MeCP2 deficiency promotes cell reprogramming by stimulating IGF1/AKT/mTOR signaling and activating ribosomal protein-mediated cell cycle gene translation

MeCP2 deficiency promotes cell reprogramming by stimulating IGF1/AKT/mTOR signaling and... Abstract The generation of induced pluripotent stem cells (iPSCs) offers a great opportunity in research and regenerative medicine. The current poor efficiency and incomplete mechanistic understanding of the reprogramming process hamper the clinical application of iPSCs. MeCP2 connects histone modification and DNA methylation, which are key changes of somatic cell reprogramming. However, the role of MeCP2 in cell reprogramming has not been examined. In this study, we found that MeCP2 deficiency enhanced reprogramming efficiency and stimulated cell proliferation through regulating cell cycle protein expression in the early stage of reprogramming. MeCP2 deficiency enhanced the expression of ribosomal protein genes, thereby enhancing reprogramming efficiency through promoting the translation of cell cycle genes. In the end, MeCP2 deficiency stimulated IGF1/AKT/mTOR signaling and activated ribosomal protein gene expression. Taken together, our data indicate that MeCP2 deficiency promoted cell reprogramming through stimulating IGF1/AKT/mTOR signaling and activating ribosomal protein-mediated cell cycle gene translation in the early stage of reprogramming. MeCP2, cell reprogramming, IGF1/AKT/mTOR signaling, ribosomal protein, cell cycle Introduction The generation of induced pluripotent stem cells (iPSCs), somatic cells reprogrammed to the pluripotent state by forced expression of nuclear transcription factors, such as Oct4, Sox2, Klf4, and c-Myc (named as OSKM hereinafter) (Takahashi and Yamanaka, 2006; Okita et al., 2007), holds great promise for research and regenerative medicine (Robinton and Daley, 2012). However, the current poor efficiency and an incomplete mechanistic understanding of the reprogramming process hamper the clinical application of iPSCs (Gonzalez et al., 2011; Vierbuchen and Wernig, 2012). The generally accepted model of the molecular mechanisms involved in iPSC reprogramming consists of three stages: initiation, maturation, and stabilization (Samavarchi-Tehrani et al., 2010; Buganim et al., 2013). Reprogramming is initiated in cells that receive reprogramming transgenes, and is characterized by increased proliferation and a metabolic switch from oxidative phosphorylation to glycolysis. The initiation stage also involves a phenotypic mesenchymal-to-epithelial transition (David and Polo, 2014). A high proliferation rate is required for cell reprogramming (Ruiz et al., 2011). c-Myc or Lin28 transgene expression and p53 knockdown increase the efficiency of iPSC reprogramming by stimulating cell proliferation (Hanna et al., 2009; Apostolou and Hochedlinger, 2013). Specifically, Lin28 has been shown to regulate cell cycle genes such as Cyclin A, Cyclin B, and Cdk4 (Xu et al., 2009). Furthermore, insulin-like growth factor (IGF) is an essential growth factor and may enhance the expression of reprogramming factors, suggesting a potential role of IGF-1 in the reprogramming process (Li and Geng, 2010). Methyl-CpG-binding protein 2 (MeCP2), is a classic methylated-DNA-binding protein, and dysfunctions in this protein lead to various neurodevelopmental disorders such as autism spectrum disorder and Rett syndrome (Amir et al., 1999; Chen et al., 2001; Guy et al., 2001; Kriaucionis and Bird, 2003). MeCP2 is regarded as a transcriptional repressor for methylated genes and interacts with Sin3A/HDAC and NCoR/SMRT co-repressor complexes (Nan et al., 1993, 1997, 1998). MeCP2 connects histone modification and DNA methylation, which are key changes of somatic cell reprogramming. However, the role of MeCP2 in cell reprogramming has not been examined. Here, we showed that MeCP2 deficiency promoted cell reprogramming through stimulating IGF1/AKT/mTOR signaling and activating ribosomal protein-mediated cell cycle gene translation in the early stage of reprogramming. Results MeCP2 deficiency enhances reprogramming efficiency To investigate whether MeCP2 plays important roles in cell fate determination, we utilized iPSC technology to examine the effects of MeCP2 deficiency on cell reprogramming. We first used MeCP2-knockout (KO) mouse embryonic fibroblasts (MEFs) expressing the four Yamanaka factors, OSKM. MeCP2-deficient MEFs exhibited significantly improved reprogramming efficiency, with almost 4-fold more alkaline phosphatase (AP)-positive iPSC colonies than that of the control (Figure 1A and B). We further determined the pluripotency of MeCP2 deficiency-induced iPSCs. The expression of pluripotency markers was indeed observed in these MeCP2-deficient iPSCs (Figure 1C). these MeCP2-deficient iPSCs exhibited the potential to develop into teratomas with three germ layers (Figure 1D), and chimeric mice could be generated from MeCP2-deficient iPSCs (Figure 1E). These results demonstrated that MeCP2-deficient iPSCs were fully reprogrammed and pluripotent. To test whether certain known mutations in MeCP2 subunits have similar promoting effects on reprogramming, we further detected the effects of two MeCP2 mutations R133C and R255X (Amir et al., 1999) on reprogramming. Our results showed that certain known mutations in MeCP2 subunits indeed have similar promoting effects on reprogramming (Figure 1F and G). Figure 1 View largeDownload slide MeCP2 deficiency enhances reprogramming efficiency. (A) Detection of iPSC colonies among control and MeCP2-deficient reprogrammed MEFs by AP staining. Successfully reprogrammed iPSC colonies are AP-positive (left panel). Bar graph showing the comparison of AP-positive colonies between control and MeCP2-deficient reprogrammed MEFs (right panel). **P < 0.01, Student’s t-test, n = 3 independent experiments. (B) Western blot for endogenous MeCP2 in control and MeCP2-deficient MEFs and iPSCs. Tubulin was used as a loading control. (C) Immunofluorescent staining of pluripotency markers in MeCP2-deficient iPSCs. (D) MeCP2-deficient iPSCs could effectively produce full teratomas, which contained differentiated cells in all three germ layers, in SCID mice. (E) MeCP2-deficient iPSCs generated mice with chimeric ability. (F) AP-positive colonies among reprogrammed MeCP2-deficient MEFs with wild-type and two mutations of MeCP2. *P < 0.05, **P < 0.01, Student’s t-test, n = 3 independent experiments. (G) Western blot for overexpression of wild-type and two mutations of MeCP2 in MEFs. Tubulin was used as a loading control. Figure 1 View largeDownload slide MeCP2 deficiency enhances reprogramming efficiency. (A) Detection of iPSC colonies among control and MeCP2-deficient reprogrammed MEFs by AP staining. Successfully reprogrammed iPSC colonies are AP-positive (left panel). Bar graph showing the comparison of AP-positive colonies between control and MeCP2-deficient reprogrammed MEFs (right panel). **P < 0.01, Student’s t-test, n = 3 independent experiments. (B) Western blot for endogenous MeCP2 in control and MeCP2-deficient MEFs and iPSCs. Tubulin was used as a loading control. (C) Immunofluorescent staining of pluripotency markers in MeCP2-deficient iPSCs. (D) MeCP2-deficient iPSCs could effectively produce full teratomas, which contained differentiated cells in all three germ layers, in SCID mice. (E) MeCP2-deficient iPSCs generated mice with chimeric ability. (F) AP-positive colonies among reprogrammed MeCP2-deficient MEFs with wild-type and two mutations of MeCP2. *P < 0.05, **P < 0.01, Student’s t-test, n = 3 independent experiments. (G) Western blot for overexpression of wild-type and two mutations of MeCP2 in MEFs. Tubulin was used as a loading control. MeCP2 deficiency enhances reprogramming efficiency in the early stage of reprogramming To determine in which stage MeCP2 deficiency promoted reprogramming, we measured SSEA-1, a marker of early reprogramming, positivity in cells during the reprogramming process and found that the SSEA-1-positive cell percentage of the MeCP2-deficient group was significantly increased compared to the percentage in the control group at reprogramming days 3, 5, 7, and 12 (Figure 2A). Notably, MeCP2 depletion during early stages of somatic reprogramming promoted iPSC derivation from MEFs (Figure 2B−D). Conversely, MeCP2 overexpression during early stages of somatic reprogramming diminished iPSC derivation from MEFs (Figure 2B and E). Figure 2 View largeDownload slide MeCP2 deficiency stimulates cell proliferation in the early stage of reprogramming. (A) SSEA1-positive cell percentages from the indicated group at reprogramming days 3, 5, 7, and 12. Wild-type cells were used for comparison. Data represent mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, Student’s t-test, n = 3 independent experiments. (B) Western blot for endogenous MeCP2 in control and MeCP2 knockdown cells. Tubulin was used as loading control (upper panel). Western blot for HA-MeCP2 in control and MeCP2-overexpressing cells. Tubulin was used as a loading control (lower panel). (C) AP-positive colonies among reprogrammed MEFs with or without MeCP2 siRNA transfection. *P < 0.05, **P < 0.01, ANOVA, together with post hoc tests, n = 3 independent experiments. (D) siRNA transfections were started either at −2, 0, +2, or +4 days relative to the expression of the four Yamanaka factors, with repeated transfections at 3-day intervals. iPSC formation was analyzed at day 14 by counting AP-positive colonies. Data represent mean ± SD of three independent experiments. ***P < 0.001, ANOVA, together with post hoc tests, n = 3 independent experiments. (E) Lenti-MeCP2 transfections were started either at −2, 0, +2, or +4 days relative to the expression of the four Yamanaka factors. iPSC formation was analyzed at day 14 by counting AP-positive colonies. Data represent mean ± SD of three independent experiments. ***P < 0.001, ANOVA, together with post hoc tests, n = 3 independent experiments. Figure 2 View largeDownload slide MeCP2 deficiency stimulates cell proliferation in the early stage of reprogramming. (A) SSEA1-positive cell percentages from the indicated group at reprogramming days 3, 5, 7, and 12. Wild-type cells were used for comparison. Data represent mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, Student’s t-test, n = 3 independent experiments. (B) Western blot