Mammalian ISWI and SWI/SNF selectively mediate binding of distinct groups of transcription factors (ATAC-seq data sets). Mammalian ISWI and SWI/SNF selectively mediate binding of distinct groups of transcription factors (ATAC-seq data sets)

Remodelers define nucleosome composition, presence and position. Mammalian imitation-switch-type (ISWI) comprise one class, mostly relying on the ATPase Snf2h for activity. We show that embryonic stem cells are viable without Snf2h, enabling to study its function and contrast it to Brg1, the ATPase of SWI/SNF. Loss of Snf2h specifically affects nucleosomal positioning and increases linker lengths genome-wide providing in vivo evidence for ISWI function in ruling nucleosomal spacing. Systematic analysis of transcription factor binding reveals selective requirement on either remodeling activity. One group, containing the transcriptional repressor REST depend on BRG1, while a non-overlapping set including the insulator protein CTCF, relies on Snf2h. Importantly localized reduction in CTCF binding decreases long-range interactions. Collectively, this links mammalian ISWI to nuclear organization, demonstrates its cellular role for nucleosomal periodicity and reveals that transcription factors rely on specific remodeling pathways for proper genomic binding. Overall design: ATAC-seq (14, 2 or 3 replicates per condition), Bis-seq (4, single replicate per condition), ChIP-seq (18 CTCF and 20 REST, 1 or 2 replicates per condition), HiC-seq (8, two replicates per condition), MNase-seq (8, 2 replicates per condition) and RNA-seq (18, 2 to 5 replicates per condition) profiling of mouse embryonic stem cells of wildtype, Snf2h ko, Snf2h ko/add-back (wildtype and mutant) and Brg1 ko genotypes.

染色质重塑因子决定核小体的组成、存在状态与定位。哺乳动物模仿开关型（imitation-switch-type, ISWI）重塑因子是其中一类，其活性主要由ATP酶Snf2h介导。本研究证实，小鼠胚胎干细胞在缺失Snf2h的情况下仍可存活，这为研究Snf2h的功能，并与SWI/SNF复合物的ATP酶Brg1进行对比分析提供了可行模型。Snf2h的缺失会特异性影响核小体定位，并在全基因组范围内增加核小体间连接DNA的长度，为ISWI调控核小体间距的体内功能提供了直接实验证据。对转录因子结合谱的系统性分析显示，不同转录因子对不同的染色质重塑活性存在选择性依赖：一类包含转录抑制因子REST的转录因子依赖BRG1，而另一组与之无重叠的、包含绝缘子蛋白CTCF的转录因子则依赖Snf2h。值得注意的是，CTCF结合位点的局部减少会降低基因组的长程染色质相互作用。综上，本研究将哺乳动物ISWI重塑因子与细胞核三维组织建立了功能关联，证实了其在维持核小体周期性方面的细胞生理功能，并揭示了转录因子需依赖特定的染色质重塑通路才能实现正常的基因组结合。

实验设计：对野生型、Snf2h敲除（Snf2h ko）、Snf2h敲除/回补（包含野生型与突变型回补两种情况）以及Brg1敲除（Brg1 ko）的小鼠胚胎干细胞进行多组学测序分析，具体包括：ATAC-seq（转座酶可及性测序，每个条件设置14、2或3次生物学重复）、Bis-seq（亚硫酸氢盐测序，每个条件1次生物学重复，共4组样本）、ChIP-seq（染色质免疫共沉淀测序，其中CTCF样本18份、REST样本20份，每个条件设置1或2次生物学重复）、HiC-seq（高通量染色体构象捕获测序，每个条件设置2次生物学重复，共8组样本）、MNase-seq（微球菌核酸酶测序，每个条件设置2次生物学重复，共8组样本）以及RNA-seq（RNA测序，每个条件设置2至5次生物学重复，共18组样本）。