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. 2017 May 3;15(5):e2001220.
doi: 10.1371/journal.pbio.2001220. eCollection 2017 May.

Smek Promotes Corticogenesis Through Regulating Mbd3's Stability and Mbd3/NuRD Complex Recruitment to Genes Associated With Neurogenesis

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Free PMC article

Smek Promotes Corticogenesis Through Regulating Mbd3's Stability and Mbd3/NuRD Complex Recruitment to Genes Associated With Neurogenesis

Byoung-San Moon et al. PLoS Biol. .
Free PMC article

Abstract

The fate of neural progenitor cells (NPCs) during corticogenesis is determined by a complex interplay of genetic or epigenetic components, but the underlying mechanism is incompletely understood. Here, we demonstrate that Suppressor of Mek null (Smek) interact with methyl-CpG-binding domain 3 (Mbd3) and the complex plays a critical role in self-renewal and neuronal differentiation of NPCs. We found that Smek promotes Mbd3 polyubiquitylation and degradation, blocking recruitment of the repressive Mbd3/nucleosome remodeling and deacetylase (NuRD) complex at the neurogenesis-associated gene loci, and, as a consequence, increasing acetyl histone H3 activity and cortical neurogenesis. Furthermore, overexpression of Mbd3 significantly blocked neuronal differentiation of NPCs, and Mbd3 depletion rescued neurogenesis defects seen in Smek1/2 knockout mice. These results reveal a novel molecular mechanism underlying Smek/Mbd3/NuRD axis-mediated control of NPCs' self-renewal and neuronal differentiation during mammalian corticogenesis.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Suppressor of Mek null (Smek) expression and function in mouse cortical development.
(A) Fixed cyroembedded coronal sections from E12.5 or E14.5 mouse forebrain stained with antibodies against Tuj1 (red), Tbr1 (green), and microtubule-associated protein 2 (MAP2) (red). Nuclear staining is shown by 4',6-diamidino-2-phenylindole (DAPI) (blue). (Right) Quantification of Fig 1A and S1D Fig. (E12.5: WT, n = 4, dKO, n = 5; E14.5: WT, n = 6, dKO, n = 5). Scale bars, 50 μm. (B) Same as Fig 1A for Pax6. (Right) Quantification of Fig 1B (E12.5: WT, n = 5, dKO, n = 4; E14.5: WT, n = 6, dKO, n = 3). Scale bar, 50 μm. (C) Immunostaining with Nestin (green) and Tuj1 (red) antibodies in WT and Smek1/2 dKO neural progenitor cells (NPCs). Nuclear staining is shown by DAPI (blue). Scale bars, 50 μm. (Lower) Quantification of anti-Tuj1–positive (WT-Un, n = 3; WT-2DIV, n = 6; dKO-Un, n = 3; dKO-2DIV, n = 6) and anti-Nestin–postive (WT-Un, n = 6; WT-2DIV, n = 6; dKO-Un, n = 6; dKO-2DIV, n = 6) cells in Fig 1C. Un, undifferentiation; 2Diff, differentiation 2 d in vitro (DIV). (D) Single cells of WT and Smek1/2 dKO NPCs were separated by serial dilution and sphere formation was induced for 8 d in vitro. Relative size of primary spheres grown up to 8 DIV were quantified by the ImageJ quantification software. Scale bars, blue (100 pixel), red (200 pixel). (E) Quantitative PCR (qPCR) analysis of indicated mRNAs. Values correspond to the average ± SD. Diff. (d), days in differentiation. Statistical t test analysis was performed to calculate significance (*p < 0.05, **p < 0.005, ***p < 0.0005; not significant (ns), p > 0.05). All quantification data underlying panels A–D can be found in S2 Data.
Fig 2
Fig 2. Suppressor of Mek null (Smek) interacts with methyl-CpG–binding domain protein 3 (Mbd3) and both colocalize in the nucleus.