for endogenous MeCP2 in control and MeCP2 knockdown cells. Tubulin was used as loading control (upper panel). Western blot for HA-MeCP2 in control and MeCP2-overexpressing cells. Tubulin was used as a loading control (lower panel). (C) AP-positive colonies among reprogrammed MEFs with or without MeCP2 siRNA transfection. *P < 0.05, **P < 0.01, ANOVA, together with post hoc tests, n = 3 independent experiments. (D) siRNA transfections were started either at −2, 0, +2, or +4 days relative to the expression of the four Yamanaka factors, with repeated transfections at 3-day intervals. iPSC formation was analyzed at day 14 by counting AP-positive colonies. Data represent mean ± SD of three independent experiments. ***P < 0.001, ANOVA, together with post hoc tests, n = 3 independent experiments. (E) Lenti-MeCP2 transfections were started either at −2, 0, +2, or +4 days relative to the expression of the four Yamanaka factors. iPSC formation was analyzed at day 14 by counting AP-positive colonies. Data represent mean ± SD of three independent experiments. ***P < 0.001, ANOVA, together with post hoc tests, n = 3 independent experiments. MeCP2 deficiency promotes cell proliferation through regulating cell cycle gene expression at the protein level Since the initiation stage of cell reprogramming is characterized by increased proliferation (David and Polo, 2014), we examined the cell proliferation of MeCP2-deficient MEFs. The growth rate of MeCP2-deficient MEFs was upregulated compared with that of control cells (Figure 3A and B). Flow cytometry (FACS) analysis showed that the percentage of cells in the G1/G0 phase of MeCP2-deficient MEFs was decreased (Figure 3C). We further examined cell proliferation and cell cycle of MeCP2-deficient MEFs expressing the four Yamanaka factors, OSKM. Consistent with the above-mentioned results, cell proliferation and FACS showed that the growth rate of MeCP2-deficient MEFs expressing the four Yamanaka factors, OSKM, was increased, and the percentage of cells in the G1/G0 phase of MeCP2-deficient MEFs expressing the four Yamanaka factors, OSKM, was decreased (Figure 3D−F). To further demonstrate the importance of MeCP2 deficiency in cell proliferation, we detected the cell cycle gene expression at the RNA and protein level. There was no difference in the expression of cell cycle genes between WT and MeCP2-deficient MEFs at the RNA level, and significantly increased expression in MeCP2-deficient MEFs at the protein level (Figure 3G and H). There was no difference of expression levels of Klf4 and c-Myc in WT and MeCP2-deficient MEFs on the second day of induction. The expression of Oct4 and Nanog was not detected in both cells, while the expression of Sox2 was only detected in MeCP2-deficient MEFs (Figure 3H). Collectively, our results suggest that MeCP2 deficiency may mediate cell proliferation through regulating the expression of cell cycle genes at the protein level. Figure 3 View largeDownload slide MeCP2 deficiency promotes cell proliferation through regulating cell cycle protein expression. (A and B) The effect of MeCP2 deficiency on cell proliferation. Cell proliferation of MeCP2-deficient and control MEFs was measured by CCK8 and CSFE assays. (C) The effect of MeCP2 deficiency on cell cycle progression. (D and E) The effect of MeCP2 deficiency on expression of the four Yamanaka factors OSKM on cell proliferation. Cell proliferation of MeCP2-deficient MEFs expressing the four Yamanaka factors OSKM and control MEFs was measured by CCK8 and CSFE assays. (F) The effect of MeCP2 deficiency in cells expressing the four Yamanaka factors on cell cycle progression. (G) qRT-PCR for the cell cycle gene expression in control and MeCP2-deficient MEFs at the RNA level. (H) Western blot for cell cycle gene and pluripotent gene expression in control and MeCP2-deficient MEFs at the protein level. Tubulin was used as a loading control. *P < 0.05, **P < 0.01, Student’s t-test, n = 3 independent experiments. Figure 3 View largeDownload slide MeCP2 deficiency promotes cell proliferation through regulating cell cycle protein expression. (A and B) The effect of MeCP2 deficiency on cell proliferation. Cell proliferation of MeCP2-deficient and control MEFs was measured by CCK8 and CSFE assays. (C) The effect of MeCP2 deficiency on cell cycle progression. (D and E) The effect of MeCP2 deficiency on expression of the four Yamanaka factors OSKM on cell proliferation. Cell proliferation of MeCP2-deficient MEFs expressing the four Yamanaka factors OSKM and control MEFs was measured by CCK8 and CSFE assays. (F) The effect of MeCP2 deficiency in cells expressing the four Yamanaka factors on cell cycle progression. (G) qRT-PCR for the cell cycle gene expression in control and MeCP2-deficient MEFs at the RNA level. (H) Western blot for cell cycle gene and pluripotent gene expression in control and MeCP2-deficient MEFs at the protein level. Tubulin was used as a loading control. *P < 0.05, **P < 0.01, Student’s t-test, n = 3 independent experiments. MeCP2 deficiency enhances the expression of ribosomal protein genes, which enhances reprogramming efficiency To elucidate the underlying mechanisms by which MeCP2 deficiency promotes reprogramming, we then performed RNA-Seq analysis using MeCP2-deficient MEFs and iPSCs (Figure 4A and B). As expected, we observed obvious differential gene expression profiles between MeCP2-deficient MEFs and iPSCs and their respective control cells. Based on the gene expression dynamics, we found that the ribosomal protein genes were reactivated and highly expressed in both MeCP2-deficient MEFs and iPSCs (Figure 4A and B). The pluripotent genes were reactivated and highly expressed in both control and MeCP2-deficient iPSCs (Figure 4B). We chose the pathways which are enriched in differentially expressed genes between WT and MeCP2-deficient MEFs and have been reported previously and found that the PI3K/AKT signaling was reactivated in MeCP2-deficient MEFs (Figure 4A). Furthermore, the cell cycle genes were highly expressed in MeCP2-deficient MEFs (Figure 4B). Figure 4 View largeDownload slide MeCP2 deficiency enhances ribosomal protein gene expression, which enhances reprogramming efficiency. (A and B) KEGG pathway (A) and heatmaps analysis (B) of upregulated genes in RNA-Seq data from MeCP2-deficient MEFs and iPSCs. (C) mRNA abundance of ribosomal protein genes in control and MeCP2-deficient MEFs. (D) Protein abundance of ribosomal proteins in control and MeCP2-deficient MEFs. Tubulin was used as loading control. (E and F) Western blot analysis of ribosomal proteins expression in the indicated cells. (G) AP-positive colonies in reprogrammed MEFs with or without transfection with siRNAs against ribosomal protein genes. (H) AP-positive colonies among reprogrammed MEFs with or without overexpression of ribosomal protein genes. *P < 0.05, ***P < 0.001, ANOVA, together with post hoc tests, n = 3 independent experiments. Figure 4 View largeDownload slide MeCP2 deficiency enhances ribosomal protein gene expression, which enhances reprogramming efficiency. (A and B) KEGG pathway (A) and heatmaps analysis (B) of upregulated genes in RNA-Seq data from MeCP2-deficient MEFs and iPSCs. (C) mRNA abundance of ribosomal protein genes in control and MeCP2-deficient MEFs. (D) Protein abundance of ribosomal proteins in control and MeCP2-deficient MEFs. Tubulin was used as loading control. (E and F) Western blot analysis of ribosomal proteins expression in the indicated cells. (G) AP-positive colonies in reprogrammed MEFs with or without transfection with siRNAs against ribosomal protein genes. (H) AP-positive colonies among reprogrammed MEFs with or without overexpression of ribosomal protein genes. *P < 0.05, ***P < 0.001, ANOVA, together with post hoc tests, n = 3 independent experiments. We further conducted quantitative real-time PCR (qRT-PCR) to detect some of these ribosomal protein genes in MeCP2-deficient MEFs. The expression of ribosomal protein genes was much higher in MeCP2-deficient MEFs, compared to expression in controls (Figure 4C). Accordingly, the abundance of ribosomal proteins was much higher in MeCP2-deficient MEFs, compared to levels in controls (Figure 4D). To investigate whether ribosomal protein genes play important roles in cell reprogramming, we first respectively downregulated three ribosomal protein genes that were highly expressed in MeCP2-deficient MEFs, into MEFs expressing the four Yamanaka factors, OSKM. Knocking down ribosomal protein genes expression using siRNAs during the reprogramming process resulted in reduced iPSC colony numbers (Figure 4E and G). Conversely, overexpressing ribosomal protein genes in MEFs significantly improved reprogramming efficiency, as seen by the number of obtained iPSC colonies (Figure 4F and H). Ribosomal protein genes promote cell cycle protein expression Since the ribosomal protein genes were highly expressed in MeCP2-deficient MEFs and MeCP2 deficiency mediated cell proliferation by regulating cell cycle protein expression, we supposed that ribosomal protein genes promote the translation of cell cycle genes. To confirm this hypothesis, we detected the protein and mRNA levels of cell cycle genes in control MEFs and MEFs overexpressing ribosomal protein genes. We found that overexpressing ribosomal protein genes increased cell cycle gene expression at the protein level (Figure 5A and B). Conversely, inhibiting ribosomal protein gene expression by knocking down ribosomal protein gene expression using siRNAs resulted in decreased cell cycle gene expression at the protein level (Figure 5C and D). To further demonstrate that ribosomal protein genes affect cell cycle gene expression at the translation level, we then performed RNA immunoprecipitation (RIP) assays to demonstrate binding between ribosomal proteins and the mRNA of cell cycle genes (Figure 5E). Our results suggested that ribosomal protein genes promote the translation of cell cycle genes. Figure 5 View largeDownload slide Ribosomal protein genes promote cell cycle protein expression. (A) Protein abundance of cell cycle genes in MEFs with or without overexpression of ribosomal protein genes. Tubulin was used as a loading control. (B) mRNA abundance of cell cycle genes in MEFs with or without overexpression of ribosomal protein genes. (C) Protein abundance of cell cycle genes in MEFs with or without