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. 2022 Oct;29(10):1000-1010.
doi: 10.1038/s41594-022-00840-5. Epub 2022 Oct 11.

Distinct roles for CKM-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

Emilia Dimitrova  1 , Angelika Feldmann  2   3 , Robin H van der Weide  4   5 , Koen D Flach  4 , Anna Lastuvkova  2 , Elzo de Wit  4 , Robert J Klose  6
Affiliations

Distinct roles for CKM-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

Emilia Dimitrova et al. Nat Struct Mol Biol. 2022 Oct.

Abstract

Precise control of gene expression underpins normal development. This relies on mechanisms that enable communication between gene promoters and other regulatory elements. In embryonic stem cells (ESCs), the cyclin-dependent kinase module Mediator complex (CKM-Mediator) has been reported to physically link gene regulatory elements to enable gene expression and also prime genes for induction during differentiation. Here, we show that CKM-Mediator contributes little to three-dimensional genome organization in ESCs, but it has a specific and essential role in controlling interactions between inactive gene regulatory elements bound by Polycomb repressive complexes (PRCs). These interactions are established by the canonical PRC1 (cPRC1) complex but rely on CKM-Mediator, which facilitates binding of cPRC1 to its target sites. Importantly, through separation-of-function experiments, we reveal that this collaboration between CKM-Mediator and cPRC1 in creating long-range interactions does not function to prime genes for induction during differentiation. Instead, we discover that priming relies on an interaction-independent mechanism whereby the CKM supports core Mediator engagement with gene promoters during differentiation to enable gene activation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. 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. 2
Fig. 2. CKM–Mediator regulates canonical PRC1 binding.
a, Heatmaps showing RING1B (PRC1) and CDK8 ChIP–seq signals at Polycomb domains (n = 2097), sorted by decreasing RING1B signal. b, A genomic snapshot of a Polycomb-bound locus, showing CDK8, RING1B, PCGF2, CBX7 and H3K27me3 ChIP–seq signal in WT (+) and CKM–MED KO (-) ESCs. c, Heatmaps showing RING1B, PCGF2, CBX7 and H3K27me3 ChIP–seq signal at Polycomb domains (n = 2,097) in WT (+) and CKM–MED KO (-) ESCs, sorted by decreasing RING1B signal. d, Metaplot analysis of RING1B, PCGF2, CBX7 and H3K27me3 enrichment at Polycomb domains (n = 2,097) in WT and CKM–MED KO ESCs.
Fig. 3
Fig. 3. 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. 4
Fig. 4. CKM–Mediator primes genes for activation during differentiation independently of cPRC1-mediated interactions.
a, A schematic of the differentiation of WT and CKM–MED KO ESCs used for cnRNA-seq. b, Boxplot analysis of the expression of CKM–MED-dependent genes (n = 631) in WT ESCs and following RA (retinoic acid) induction (WT and CKM–MED KO). Boxes show IQR, center lines represent the median, whiskers extend by 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. c, A schematic of the differentiation of WT and cPRC1 KO ESCs for cnRNA-seq. d, As in b but for cPRC1 cKO cells. e, A screenshot showing the expression of genes within the HoxB cluster following RA induction of CKM–MED cKO or cPRC1 KO cells. Forward strand is shown on top and reverse strand is shown at the bottom of each track. ChIP–seq tracks for CDK8 and cPRC1 (PCGF2) enrichment are shown. f, Boxplot analysis of the expression of RA-induced (RA-ind) genes from the Polycomb (PcG) network (top, n=482) and CKM–Med-dependent genes from the PcG network (bottom, n=184) following RA induction of CKM–MED cKO or cPRC1 KO cells. Boxes are defined as in a.
Fig. 5
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.
Extended Data Fig. 1
Extended Data Fig. 1. CKM-Mediator has a limited role in 3D genome organisation but is essential for Polycomb domain interactions.