(A) Numerical result of clones observed by β-galactosidase colony-lift filter assay using bait (Smek2) and prey (Mbd3). (B) Coimmunoprecipitation (co-IP) of human influenza hemagglutinin (HA)-tagged Mbd3 or Flag-tagged Smek2 in HEK293T cells transfected with Mbd3 and/or Smek (n = 2). IB, immunoblot. (C) Immunostaining with Mbd3 (red) and Smek2 (green) antibodies in NPCs. Nuclear staining is shown by 4',6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 50 μm. (D) Fixed, cyroembedded coronal sections from E15.5 mouse forebrain stained with antibodies against Mbd3 (green), Smek1 (red), and Nestin (white). Nuclear staining is shown by DAPI (blue). Yellow arrows indicate perinuclear localization of Smek1 in ventricular zone progenitor cells. (E) Co-IP of endogenous Smek2 and Mbd3 in undifferentiated (left) and differentiated (right) NPCs using anti- immunoglobulin G (IgG) (negative control), anti-Mbd3, or anti-Smek2 antibodies (n = 2). Un, undifferentiation; Diff, differentiation. (F) (Upper) Schematic of Smek1 mutants used in deletion analysis. (Lower) Co-IP of these Smek1 mutants with HA-tagged Mbd3 in the cells expressing full-length HA-tagged Mbd3 and Flag-tagged deletion Smek1 mutants (n = 2). Immunoprecipitation was performed using an anti-Flag antibody and immunoblotting was done using an anti-HA antibody. WCL, whole cell lysate. (G) (Upper) Schematic showing Mbd3 deletion mutants used in domain mapping analysis. (Lower) Glutathione-S-transferase (GST) pull-down assay using of purified GST, GST-Mbd3, or deletion mutants that were mixed with Smek2-overexpressing cell lysate. Immunoblotting was performed using Smek2 and Mbd3 antibodies. Blue arrows indicate major bands of each GST-fused Mbd3 mutant protein expression. (H) Co-IP of Smek with full-length or mutant Mbd3 (n = 1). Black arrows indicate band of each full-length or mutant (ΔN92) Mbd3. All quantification data underlying panel A can be found in S2 Table and quantification data for panel E can be found in S2 Data.
Fig 3
Fig 3. Both endogenous and overexpressed methyl-CpG–binding domain protein 3 (Mbd3) are degraded as neural progenitor cells (NPCs) differentiate.
(A) Immunostaining to detect Mbd3 expression in wild-type (WT), Smek1 knockout (S1-KO), Smek2 knockout (S2-KO) and Smek1/2 double knockout (S1/2-dKO) cells. Nuclear staining is shown by 4',6-diamidino-2-phenylindole (DAPI) (blue). Mbd3 is shown in green. Scale bar, 50 μm. (Lower) Quantification of Fig 3A. Scale bar, 50 μm. Diff. (d), differentiation days in vitro. (B) Western blot of Mbd3 in WT or Smek knockout NPCs that are under differentiation for 0, 1, or 3 d (n = 3). α-tubulin was used as an internal loading control. Arrows indicate multiple Mbd3 isoforms around 30–40 kDa (upper, Mbd3A; middle, Mbd3B; bottom, Mbd3C). (C) Quantification of Fig 3B. (D) Reverse transcription PCR (RT-PCR) analysis of Mbd3 and Gapdh transcript levels at indicated days (d) of differentiation (n = 2). (E) Quantification of data shown in Fig 3D. (F) (left panel) Immunoblot (IB) analysis of Mbd3 and α-tubulin in cells treated with cycloheximide (CHX) for indicated times (h, hours) with or without MG132 in NPCs (n = 3) or 293T cells (n = 5). (Right panel) Quantification of band intensity in Fig 3F, left panel using the ImageJ software. (G) NPCs were treated with MG132 for 6 h, harvested, and the lysates were immunoblotted with anti-Mbd3, anti–α-tubulin and anti–β-catenin antibodies. β-catenin was used as a positive control (n = 2). Quantification of band intensity in Fig 3G, bottom panel using the ImageJ software. (H) Ubiquitylation of overexpressed Mbd3 in NPCs (n > 3). (I) Ubiquitylation of overexpressed Mbd3 in HEK293T transfected with Flag-Mbd3 and HA-Ub expression vectors and treated 1 d later with MG132 were detected by immunoprecipitation with Flag antibody followed by immunoblotting with HA antibody (upper panel) (n > 2). Levels of each protein in the whole cell lysate are shown with western blot. (J) Same as panels H and I except that endogenous Mbd3 polyubiquitylation was shown (n = 3). Values correspond to the average ± SD. Statistical t test analysis was performed to calculate significance (*p < 0.05, **p < 0.005, ***p < 0.0005; not significant (ns), p > 0.05). All quantification data underlying panels A, C, E, F, and G can be found in S2 Data.
