Fig. 2

Structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) and DNA methylation synergistically maintain inactive X chromosome (Xi) silencing. a An RNA-sequencing (RNA-seq) MA plot comparing the gene expression profiles of wild-type (WT) and Smchd1^−/− mouse embryonic fibroblasts (MEFs) treated with dimethyl sulfoxide (DMSO). Gray dots, genes not differentially expressed. Blue dots, autosomal differentially expressed genes. Red dots, X-linked differentially expressed genes. b Workflow showing the identification of genes subject to X-chromosome inactivation (XCI). Please see Supplementary Data 1 for the full list of genes subject to XCI and escapees. c Cumulative distribution plots (CDPs) of %mus for genes subject to XCI in DMSO-treated WT and Smchd1^−/− MEFs. P values are given by the Wilcoxon’s rank-sum test (paired, one-sided). d Cumulative distribution plots (CDPs) of %mus for genes subject to XCI in DMSO/5-aza-2′-deoxycytidine (Aza)-treated WT and Smchd1^−/− MEFs. P values are given by the Wilcoxon’s rank-sum test (paired, one-sided). e A heatmap showing the %mus of genes subject to XCI in eight different RNA-seq datasets, with unsupervised hierarchical clustering accurately grouping clones with the same genotype and treatment together. f Allele-specific RNA-seq coverage tracks of Flna and Ftsj1, two representative genes subject to XCI. cas, cas-specific reads (active X chromosome; Xa); mus, mus-specific reads (Xi). comp, all reads. To visualize rare mus reads originating from the Xi, the scales of mus tracks were set differently from the cas tracks. For simplicity, only the minus strand was shown. g CDPs comparing %mus of Class I genes vs. other genes subject to XCI. P values are given by the Wilcoxon’s rank-sum test (unpaired, one-sided). Please see Supplementary Data 2 for the full list of Class I genes defined previously²⁸