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 (?RBR-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. Overall design: 1) ChIP-Seq of CTCF and Smc1a cohesin subunit in mouse ES cells homozygous for FLAG-Halo-mCTCF and mRad21-SNAPf-V5 (C59: wt-CTCF) and in the same ES cell line where we deleted CTCF RNA binding region (C59D2: ?RBR-CTCF). ChIP-Seq libraries were prepared independently from two ChIP biological replicates, using normal serum IgGs as a control, and including total inputs as reference datasets. 2) Three replicates of Micro-C data were generated in the same cell lines as ChIP-seq. Two replicates (named as “QC”) were initially generated for evaluating library quality and improving experimental protocol. These samples qualitatively and quantitatively reproduce the results by the optimized protocol, regardless a higher percentage of PCR duplicates. High quality and coverage of Micro-C data (named as “highCov”) were generated with an optimized protocol producing over 650M unique pairs per sample. 3) RNA-Seq of total RNA (ribosomal RNA depleted) in mouse ES cells homozygous for FLAG-Halo-mCTCF and mRad21-SNAPf-V5 (C59: wt-CTCF) and in the same ES cell line where we deleted CTCF RNA binding region (C59D2: ?RBR-CTCF). RNA-Seq libraries were prepared from two biological replicates.