Fig 4
Fig 4. Suppressor of Mek null (Smek) promotes methyl-CpG–binding domain protein 3 (Mbd3) degradation and polyubiquitylation.
(A) HEK293 cells and cell lines stably expressing Smek1 or Smek2 were treated with cycloheximide (CHX) for indicated times and whole cell lysates were prepared for immunoblotting (n = 4). (Lower) Quantification of band intensities is shown in the lower panel. (B) Wild-type and Smek1/2 dKO NPCs were treated with CHX for indicated times (h, hours) and harvested, and then lysates were immunoblotted (n = 3). (Lower) Quantification of band intensities. OE, overexpression; Ub, ubiquitin. (C) Ubiquitylation assay of Mbd3 using control and Smek1 or Smek2 stable HEK293T transfected with Mbd3-myc and HA-Ub expression vectors and treated 1 d later with MG132 for 6 h (upper blot) and IB for indicated proteins (lower blot). (Lower) Quantification of band intensities (n = 3). (D) Same as panel C except using WT or Smek1/2 dKO NPCs (n = 2). (E) Same as panel D except using cells transfected with Mbd3(ΔN92)-myc and HA-Ub expression vectors at control and Smek1 or Smek2 stable cell lines (n = 2). In (A–C and E), quantification of band intensity was done using ImageJ. Data are presented as average ± SD. Statistical t test analysis was performed (*p < 0.05, **p < 0.005, ***p < 0.005; not significant (ns), p > 0.05). All quantification data underlying panels A, B, C, and E can be found in S2 Data.
Fig 5
Fig 5. Suppressor of Mek null (Smek) and methyl-CpG–binding domain protein 3 (Mbd3) colocalize on neuronal gene promoters.
(A) Smek1-chromatin immunoprecipitation sequencing (ChIP-seq) analysis in NPCs. Distribution of Smek1-binding peaks in NPCs. Proximal promoter regions are defined as sequences within 3 kb of the transcription starting site (TSS) of annotated genes. (B) Gene ontology (GO) analysis of annotated genes at Smek1-binding sites. (C) Smek1-binding peaks in NPCs in differentiation genes such as Dlx1, Tlx3, NeuroD1, Ascl1, and Lbx1. (D) ChIP-quantitative PCR (qPCR) analysis of Mbd3 occupancy at a Smek-binding locus in undifferentiated or differentiated conditions in WT (n = 3) and Smek1/2 dKO (n = 3) NPCs. (E) ChIP-qPCR analysis of Smek1 and Mbd3 occupancy at a Smek-binding locus in 0.2 or 2 d differentiated conditions in NPCs knocked down by shScramble (n = 3) and shMbd3 (n = 3) NPCs. (F) ChIP-qPCR analysis of HDAC1, HDAC2, MTA1, and acetyl histone H3 occupancy at a Smek-binding locus in undifferentiated or differentiated conditions in WT (n = 3) and Smek1/2 dKO (n = 3) NPCs. In panels D–F, immunoglobulin G (IgG) ChIP served as a negative control. Values are normalized to input control and represent average ± SD. t test analysis was performed to calculate the statistical significance (*p < 0.05, **p < 0.005). The ChIP-seq dataset for panels A–C can be found in S1 Data and all individual quantification data for panels D–F can be found in S2 Data.
Fig 6
Fig 6. Effects of methyl-CpG–binding domain protein 3 (Mbd3) overexpression on neuronal gene expression and promoter occupancy during differentiation of neural progenitor cells (NPCs).
