Cell Biology

Mecp2 Fine-tunes Quiescence Exit by Targeting Nuclear Receptors

Jun Yang
Shitian Zou
Zeyou Qiu
Mingqiang Lai
Qing Long
Huan Chen
Sheng Zhang
Xiaoling Xie
Yan Gong
Anling Liu author has email address
Mangmang Li author has email address
Xiaochun Bai author has email address

Guangdong Provincial Key Laboratory of Bone and Joint Degenerative Diseases, The Third Affiliated Hospital of Southern Medical University, Guangzhou, 510630, China
State Key Laboratory of Organ Failure Research, Department of Cell Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
Department of Biochemistry, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China

https://doi.org/10.7554/eLife.89912.1

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Figures and data

Mecp2 is immediately decreased in the liver after PHx.
(A) Real-time PCR to evaluate mRNA levels of Mecp2 at different time points after PHx. Data are presented as means ±SEM; n = 3. n.s., not significant; ****p < 0.0001 by one-way ANOVA.
(B) WB showing the time course of protein levels of Mecp2, pRb, Cyclin D1, Cyclin E1, Cyclin A1, Cyclin B1 and β-actin in mouse livers after PHx. Right panel, quantification of Mecp2. Data are presented as means ±SEM; n = 3. n.s., not significant; ****p < 0.0001; *p < 0.05 by one-way ANOVA.
(C) Representative IF staining of Mecp2 (red) and Alb (green), together with DAPI (blue) for nuclei in liver sections at different time points after PHx. Lower panels, higher-magnification images.
(D) Representative IHC images of liver tissues stained for Mecp2 or Ki67 at the indicated time points after PHx.

Mecp2 fine-tunes quiescence exit in hepatocytes after PHx in vivo.
(A-D) Liver regeneration in Mecp^fl/fl and Mecp2 cKO mice after PHx. (A) Real-time PCR to measure mRNA levels of Mecp2. The effects of Mecp2 KO on quiescence exit and liver regeneration were assessed by WB of Mecp2, pRb and Cyclin D1 (B), IF staining of Mecp2 (red) and Ki67 (green) in liver sections (C), and liver index of control and Mecp2 cKO mice
(D) at the indicated time points.
(E-H) Liver regeneration in Mecp2^fl/fl livers without (AAV-EV) or with AAV-mediated Mecp2 OE (AAV-Mecp2) after PHx. EV, empty vector. (E) Real-time PCR to measure mRNA levels of Mecp2. (F-H) The effects of Mecp2 OE on quiescence exit and liver regeneration were assessed by WB of Mecp2, pRb and Cyclin D1 (F), IF staining of Mecp2 (red) and Ki67 (green) in liver sections (G), and liver index at the indicated time points (H).
(I-L) Liver regeneration in Mecp2 cKO livers without (Mecp2 cKO/AAV-EV) or with AAV-mediated Mecp2 restoration (Mecp2 cKO/AAV-Mecp2) after PHx. (I) Real-time PCR to measure mRNA levels of Mecp2. (J-L) The effects of Mecp2 restoration on quiescence exit and liver regeneration in Mecp2 cKO livers were assessed by WB of Mecp2, pRb and Cyclin D1 (J), IF staining of Mecp2 (red) and Ki67 (green) in liver sections (K), and liver index (L) at the indicated time points. Data are presented as means ±SEM. In (A), (B), (E), (F), (I) and (J), n = 3; in (D), (H) and (L), n = 5 mice/group. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by two-way ANOVA.

