Fig 2: CA suppresses AML cell proliferation by inhibiting Mediator kinases(a) CA structure with N,N-dimethylamine red, C5–C9 ethano bridge magenta, C13-methyl green and isoquinoline blue. (b) Phosphorylation of the RNA pol II C-terminal domain (mean ± s.e.m., n=3 biological replicates, one of two experiments shown, autorad in Supplementary Figure 1). (c) Kinome profiling in MOLM-14 lysate (mean, n=2 biological replicates, experiment performed once, values < 35% indicate no change). (d) CA binding pocket of CDK8 from CA-CDK8-CCNC crystal structure (semi-transparent surface; CA in gold, CDK8 in grey) with contact residues and CA in stick representation. Dotted red lines indicate H-bonds. (e) Effect of CA on growth of indicated cell lines (mean ± s.e.m., n=3 biological replicates, one of two experiments shown). (f) Sensitivity of MOLM-14 cells to CA upon expression of indicated kinases (mean ± s.e.m., n=3 biological replicates, one of two experiments shown). (g) Immunoblot showing IFN-?-stimulated STAT1 S727 phosphorylation in MOLM-14 cells expressing indicated kinases and treated with CA (one of two experiments shown, full scan in Supplementary Figure 1).

Fig 3: Removing the CpG island-binding domain of FBXL19 results in a failure to induce developmental genes during ES cell differentiation.(A) A schematic illustrating the OHT treatment and differentiation approach. (B) RT-qPCR gene expression analysis of genes showing decreased CDK8 binding in Fbxl19ΔCXXC ES cells before (ESC) and after RA induction. Expression is relative to the average of two house-keeping genes. Error bars show SEM of three biological replicates, asterisks represent statistical significance calculated by Student T-test: *p<0.05, **p<0.01, ***p<0.001. (C) Volcano plots showing differential expression (log2 fold change) comparing WT and Fbxl19ΔCXXC cells in the ES cell (top) and RA-induced state (bottom). Differentially expressed genes (log2FC < −0.5 or log2FC > 0.5, padj <0.1) are shown in red. The number of genes considered to be significantly altered in expression are indicated. (D) Gene ontology analysis of genes with decreased expression in Fbxl19ΔCXXC ES cells following RA treatment (n = 552). (E) A boxplot indicating the expression level (FPKM) based on 4sU RNA-seq in wild-type ES cells of the gene groupings in (C): n.c. genes n = 17869 (no significant change), up genes = 889, down genes = 552. p-values calculated using a Wilcoxon rank sum test are indicated. (F) A boxplot indicating the log2 fold change in gene expression of the gene groupings in (E) upon RA differentiation of ES cells. p-values calculated using a Wilcoxon rank sum test are indicated. (G) A boxplot indicating the change in CDK8 binding (log2FC) at the promoters of the gene groupings in (E) in ES cells. p-values calculated using a Wilcoxon rank sum test are indicated.10.7554/eLife.37084.015Figure 6—source data 1.Differential gene expression analysis.

Fig 4: cPRC1 creates interactions between Polycomb domains.a, A schematic of the integrated TetO site and experimental setup. b, A snapshot showing Capture-C read count signal from TetR-PCGF2, TetR-CDK8 and TetR-GFP lines at the TetO array. CDK8 and PCGF2 (cPRC1) ChIP–seq signal is given as a reference. The TetO bait is shown as a triangle and interactions created with surrounding cPRC1-bound sites are represented with arrowheads. c, A schematic of the cPRC1 (Pcgf4-/-Pcgf2fl/fl) conditional KO line. d, A snapshot showing Capture-C read count signal from WT and cPRC1 KO ESCs. Interactions between the Nkx2-1 promoter bait (triangle) and surrounding Polycomb domain sites are shown with arrowheads. cPRC1 binding (PCGF2 ChIP–seq) is shown as a reference. e, Boxplot analysis of normalized read counts from WT and cPRC1 KO ESCs showing interactions between Polycomb gene promoters and other Polycomb domains (left), or non-Polycomb gene promoters and active sites (H3K27ac, right). Boxes show IQR, center lines represent the median, whiskers extend 1.5 × IQR or the most extreme point (whichever is closer to the median), whereas notches extend by 1.58 x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians.

Fig 5: CKM–Mediator enables gene induction via recruitment of the Mediator complex.a, A genomic snapshot of two CKM–Mediator-dependent genes, showing CDK8 and T7-MED14 ChIP–seq and cnRNA-seq in WT (+) and CKM–MED KO (-) ESCs (top) and following RA induction (bottom). b, Heatmaps showing CDK8 and T7-MED14 ChIP–seq signal at promoters (TSS±2.5 kb) of CKM–MED-dependent genes in ESCs and following RA induction (n = 631). T7-MED14 signals are shown for WT and CKM–Mediator KO RA-induced cells. Genes are sorted by decreasing T7-MED14 signal in RA-treated cells. Metaplots showing read density are shown on the top of each heatmap.