Volume 31, Issue 3, Pages 347-359 (August 2008) Mediator Links Epigenetic Silencing of Neuronal Gene Expression with X-Linked Mental Retardation  Ning Ding, Haiying Zhou, Pierre-Olivier Esteve, Hang Gyeong Chin, Seokjoong Kim, Xuan Xu, Sumy M. Joseph, Michael J. Friez, Charles E. Schwartz, Sriharsa Pradhan, Thomas G. Boyer  Molecular Cell  Volume 31, Issue 3, Pages 347-359 (August 2008) DOI: 10.1016/j.molcel.2008.05.023 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 MED12 and G9a Interact In Vitro and In Vivo (A) Schematic summary of reciprocal binding domains on MED12 and G9a. A proline-, glutamine-, and leucine-rich (PQL) domain on MED12 (aa 1616–2050) interacts more strongly with an ankyrin-repeat domain (aa 486–689) than a cysteine-rich domain (aa 250–332) on G9a. (B) FLAG-MED12 and HA-G9a were expressed with or without one another in HeLa cells prior to immunoprecipitation (IP) of whole-cell lysates using antibodies specific for the FLAG or HA epitopes as indicated. Immunoprecipitates were resolved by SDS-PAGE and processed by western blot (WB) analysis using FLAG- or HA-specific antibodies as indicated. Input, 10% of the nuclear lysate used for IP reactions. Molecular Cell 2008 31, 347-359DOI: (10.1016/j.molcel.2008.05.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 MED12 Is a Likely Mediator Interface for G9a HMTase (A) HeLa nuclear lysate was subjected to IP using IgG or antibodies specific for MED30 as indicated. Immunoprecipitates were washed with 300 mM KCl and 0.2% NP-40 prior to resolution by SDS-PAGE and processing by WB analysis using the specified antibodies. Input, 10% of the nuclear lysate used for IP reactions. (B) DsRed-G9a and FLAG-Mediator subunits as indicated were monitored for colocalization following their transient expression in COS-7 cells. DNA was visualized with Hoechst stain. (C and D) HeLa nuclear lysate was subjected to IP with IgG or antibodies specific for G9a, MED4, or MED30 as indicated. Immunoprecipitates were washed as in (A) prior to incubation with S-adenosyl-L-[methyl-3H] methionine and either core histones (C) or a panel of recombinant GST-H3 N-termini (aa 1–84) bearing the indicated lysine to arginine substitution mutations (D). Reaction products were resolved by SDS-PAGE and visualized by autoradiography, WB, or Coomassie blue staining as indicated. H3me, methylated H3. (E and F) Nuclear lysates from HeLa cells transfected with control (CNTL), G9a-, MED12-, MED23-, or CDK8-specific siRNAs were subjected to IP using IgG or antibodies specific for MED30 or MED4 as indicated. Immunoprecipitates were washed as in (A) prior to resolution by SDS-PAGE and processing by WB analysis using the specified antibodies or incubation with S-adenosyl-L-[methyl-3H] methionine and core histones (HMTase assay). Input, 10% of the nuclear lysates used for IP reactions. H3me, methylated H3. Molecular Cell 2008 31, 347-359DOI: (10.1016/j.molcel.2008.05.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 MED12 Is Required for REST- and G9a-Dependent Neuronal Gene Repression (A) MED30-specific Mediator immunoprecipitates from nuclear lysates of HEK293 cells transfected without (−) or with (+) Myc-tagged REST were resolved by SDS-PAGE and processed by WB analysis using the specified antibodies. Input, 5% of the nuclear lysates used for IP reactions. (B) MED30-specific Mediator immunoprecipitates bearing G9a (from Figure 2A and Figure S6) were reblotted with antibodies specific for REST. (C) Terminal immunoprecipitates derived from successive rounds of IP from MED31/HeLa nuclear lysate with FLAG- followed by G9a-specific antibodies were resolved by SDS-PAGE and processed by WB analysis using the specified antibodies. Input, 5% of the nuclear lysate used in first-round IP reactions. (D) Whole-cell lysates from BG1 cells serially transfected with control, REST-, G9a-, MED12-, CDK8-, or MED23-specific siRNA as indicated and subsequently with TATA-Luc or RE1-TATA-Luc reporter plasmids were assayed for normalized luciferase activities using a dual luciferase assay system. Luciferase activities are expressed relative to the luciferase activity obtained in cells transfected with the TATA-Luc reporter plasmid, which was arbitrarily assigned a value of 1. Data represent the mean ± SEM of at least three independent experiments performed in