knockdown of ribosomal protein genes. Tubulin was used as a loading control. (D) mRNA abundance of cell cycle genes in MEFs with or without knockdown of ribosomal protein genes. (E) RIP assays for the recruitment of ribosomal proteins to the cell cycle gene mRNA. **P < 0.01, ***P < 0.001, Student’s t-test, n = 3 independent experiments. Figure 5 View largeDownload slide Ribosomal protein genes promote cell cycle protein expression. (A) Protein abundance of cell cycle genes in MEFs with or without overexpression of ribosomal protein genes. Tubulin was used as a loading control. (B) mRNA abundance of cell cycle genes in MEFs with or without overexpression of ribosomal protein genes. (C) Protein abundance of cell cycle genes in MEFs with or without knockdown of ribosomal protein genes. Tubulin was used as a loading control. (D) mRNA abundance of cell cycle genes in MEFs with or without knockdown of ribosomal protein genes. (E) RIP assays for the recruitment of ribosomal proteins to the cell cycle gene mRNA. **P < 0.01, ***P < 0.001, Student’s t-test, n = 3 independent experiments. MeCP2 deficiency enhances reprogramming efficiency through stimulating IGF1/AKT/mTOR signaling and activating ribosomal protein genes Since the PI3K/AKT signaling and ribosomal protein genes were activated in MeCP2-deficient MEFs, we supposed that the PI3K/AKT signaling-mediated ribosomal protein genes activation play important roles in reprogramming. To confirm this hypothesis, we examined IGF1/AKT/mTOR signaling, a traditional ribosomal protein gene regulatory pathway, and found that levels of IGF1/AKT/mTOR signaling proteins P-AKT and P-S6 were significantly increased in MeCP2-deficient MEFs (Figure 6A). Furthermore, IGF1/AKT/mTOR signaling was inhibited by treating cells with rapamycin and AZD8055, inhibitors of IGF1/AKT/mTOR signaling, during the reprogramming process. Inhibiting this signaling pathway reduced iPSC colony numbers (Figure 6B). Meanwhile, inhibiting IGF1/AKT/mTOR signaling reduced the expression of ribosomal proteins and cell cycle proteins in MeCP2-deficient MEFs (Figure 6C). Figure 6 View largeDownload slide MeCP2 deficiency enhances reprogramming efficiency through stimulating IGF1/AKT/mTOR signaling and activating ribosomal protein genes. (A) Western blot for IGF1/AKT/mTOR signaling protein levels in control and MeCP2-deficient MEFs. Tubulin was used as loading control. (B) Detection of iPSC colonies among control and MeCP2-deficient reprogrammed MEFs treated with the IGF1/AKT/mTOR signaling inhibitors rapamycin and AZD8055 by AP staining. ***P < 0.001, ANOVA, together with post hoc tests, n = 3 independent experiments. (C) Western blot for ribosomal protein and cell cycle protein expression in control and MeCP2-deficient MEFs treated with IGF1/AKT/mTOR signaling inhibitors rapamycin and AZD8055. Tubulin was used as a loading control. (D) The recruitment of HDAC2 to the IGF1 promoter. Soluble chromatin from control and MeCP2-deficient MEFs was immunoprecipitated with anti-HDAC2 or a control rabbit normal IgG. The extracted DNA was amplified by qRT-PCR using primers that cover the proximal promoter region of the IGF1 gene. ***P < 0.001, Student’s t-test, n = 3 independent experiments. (E) The recruitment of MeCP2 to the IGF1, CDK1, and CDK2 promoter. Soluble chromatin from MEFs was immunoprecipitated with anti-MeCP2 or a control rabbit normal IgG. The extracted DNA was amplified by qRT-PCR using primers that cover the proximal promoter region of the IGF1, CDK1, and CDK2 genes. ***P < 0.001, Student’s t-test, n = 3 independent experiments. (F) mRNA abundance of IGF1 in control and MeCP2-deficient MEFs. ***P < 0.001, Student’s t-test, n = 3 independent experiments. (G) Protein abundance of IGF1 in control and MeCP2-deficient MEFs. The supernatant of control and MeCP2-deficient MEFs was collected, and the amounts of IGF1 were measured using Mouse/Rat IGF1 Quantikine ELISA kit (R&D) according to the manufacturer’s instructions. ***P < 0.001, Student’s t-test, n = 3 independent experiments. (H) co-IP assays for the interaction between HDAC2 and MeCP2. (I) AP-positive colonies in reprogrammed MeCP2-deficient MEFs with IGF1 overexpression and knockdown. (J) Western blot for IGF1 in MEFs with IGF1 overexpression and knockdown. Figure 6 View largeDownload slide MeCP2 deficiency enhances reprogramming efficiency through stimulating IGF1/AKT/mTOR signaling and activating ribosomal protein genes. (A) Western blot for IGF1/AKT/mTOR signaling protein levels in control and MeCP2-deficient MEFs. Tubulin was used as loading control. (B) Detection of iPSC colonies among control and MeCP2-deficient reprogrammed MEFs treated with the IGF1/AKT/mTOR signaling inhibitors rapamycin and AZD8055 by AP staining. ***P < 0.001, ANOVA, together with post hoc tests, n = 3 independent experiments. (C) Western blot for ribosomal protein and cell cycle protein expression in control and MeCP2-deficient MEFs treated with IGF1/AKT/mTOR signaling inhibitors rapamycin and AZD8055. Tubulin was used as a loading control. (D) The recruitment of HDAC2 to the IGF1 promoter. Soluble chromatin from control and MeCP2-deficient MEFs was immunoprecipitated with anti-HDAC2 or a control rabbit normal IgG. The extracted DNA was amplified by qRT-PCR using primers that cover the proximal promoter region of the IGF1 gene. ***P < 0.001, Student’s t-test, n = 3 independent experiments. (E) The recruitment of MeCP2 to the IGF1, CDK1, and CDK2 promoter. Soluble chromatin from MEFs was immunoprecipitated with anti-MeCP2 or a control rabbit normal IgG. The extracted DNA was amplified by qRT-PCR using primers that cover the proximal promoter region of the IGF1, CDK1, and CDK2 genes. ***P < 0.001, Student’s t-test, n = 3 independent experiments. (F) mRNA abundance of IGF1 in control and MeCP2-deficient MEFs. ***P < 0.001, Student’s t-test, n = 3 independent experiments. (G) Protein abundance of IGF1 in control and MeCP2-deficient MEFs. The supernatant of control and MeCP2-deficient MEFs was collected, and the amounts of IGF1 were measured using Mouse/Rat IGF1 Quantikine ELISA kit (R&D) according to the manufacturer’s instructions. ***P < 0.001, Student’s t-test, n = 3 independent experiments. (H) co-IP assays for the interaction between HDAC2 and MeCP2. (I) AP-positive colonies in reprogrammed MeCP2-deficient MEFs with IGF1 overexpression and knockdown. (J) Western blot for IGF1 in MEFs with IGF1 overexpression and knockdown. To determine if MeCP2 deficiency regulates IGF1 gene transcription, we performed chromatin immunoprecipitation (ChIP) assays to determine the recruitment of HDAC2 to the IGF1 promoter in MeCP2-deficient and control MEFs. As shown in Figure 6D, the recruitment of HDAC2 to the IGF1 promoter decreased in MeCP2-deficient MEFs. We further detected the binding ability of MeCP2 on IGF1, CDK1, and CDK2 promoter and found that MeCP2 binding on IGF1 promoter but not on CDK1 and CDK2 promoter (Figure 6E). The RNA and protein expression of IGF1 was elevated in MeCP2-deficient MEFs (Figure 6F and G). Moreover, the interaction between HDAC2 and MeCP2 was detected in this system using a co-immunoprecipitation (co-IP) assay (Figure 6H). To verify whether IGF1 affected reprogramming, the effects of IGF1 overexpression and knockdown on cell reprogramming were observed, and the results showed that IGF1 promotes reprogramming (Figure 6I and J). These results demonstrated that MeCP2 deficiency stimulated IGF1/AKT/mTOR signaling by decreasing the binding of HDAC2 to the IGF1 promoter. Discussion The generation of iPSCs offers a great opportunity in research and regenerative medicine. The current poor efficiency and incomplete mechanistic understanding of the reprogramming process hamper clinical application of iPSCs. In this study, we showed that MeCP2-deficient MEFs exhibited significantly improved reprogramming efficiency, and we revealed the mechanism of MeCP2 in cell reprogramming. A key issue in dealing with the generation of iPSCs is how to maintain a high proliferation rate and activate G1-S phase progression (Yamanaka, 2009). Since IGF-1 activates G1-S progression, this suggests a potential role of IGF-1 in promoting the generation of iPSCs (Bendall et al., 2007; Nguyen et al., 2007; Huang et al., 2009). In this study, we showed that MeCP2 deficiency enhanced IGF-1 expression, promoting cell proliferation and activating G1-S phase progression in the early stage of reprogramming. The mTOR/S6K pathway regulates multiple cellular functions, including cell cycle, protein synthesis, apoptosis, and autophagy. Activation of the mTOR/S6K axis stimulates protein synthesis and cell growth (Cruz et al., 2005; Goh et al., 2010; Chauvin et al., 2014). Recently, multiple studies have identified the role of mTOR-S6K in regulating the self-renewal of leukemia stem cells and neural stem cells (Hartman et al., 2013; Ghosh et al., 2016). Furthermore, IGF1 promotes cell proliferation by activating PI3K/AKT/mTOR signaling in human ovarian cancer cells (Lau and Leung, 2012). A recent study showed that IGF-1 activates mTOR and stimulates the rate of mRNA translation in osteoblasts (Bakker et al., 2016). Moreover, ribosomal proteins affect cell cycle progression via various mechanisms (Wang et al., 2006; Jang et al., 2012). We demonstrated that IGF1/AKT/mTOR signaling plays an important role in promoting the generation of iPSCs through activating ribosomal protein-mediated cell cycle gene translation. Our findings suggest that MeCP2 connects IGF1/AKT/mTOR signaling with protein synthesis and cell proliferation in somatic cell reprogramming. The increase in the number of AP-positive colonies owing to ribosomal proteins in WT MEFs was not as dramatic as that in the case of the MeCP2 knockout MEFs; there may be other mechanisms mediating the effect of MeCP2 knockout on reprogramming. Transcriptional repression of methylated genes by MeCP2 involves the binding of MeCP2 to Sin3A-HDAC complexes and HDAC2 is the main histone deacetylase of the co-repressor complex that interacts with MeCP2 (Jones et al., 1998; Nan et al., 1998). In this study, we showed that MeCP2 deficiency decreased the binding of HDAC2 to the IGF1 promoter region, which then increased IGF1 transcription and activated IGF1/AKT/mTOR signaling. Our finding connected histone modification with IGF1/AKT/mTOR signaling in somatic cell reprogramming