Mecp2 is immediately decreased during quiescence exit in cellular models.
(A-B) Representative histograms of PI staining of either asynchronized (Asyn) proliferating or starvation-induced quiescent 3T3 cells after S.R. in (A) and statistical analysis of cell cycle distribution (B).
(C) Real-time PCR to examine mRNA levels of Mecp2. Data are presented as means ±SEM; n = 3. n.s., not significant; ****p < 0.0001 by one-way ANOVA.
(D) WB of Mecp2, pRb, Cyclin D1, Cyclin E1, Cyclin A1 and Cyclin B1 in quiescent 3T3 cells upon S.R.. Right panel, quantification of Mecp2. Data are presented as means ±SEM; n = 3. n.s., not significant; ****p < 0.0001 by one-way ANOVA.
(E) Representative IF staining of Mecp2 (red) and Ki67 (green), together with DAPI (blue) in Asyn and quiescent 3T3 cells upon S.R..
(F-G) Representative histograms of PI staining of C.I.-induced quiescent 3T3 cells after C.I.L.
(F) and statistical analysis of cell cycle distribution (G) at the indicated time points.
(H) Real-time PCR to examine mRNA levels of Mecp2 in 3T3 cells released from C.I.-induced quiescence. Data are presented as means ± SEM; n = 3. n.s., not significant; ****p < 0.0001 by one-way ANOVA.
(I) WB of Mecp2, pRb, Cyclin D1, Cyclin E1, Cyclin A1 and Cyclin B1 in quiescent 3T3 cells upon C.I.L.. Right panel, quantification of Mecp2. Data are presented as means ±SEM; n = 3. n.s., not significant; ****p < 0.0001 by one-way ANOVA.
(J) Representative IF staining of Mecp2 and Ki67 in 3T3 cells released from C.I.-induced quiescence. See also Figure S3.

Mecp2 negatively regulates the G0/G1 transition in the cellular model of S.R.-induced quiescence exit.
(A) Real-time PCR showing the mRNA levels of Mecp2 in 3T3 cells transfected with negative control siRNA (NC si) or Mecp2 siRNA (Mecp2 si) at the early stages of S.R.-induced cell cycle reentry. Data are presented as means ±SEM; n = 3. n.s. not significant; ***p < 0.001; ****p < 0.0001 by two-way ANOVA.
(B) WB of Mecp2, pRb and Cyclin D1 in control and Mecp2 KD 3T3 cells released from S.S.-induced quiescence at the indicated time points.
(C) Representative IF staining of Mecp2 and Ki67 in control and Mecp2 KD 3T3 cells upon S.R.-induced quiescent exit.
(D) Ki67 and PI double staining followed by flow cytometry showing cell cycle profiles of 3T3 cells transfected with NC or Mecp2 siRNA upon S.R.-induced quiescence exit. Cells in G0, G1 and S/G2/M phases were defined by Ki67−/2N DNA content, Ki67+/2N DNA content and >2N DNA content population, respectively. Lower panel, quantification of the percentage of 3T3 cells in the G0 phase. Data are presented as means ±SEM; n = 3. n.s. not significant; ****p < 0.0001 by two-way ANOVA.
(E) Real-time PCR showing the mRNA levels of Mecp2 in 3T3 cells transduced with the EV or the vector overexpressing Mecp2 (Mecp2 OE) at the early stages of quiescence exit. Data are presented as means ±SEM; n = 3. ****p < 0.0001 by two-way ANOVA.
(F) WB of Mecp2, pRb and Cyclin D1 in control and Mecp2 OE 3T3 cells released from S.S.-induced quiescence at the indicated time points.
(G) Representative IF staining of Mecp2 and Ki67 in quiescent control and Mecp2 OE 3T3 cells upon S.R..
(H) Representative flow cytometry plots of Ki67/PI double staining in control and Mecp2 OE 3T3 cells upon S.R.-induced quiescence exit. Lower panel, quantification of proportion of 3T3 cells in the G0 phase. Data are presented as means ± SEM; n = 3. n.s. not significant; ***p < 0.001; ****p < 0.0001 by two-way ANOVA.