duplicate. Asterisks denote statistically significant values relative to control siRNA (Student's t test, ∗p < 0.1, ∗∗p < 0.05, ∗∗∗p < 0.01). (E) RNA from HeLa cells transfected with control, REST-, MED12-, G9a-, MED23-, or CDK8-specific siRNAs as indicated was used for RT-qPCR. mRNA levels are expressed relative to mRNA levels in control siRNA-transfected cells, which was arbitrarily assigned a value of 1. Data represent the mean +/− SEM of at least three independent experiments performed in duplicate. Asterisks denote statistically significant values relative to control siRNA (Student's t test, ∗p < 0.1, ∗∗p < 0.05, ∗∗∗p < 0.01). (F) Nuclear lysates from cells analyzed in (D) were resolved by SDS-PAGE and processed by WB analysis using the indicated antibodies to validate knockdown efficiencies. Molecular Cell 2008 31, 347-359DOI: (10.1016/j.molcel.2008.05.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 MED12 Is Required for REST-Directed G9a Recruitment to RE1 Silencing Elements (A–D) Soluble chromatin prepared from untransfected HeLa cells (A and B) or HeLa cells transfected with control, REST-, MED12-, G9a-, MED23-, or CDK8-specific siRNAs as indicated (C and D) was subjected to one (A, C, and D) or two (B) rounds of IP using the indicated antibodies (IgG, mix of goat [g] and rabbit [r] IgGs). Immunoprecipitated chromatin was analyzed by semiquantitative (A and B) or quantitative (C and D) PCR using primers flanking RE1 elements or upstream gene sequences within the M4, SNAP25, and Synapsin1 genes. In (C and D), the level of RE1 site occupancy for each protein is expressed relative to its level of occupancy in control siRNA-transfected cells, which was arbitrarily assigned a value of 100%. Data represent the mean ± SEM of at least three independent experiments performed in triplicate. Asterisks denote statistically significant values relative to control siRNA (Student's t test, ∗p < 0.1, ∗∗p < 0.05, ∗∗∗p < 0.01). Molecular Cell 2008 31, 347-359DOI: (10.1016/j.molcel.2008.05.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Direct Pairwise Interactions between REST, Mediator, and G9a (A) Diagram of REST fragments expressed as GST-fusion proteins. Numbers refer to amino acid coordinates. NTRD, CTRD, 8 × Zinc Fingers, K, and Pro refer to N- and C-terminal repression domains, zinc finger DNA-binding domain, and lysine- or proline-rich domains, respectively. (B) Purified E. coli-expressed GST-REST, HeLa cell Mediator (MED), and baculovirus-expressed G9a protein preparations used for in vitro binding assays. Mediator was purified from HeLa cells stably expressing an HA epitope-tagged MED6 subunit (Kim et al., 2006; Zhou et al., 2006). (C and D) Immobilized GST or GST-REST derivatives as indicated were incubated with purified Mediator (C) or G9a (D) and specifically bound proteins resolved by SDS-PAGE prior to WB analysis using the specified antibodies. Input, 10% of Mediator or G9a used in binding reactions. (E) Purified Mediator and G9a were incubated with or without one another prior to IP with antibodies specific for either G9a or the HA epitope on MED6 as indicated. Immunoprecipitates were resolved by SDS-PAGE and processed by WB analysis using the specified antibodies. Molecular Cell 2008 31, 347-359DOI: (10.1016/j.molcel.2008.05.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Delineation of a MED12/Mediator- and G9a-Dependent Internal Repression Domain in REST (A) Immobilized GST or GST-REST derivatives as indicated were incubated with HeLa nuclear lysate and specifically bound proteins resolved by SDS-PAGE prior to WB analysis using the specified antibodies. Input, 10% of nuclear lysate used in binding reactions. (B) Whole-cell lysates from representative transient repression assays in (C) were resolved by SDS-PAGE and processed by WB analysis using antibodies specific for the Gal4 DNA-binding domain or the p89 subunit of TFIIH as an internal loading control. Arrows indicate the relative positions of Gal4-REST derivatives, which are expressed at roughly equivalent levels. (C) HeLa cells were cotransfected with a G5TK-Luc reporter bearing five Gal4 DNA-binding sites upstream of the TK promoter along with the Gal4 DNA-binding domain (Gal4) or the indicated Gal4-REST fragments. Transfected whole-cell lysates were assayed for normalized luciferase activities