RRBS in Smchd1 control and Smchd1 deleted 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. Neural stem cells were generated from Smchd1 del/fl Xist 129-KO/Xist WT-Cast E14.5 embryos, as previously described (Chen et al., 2015 PNAS). After these lines were established, they were split in two - half were transduced with a retrovirus carrying Cre recombinase and a puromycin selection cassette (MSCV-Cre-puro), half were retained untransduced as controls. 24h post transduction puromycin was added to select transduced cells, and 7 days following transduction cells were harvested for genomic analyses.