RRBS in Smchd1 control and Smchd1 deleted male and female neural stem cells.. 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. Overall design: 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.