by MeCP2. The ongoing dogma that patients with Rett syndrome with MeCP2 mutations undergo growth arrest somewhat contradicts the work described here. This inconsistency may be caused because of the following reasons. First, genes affected by MeCP2 knockout differ markedly between cell types (Sugino et al., 2014). Second, the function of genes in the whole body may not be consistent with their functions at the cellular level. Lastly, mutations in patients with RTT may not give rise to a complete loss of function of the MeCP2 protein. In summary, we demonstrated that MeCP2 deficiency promotes cell reprogramming through stimulating IGF1/AKT/mTOR signaling and activating ribosomal protein-mediated cell cycle gene translation in the early stage of reprogramming. These findings provided a strategy to regulate cell reprogramming by modulating MeCP2 and revealed the mechanism of MeCP2 in cell fate determination. Materials and methods Knocking out MeCP2 in mice sgRNAs targeting the MeCP2 gene were designed using CRISPR Design tool (http://crispr.mit.edu/), and inserted into a pMD-18T-modified plasmid containing an sgRNA-expressing skeleton. Cas9 and sgRNA mRNAs needed for microinjection were in vitro transcribed by the T7 in Vitro Transcription Kit (NEB). The one-cell-stage embryos were collected 12 h post coitum, and sgRNA and Cas9 mRNA were microinjected into their cytoplasms. After microinjection, the embryos were implanted in the oviducts of pseudopregnant female mice to obtain full-term pups. Cell lines and cell culture MEFs were isolated from WT and MeCP2 knockout embryos at E13.5 and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1 × penicillin/streptomycin. iPSCs were cultured with DMEM supplemented with 5% knockout serum replacement (KOSR), 0.1 mM β-mercaptoethanol, 1000 units/ml leukemia inhibitory factor, and 0.1 mM nonessential amino acids. Lentiviral production Viral particles were generated by transfecting plated 293T cells with pHIV vectors encoding MeCP2, Rps18, Rpl21, Rpl35a, and IGF1 along with pMD.2G and psPAX2 vectors. Supernatants from the transfected cells were collected 48 h after transfection. The viral suspension was mixed with 10 mg/ml polybrene (Millipore) and used to infect cells. Generation of iPSC and reprogramming efficiency evaluation Generation of pluripotent iPSC lines was performed as described previously (Carey et al., 2009). MEFs were isolated from E13.5 embryos cultured under established iPSC conditions and the four Yamanaka factors OSKM were expressed. The efficiency of iPSC formation is estimated according to the presence of AP-positive colonies after 14 days of reprogramming. The number of iPSC colonies per well was counted in triplicate. Immunofluorescence staining iPSCs growing on coverslips were rinsed briefly in PBS and fixed in 4% paraformaldehyde in PBS for 10 min on ice. After 10 min of permeabilization in PBS containing 0.2% Triton X-100 on ice, cells were subsequently washed with PBS three times. After blocking in 5% milk for 30 min at 37°C, anti-Oct4 (Santa Cruz), anti-Nanog (Proteintech), or anti-SSEA1 (Santa Cruz) were added at the dilution ratio suggested by the manufacturer. Cells were then counterstained with DAPI (Invitrogen). Fluorescent images were acquired using a Leica TCS SP8 confocal microscope. Point mutation of MeCP2 The point mutations of MeCP2 were constructed by QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s protocol. Primers used in the mutations are listed in Supplementary Table S1. RNA extraction and qRT-PCR Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Afterward, 1 μg of RNA from each sample was extracted and reverse-transcribed into cDNA using random primers and was subjected to qRT-PCR. qRT-PCR was performed using a Stratagene Mx3000P quantitative PCR system (Genetimes Technology). The reactions were incubated in a 96-well plate at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. All reactions were run in triplicate. Primers used in the study are listed in Supplementary Table S1. Western blot analysis Western blotting was performed as described previously (Wu et al., 2005). The commercial antibodies used were anti-MeCP2, anti-Rps18, anti-Rpl21, and anti-Rpl35a, which were all purchased from Abcam. Anti-CDK1, anti-CDK2, anti-Cyclin A2, anti-Cyclin B1, anti-Cyclin D1, anti-Cyclin E1, anti-AKT, anti-phosphor-AKT (Ser473), anti-S6, and anti-P-S6 were purchased from Cell Signaling Technology. Anti-Oct4, anti-Nanog, and anti-SSEA1 were purchased from Millipore. Anti-Tubulin and anti-HA tag antibodies were purchased from Sigma. Anti-IGF1, anti-Klf4, and anti-c-Myc were purchased from Abcam. Anti-Sox2 was purchased from Merck Millipore. Teratoma formation and histological analysis iPSCs were digested into single cells and cultured in gelatin-coated dishes to discard the attached feeder cells. The cells were collected and resuspended in PBS and 1 × 107 cells were injected under the skin of 5-week-old BALB/c SCID mice. The teratomas harvested for histological analysis after 4 weeks were fixed and sliced into sections that were then stained with hematoxylin and eosin. Diploid blastocyst injection Diploid blastocysts were collected from E3.5 time-pregnant mice. iPSCs were injected into diploid blastocysts that were transferred to CD-1 pseudopregnant recipient females. Then, the adult chimeric offspring were mated to a CD-1 mouse. siRNAs and RNA interference All siRNAs were designed and synthesized by RiboBio Co. Ltd. and listed in Supplementary Table S1. siRNAs were transfected into mouse MEFs using Lipo3000 reagent (Invitrogen) according to the manufacturer’s instructions. Cell counting kit-8 and CFSE cell proliferation assay MEFs were harvested by trypsinization and then plated into 96-well plates at a density of 10000/well. The cells were cultured and subjected to a cellular viability assay using a Cell counting kit-8 (Promega). CCK8 assays aim to quantify viable cells in proliferation assays. CFSE is also widely used for cell proliferation assays; the decrease in fluorescence intensity due to cell division is measured. To detect the proliferation ability of cells, a CFSE assay was performed using the CellTrace CFSE cell proliferation kit (Thermo) according to the manufacturer’s protocol. Flow cytometry SSEA1 positivity in cells was analyzed by FACS. Briefly, cells were trypsinized and washed once in PBS. For APC-conjugated SSEA1 (R&D), 1 × 105 cells were washed once in PBS and 0.5% BSA, and incubated with SSEA1 in 2 ml of 0.5% BSA in PBS for 30 min at 37°C. Afterwards, cells were subjected to FACS analysis. The cell cycle phase was detected by a Ki67-ICF/FACS kit. A FACSCanto II (BD Biosciences) instrument was used for all FACS analysis. ChIP assays WT and MeCP2 KO MEFs were maintained in MEF medium. Approximately 1 × 107 cells were used for each ChIP assay. ChIP assays were performed according to a previously described protocol (Shang et al., 2000). Chromatin was precipitated with either normal rabbit IgG (control) or anti-HDAC2 (CST) and anti-MeCP2 (Abcam), and was purified with a Qiagen PCR purification kit. The samples were analyzed by qRT-PCR using primers described in Supplementary Table S1. RNA immunoprecipitation WT and MeCP2 KO MEFs were crosslinked with 1% formaldehyde for 15 min. Cells were lysed by RIP lysis buffer containing a protease inhibitor cocktail and RNase inhibitor. The antibodies used for immunoprecipitation were Rps18 (Abcam) and Rpl21 (Abcam). Immune complexes were subjected to magnetic beads and washed sequentially with low-salt immune complex wash buffer, high-salt immune complex wash buffer, and then samples were washed with cold RIP wash buffer three times. For RNA purification, proteinase K buffer was added to samples at 55°C for 30 min. RNA was extracted in the supernatant by the phenol : chloroform : isoamyl alcohol method. The collected RNA was reverse transcribed to cDNA and subjected to qRT-PCR analysis. The primers used for detecting ribosomal protein binding sites are listed in Supplementary Table S1. Co-IP assay To explore the endogenous interaction between MeCP2 and HDAC2, MEF cells were collected and lysis for protein extraction with lysis buffer (100 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, and 0.01% Triton X-100, pH 8.0). The supernatant was collected and dithiobis(succinimidyl propionate) (DSP) (22585, Thermo Fisher Scientific) was added into supernatant for 1 h at 4°C. Then, the supernatant was immunoprecipitated overnight at 4°C with anti-MeCP2 antibody. The complexes were immunoprecipitated with Protein G Agarose Beads (CST) for 1 h at 4°C. Then agarose was washed three times with IP washing buffer (50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, and 0.01% Triton X-100, pH 8.0), then detected by western blotting with anti-HDAC2 antibody. Accession numbers Sequencing data generated by this work have been deposited into the Genome Sequence Archive (Wang et al., 2017) in the BIG Data Center (BIG Data Center Members, 2018), Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under the accession number CRA000764 (released from June 1, 2018), which are publicly accessible at http://bigd.big.ac.cn/gsa. Supplementary material Supplementary material is available at Journal of Molecular Cell Biology online. Acknowledgements The authors would like to thank all members of our group for discussion and help. We thank Ms Shiwen Li (Institute of Zoology, Chinese Academy of Sciences) for her kind technique support on confocal experiments. Funding This study was supported by the National Natural Science Foundation of China (31471395 to Q.Z.), the Key Research Projects of the Frontier Science of the Chinese Academy of Sciences (QYZDY-SSW-SMC002), and the National Basic Research Program of China (2014CB964903). 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Nature 460 , 49 – 52 . Google Scholar CrossRef Search ADS PubMed Author notes Edited by Jiarui Wu © The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Cell Biology Oxford University Press

MeCP2 deficiency promotes cell reprogramming by stimulating IGF1/AKT/mTOR signaling and activating ribosomal protein-mediated cell cycle gene translation

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Oxford University Press
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© The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.
