Fig 2: Loss of SMCHD1 leads to changes of modified cytosine levels at the Dux locus.(A) Single CpG modification levels of WT and SMCHD1 KO samples at the Dux locus, as determined by WGBS. The differential methylation region is shaded in purple. CpGs are denoted with tick marks. Red circles, WT; blue circles, SMCHD1 KO. Circle size is proportional to coverage. A smoothed line is shown for each sample. (B) Manual bisulfite sequencing of the Dux promoter in WT and SMCHD1 KO cells. Solid black circles indicate modified CpG sites; open circles indicate unmodified CpG sites. Total percentages of modified cytosines (%Me) are shown. The primers are also indicated in (A). (C) Representative fluorescence image and FACS plot of 2C::tdTomato+ cells in the Smchd1 KO ES cell populations. (D) Methylation of the Dux promoter in the dTomato-expressing (FACS-sorted) Smchd1 KO cell population is further reduced. The data show bisulfite sequencing analysis of the Dux promoter in reporter-expressing WT and SMCHD1 KO ES cells. Total percentages of modified cytosines (%Me) are shown. (E) Percentages of modified cytosines at the Dux promoter determined from (B) and (D). One-way ANOVA was performed (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). Error bars indicate SEM from triplicate clones (WT and Smchd1-KO) or duplicate samples (FACS-sorted 2c-Smchd1-KO cells).

Fig 3: Transcription activation and 2c-like gene signature in the absence of SMCHD1.(A) Absence of SMCHD1 protein in three CRISPR-Cas9 KO ES cell clones. (B) Heatmap of RNA-seq data indicates differentially expressed genes between WT (n = 3 clones) and SMCHD1 KO (n = 3 clones) ES cells. (C) Gene set enrichment analysis (GSEA) of the 2c-like ES cell signature. The gene set represents genes activated during zygotic genome activation in 2c mouse embryos and enriched in 2C::tomato+ cells (42). The x axis shows the log2 fold change of the KO/WT-ranked transcriptome. GSEA analysis was performed as previously described (49). (D) The heatmap indicates the differentially expressed 2c-like genes between WT (n = 6) and SMCHD1 KO (n = 6) ES cells including two technical replicates for each clone. Typical 2c-like genes, such as Dux (indicated by red arrow), Zscan4c, Dub1, and Usp17l family members (indicated by purple arrows) are indicated. (E) The density plot indicates activation of repeat elements in SMCHD1 KO cells. The x axis shows the log2 (fold change of KO/WT) of repeat element expression. The y axis shows the density.

Fig 4: The 2c-like transcriptome in the absence of SMCHD1 is partially dependent on Dux.(A) The small guide RNA (gRNA) targeting region is immediately downstream of the start codon (ATG) of the Dux gene. Sanger sequencing confirmed frameshift mutations. Sequences targeted by the gRNA are in blue, and the PAM (protospacer adjacent motif) sequence is shown in red. Biallelic frameshift mutation was shown for each clone. The gRNA was applied in WT and in SMCHD1 KO ES cells to obtain the Dux single-knockout and Smchd1/Dux double-knockout ES cell clones, respectively. (B) A heatmap indicates the differentially expressed 2c-like genes between Dux/Smchd1 double-knockout (n = 3 clones) and Smchd1 single-knockout (n = 3 clones, two replicates each) ES cells. The blue color indicates genes no longer up-regulated in the double knockouts. (C) The pie chart shows that 47 of the 136 single-KO up-regulated 2c-like genes were no longer up-regulated in the absence of Dux in the Smchd1/Dux double KO. (D) RNA-seq tracks generated by the GVIZ package across the Zscan4 gene family members in WT ES cells (black), Smchd1 KO ES cells (red), Dux KO ES cells (blue), and Smchd1 plus Dux (double-KO) ES cells (green).

Fig 5: Loss of SMCHD1 causes up-regulation of Dux and the Zscan4 gene cluster, leading to the appearance of 2c-like cells.(A) Integrative Genomics Viewer screenshots of RNA-seq track peaks across all Zscan4 family members in WT and SMCHD1 KO ES cells. (B) Up-regulation of ZSCAN4 protein in SMCHD1 KO cell lines. (C) The fraction of ZSCAN4-positive cells in the ES cell population is increased in the absence of SMCHD1. ES cells were immunostained for ZSCAN4 (green). DNA was counterstained with DAPI (4',6-diamidino-2-phenylindole) (blue). Scale bars, 50 µm. (D) Fractions of ZSCAN4+ cells in WT ES cells and Smchd1-KO ES cells. t test was performed for statistical analysis (P < 0.001). Error bars indicate SEM (six independent experiments). (E) Browser view of RNA-seq tracks across the Dux locus in WT and SMCHD1 KO ES cells. The Dux gene itself is shaded in yellow. (F) Quantitative real-time polymerase chain reaction (qRT-PCR) data confirm Dux activation upon SMCHD1 loss. ß-Actin was used as a control. One-way ANOVA was performed for statistical analysis, comparing the mean of each group with the mean of the WT group (***P < 0.001). Data are for means ± SEM of three independent KO clones.