Nedd4 interacts with Mecp2 and affects quiescence exit by facilitating Mecp2 degradation.
(A) Reciprocal IP-WB analysis to validate the interaction between endogenous Mecp2 and Nedd4.
(B) Co-IP of Mecp2, ubiquitin and Nedd4 in quiescent 3T3 cells during S.R.-induced quiescence exit.
(C) Real-time PCR showing siRNA-mediated Nedd4 KD in 3T3 cells upon S.R.-induced quiescence exit. Data are presented as means ± SEM; n = 3. ****p < 0.0001 by two-way ANOVA.
(D-F) The effect of Nedd4 KD on quiescent exit in 3T3 cells determined by WB (D), IF staining of Ki67 and Nedd4 (E), and Ki67/PI staining followed by flow cytometry (F) at the indicated time points. Lower panel in (F): quantification of the percentage of 3T3 cells in the G0 phase. Data are presented as means ± SEM; n = 3. n.s. not significant; ***p < 0.001, ****p < 0.0001 by two-way ANOVA.
(G) Real-time PCR showing Nedd4 OE in 3T3 cells upon S.R.-induced quiescence exit. Data are presented as means ±SEM; n = 3. ****p < 0.0001 by two-way ANOVA.
(H-J) The effect of Nedd4 OE on quiescent exit in 3T3 cells determined by WB (H), IF staining of Ki67 and Nedd4 (I), and Ki67/PI staining (J) at the indicated time points. Data are presented as means ±SEM; n = 3. n.s. not significant; ****p < 0.0001 by two-way ANOVA.

Mecp2 transcriptionally regulates quiescence exit.
(A) Heatmap of Mecp2 direct target genes at the early stage of liver regeneration rank-ordered by their gene expression fold change.
(B) The top ten most significantly overrepresented GO terms for the PHx-activated (red) and PHx-repressed (green) Mecp2 target genes.
(C) Heatmaps depicting ChIP-seq enrichment of Mecp2 at the promoter-proximal region (3 kb away from TSS) and the defined gene region of Mecp2 target genes in Mecp^fl/fl and Mecp2 cKO livers before and 6 h post-PHx. Genes are rank-ordered according to the fold change of expression.
(D) Real-time PCR validation of PHx-repressed NRs in Mecp2^fl/fl and Mecp2 cKO livers upon PHx. Data are presented as means ±SEM; n = 3. n.s. not significant; **p < 0.01; ****p < 0.0001 by two-way ANOVA.

Depletion of either Nr1h3 or Rara mimics the Mecp2 KD phenotype during quiescence exit.
(A) ChIP-qPCR analyses of Mecp2 at the promoter-proximal regions of Rara and Nr1h3 in Mecp2^fl/fl and Mecp2-cKO livers before and 6 h post-PHx.
(B-D) Either Nr1h3 or Rara KD promotes S.R.-induced quiescence exit in 3T3 cells. (B) Real-time PCR showing lentivirus-mediated knockdown of either Nr1h3 or Rara. shLuc served as a negative control. (C-D) The effect of Nr1h3 or Rara KD on quiescent exit in 3T3 cells determined by WB (C) and Ki67/PI staining followed by flow cytometry (D) at the indicated time points. Data are presented as means ± SEM; n = 3. n.s. not significant; *p < 0.05; ***p < 0.001; ****p < 0.0001 by two-way ANOVA.
(E-H) Either Nr1h3 or Rara KD further enhances quiescence exit in Mecp2 cKO livers. (E) Real-time PCR showing AAV-mediated knockdown of either Nr1h3 or Rara. shLuc served as a negative control. (F-H) The effect of Nr1h3 or Rara KD on quiescent exit in Mecp2 cKO livers determined by WB (F), IF (G) and liver index (H) before and 6 h post-PHx. Data are presented as means ±SEM. In (E), n = 3; ***p < 0.001; ****p < 0.0001 by one-way ANOVA. In (H), n = 5 mice/group; n.s. not significant; *p < 0.05, by two-way ANOVA.
(I) Model of the negative regulatory role for Mecp2 in fine-tuning quiescence exit.

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