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Abstract

Abstract The generation of induced pluripotent stem cells (iPSCs) offers a great opportunity in research and regenerative medicine. The current poor efficiency and incomplete mechanistic understanding of the reprogramming process hamper the clinical application of iPSCs. MeCP2 connects histone modification and DNA methylation, which are key changes of somatic cell reprogramming. However, the role of MeCP2 in cell reprogramming has not been examined. In this study, we found that MeCP2 deficiency enhanced reprogramming efficiency and stimulated cell proliferation through regulating cell cycle protein expression in the early stage of reprogramming. MeCP2 deficiency enhanced the expression of ribosomal protein genes, thereby enhancing reprogramming efficiency through promoting the translation of cell cycle genes. In the end, MeCP2 deficiency stimulated IGF1/AKT/mTOR signaling and activated ribosomal protein gene expression. Taken together, our data indicate that MeCP2 deficiency promoted cell reprogramming through stimulating IGF1/AKT/mTOR signaling and activating ribosomal protein-mediated cell cycle gene translation in the early stage of reprogramming. MeCP2, cell reprogramming, IGF1/AKT/mTOR signaling, ribosomal protein, cell cycle Introduction The generation of induced pluripotent stem cells (iPSCs), somatic cells reprogrammed to the pluripotent state by forced expression of nuclear transcription factors, such as Oct4, Sox2, Klf4, and c-Myc (named as OSKM hereinafter) (Takahashi and Yamanaka, 2006; Okita et al., 2007), holds great promise for research and regenerative medicine (Robinton and Daley, 2012). However, the current poor efficiency and an incomplete mechanistic understanding of the reprogramming process hamper the clinical application of iPSCs (Gonzalez et al., 2011; Vierbuchen and Wernig, 2012). The generally accepted model of the molecular mechanisms involved in iPSC reprogramming consists of three stages: initiation, maturation, and stabilization (Samavarchi-Tehrani et al., 2010; Buganim et al., 2013). Reprogramming is initiated in cells that receive reprogramming transgenes, and is characterized by increased proliferation and a metabolic switch from oxidative phosphorylation to glycolysis. The initiation stage also involves a phenotypic mesenchymal-to-epithelial transition (David and Polo, 2014). A high proliferation rate is required for cell reprogramming (Ruiz et al., 2011). c-Myc or Lin28 transgene expression and p53 knockdown increase the efficiency of iPSC reprogramming by stimulating cell proliferation (Hanna et al., 2009; Apostolou and Hochedlinger, 2013). Specifically, Lin28 has been shown to regulate cell cycle genes such as Cyclin A, Cyclin B, and Cdk4 (Xu et al., 2009). Furthermore, insulin-like growth factor (IGF) is an essential growth factor and may enhance the expression of reprogramming factors, suggesting a potential role of IGF-1 in the reprogramming process (Li and Geng, 2010). Methyl-CpG-binding protein 2 (MeCP2), is a classic methylated-DNA-binding protein, and dysfunctions in this protein lead to various neurodevelopmental disorders such as autism spectrum disorder and Rett syndrome (Amir et al., 1999; Chen et al., 2001; Guy et al., 2001; Kriaucionis and Bird, 2003). MeCP2 is regarded as a transcriptional repressor for methylated genes and interacts with Sin3A/HDAC and NCoR/SMRT co-repressor complexes (Nan et al., 1993, 1997, 1998). MeCP2 connects histone modification and DNA methylation, which are key changes of somatic cell reprogramming. However, the role of MeCP2 in cell reprogramming has not been examined. Here, we showed that MeCP2 deficiency promoted cell reprogramming through stimulating IGF1/AKT/mTOR signaling and activating ribosomal protein-mediated cell cycle gene translation in the early stage of reprogramming. Results MeCP2 deficiency enhances reprogramming efficiency To investigate whether MeCP2 plays important roles in cell fate determination, we utilized iPSC technology to examine the effects of MeCP2 deficiency on cell reprogramming. We first used MeCP2-knockout (KO) mouse embryonic fibroblasts (MEFs) expressing the four Yamanaka factors, OSKM. MeCP2-deficient MEFs exhibited significantly improved reprogramming efficiency, with almost 4-fold more alkaline phosphatase (AP)-positive iPSC colonies than that of the control (Figure 1A and B). We further determined the pluripotency of MeCP2 deficiency-induced iPSCs. The expression of pluripotency markers was indeed observed in these MeCP2-deficient iPSCs (Figure 1C). these MeCP2-deficient iPSCs exhibited the potential to develop into teratomas with three germ layers (Figure 1D), and chimeric mice could be generated from MeCP2-deficient iPSCs (Figure 1E). These results demonstrated that MeCP2-deficient iPSCs were fully reprogrammed and pluripotent. To test whether certain known mutations in MeCP2 subunits have similar promoting effects on reprogramming, we further detected the effects of two MeCP2 mutations R133C and R255X (Amir et al., 1999) on reprogramming. Our results showed that certain known mutations in MeCP2 subunits indeed have similar promoting effects on reprogramming (Figure 1F and G). Figure 1 View largeDownload slide MeCP2 deficiency enhances reprogramming efficiency. (A) Detection of iPSC colonies among control and MeCP2-deficient reprogrammed MEFs by AP staining. Successfully reprogrammed iPSC colonies are AP-positive (left panel). Bar graph showing the comparison of AP-positive colonies between control and MeCP2-deficient reprogrammed MEFs (right panel). **P < 0.01, Student’s t-test, n = 3 independent experiments. (B) Western blot for endogenous MeCP2 in control and MeCP2-deficient MEFs and iPSCs. Tubulin was used as a loading control. (C) Immunofluorescent staining of pluripotency markers in MeCP2-deficient iPSCs. (D) MeCP2-deficient iPSCs could effectively produce full teratomas, which contained differentiated cells in all three germ layers, in SCID mice. (E) MeCP2-deficient iPSCs generated mice with chimeric ability. (F) AP-positive colonies among reprogrammed MeCP2-deficient MEFs with wild-type and two mutations of MeCP2. *P < 0.05, **P < 0.01, Student’s t-test, n = 3 independent experiments. (G) Western blot for overexpression of wild-type and two mutations of MeCP2 in MEFs. Tubulin was used as a loading control. Figure 1 View largeDownload slide MeCP2 deficiency enhances reprogramming efficiency. (A) Detection of iPSC colonies among control and MeCP2-deficient reprogrammed MEFs by AP staining. Successfully reprogrammed iPSC colonies are AP-positive (left panel). Bar graph showing the comparison of AP-positive colonies between control and MeCP2-deficient reprogrammed MEFs (right panel). **P < 0.01, Student’s t-test, n = 3 independent experiments. (B) Western blot for endogenous MeCP2 in control and MeCP2-deficient MEFs and iPSCs. Tubulin was used as a loading control. (C) Immunofluorescent staining of pluripotency markers in MeCP2-deficient iPSCs. (D) MeCP2-deficient iPSCs could effectively produce full teratomas, which contained differentiated cells in all three germ layers, in SCID mice. (E) MeCP2-deficient iPSCs generated mice with chimeric ability. (F) AP-positive colonies among reprogrammed MeCP2-deficient MEFs with wild-type and two mutations of MeCP2. *P < 0.05, **P < 0.01, Student’s t-test, n = 3 independent experiments. (G) Western blot for overexpression of wild-type and two mutations of MeCP2 in MEFs. Tubulin was used as a loading control. MeCP2 deficiency enhances reprogramming efficiency in the early stage of reprogramming To determine in which stage MeCP2 deficiency promoted reprogramming, we measured SSEA-1, a marker of early reprogramming, positivity in cells during the reprogramming process and found that the SSEA-1-positive cell percentage of the MeCP2-deficient group was significantly increased compared to the percentage in the control group at reprogramming days 3, 5, 7, and 12 (Figure 2A). Notably, MeCP2 depletion during early stages of somatic reprogramming promoted iPSC derivation from MEFs (Figure 2B−D). Conversely, MeCP2 overexpression during early stages of somatic reprogramming diminished iPSC derivation from MEFs (Figure 2B and E). Figure 2 View largeDownload slide MeCP2 deficiency stimulates cell proliferation in the early stage of reprogramming. (A) SSEA1-positive cell percentages from the indicated group at reprogramming days 3, 5, 7, and 12. Wild-type cells were used for comparison. Data represent mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, Student’s t-test, n = 3 independent experiments. (B) Western blot for endogenous MeCP2 in control and MeCP2 knockdown cells. Tubulin was used as loading control (upper panel). Western blot for HA-MeCP2 in control and MeCP2-overexpressing cells. Tubulin was used as a loading control (lower panel). (C) AP-positive colonies among reprogrammed MEFs with or without MeCP2 siRNA transfection. *P < 0.05, **P < 0.01, ANOVA, together with post hoc tests, n = 3 independent experiments. (D) siRNA transfections were started either at −2, 0, +2, or +4 days relative to the expression of the four Yamanaka factors, with repeated transfections at 3-day intervals. iPSC formation was analyzed at day 14 by counting AP-positive colonies. Data represent mean ± SD of three independent experiments. ***P < 0.001, ANOVA, together with post hoc tests, n = 3 independent experiments. (E) Lenti-MeCP2 transfections were started either at −2, 0, +2, or +4 days relative to the expression of the four Yamanaka factors. iPSC formation was analyzed at day 14 by counting AP-positive colonies. Data represent mean ± SD of three independent experiments. ***P < 0.001, ANOVA, together with post hoc tests, n = 3 independent experiments. Figure 2 View largeDownload slide MeCP2 deficiency stimulates cell proliferation in the early stage of reprogramming. (A) SSEA1-positive cell percentages from the indicated group at reprogramming days 3, 5, 7, and 12. Wild-type cells were used for comparison. Data represent mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, Student’s t-test, n = 3 independent experiments. (B) Western blot for endogenous MeCP2 in control and MeCP2 knockdown cells. Tubulin was used