using a dual luciferase assay system. Luciferase activities (RLU) are expressed relative to the luciferase activity obtained in cells transfected with Gal4, which was arbitrarily assigned a value of 1. Data represent the mean ± SEM of at least three independent transfections performed in duplicate. Asterisks denote statistically significant differences relative to Gal4 (Student's t test, ∗p < 0.1, ∗∗p < 0.05, ∗∗∗p < 0.01). (D) HeLa cells were transfected with control, MED12-, or G9a-specific siRNAs as indicated 48 hr prior to cotransfection with pG5TK-Luc along with Gal4 or the indicated Gal4-REST fragments and subsequent assay of transfected whole-cell lysates for normalized luciferase activities. Luciferase activities are expressed relative to the luciferase activity obtained in cells transfected with control siRNA and Gal4. Data represent the mean ± SEM of at least three independent transfections performed in duplicate. Asterisks denote statistically significant differences (Student's t test, ∗p < 0.1, ∗∗p < 0.05, ∗∗∗p < 0.01). (E) HeLa cells cotransfected with pG5TK-Luc along with Gal4 or the indicated Gal4-REST fragments were treated with DMSO or the HDAC inhibitor TSA (330 nM) prior to assay of transfected whole-cell lysates for normalized luciferase activites. Luciferase activities are expressed relative to the luciferase activity obtained in DMSO-treated cells transfected with Gal4. Data represent the mean ± SEM of at least three independent transfections performed in duplicate. Asterisks denote statistically significant differences (Student's t test, ∗p < 0.1, ∗∗p < 0.05, ∗∗∗p < 0.01). Molecular Cell 2008 31, 347-359DOI: (10.1016/j.molcel.2008.05.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 XLMR-Associated Missense Mutations in MED12 Disrupt Its REST-Specific Corepressor Function (A and B) HeLa cells were transfected with control (CNTL) or MED12-specific siRNA and subsequently with an internal control SV40-β-galactosidase expression plasmid along with pG5TK-Luc and either Gal4, Gal4-REST 141–600 (A), or Gal4-β-catenin (B). Where indicated (MED12r), DNA transfections also included siRNA-resistant WT, FG, Lujan, or ΔPQL mutant MED12 derivatives. Normalized luciferase activities are expressed relative to the luciferase activity obtained in cells transfected with control siRNA and Gal4, which was arbitrarily assigned a value of 1. In (B), the relative luciferase activity of β-catenin in control siRNA-transfected cells was set at a value of 100%, yielding a relative transactivation level (RTL); its corresponding transactivation levels in MED12 siRNA-transfected cells are expressed relative to this value. Data represent mean ± SEM of at least three independent transfections performed in duplicate. Asterisks denote statistically significant differences compared to MED12r WT (Student's t test, ∗p < 0.1, ∗∗p < 0.05, ∗∗∗p < 0.01). (C) Whole-cell lysates from representative transient knockdown/rescue assays in (A) were resolved by SDS-PAGE and processed by WB analysis using antibodies specific for either MED12, the FLAG epitope on MED12r derivatives, or the p89 subunit of TFIIH. (D–F) HeLa cells were transfected with control (siCNTL) or MED12-specific (siMED12) siRNAs. Where indicated (MED12r), MED12 knockdown cells were transfected with siRNA-resistant WT, FG, or Lujan mutant MED12 expression plasmids. (D) RNA from transfected cells was subjected to RT-qPCR using Synapsin1- and M4-specific primers. mRNA levels are expressed relative to mRNA levels in control siRNA-transfected cells, which was arbitrarily assigned a value of 1. (E and F) Soluble chromatin from transfected cells was subjected to IP using the indicated antibodies. Immunoprecipitated chromatin was analyzed by qPCR using primers flanking RE1 elements within the Synapsin1 and M4 genes. The level of RE1 site occupancy for each protein is expressed relative to its level of occupancy in control siRNA-transfected cells, which was arbitrarily assigned a value of 100%. Data represent the mean ± SEM of at least three independent experiments performed in duplicate. Asterisks denote statistically significant differences compared to MED12r WT (Student's t test, ∗p < 0.1, ∗∗p < 0.05, ∗∗∗p < 0.01). Molecular Cell 2008 31, 347-359DOI: (10.1016/j.molcel.2008.05.023) Copyright © 2008 Elsevier Inc. Terms and Conditions