Investigating the role of SMCHD1 in de novo X chromosome inactivation. Mus musculus

To investigate the effect of Smchd1 ablation on de novo X chromosome inactivation (XCI), we derived clonal neural progenitor cells (NPCs) from Smchd1+/+ (wild-type, WT) and Smchd1-/- mouse embryonic stem cells (ES cells; ESC). We generated allele-specific RNA-seq datasets from WT and Smchd1-/- NPCs to examine the effect of Smchd1 ablation on gene silencing. In addition, we produced allele-specific ChIP-seq profiles of H3K4me3, H3K27me3, CTCF, and RAD21 and Xist CHART-seq profiles in WT and Smchd1-/- NPCs to investigate the role of SMCHD1 on the distribution of euchromatin, facultative heterochromatin, architectural proteins, and Xist RNA on the inactive X chromosome (Xi). To determine the localization of SMCHD1 on the Xi, we employed allele-specific DamID-seq to map SMCHD1-binding regions in female mouse embryonic fibroblasts. Furthermore, we performed in situ Hi-C on WT NPCs, Smchd1-/- NPCs, and female ES cells undergoing XCI, in order to explore the role of SMCHD1 in regulating the higher-order structure of the Xi. Overall design: RNA-seq, ChIP-seq for H3K4me3, H3K27me3, CTCF, and RAD21, Xist CHART-seq, and in situ Hi-C in neural progenitor cell clones derived from WT and Smchd1-/- female mouse embryonic stem cells (ES cells). DamID-seq for SMCHD1 and CBX1 in female mouse embryonic fibroblasts. In situ Hi-C on WT female mouse ES cells at different stages of X chromosome inactivation.