as loading control (upper panel). Western blot for HA-MeCP2 in control and MeCP2-overexpressing cells. Tubulin was used as a loading control (lower panel). (C) AP-positive colonies among reprogrammed MEFs with or without MeCP2 siRNA transfection. *P < 0.05, **P < 0.01, ANOVA, together with post hoc tests, n = 3 independent experiments. (D) siRNA transfections were started either at −2, 0, +2, or +4 days relative to the expression of the four Yamanaka factors, with repeated transfections at 3-day intervals. iPSC formation was analyzed at day 14 by counting AP-positive colonies. Data represent mean ± SD of three independent experiments. ***P < 0.001, ANOVA, together with post hoc tests, n = 3 independent experiments. (E) Lenti-MeCP2 transfections were started either at −2, 0, +2, or +4 days relative to the expression of the four Yamanaka factors. iPSC formation was analyzed at day 14 by counting AP-positive colonies. Data represent mean ± SD of three independent experiments. ***P < 0.001, ANOVA, together with post hoc tests, n = 3 independent experiments. MeCP2 deficiency promotes cell proliferation through regulating cell cycle gene expression at the protein level Since the initiation stage of cell reprogramming is characterized by increased proliferation (David and Polo, 2014), we examined the cell proliferation of MeCP2-deficient MEFs. The growth rate of MeCP2-deficient MEFs was upregulated compared with that of control cells (Figure 3A and B). Flow cytometry (FACS) analysis showed that the percentage of cells in the G1/G0 phase of MeCP2-deficient MEFs was decreased (Figure 3C). We further examined cell proliferation and cell cycle of MeCP2-deficient MEFs expressing the four Yamanaka factors, OSKM. Consistent with the above-mentioned results, cell proliferation and FACS showed that the growth rate of MeCP2-deficient MEFs expressing the four Yamanaka factors, OSKM, was increased, and the percentage of cells in the G1/G0 phase of MeCP2-deficient MEFs expressing the four Yamanaka factors, OSKM, was decreased (Figure 3D−F). To further demonstrate the importance of MeCP2 deficiency in cell proliferation, we detected the cell cycle gene expression at the RNA and protein level. There was no difference in the expression of cell cycle genes between WT and MeCP2-deficient MEFs at the RNA level, and significantly increased expression in MeCP2-deficient MEFs at the protein level (Figure 3G and H). There was no difference of expression levels of Klf4 and c-Myc in WT and MeCP2-deficient MEFs on the second day of induction. The expression of Oct4 and Nanog was not detected in both cells, while the expression of Sox2 was only detected in MeCP2-deficient MEFs (Figure 3H). Collectively, our results suggest that MeCP2 deficiency may mediate cell proliferation through regulating the expression of cell cycle genes at the protein level. Figure 3 View largeDownload slide MeCP2 deficiency promotes cell proliferation through regulating cell cycle protein expression. (A and B) The effect of MeCP2 deficiency on cell proliferation. Cell proliferation of MeCP2-deficient and control MEFs was measured by CCK8 and CSFE assays. (C) The effect of MeCP2 deficiency on cell cycle progression. (D and E) The effect of MeCP2 deficiency on expression of the four Yamanaka factors OSKM on cell proliferation. Cell proliferation of MeCP2-deficient MEFs expressing the four Yamanaka factors OSKM and control MEFs was measured by CCK8 and CSFE assays. (F) The effect of MeCP2 deficiency in cells expressing the four Yamanaka factors on cell cycle progression. (G) qRT-PCR for the cell cycle gene expression in control and MeCP2-deficient MEFs at the RNA level. (H) Western blot for cell cycle gene and pluripotent gene expression in control and MeCP2-deficient MEFs at the protein level. Tubulin was used as a loading control. *P < 0.05, **P < 0.01, Student’s t-test, n = 3 independent experiments. Figure 3 View largeDownload slide MeCP2 deficiency promotes cell proliferation through regulating cell cycle protein expression. (A and B) The effect of MeCP2 deficiency on cell proliferation. Cell proliferation of MeCP2-deficient and control MEFs was measured by CCK8 and CSFE assays. (C) The effect of MeCP2 deficiency on cell cycle progression. (D and E) The effect of MeCP2 deficiency on expression of the four Yamanaka factors OSKM on cell proliferation. Cell proliferation of MeCP2-deficient MEFs expressing the four Yamanaka factors OSKM and control MEFs was measured by CCK8 and CSFE assays. (F) The effect of MeCP2 deficiency in cells expressing the four Yamanaka factors on cell cycle progression. (G) qRT-PCR for the cell cycle gene expression in control and MeCP2-deficient MEFs at the RNA level. (H) Western blot for cell cycle gene and pluripotent gene expression in control and MeCP2-deficient MEFs at the protein level. Tubulin was used as a loading control. *P < 0.05, **P < 0.01, Student’s t-test, n = 3 independent experiments. MeCP2 deficiency enhances the expression of ribosomal protein genes, which enhances reprogramming efficiency To elucidate the underlying mechanisms by which MeCP2 deficiency promotes reprogramming, we then performed RNA-Seq analysis using MeCP2-deficient MEFs and iPSCs (Figure 4A and B). As expected, we observed obvious differential gene expression profiles between MeCP2-deficient MEFs and iPSCs and their respective control cells. Based on the gene expression dynamics, we found that the ribosomal protein genes were reactivated and highly expressed in both MeCP2-deficient MEFs and iPSCs (Figure 4A and B). The pluripotent genes were reactivated and highly expressed in both control and MeCP2-deficient iPSCs (Figure 4B). We chose the pathways which are enriched in differentially expressed genes between WT and MeCP2-deficient MEFs and have been reported previously and found that the PI3K/AKT signaling was reactivated in MeCP2-deficient MEFs (Figure 4A). Furthermore, the cell cycle genes were highly expressed in MeCP2-deficient MEFs (Figure 4B). Figure 4 View largeDownload slide MeCP2 deficiency enhances ribosomal protein gene expression, which enhances reprogramming efficiency. (A and B) KEGG pathway (A) and heatmaps analysis (B) of upregulated genes in RNA-Seq data from MeCP2-deficient MEFs and iPSCs. (C) mRNA abundance of ribosomal protein genes in control and MeCP2-deficient MEFs. (D) Protein abundance of ribosomal proteins in control and MeCP2-deficient MEFs. Tubulin was used as loading control. (E and F) Western blot analysis of ribosomal proteins expression in the indicated cells. (G) AP-positive colonies in reprogrammed MEFs with or without transfection with siRNAs against ribosomal protein genes. (H) AP-positive colonies among reprogrammed MEFs with or without overexpression of ribosomal protein genes. *P < 0.05, ***P < 0.001, ANOVA, together with post hoc tests, n = 3 independent experiments. Figure 4 View largeDownload slide MeCP2 deficiency enhances ribosomal protein gene expression, which enhances reprogramming efficiency. (A and B) KEGG pathway (A) and heatmaps analysis (B) of upregulated genes in RNA-Seq data from MeCP2-deficient MEFs and iPSCs. (C) mRNA abundance of ribosomal protein genes in control and MeCP2-deficient MEFs. (D) Protein abundance of ribosomal proteins in control and MeCP2-deficient MEFs. Tubulin was used as loading control. (E and F) Western blot analysis of ribosomal proteins expression in the indicated cells. (G) AP-positive colonies in reprogrammed MEFs with or without transfection with siRNAs against ribosomal protein genes. (H) AP-positive colonies among reprogrammed MEFs with or without overexpression of ribosomal protein genes. *P < 0.05, ***P < 0.001, ANOVA, together with post hoc tests, n = 3 independent experiments. We further conducted quantitative real-time PCR (qRT-PCR) to detect some of these ribosomal protein genes in MeCP2-deficient MEFs. The expression of ribosomal protein genes was much higher in MeCP2-deficient MEFs, compared to expression in controls (Figure 4C). Accordingly, the abundance of ribosomal proteins was much higher in MeCP2-deficient MEFs, compared to levels in controls (Figure 4D). To investigate whether ribosomal protein genes play important roles in cell reprogramming, we first respectively downregulated three ribosomal protein genes that were highly expressed in MeCP2-deficient MEFs, into MEFs expressing the four Yamanaka factors, OSKM. Knocking down ribosomal protein genes expression using siRNAs during the reprogramming process resulted in reduced iPSC colony numbers (Figure 4E and G). Conversely, overexpressing ribosomal protein genes in MEFs significantly improved reprogramming efficiency, as seen by the number of obtained iPSC colonies (Figure 4F and H). Ribosomal protein genes promote cell cycle protein expression Since the ribosomal protein genes were highly expressed in MeCP2-deficient MEFs and MeCP2 deficiency mediated cell proliferation by regulating cell cycle protein expression, we supposed that ribosomal protein genes promote the translation of cell cycle genes. To confirm this hypothesis, we detected the protein and mRNA levels of cell cycle genes in control MEFs and MEFs overexpressing ribosomal protein genes. We found that overexpressing ribosomal protein genes increased cell cycle gene expression at the protein level (Figure 5A and B). Conversely, inhibiting ribosomal protein gene expression by knocking down ribosomal protein gene expression using siRNAs resulted in decreased cell cycle gene expression at the protein level (Figure 5C and D). To further demonstrate that ribosomal protein genes affect cell cycle gene expression at the translation level, we then performed RNA immunoprecipitation (RIP) assays to demonstrate binding between ribosomal proteins and the mRNA of cell cycle genes (Figure 5E). Our results suggested that ribosomal protein genes promote the translation of cell cycle genes. Figure 5 View largeDownload slide Ribosomal protein genes promote cell cycle protein expression. (A) Protein abundance of cell cycle genes in MEFs with or without overexpression of ribosomal protein genes. Tubulin was used as a loading control. (B) mRNA abundance of cell cycle genes in MEFs with or without overexpression of ribosomal protein genes. (C) Protein abundance of cell cycle