ChIP-seq for Smchd1 in male and female neural stem cells.. ChIP-seq for Smchd1 in male and female neural stem cells.

We sought to examine whether the non-canonical SMC protein Smchd1 plays a role in chromosome conformation. We used in situ Hi-C to analyse chromosome conformation changes upon deletion of the epigenetic regulator Smchd1 in female neural stem cells. In parallel, we analysed nucleosome accessibility using ATAC-seq, gene expression using RNA-seq, chromatin marks H3K27me3 and H3K27ac and Ctcf binding using ChIP-seq. We additionally analysed Smchd1 binding genome-wide using ChIP-seq. Together, we find that deletion of Smchd1 alters chromosome conformation at Smchd1 target genes including the inactive X chromosome, Hox genes and imprinted loc. Smchd1 deletion results in gain in Ctcf binding and activation of enhancers. We propose Smchd1 functions by limiting Ctcf-mediated chromosome looping. Overall design: n=2 Smchd1 GFP/GFP vs n=2 Smchd1+/+ female neural stem cell lines + Whole cell extract controls, to test binding of Smchd1 to the inactive X, using long sonication to release the inactive X heterochromatin. n=1 Smchd1GFP/GFP female + n=1 Smchd1 GFP/GFP male NSCs vs n=1 Smchd1+/+ female and n=1 Smchd1+/+ male neural stem cell lines plus whole cell extract controls, using MNase digestion for fragmentation, to assess autosomal binding of Smchd1.

本研究旨在探究非经典SMC（non-canonical SMC）蛋白Smchd1是否在染色体构象调控中发挥作用。本研究采用原位Hi-C（in situ Hi-C）技术，分析雌性神经干细胞中表观遗传调控因子Smchd1敲除后染色体构象的变化情况。同时，本研究采用转座酶可及性染色质测序（ATAC-seq）分析核小体可及性，采用RNA测序（RNA-seq）分析基因表达水平，采用染色质免疫共沉淀测序（ChIP-seq）检测染色质修饰组蛋白H3第27位三甲基化（H3K27me3）、组蛋白H3第27位乙酰化（H3K27ac）以及Ctcf（CCCTC结合因子）的结合情况。此外，本研究还采用ChIP-seq技术对全基因组范围内的Smchd1结合情况进行了分析。
综合分析结果显示，Smchd1敲除会改变Smchd1靶基因区域的染色体构象，这些靶基因包括失活X染色体、Hox基因以及印记基因座。Smchd1敲除会导致Ctcf结合增强以及增强子激活。本研究提出，Smchd1通过抑制Ctcf介导的染色体环形成发挥功能。
总体实验设计：第一组实验设置2株Smchd1 GFP/GFP雌性神经干细胞系与2株Smchd1+/+雌性神经干细胞系作为对照，并增设全细胞提取物对照，采用长片段超声破碎法释放失活X染色体异染色质，以检测Smchd1在失活X染色体上的结合情况。第二组实验设置1株Smchd1GFP/GFP雌性神经干细胞、1株Smchd1 GFP/GFP雄性神经干细胞，分别与1株Smchd1+/+雌性神经干细胞、1株Smchd1+/+雄性神经干细胞进行对照，并增设全细胞提取物对照，采用微球菌核酸酶（MNase）酶解法进行片段化处理，以检测Smchd1在常染色体上的结合情况。