出现功能异常，表现为黏连蛋白（cohesin）及其装载因子的结合占有率降低，且上述位点的绝缘子功能也随之减弱，这凸显了NURF介导的染色质重塑对于开放染色质形成及CTCF正常功能的必要性。本项全面分析揭示了NURF在结合状态的CTCF位点介导Snf2h定位与染色质开放过程中的特异性作用，证实局部染色质开放性对于黏连蛋白结合及绝缘子功能至关重要。实验整体设计：在小鼠胚胎干细胞（mouse Embryonic Stem Cells）中采用基于扩增子的单分子足迹法（Single Molecule Footprinting, SMF）进行检测，样本涵盖野生型与Bptf基因敲除（Bptf knock-out）组，每组设置三次生物学重复。