genes in MEFs with or without knockdown of ribosomal protein genes. Tubulin was used as a loading control. (D) mRNA abundance of cell cycle genes in MEFs with or without knockdown of ribosomal protein genes. (E) RIP assays for the recruitment of ribosomal proteins to the cell cycle gene mRNA. **P < 0.01, ***P < 0.001, Student’s t-test, n = 3 independent experiments. Figure 5 View largeDownload slide Ribosomal protein genes promote cell cycle protein expression. (A) Protein abundance of cell cycle genes in MEFs with or without overexpression of ribosomal protein genes. Tubulin was used as a loading control. (B) mRNA abundance of cell cycle genes in MEFs with or without overexpression of ribosomal protein genes. (C) Protein abundance of cell cycle genes in MEFs with or without knockdown of ribosomal protein genes. Tubulin was used as a loading control. (D) mRNA abundance of cell cycle genes in MEFs with or without knockdown of ribosomal protein genes. (E) RIP assays for the recruitment of ribosomal proteins to the cell cycle gene mRNA. **P < 0.01, ***P < 0.001, Student’s t-test, n = 3 independent experiments. MeCP2 deficiency enhances reprogramming efficiency through stimulating IGF1/AKT/mTOR signaling and activating ribosomal protein genes Since the PI3K/AKT signaling and ribosomal protein genes were activated in MeCP2-deficient MEFs, we supposed that the PI3K/AKT signaling-mediated ribosomal protein genes activation play important roles in reprogramming. To confirm this hypothesis, we examined IGF1/AKT/mTOR signaling, a traditional ribosomal protein gene regulatory pathway, and found that levels of IGF1/AKT/mTOR signaling proteins P-AKT and P-S6 were significantly increased in MeCP2-deficient MEFs (Figure 6A). Furthermore, IGF1/AKT/mTOR signaling was inhibited by treating cells with rapamycin and AZD8055, inhibitors of IGF1/AKT/mTOR signaling, during the reprogramming process. Inhibiting this signaling pathway reduced iPSC colony numbers (Figure 6B). Meanwhile, inhibiting IGF1/AKT/mTOR signaling reduced the expression of ribosomal proteins and cell cycle proteins in MeCP2-deficient MEFs (Figure 6C). Figure 6 View largeDownload slide MeCP2 deficiency enhances reprogramming efficiency through stimulating IGF1/AKT/mTOR signaling and activating ribosomal protein genes. (A) Western blot for IGF1/AKT/mTOR signaling protein levels in control and MeCP2-deficient MEFs. Tubulin was used as loading control. (B) Detection of iPSC colonies among control and MeCP2-deficient reprogrammed MEFs treated with the IGF1/AKT/mTOR signaling inhibitors rapamycin and AZD8055 by AP staining. ***P < 0.001, ANOVA, together with post hoc tests, n = 3 independent experiments. (C) Western blot for ribosomal protein and cell cycle protein expression in control and MeCP2-deficient MEFs treated with IGF1/AKT/mTOR signaling inhibitors rapamycin and AZD8055. Tubulin was used as a loading control. (D) The recruitment of HDAC2 to the IGF1 promoter. Soluble chromatin from control and MeCP2-deficient MEFs was immunoprecipitated with anti-HDAC2 or a control rabbit normal IgG. The extracted DNA was amplified by qRT-PCR using primers that cover the proximal promoter region of the IGF1 gene. ***P < 0.001, Student’s t-test, n = 3 independent experiments. (E) The recruitment of MeCP2 to the IGF1, CDK1, and CDK2 promoter. Soluble chromatin from MEFs was immunoprecipitated with anti-MeCP2 or a control rabbit normal IgG. The extracted DNA was amplified by qRT-PCR using primers that cover the proximal promoter region of the IGF1, CDK1, and CDK2 genes. ***P < 0.001, Student’s t-test, n = 3 independent experiments. (F) mRNA abundance of IGF1 in control and MeCP2-deficient MEFs. ***P < 0.001, Student’s t-test, n = 3 independent experiments. (G) Protein abundance of IGF1 in control and MeCP2-deficient MEFs. The supernatant of control and MeCP2-deficient MEFs was collected, and the amounts of IGF1 were measured using Mouse/Rat IGF1 Quantikine ELISA kit (R&D) according to the manufacturer’s instructions. ***P < 0.001, Student’s t-test, n = 3 independent experiments. (H) co-IP assays for the interaction between HDAC2 and MeCP2. (I) AP-positive colonies in reprogrammed MeCP2-deficient MEFs with IGF1 overexpression and knockdown. (J) Western blot for IGF1 in MEFs with IGF1 overexpression and knockdown. Figure 6 View largeDownload slide MeCP2 deficiency enhances reprogramming efficiency through stimulating IGF1/AKT/mTOR signaling and activating ribosomal protein genes. (A) Western blot for IGF1/AKT/mTOR signaling protein levels in control and MeCP2-deficient MEFs. Tubulin was used as loading control. (B) Detection of iPSC colonies among control and MeCP2-deficient reprogrammed MEFs treated with the IGF1/AKT/mTOR signaling inhibitors rapamycin and AZD8055 by AP staining. ***P < 0.001, ANOVA, together with post hoc tests, n = 3 independent experiments. (C) Western blot for ribosomal protein and cell cycle protein expression in control and MeCP2-deficient MEFs treated with IGF1/AKT/mTOR signaling inhibitors rapamycin and AZD8055. Tubulin was used as a loading control. (D) The recruitment of HDAC2 to the IGF1 promoter. Soluble chromatin from control and MeCP2-deficient MEFs was immunoprecipitated with anti-HDAC2 or a control rabbit normal IgG. The extracted DNA was amplified by qRT-PCR using primers that cover the proximal promoter region of the IGF1 gene. ***P < 0.001, Student’s t-test, n = 3 independent experiments. (E) The recruitment of MeCP2 to the IGF1, CDK1, and CDK2 promoter. Soluble chromatin from MEFs was immunoprecipitated with anti-MeCP2 or a control rabbit normal IgG. The extracted DNA was amplified by qRT-PCR using primers that cover the proximal promoter region of the IGF1, CDK1, and CDK2 genes. ***P < 0.001, Student’s t-test, n = 3 independent experiments. (F) mRNA abundance of IGF1 in control and MeCP2-deficient MEFs. ***P < 0.001, Student’s t-test, n = 3 independent experiments. (G) Protein abundance of IGF1 in control and MeCP2-deficient MEFs. The supernatant of control and MeCP2-deficient MEFs was collected, and the amounts of IGF1 were measured using Mouse/Rat IGF1 Quantikine ELISA kit (R&D) according to the manufacturer’s instructions. ***P < 0.001, Student’s t-test, n = 3 independent experiments. (H) co-IP assays for the interaction between HDAC2 and MeCP2. (I) AP-positive colonies in reprogrammed MeCP2-deficient MEFs with IGF1 overexpression and knockdown. (J) Western blot for IGF1 in MEFs with IGF1 overexpression and knockdown. To determine if MeCP2 deficiency regulates IGF1 gene transcription, we performed chromatin immunoprecipitation (ChIP) assays to determine the recruitment of HDAC2 to the IGF1 promoter in MeCP2-deficient and control MEFs. As shown in Figure 6D, the recruitment of HDAC2 to the IGF1 promoter decreased in MeCP2-deficient MEFs. We further detected the binding ability of MeCP2 on IGF1, CDK1, and CDK2 promoter and found that MeCP2 binding on IGF1 promoter but not on CDK1 and CDK2 promoter (Figure 6E). The RNA and protein expression of IGF1 was elevated in MeCP2-deficient MEFs (Figure 6F and G). Moreover, the interaction between HDAC2 and MeCP2 was detected in this system using a co-immunoprecipitation (co-IP) assay (Figure 6H). To verify whether IGF1 affected reprogramming, the effects of IGF1 overexpression and knockdown on cell reprogramming were observed, and the results showed that IGF1 promotes reprogramming (Figure 6I and J). These results demonstrated that MeCP2 deficiency stimulated IGF1/AKT/mTOR signaling by decreasing the binding of HDAC2 to the IGF1 promoter. Discussion The generation of iPSCs offers a great opportunity in research and regenerative medicine. The current poor efficiency and incomplete mechanistic understanding of the reprogramming process hamper clinical application of iPSCs. In this study, we showed that MeCP2-deficient MEFs exhibited significantly improved reprogramming efficiency, and we revealed the mechanism of MeCP2 in cell reprogramming. A key issue in dealing with the generation of iPSCs is how to maintain a high proliferation rate and activate G1-S phase progression (Yamanaka, 2009). Since IGF-1 activates G1-S progression, this suggests a potential role of IGF-1 in promoting the generation of iPSCs (Bendall et al., 2007; Nguyen et al., 2007; Huang et al., 2009). In this study, we showed that MeCP2 deficiency enhanced IGF-1 expression, promoting cell proliferation and activating G1-S phase progression in the early stage of reprogramming. The mTOR/S6K pathway regulates multiple cellular functions, including cell cycle, protein synthesis, apoptosis, and autophagy. Activation of the mTOR/S6K axis stimulates protein synthesis and cell growth (Cruz et al., 2005; Goh et al., 2010; Chauvin et al., 2014). Recently, multiple studies have identified the role of mTOR-S6K in regulating the self-renewal of leukemia stem cells and neural stem cells (Hartman et al., 2013; Ghosh et al., 2016). Furthermore, IGF1 promotes cell proliferation by activating PI3K/AKT/mTOR signaling in human ovarian cancer cells (Lau and Leung, 2012). A recent study showed that IGF-1 activates mTOR and stimulates the rate of mRNA translation in osteoblasts (Bakker et al., 2016). Moreover, ribosomal proteins affect cell cycle progression via various mechanisms (Wang et al., 2006; Jang et al., 2012). We demonstrated that IGF1/AKT/mTOR signaling plays an important role in promoting the generation of iPSCs through activating ribosomal protein-mediated cell cycle gene translation. Our findings suggest that MeCP2 connects IGF1/AKT/mTOR signaling with protein synthesis and cell proliferation in somatic cell reprogramming. The increase in the number of AP-positive colonies owing to ribosomal proteins in WT MEFs was not as dramatic as that in the case of the MeCP2 knockout MEFs; there may be other mechanisms mediating the effect of MeCP2 knockout on reprogramming. Transcriptional repression of methylated genes by MeCP2 involves the binding of MeCP2 to Sin3A-HDAC complexes and HDAC2 is the main histone deacetylase of the co-repressor complex that interacts with MeCP2 (Jones et al., 1998; Nan et al., 1998). In this study, we showed that MeCP2 deficiency decreased the binding of HDAC2 to the IGF1 promoter region, which then increased IGF1 transcription