a, A representative Western blot analysis of CDK8 immunoprecipitation (n = 2) from nuclear extracts from Med13/13lfl/fl (WT) and Med13/13l−/− (CKM-Mediator KO) ESCs, probed with the indicated antibodies. b, Quality control metrics of the Hi-C data, showing total sequenced read-pairs in millions, total valid contacts in millions and percentages in cis contacts for WT and CKM-Mediator KO ESCs. c, Aggregate analysis of super enhancer interactions in WT and CKM-Mediator KO ESCs. The difference between WT and KO is shown. d, Aggregate analysis of Hi-C signal (10 kb resolution) at pairs of Polycomb domains at the indicated distance ranges in Med13/13lfl/fl (WT) and Med13/13l−/− (CKM-Mediator KO) ESCs, with 200 kb flanking regions. Interactions of inactive non-Polycomb gene promoters subsampled to match regions as in Fig. 1f (n = 2096), are included as a negative control (bottom). The difference between WT and KO is shown. e, Capture-C interaction scores for interactions between Polycomb domains in WT and CKM-Mediator KO ESCs (number of promoters = 51, number of interactions = 148). f, Boxplot analysis of Capture-C interaction scores from WT and CKM-Mediator KO ESCs showing interactions between Polycomb gene promoters and other Polycomb-domains (left), or non-Polycomb gene promoters with active sites (H3K27ac, right). Number of promoters (P) and interactions (int) is shown. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians. Source data
Extended Data Fig. 2
Extended Data Fig. 2. CKM-Mediator regulates canonical PRC1 binding.
a, A Venn diagram showing the overlap between CDK8 peaks and Polycomb domains. Number of peaks and percent overlap are indicated. b, Metaplot analysis of CDK8 enrichment at Polycomb domains (n = 2097) in WT and CKM-Mediator (CKM-MED) KO ESCs. c, Heatmaps showing CDK8 ChIPseq signal at Polycomb domains (n = 2097) in WT and CKM-Mediator KO ESCs, sorted by decreasing RING1B signal. d, A representative Western blot analysis (n = 6) of nuclear extracts from WT and CKM-MED KO ESCs probed with the indicated antibodies. TBP and HDAC1 are used as loading controls. e, Comparison between loss of Hi-C signal (difference between CKM-MED-KO and WT) and loss of cPRC1 (PCGF2) binding (log2 fold change) at Polycomb domains. Polycomb domains were divided into equal bins (261 domains each) based on log2 fold change in cPRC1 binding. f, Comparison between loss of Hi-C signal (difference between CKM-MED-KO and WT) and levels of CDK8 binding in WT cells (log2RPKM). Domains were divided into eight bins based on CDK8 RPKM levels. Source data
Extended Data Fig. 3
Extended Data Fig. 3. cPRC1 creates interactions between Polycomb domains.
a, A representative Western blot analysis (n = 3) of nuclear extracts from the TetR-fusion lines used for Capture-C analysis probed with anti-Flag antibody to detect expression of the fusion proteins. HDAC1 is used as a loading control. b, ChIP-qPCR analysis of binding of the different TetR-fusion lines to the TetO array. Data are presented as mean value (n = 2) ±SD. Data points for individual replicates are shown. c, ChIP-qPCR analysis of binding of the CKM-Mediator complex to the TetO array in the TetR-CDK8, TetR-PCGF2, and TetR-GFP lines.. Data are presented as mean value (n = 2 for TetR-CDK8 and n = 3 for TetR-PCGF2 and TetR-GFP) ± SD. Data points for individual replicates are shown. d, Boxplot analysis of Capture-C mean normalised read counts and interaction scores in the TetR-fusion lines, looking at interactions with Polycomb domains (PCGF2-bound). Number of interactions is shown. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians. e, Snapshots showing Capture-C read count signal from TetR-CDK8, TetR-PCGF2 and TetR-GFP lines at a control locus. CDK8 and PCGF2 (cPRC1) ChIPseq signal is given as a reference. The Fli1 promoter bait is shown as a triangle and interactions created with surrounding cPRC1-bound sites are represented with arrowheads. f, A representative Western blot analysis of nuclear extracts (n = 3) from WT and cPRC1 KO ESCs probed with the indicated antibodies. TBP is used as a loading control. g, Metaplot analysis of CDK8 enrichment at CDK8 peaks (n = 24275) and Polycomb domains (n = 2097) in WT and cPRC1 KO ESCs. h, Heatmaps showing CDK8 ChIPseq signal at CDK8 peaks (n = 24275) and Polycomb domains (n = 2097) in WT and cPRC1 KO ESCs, sorted by decreasing CDK8 or RING1B signal, respectively. i, Boxplot analysis of Capture-C interaction scores 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). Number of promoters (P) and interactions (int) is shown. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians. Source data
Extended Data Fig. 4
Extended Data Fig. 4. CKM-Mediator primes genes for activation during differentiation independently of cPRC1-mediated interactions.
a, An MA plot of log2 fold changes in gene expression (cnRNA-seq) WT ESCs and RA-treated cells, determined using DESeq2. Significant expression changes (>1.5 fold change and padj<0.05) are shown in red and number of genes is indicated. Distribution of gene expression changes is shown on the right as a density. b, An MA plot of log2 fold changes in gene expression (cnRNAseq) WT and CKM-Mediator KO RA-treated cells, determined using DESeq2. Significant expression changes (>1.5 fold change and padj<0.05) are shown in red and number of genes is indicated. Distribution of gene expression changes is shown on the right as a density. c, A Venn diagram showing the overlap between RA-induced genes as defined in a and genes downregulated in CKM-Mediator KO cells, following RA treatment, as defined in b. d, A metaplot showing enrichment of cPRC1 (PCGF2) over the transcription start site (TSS) of the indicated different classes of genes. All=20633, ES-specific=2617; RA-induced=3320; CKM-Mediator-dependent=631. e, ChIP-qPCR showing enrichment of RING1B at promoters of developmental genes in WT ESCs and RA-induced cells (top). Data are presented as mean value (n = 3) ±SD. Data points for individual replicates are shown. Gene desert (g.d.) is included as a negative control region. Expression of the corresponding genes (RPKM) is shown below. Bpm7 is a control, non-induced gene. f, As in a for cPRC1 cKO cells. g, As in b for cPRC1 cKO cells. h, A Venn diagram showing the overlap between CKM-Mediator-dependent and cPRC1-dependent genes. Gene numbers are indicated. i, Boxplot analysis of the expression of cPRC1-dependent genes (n = 34), as defined in Extended Data Fig. 4f. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians. j, Boxplot analysis of the expression of RA-induced cPRC1 (PCGF2) target genes (n = 1201) in WT and cPRC1 KO ESCs and following RA-induction. Boxes are defined as in i. k, Boxplot analysis of the expression of genes within the Polycomb network in ESCs and following RA induction (all = 1974; RA-induced=482). Boxes are defined as in i. i, Boxplot analysis of Capture-C mean normalised read counts (left) and interaction score (right) from CKM-Mediator cKO and cPRC1 cKO ESCs showing interactions between promoters of genes within the Polycomb (PcG) network and Polycomb domains. Number of promoters (p) and interactions (int) is shown. Boxes are defined as in i. m, Boxplot analysis of Capture-C mean normalised read counts (left) and interaction score (right) from CKM-Mediator cKO and cPRC1 cKO ESCs showing interactions between gene promoters and poised enhancers (PE). Genes were divided into non-Polycomb targets (left set), Polycomb targets (middle set) and Polycomb targets induced by RA (right set). Number of promoters (P) and interactions (int) is shown. Boxes are defined as in i. n, Boxplot analysis of the expression of RA-induced genes that interact with a poised enhancer (n = 55) in CKM-Mediator cKO and cPRC1 cKO cells. The difference between WT RA cells and ESCs (left), as well as KO and WT cells (right), is shown as log2FC. Boxes are defined as in i. Source data
Extended Data Fig. 5
Extended Data Fig. 5. CKM-Mediator enables gene induction via recruitment of the Mediator complex.
a, A schematic illustration of the generation of the T7-MED14 expressing Med13/13lfl/fl ESC line. b, PCR showing amplification of homozygously-tagged T7-Med14 alleles (n = 2). c, A representative Western blot analysis of nuclear extracts from the T7-MED14 Med13/13lfl/fl ESC line, following tamoxifen (TAM) treatment (n = 3). Extract from an untagged ESC line was used as a control. HDAC1 was used as a loading control. d, A representative immunoprecipitation (IP) of endogenously T7-tagged MED14 with T7 antibody using nuclear extracts from Med13/13lfl/f ESCs before (UNT) and after tamoxifen (TAM) treatment (n = 2). The IPs were probed with the indicated antibodies. An IP from an untagged ESC line was performed as a negative control and a Western blot for SUZ12 was included as a control protein that does not interact with Mediator. e, Heatmaps of CDK8 and T7-MED14 ChIPseq signal at Polycomb domains (n = 2097) and H3K27ac peaks (n = 4037), sorted by decreasing CDK8 signal. f, Boxplots showing gene expression change (log2FC) of CKM-Mediator-dependent (n = 631) and CKM-Mediator-independent (n = 2689) RA-induced genes following RA differentiation of WT ESCs. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians. g, Boxplots showing T7-MED14 ChIPseq signal at the TSS (1000 bp) of the different classes of RA-induced gene classes as defined in e in ESCs and RA-induced cells (WT and CKM-Mediator KO). Boxes are defined as in f. Signal is an average from three independent biological experiments. Source data
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