An RNA binding region in CTCF regulates chromatin looping and CTCF nuclear organization

The study investigates CTCF/cohesin binding and chromatin looping by ChIP-seq and Micro-C. By ChIP-seq, we determined the genome-wide binding profiles of CTCF and Smc1a cohesin subunit in a knock-in mouse ES cell line (wt-CTCF; clone C59) with endogenously tagged wild type CTCF (FLAG-Halo-mCTCF) and Rad21 (mRad21-SNAPf-V5), and compared them to the same ES line expressing a mutant CTCF (ΔRBR-CTCF; clone C59D2), where we replaced a previously described RNA binding region with a short linker (GDGAGLINS) followed by a 3xHA tag (N576_D611del::3xHA). By Micro-C, we compared nucleosome-resolution chromosome folding maps of the same ES cell lines C59 and C59D2 described above, to determine the effect of deleting CTCF RNA binding region on chromatin looping.

本研究借助染色质免疫共沉淀测序（ChIP-seq）与Micro-C技术，探究CCCTC结合因子（CTCF）/黏连蛋白（cohesin）的结合状态及染色质环形成过程。通过ChIP-seq实验，我们在一株内源标记野生型CTCF（FLAG-Halo-mCTCF）与Rad21（mRad21-SNAPf-V5）的敲入型小鼠胚胎干细胞（ES细胞）系（wt-CTCF；克隆C59）中，测定了CTCF与Smc1a黏连蛋白亚基的全基因组结合谱，并将其与另一株表达突变型CTCF的ES细胞系（ΔRBR-CTCF；克隆C59D2）进行对比；后者通过将已报道的RNA结合区域替换为短连接肽GDGAGLINS，后续连接3xHA标签（具体突变为N576_D611del::3xHA）构建获得。此外，我们通过Micro-C技术，对比了上述C59与C59D2两株ES细胞系的核小体分辨率级染色体折叠图谱，以明确CTCF RNA结合区域缺失对染色质环形成的影响。