and activated IGF1/AKT/mTOR signaling. Our finding connected histone modification with IGF1/AKT/mTOR signaling in somatic cell reprogramming by MeCP2. The ongoing dogma that patients with Rett syndrome with MeCP2 mutations undergo growth arrest somewhat contradicts the work described here. This inconsistency may be caused because of the following reasons. First, genes affected by MeCP2 knockout differ markedly between cell types (Sugino et al., 2014). Second, the function of genes in the whole body may not be consistent with their functions at the cellular level. Lastly, mutations in patients with RTT may not give rise to a complete loss of function of the MeCP2 protein. In summary, we demonstrated that MeCP2 deficiency promotes cell reprogramming through stimulating IGF1/AKT/mTOR signaling and activating ribosomal protein-mediated cell cycle gene translation in the early stage of reprogramming. These findings provided a strategy to regulate cell reprogramming by modulating MeCP2 and revealed the mechanism of MeCP2 in cell fate determination. Materials and methods Knocking out MeCP2 in mice sgRNAs targeting the MeCP2 gene were designed using CRISPR Design tool (http://crispr.mit.edu/), and inserted into a pMD-18T-modified plasmid containing an sgRNA-expressing skeleton. Cas9 and sgRNA mRNAs needed for microinjection were in vitro transcribed by the T7 in Vitro Transcription Kit (NEB). The one-cell-stage embryos were collected 12 h post coitum, and sgRNA and Cas9 mRNA were microinjected into their cytoplasms. After microinjection, the embryos were implanted in the oviducts of pseudopregnant female mice to obtain full-term pups. Cell lines and cell culture MEFs were isolated from WT and MeCP2 knockout embryos at E13.5 and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1 × penicillin/streptomycin. iPSCs were cultured with DMEM supplemented with 5% knockout serum replacement (KOSR), 0.1 mM β-mercaptoethanol, 1000 units/ml leukemia inhibitory factor, and 0.1 mM nonessential amino acids. Lentiviral production Viral particles were generated by transfecting plated 293T cells with pHIV vectors encoding MeCP2, Rps18, Rpl21, Rpl35a, and IGF1 along with pMD.2G and psPAX2 vectors. Supernatants from the transfected cells were collected 48 h after transfection. The viral suspension was mixed with 10 mg/ml polybrene (Millipore) and used to infect cells. Generation of iPSC and reprogramming efficiency evaluation Generation of pluripotent iPSC lines was performed as described previously (Carey et al., 2009). MEFs were isolated from E13.5 embryos cultured under established iPSC conditions and the four Yamanaka factors OSKM were expressed. The efficiency of iPSC formation is estimated according to the presence of AP-positive colonies after 14 days of reprogramming. The number of iPSC colonies per well was counted in triplicate. Immunofluorescence staining iPSCs growing on coverslips were rinsed briefly in PBS and fixed in 4% paraformaldehyde in PBS for 10 min on ice. After 10 min of permeabilization in PBS containing 0.2% Triton X-100 on ice, cells were subsequently washed with PBS three times. After blocking in 5% milk for 30 min at 37°C, anti-Oct4 (Santa Cruz), anti-Nanog (Proteintech), or anti-SSEA1 (Santa Cruz) were added at the dilution ratio suggested by the manufacturer. Cells were then counterstained with DAPI (Invitrogen). Fluorescent images were acquired using a Leica TCS SP8 confocal microscope. Point mutation of MeCP2 The point mutations of MeCP2 were constructed by QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s protocol. Primers used in the mutations are listed in Supplementary Table S1. RNA extraction and qRT-PCR Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Afterward, 1 μg of RNA from each sample was extracted and reverse-transcribed into cDNA using random primers and was subjected to qRT-PCR. qRT-PCR was performed using a Stratagene Mx3000P quantitative PCR system (Genetimes Technology). The reactions were incubated in a 96-well plate at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. All reactions were run in triplicate. Primers used in the study are listed in Supplementary Table S1. Western blot analysis Western blotting was performed as described previously (Wu et al., 2005). The commercial antibodies used were anti-MeCP2, anti-Rps18, anti-Rpl21, and anti-Rpl35a, which were all purchased from Abcam. Anti-CDK1, anti-CDK2, anti-Cyclin A2, anti-Cyclin B1, anti-Cyclin D1, anti-Cyclin E1, anti-AKT, anti-phosphor-AKT (Ser473), anti-S6, and anti-P-S6 were purchased from Cell Signaling Technology. Anti-Oct4, anti-Nanog, and anti-SSEA1 were purchased from Millipore. Anti-Tubulin and anti-HA tag antibodies were purchased from Sigma. Anti-IGF1, anti-Klf4, and anti-c-Myc were purchased from Abcam. Anti-Sox2 was purchased from Merck Millipore. Teratoma formation and histological analysis iPSCs were digested into single cells and cultured in gelatin-coated dishes to discard the attached feeder cells. The cells were collected and resuspended in PBS and 1 × 107 cells were injected under the skin of 5-week-old BALB/c SCID mice. The teratomas harvested for histological analysis after 4 weeks were fixed and sliced into sections that were then stained with hematoxylin and eosin. Diploid blastocyst injection Diploid blastocysts were collected from E3.5 time-pregnant mice. iPSCs were injected into diploid blastocysts that were transferred to CD-1 pseudopregnant recipient females. Then, the adult chimeric offspring were mated to a CD-1 mouse. siRNAs and RNA interference All siRNAs were designed and synthesized by RiboBio Co. Ltd. and listed in Supplementary Table S1. siRNAs were transfected into mouse MEFs using Lipo3000 reagent (Invitrogen) according to the manufacturer’s instructions. Cell counting kit-8 and CFSE cell proliferation assay MEFs were harvested by trypsinization and then plated into 96-well plates at a density of 10000/well. The cells were cultured and subjected to a cellular viability assay using a Cell counting kit-8 (Promega). CCK8 assays aim to quantify viable cells in proliferation assays. CFSE is also widely used for cell proliferation assays; the decrease in fluorescence intensity due to cell division is measured. To detect the proliferation ability of cells, a CFSE assay was performed using the CellTrace CFSE cell proliferation kit (Thermo) according to the manufacturer’s protocol. Flow cytometry SSEA1 positivity in cells was analyzed by FACS. Briefly, cells were trypsinized and washed once in PBS. For APC-conjugated SSEA1 (R&D), 1 × 105 cells were washed once in PBS and 0.5% BSA, and incubated with SSEA1 in 2 ml of 0.5% BSA in PBS for 30 min at 37°C. Afterwards, cells were subjected to FACS analysis. The cell cycle phase was detected by a Ki67-ICF/FACS kit. A FACSCanto II (BD Biosciences) instrument was used for all FACS analysis. ChIP assays WT and MeCP2 KO MEFs were maintained in MEF medium. Approximately 1 × 107 cells were used for each ChIP assay. ChIP assays were performed according to a previously described protocol (Shang et al., 2000). Chromatin was precipitated with either normal rabbit IgG (control) or anti-HDAC2 (CST) and anti-MeCP2 (Abcam), and was purified with a Qiagen PCR purification kit. The samples were analyzed by qRT-PCR using primers described in Supplementary Table S1. RNA immunoprecipitation WT and MeCP2 KO MEFs were crosslinked with 1% formaldehyde for 15 min. Cells were lysed by RIP lysis buffer containing a protease inhibitor cocktail and RNase inhibitor. The antibodies used for immunoprecipitation were Rps18 (Abcam) and Rpl21 (Abcam). Immune complexes were subjected to magnetic beads and washed sequentially with low-salt immune complex wash buffer, high-salt immune complex wash buffer, and then samples were washed with cold RIP wash buffer three times. For RNA purification, proteinase K buffer was added to samples at 55°C for 30 min. RNA was extracted in the supernatant by the phenol : chloroform : isoamyl alcohol method. The collected RNA was reverse transcribed to cDNA and subjected to qRT-PCR analysis. The primers used for detecting ribosomal protein binding sites are listed in Supplementary Table S1. Co-IP assay To explore the endogenous interaction between MeCP2 and HDAC2, MEF cells were collected and lysis for protein extraction with lysis buffer (100 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, and 0.01% Triton X-100, pH 8.0). The supernatant was collected and dithiobis(succinimidyl propionate) (DSP) (22585, Thermo Fisher Scientific) was added into supernatant for 1 h at 4°C. Then, the supernatant was immunoprecipitated overnight at 4°C with anti-MeCP2 antibody. The complexes were immunoprecipitated with Protein G Agarose Beads (CST) for 1 h at 4°C. Then agarose was washed three times with IP washing buffer (50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, and 0.01% Triton X-100, pH 8.0), then detected by western blotting with anti-HDAC2 antibody. Accession numbers Sequencing data generated by this work have been deposited into the Genome Sequence Archive (Wang et al., 2017) in the BIG Data Center (BIG Data Center Members, 2018), Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under the accession number CRA000764 (released from June 1, 2018), which are publicly accessible at http://bigd.big.ac.cn/gsa. Supplementary material Supplementary material is available at Journal of Molecular Cell Biology online. Acknowledgements The authors would like to thank all members of our group for discussion and help. We thank Ms Shiwen Li (Institute of Zoology, Chinese Academy of Sciences) for her kind technique support on confocal experiments. Funding This study was supported by the National Natural Science Foundation of China (31471395 to Q.Z.), the Key Research Projects of the Frontier Science of the Chinese Academy of Sciences (QYZDY-SSW-SMC002), and the National Basic Research Program of China (2014CB964903). 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Nature 460 , 49 – 52 . Google Scholar CrossRef Search ADS PubMed Author notes Edited by Jiarui Wu © The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Journal of Molecular Cell BiologyOxford University Press

Published: Mar 19, 2018

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