Fig 2: CARM1-mediated MED12 methylation is involved in estrogen-induced gene transcriptional activation. (A) Heat map representation of CARM1 and MED12 ChIP-seq tag density centered on estrogen-induced CARM1 sites (± 3,000 bp). (B) Correlation between the ChIP-seq tag density (log2) of CARM1 and MED12 on estrogen-induced CARM1 sites. (C) MCF7 cells were transfected with siCTL, siCARM1 or siMED12, and treated with or without estrogen (E2, 10-7 M, 6 hrs) followed by RNA-seq analysis. Estrogen-induced genes which were dependent on both CARM1 and MED12 were shown by Pie chart. (D, E) Heat map (D) and box plot (E) representation of the expression levels (FPKM) for genes induced by estrogen and dependent on both CARM1 and MED12 as described in (C). Heat map: z-score normalized FPKM; box plot: FPKM (log2). (F) MCF7 cells were transfected with siCTL or siMED12 in stripping medium for three days, and treated with or without estrogen (E2, 10-7 M, 6 hrs), followed by RNA extraction and RT-qPCR analysis to examine the expression of selected estrogen-induced coding genes as indicated (± s.e.m., **P<0.01, ***P<0.001). (G) MCF7 cells were transfected with siCTL or siCARM1 in stripping medium for three days, and treated with or without estrogen (E2, 10-7 M, 1 hr) followed by ChIP-seq with anti-MED12 antibody. MED12 ChIP-seq tag density distribution centered on estrogen-induced CARM1 sites was shown (± 3,000 bp). (H, I) The binding of MED12 as described in (G) was shown for specific genes, as indicated. (J) MCF7 cells were infected with lenti-viral vectors expressing shRNA targeting MED12 together with or without Flag-tagged wild type (WT) MED12 or MED12 mutants with arginine 1899 replaced by alanine (R1899A), and then treated with or without estrogen (E2, 10-7 M, 6 hrs) followed by RT-qPCR analysis to examine the expression of selected estrogen-induced genes as indicated (± s.e.m., **P<0.01, ***P<0.001). Data was presented as fold induction by estrogen.

Fig 3: Mediator-CDK subunits control canonical CTCF-cohesin binding sites.A Two example regions showing CTCF binding in WT, ?MED12 and ?CCNC cells. Gray rectangle indicates position of HCD B Venn diagram showing the overlap between CTCF and SCC1 peaks in WT, ?MED12 and ?CCNC cells. C Aggregate alignment of CTCF ChIPseq signal on canonical (i.e. CTCF/SCC1 co-bound sites) stratified for inside and outside a H3K9me3 domain. D Virtual loop anchorpoints were selected as intersection of CTCF and SCC1 sites that were in the top 10% with the strongest decrease in signal. Pairwise alignment of Hi-C signal (PE-SCAn) on virtual anchorpoints. E Relative enrichment of lost virtual anchorpoints inside H3K9Me3 domains versus expected based on circular permutation (shuffled).

Fig 4: Mediator-CDK subunits restrict heterochromatin.A Left panel shows schematic explanation of the haploid genetic gene trap assay. Sense integrations with respect to a protein coding gene result in truncating deletion. Anti-sense integrations are largely inconsequential and serve as a control for accessibility of the gene. Right panel shows percentage of sense integrations (over a total of sense and anti-sense) shown for WT and ?WAPL cells for the Mediator subunits Cyclin-C (CCNC) and MED12. Boxplot shows the result of n = 3 haploid genetic gene trap assays for WT and ?WAPL cells. Boxes indicate the interquartile range (IQR) of the data (25–75%) and box center line indicates the median. Whiskers extend to the minimum or maximum value that lies no further than 1.5 times the IQR from the bottom or top of the box, respectively. B Western blot analysis confirming knock-out of MED12 and Cyclin-C. HSP90 and actin represent loading controls for MED12 and Cyclin-C panels directly above. Representative images are shown from experiments that have been performed twice. C ChromHMM analysis segmenting the genome in 15 chromatin states shown for WT and MED12 cells. Table shows the percentage of genome coverage. Red/blue color scale indicates the relative increase or decrease in ?MED12 compared to the WT cells. D Barplot showing the coverage of H3K9me3 domains over the genome and (E) the absolute number of H3K9me3 peaks. F Example region showing the H3K4me1 (green) and H3K9me3 (orange) ChIPseq profiles in WT, ?MED12 and ?CCNC cells. G Aggregate plots showing the average H3K9me3 signal over all the H3K9me3 domains for WT and mutant cells. Heatmaps show the signal in and around individual domains. H Immunofluorescence analysis of H3K9me3 levels in WT, ?MED12 and ?CCNC cells. Representative images are shown from experiments that have been performed twice (see Supplementary Fig. 2e).

Fig 5: Mediator complex interactor validation. a Immunoprecipitation (IP) of Carm1 and Med12 by a Carm1 antibody from NSC nuclear extract. Western blots are probed with the indicated antibodies. Control IP by rabbit IgG and 5% input are also shown. Source data are provided as a Source Data file. b Immunoprecipitation (IP) of Jmjd1c and Med12 by Jmjd1c antibody from NSC nuclear extract. Western blots are probed with the indicated antibodies. Control IP by rabbit IgG and 5% input are also shown. Source data are provided as a Source Data file. c Function in neural development of identified Mediator-interacting transcription factors in NSCs. References are provided in the Methods. d mRNA levels in NSCs of Mediator-interacting transcription factors (TFs) and the Top 600 highest expressed TFs in NSCs. The average emPAI scores, a semi-quantitative mass spectrometry-based measure of molar amounts, in the four Mediator complex purifications is shown for Mediator–interacting TFs