Fig 2: FMRP targets encoding large proteins are downregulated in the mouse cortex. a, a′) Profiles and quantification of capillary electrophoresis western experiments showing a partial reduction in the levels of FMRP targets Auts2 and Med13L and complete loss of FMRP in the Fmr1 KO mouse cortex as compared to wild-type controls. Insets show standard curves of western signal as a function of total protein concentration. b) Total protein levels of all biotin-labeled proteins in mouse cortex extracts. b′) A zoom-in of (b) showing large proteins >300 kDa. b″) Quantification of (b) showing a decrease in the levels of large proteins but not total proteins in Fmr1 KO mouse cortex samples. c) Quantification of capillary electrophoresis western experiments of indicated proteins performed on mouse cortex extracts from wild-type or Fmr1 KO animals. *P < 0.05, #P < 0.1.

Fig 3: CKM–Mediator has a limited role in 3D genome organization but is essential for Polycomb domain interactions.a, A schematic of Med13/13lfl/fl ESCs. 4-Hydroxytamoxifen (TAM) induces conditional disruption of the CKM–Mediator complex (CKM–MED). b, A representative western blot analysis (n = 6) of nuclear extracts from Med13/13lfl/fl (wild type, WT) and Med13/13l-/- (CKM–MED KO) ESCs showing depletion of MED13 and MED13L proteins. HDAC1 is shown as a loading control. c, Hi-C contact matrices of WT and CKM–MED KO ESCs at 10 kb resolution. Genomic coordinates are indicated. d, Aggregate analysis of TADs and loops in WT and CKM–MED KO ESCs at 10 kb resolution. e, Hi-C contact matrices of WT and CKM–MED KO ESCs at 5 kb resolution. Interactions between Polycomb domains are indicated with a red circle. The blue track shows binding of PRC1 (RING1B ChIP–seq). Genomic coordinates are indicated. f, Aggregate analysis of Hi-C signal (10 kb resolution) at pairs of Polycomb domains in Med13/13lfl/fl (WT) and Med13/13l-/- (CKM–MED KO) ESCs, with 200 kb flanking regions. The difference between WT and KO is shown. g, A snapshot showing Capture-C read count signal in WT and CKM–MED KO ESCs. Interactions between the Nkx2-1 promoter bait (triangle) and surrounding Polycomb-bound sites are shown with arrowheads. PRC1 binding (RING1B ChIP–seq) is shown as a reference. h, Boxplot analysis of mean normalized read counts from WT and CKM–MED KO ESCs showing interactions between Polycomb gene promoters and other Polycomb domains (left), or non-Polycomb gene promoters and active sites (H3K27ac, right). Interactions were not distance-matched due to differences in the interaction ranges for the two promoter types. Boxes show IQRs, center line represents median, whiskers extend by 1.5 × IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58 x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians.Source data

Fig 4: PPARβ/δ inverse agonists prevent TGFβ-stimulated recruitment of Mediator subunits to the ANGPTL4 promoter. Caki-1 cells were treated for 30 min as indicated and subjected to scanning ChIP-qPCR analysis with primer pairs that amplify regions close to the TSS of ANGPTL4. Antibodies against MED1(A), MED26 (B) and MED13L (D) were used. Panels (C and F) show ChIP-qPCR data from these samples using a negative control region primer pair. In (E) RNAPII binding is shown as an additional control. Commercially available antibodies are denominated by the terms in parentheses. Means and standard deviations of three technical replicates from representative ChIP assays are plotted.

Fig 5: (A) MED13L gene expression analysis. The results indicate a mean fold change (FC) in target gene expression values of the control group (n = 3) versus our proband at three different locations along MED13L: exons 1–2 (marked as MED13L_1-2), exons 3–4 (MED13L_3-4), and exons 16–17 (MED13L_16-17). (B) Schematic representation of the gel electrophoresis of the proband’s cDNA synthesised from the RNA sample. Lanes 1, 3, and 5 show the results corresponding to amplicons #A, #B, and #C, respectively. Lanes 2, 4, and 6 correspond to PCR reaction products of the negative control. The green arrows indicate the wild-type alleles and the red arrow indicates the allele with the intragenic MED13L deletion. (C) The chromatogram represents the Sanger sequencing results of the MED13L coding sequence. (D) Model of the MED13L protein (UniProtKB: Q71F56) and its functional domains. (E) Results of the Western blot analysis.