Mammalian SWI/SNF continuously restores local accessibility to chromatin [MNase-Seq]

Open chromatin is a hallmark of regulatory regions and serves as a feature for their identification. Accessible genomic sites are mediated by the activity of transcription factors (TFs) that can engage with nucleosomes to enable stable gene expression changes that underlie development. While the variability of open chromatin between tissues has been studied in large detail, we have limited knowledge on the dynamics of accessible chromatin in any given cell. Answering this question is critical to better understand cellular memory but also to discern actual kinetics of TF binding and its consequence on local nucleosomal organization. Here we use small molecule inhibition of the catalytic subunit of the mouse SWI/SNF remodeler complex to show that accessibility at sites of TF binding critically relies on persistent activity of nucleosome remodelers. Within minutes of remodeler inhibition, accessibility decreases excluding that remodeler activity is only necessary in defined cell cycle transitions. Moreover, this surprisingly fast response is irrespective of TF function as it is similar for the pluripotency factor and activating TF Oct4 and the repressive TF Rest. Importantly, accessibility is rapidly restored upon inhibitor removal demonstrating that open chromatin is regenerated continuously and in a cell-autonomous fashion. Combined, this establishes a model where TF binding to chromatin and remodeler-mediated nucleosomal removal do not represent an initial opening event leading to a stable situation but instead open chromatin reflects an average of a highly dynamic situation that requires continued reestablishment. This argues against a model where open chromatin can persist in absence of continuous TF binding and remodeler activity. MNase-Seq (2 replicates per condition) profiling of wildtype mouse embryonic stem cells treated with BRM014 compound, DMSO (control), or with BRM014 followed by wash-out of the compound.

开放染色质（open chromatin）是基因组调控区域的标志性特征，亦是调控区域识别的重要分子标记。基因组位点的染色质可及性由转录因子（transcription factors, TFs）的活性介导，此类转录因子可与核小体（nucleosomes）结合，从而介导稳定的基因表达改变，这一过程为个体发育提供了基础。尽管不同组织间开放染色质的差异已得到较为深入的研究，但我们对单个细胞内可及染色质的动态变化却知之甚少。解答这一问题，不仅有助于深入理解细胞记忆，亦能帮助我们解析转录因子结合的真实动力学过程，以及其对局部核小体结构的调控效应。本研究通过小分子抑制剂抑制小鼠SWI/SNF染色质重塑复合物的催化亚基，证实转录因子结合位点的染色质可及性严格依赖于核小体重塑酶的持续活性。在抑制重塑酶活性后的数分钟内，染色质可及性便迅速下降，这表明重塑酶的活性并非仅在特定细胞周期阶段才是必需的。此外，这一意外快速的响应与转录因子的功能无关：无论是多能性维持相关的激活型转录因子Oct4，还是抑制型转录因子Rest，其结合位点的可及性变化均呈现相似的动力学特征。值得注意的是，移除抑制剂后，染色质可及性可快速恢复，这表明开放染色质处于持续的、细胞自主的重建过程中。综合以上结果，本研究提出了全新的模型：转录因子与染色质的结合以及重塑酶介导的核小体移除，并非是启动染色质开放并维持稳定状态的初始事件；相反，开放染色质其实是高度动态过程的平均状态，这一过程需要持续的重建。这一结论与“在缺乏持续的转录因子结合和重塑酶活性的情况下，开放染色质仍可维持稳定”的模型相悖。本研究对经BRM014小分子化合物、二甲基亚砜（dimethyl sulfoxide, DMSO，对照组）处理，或经BRM014处理后洗脱化合物的野生型小鼠胚胎干细胞，开展了微球菌核酸酶测序（MNase-Seq）分析，每个处理条件设置2次生物学重复。