The chromosomal protein SMCHD1 regulates DNA methylation and the 2c-like state of embryonic stem cells by antagonizing TET proteins

Abstract

5-Methylcytosine (5mC) oxidases, the ten-eleven translocation (TET) proteins, initiate DNA demethylation, but it is unclear how 5mC oxidation is regulated. We show that the protein SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1) is found in complexes with TET proteins and negatively regulates TET activities. Removal of SMCHD1 from mouse embryonic stem (ES) cells induces DNA hypomethylation, preferentially at SMCHD1 target sites and accumulation of 5-hydroxymethylcytosine (5hmC), along with promoter demethylation and activation of the Dux double-homeobox gene. In the absence of SMCHD1, ES cells acquire a two-cell (2c) embryo-like state characterized by activation of an early embryonic transcriptome that is substantially imposed by Dux Using Smchd1/Tet1/Tet2/Tet3 quadruple-knockout cells, we show that DNA demethylation, activation of Dux, and other genes upon SMCHD1 loss depend on TET proteins. These data identify SMCHD1 as an antagonist of the 2c-like state of ES cells and of TET-mediated DNA demethylation.

Fig. 1. Interaction of SMCHD1 and TET proteins.

(A) Flag purification of TET3FL and TET3S from 293T cells. The purified samples were subjected to Coomassie blue staining (left) and Western blotting (right). The gel segments indicated were analyzed by MS. M, molecular weight markers; IB, immunoblot. (B) Identification of SMCHD1 as a binding partner of TET3 by MS. TET3S was expressed in 293T cells and immunoprecipitated with anti-FLAG beads. Gel segment 2 (A) was subjected to LC-MS/MS (liquid chromatography–tandem MS) analysis (see Materials and Methods and table S1). The top eight highest-scoring proteins are shown. M.W., molecular weight. (C) Endogenous coimmunoprecipitation (co-IP) of SMCHD1 with TET1, TET2, and TET3FL. (D) Interaction between TET proteins and SMCHD1 by co-IP using expression of tagged proteins in 293T cells. (E) Different domains of TET3 were cotransfected with full-length SMCHD1 into 293T cells. After IP, the interacting proteins were identified by Western blotting. Stars indicate IgG (immunoglobulin G) bands. aa, amino acids. (F) Different domains of SMCHD1 were cotransfected with TET3FL into 293T cells. After IP, the interacting proteins were identified by Western blotting. Stars indicate IgG bands. HATPase, histidine kinase-like ATPase domain.

Fig. 2. Inhibition of TET activity by SMCHD1.

(A) Reduction in 5hmC levels by coexpression of SMCHD1 with TET3 in 293T cells. 5hmC and 5mC contents were assessed using antibody-based dot blots. One-way analysis of variance (ANOVA) was performed comparing the mean of each group with the mean of the second group (**P < 0.01 and ***P < 0.001; mean ± SEM). ns, not significant. (B) Inhibition of TET3S-induced reactivation of a methylation-silenced luciferase construct by SMCHD1 in 293T cells (top). One-way ANOVA was performed (**P < 0.01 and ****P < 0.0001). Data are for means ± SEM of three independent experiments. An unmethylated luciferase vector was used as a control (bottom). (C) FLAG purification of TET2-CD and SMCHD1 full length (SMCHD1-FL) from Sf9 insect cells. Coomassie blue staining. (D) Inhibition of TET2-CD activity on fully methylated DNA in the presence of SMCHD1 as shown by combined bisulfite restriction analysis (COBRA) assay (BstU I cleavage indicates methylation). P.C., positive control with excess TET protein (18 μg); N.C., negative control without TET treatment. Different molar ratios of SMCHD1 and TET protein (1.15 μg) are shown. The H19 imprinting control region was analyzed. (E) Bisulfite sequencing analysis of H19 methylation analyzed in duplicates. Solid black circles indicate modified CpGs; open circles indicate TET-oxidized mCpGs. The purple arrows indicate BstU I sites. (F) Percentages of modified cytosines (%Me) of the different samples. P values were determined by Fisher’s exact test (two sided).

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. 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. β-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.

Fig. 5. 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. 6. 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. 7. The aberrant transcriptome and DNA hypomethylation in the absence of SMCHD1 depend on the presence of TET proteins.

(A) Absence of SMCHD1 protein in three CRISPR-Cas9–targeted Tet triple-knockout ES cell clones. (B) RNA-seq tracks across Dux in WT, Tet triple-knockout ES cells, and Tet-Smchd1 quadruple-KO cells. (C) Quantitative RT-PCR analysis of Dux expression in WT, Tet triple-knockout and Tet-Smchd1 quadruple-knockout cells. One-way ANOVA was performed. Data are for means ± SEM of three independent clones. (D) RNA-seq analysis across different genes in WT, Smchd1 KO, Dux KO, Smchd1 and Dux (double) KO, Tet triple-knockout, and Tet-Smchd1 quadruple-knockout ES cells. One-way ANOVA was performed, comparing the mean of each group (n = 3 clones each) with the mean of the Smchd1-KO group (**P < 0.01 and ****P < 0.0001). Error bars indicate SEM. (E) The number of up-regulated 2c-like genes is decreased in the absence of TET proteins. (F) Bisulfite sequencing analysis of the Dux promoter in Tet-TKO cells and quadruple-knockout cells. Percentages of modified cytosines (%Me) are shown. (G) Eighty-nine percent (75 and 14%) of significantly up-regulated genes in the SMCHD1 single KO are no longer up-regulated in the absence of TET proteins in the Tet-Smchd1 quadruple-KO (qKO) cells. (H) Model of SMCHD1 as a negative regulator of TET proteins at the Dux promoter. Black circles, 5mC; light blue circles, 5hmC; white circles, unmethylated CpGs.