(A) NPCs were electroporated with either pUltra-hot-control-mcherry or pUltra-hot-Mbd3-mcherry lentiviral vector and grown for 0.2 or 2 d in N2 medium with basic fibroblast growth factor (bFGF). Quantitative PCR (qPCR) analysis was used to measure the indicated transcript levels. (B) Mbd3 overexpression in WT NPCs increases Mbd3 occupancy of neuronal gene promoters but not that of Gfap, as determined by chromatin immunoprecipitation (ChIP)-qPCR. (C) Immunostaining to detect Tuj1 (green) and Mbd3-mcherry (red) expression. Nuclear staining is shown by 4',6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 100 μm. Yellow arrows indicate Tuj1/mcherry double-positive cells, and white arrows indicate Tuj1-positive cells. (D) Quantification of panel C. Data are presented as average ± SD. t test analysis was performed to calculate significance (*p < 0.05, ***p < 0.0005; not significant (ns), p > 0.05). All individual quantification data underlying panels A, B, and D can be found in S2 Data.
Fig 7
Fig 7. Effect of methyl-CpG–binding domain protein 3 (Mbd3) knockdown on neuronal gene expression and promoter occupancy over the course of differentiation of Suppressor of Mek null double knockout (Smek dKO) neural progenitor cells (NPCs).
(A) Wild-type (WT) or Smek1/2 dKO NPCs were electroporated with either control pLKO3G-shScramble or pLKO3G-shMbd3 lentiviral vector and grown for 2 d in N2 medium with basic fibroblast growth factor (bFGF). qPCR analysis was performed to detect indicated mRNAs (n = 3 or 6). (B) Mbd3 knockdown in Smek1/2 dKO NPCs decreases Mbd3 occupancy of Dlx1, Tlx3, NeuroD1, Ascl1, and Lbx1 promoters but not that of Gfap, as determined by chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) (n = 3). (C) Immunostaining to detect Tuj1 (red) and enhanced green fluorescent protein (EGFP) (green) expression. Nuclear staining is shown by 4',6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 100 μm. Red arrows indicate Tuj1/EGFP double-positive cells, and white arrows indicate EGFP-positive cells. (D) Quantification of panel C. All data are presented as average ± SD. t test analysis was performed to calculate significance (*p < 0.05, **p < 0.005; not significant (ns), p > 0.05). All individual quantification data underlying panels A, B, and D can be found in S2 Data.
Fig 8
Fig 8. Effect of methyl-CpG–binding domain protein 3 (Mbd3) knockdown on the embryonic mouse cortex.
(A) Schematic of injection site (green) and electrode position for in utero electroporation. Green fluorescent images indicates enhanced green fluorescent protein (EGFP)-labelled shScramble or shMbd3 expression in electroporated embryo brain (right panel). Fb, forebrain. (B) Confocal images showing immunofluorescent labeling of Tuj1 (red) and EGFP (green) in sections of the embryonic brain electroporated in utero. Nuclear staining is shown by 4',6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 50 μm. (C) Analysis of the percentage of migrating cells expressing EGFP in the brain’s ventricular zone (VZ)/subventricular zone (SVZ), intermediate zone (IZ), and cortical plate (CP) compartments in embryos electroporated either with control shScramble (n = 3) or shMbd3 (n = 4). (D) The effect of Mbd3 knockdown on neurogenesis was evaluated by counting Tuj1/EGFP double-positive cells versus total EGFP-positive cells in VZ/SVZ, IZ, and CP compartments. (E) A model on the role of the Smek-Mbd3-NuRD axis in cell-fate determination in cortical progenitor cells to neuronal cells during neurogenesis. Data are presented as average ± SD. Ac, acetylation; TSS, transcription starting site. t test analysis was performed to calculate statistical significance (*p < 0.05, **p < 0.005). All individual quantification data underlying panels C and D can be found in S2 Data.

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California Institute for Regenerative Medicine (CIRM) https://www.cirm.ca.gov/grants (grant number TG2-01161). Received by BSM. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National Institutes of Health https://www.nih.gov/grants-funding (grant number 5R01NS06721305). Received by WL. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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