Precise modulation of mSWI/SNF identified dosage-sensitive chromatin regulation [CUT&Run]

ATP-dependent chromatin remodelers establish and arrange nucleosome distribution, modifying accessibility of DNA. Brg1 is exclusive ATPase subunit of mSWI/SNF complex. To understand the dosage dependent function of mSWI/SNF complex. We take an advantage of dTag system to precisely control abundancy of BRG1. We found overall Brg1-binding is sensitive to its abundance nevertheless OCT4-target or H3K27ac region, suggesting Brg1 genome-wide distribution rather than being actively recruited. We also reveal open chromatin dependency on BRG1. The chromatin accessibility of super enhancer shows buffered response on depletion of BRG1, whereas weak enhancer is sensitive. Finally, transcriptomic analysis identified that transcription shows buffered dependencies on BRG1 loss. Overall, our results highlight kinetics differences of BRG1-binding to transcriptome underline BRG1 dosage-sensitive mechanism. Overall design: To study the kinetics of BRG1 binding, chromatin accessibility, and gene expression, we employed the dTAG system to precisely control Brg1 protein levels in mESCs. We then used Cut&Run, ATAC-seq, and RNA-seq to examine the kinetic changes under different Brg1 protein levels.

ATP依赖的染色质重塑因子（ATP-dependent chromatin remodelers）通过建立与排布核小体分布，调控DNA的可及性。BRG1是mSWI/SNF复合物（mSWI/SNF complex）独有的ATP酶亚基。为阐明mSWI/SNF复合物的剂量依赖性功能，我们借助dTag系统（dTag system）精准调控BRG1的蛋白丰度。
研究发现，整体BRG1结合信号对其自身丰度极为敏感，即便在OCT4靶位点或H3K27ac富集区域亦是如此，这提示BRG1的全基因组分布模式更倾向于被动分布而非主动招募。我们还揭示了开放染色质对BRG1的依赖性：超级增强子的染色质可及性在BRG1缺失后表现出缓冲性响应，而弱增强子则对BRG1丰度变化更为敏感。最后，转录组分析显示，基因转录对BRG1缺失的依赖性同样存在缓冲效应。综上，本研究结果揭示了BRG1结合位点与转录组之间的动力学差异，阐明了BRG1的剂量敏感性调控机制。
总体实验设计：为探究BRG1结合、染色质可及性与基因表达的动力学特征，我们在小鼠胚胎干细胞（mESCs）中通过dTag系统精准调控BRG1的蛋白丰度，随后利用切割与释放技术（Cut&Run）、转座酶可及性染色质测序（ATAC-seq）及RNA测序（RNA-seq），检测不同BRG1蛋白水平下的动态变化。