Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: (A) Schematic representation of CBX2-PRC1. (B) A hypothetical model describing how CBX2-PRC1 is assembled into condensates through phase separation. (C) Representative epi-fluorescence images of condensates of individual CBX2-PRC1 components. Scale bars, 5.0 μm. (D) Condensed fraction of CBX2 and PHC1/2/3 quantified from (C). Error bars denote SD. (E–G) Representative epi-fluorescence images of the scaffold CBX2 and the clients. CBX2, at a fixed concentration of 0.5 μM, was mixed with serial dilutions of the clients RING1B (E), MEL18 (F), and PHC1 (F). Scale bars, 5.0 μm. (H–M) Condensed fraction (H–J) and condensate size (K–M) of the scaffold CBX2 and the clients quantified from (E)–(G). Error bars denote SD.

Article Snippet: Anti-RING1B mouse mAb , MBL , Cat#D139-3; RRID: AB_592650.

Techniques: Fluorescence, Concentration Assay

Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: (A) Schematic representation of the CBX-PRC1 complexes. (B) Condensed fraction of the CBX proteins quantified from . Error bars denote SD. (C) Representative epi-fluorescence images of the CBX-PRC1 components. Panels on left: RING1B was unlabeled and not shown. Panels on right: only the RING1B images are shown. Scale bars, 5.0 μm. (D) Box plot of condensed fraction quantified from (C). (E) A hypothetical model describing how individual CBX-PRC1 complexes are assembled to condensates in vitro . (F) Live-cell epi-fluorescence images showing subnuclear localization of the CBX proteins fused with HaloTag treated with and without Dox. Scale bars, 5.0 μm. (G) Box plot of condensed fraction of the CBX proteins quantified from (F). p value is calculated using Student’s t test (**p < 0.01).

Article Snippet: Anti-RING1B mouse mAb , MBL , Cat#D139-3; RRID: AB_592650.

Techniques: Fluorescence, In Vitro

Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: (A) Schematic representation for CRISPR-Cas9-mediated homologous recombination to insert HaloTag to the C terminus of Cbx2 in mESCs. Scissors indicate the sgRNA-targeted location. Red arrows indicate the primers used to verify the insertion. LHA, left homology arm; RHA, right homology arm; HT, HaloTag. (B) Agarose gel analysis of PCR amplicons from homozygous HaloTag ( Cbx2 HT/HT ) and heterozygous HaloTag ( Cbx2 WT/HT ) knockin mESC lines. Arrows show the correct size of PCR amplicons. (C) Western blots for HaloTag comparing wild-type (WT), homozygous CBX2-HT, and heterozygous CBX2-HT mESC lines. (D) Live-cell imaging showing subnuclear distribution of CBX2-HT in Cbx2 HT/HT (left) and Cbx2 HT/WT (right) mESC lines. The number of condensates is shown to the right, along with the intensity ratio of condensates to non-condensed regions and the condensed fraction. Scale bars, 5.0 μm. (E) Co-immunostaining analysis of CBX2-HT as well as endogenous RING1B and PHC1 in Cbx2 HT/HT cell line. CBX2-HT was stained by an anti-HaloTag antibody. RING1B and PHC1 were stained by anti-RING1B and anti-PHC1 antibodies, respectively. Scale bars, 5.0 μm. (F) Live-cell imaging showing subnuclear localization of the CBX2-PRC1 components. The expression level is controlled by a tetracycline-response element (TETp, top panel) and induced by doxycycline (Dox). The thinness of the arrow corresponds to the level of expression. Scale bars, 5.0 μm. (G) Box plot of condensed fraction of the CBX2-PRC1 components quantified from (F). p value is calculated using Student’s t test (*p < 0.05; **p < 0.01).

Article Snippet: Anti-RING1B mouse mAb , MBL , Cat#D139-3; RRID: AB_592650.

Techniques: CRISPR, Homologous Recombination, Agarose Gel Electrophoresis, Knock-In, Western Blot, Live Cell Imaging, Immunostaining, Staining, Expressing

Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: (A) A hypothetical model describing how condensate composition regulates the partitioning of CBX2-PRC1 components and nucleosomes and the exchange properties of the scaffold CBX2. Colored hexagons are the CBX2-PRC1 clients (magenta) and nucleosomes (green). (B) Representative epi-fluorescence images of CBX2-PRC1 subunits in the four-component (CBX2, RING1B [R], MEL18 [M], and PHC1 [P]) system. Scale bars, 5.0 μm. (C) Box plot of condensed fraction in the four-component system quantified from (B). p value is calculated using Student’s t test (*p < 0.05; **p < 0.01). (D) FRAP curves of CBX2 in the single-component, two-component, three-component, and four-component systems. Error bars denote SD. (E) Example confocal fluorescence images of CBX2 and nucleosomes (Nuc.) in the two-component, three-component, four-component, and five-component systems. Scale bars, 5.0 μm. (F) Box plot of condensed fraction of CBX2 and nucleosomes quantified from (E). p value is calculated using Student’s t test (**p < 0.01). (G) FRAP curves of YFP-CBX2 in the two-, three-, four-, and five-component systems. Error bars denote SD. (H) Representative live-cell epi-fluorescence images of HT-CBX2 in wild-type (WT), Ring1a −/− /b −/− , and Bmi1 −/− /Mel18 −/− mESC lines. Scale bars, 5.0 μm. (I and J) Box plots of condensed fraction (I) and size (J) of HT-CBX2 condensates quantified from (H). p value is calculated using Student’s t test (**p < 0.01). Error bars denote SD. (K) Example confocal images of FRAP of HT-CBX2 in wild-type (WT), Ring1a −/− /b −/− , and Bmi1 −/− /Mel18 −/− mESC lines. Red arrows show condensates to be bleached. Scale bar, 5.0 μm. (L) FRAP curves of HT-CBX2 within and outside condensates in wild-type (WT), Ring1a −/− /b −/− , and Bmi1 −/− /Mel18 −/− mESC lines. Error bars denote SD.

Article Snippet: Anti-RING1B mouse mAb , MBL , Cat#D139-3; RRID: AB_592650.

Techniques: Fluorescence

Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: (A) Residue-resolution coarse-grained model of CBX2 and RING1B. f, folded regions; d, disordered regions. (B) Minimal coarse-grained model of CBX2, RING1B, MEL18, and PHC1 that describes the proteins as patchy colloids. CBX2 and PHC1 are represented as 4-valency patchy particles, while RING1B and MEL18 are represented as 3-valency patchy particles. The interaction matrix shows the relative pairwise interaction strengths between the patches on the four different proteins in the minimal model at reduced temperature T = 1 . (C) Contact maps showing the frequency of contacts between the different regions of the proteins, for a pure CBX2 system and a 1:1 CBX2/RING1B mixture. For the regions with a larger relative contribution of contacts highlighted by the red square, a residue-resolution contact map is shown. (D) