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).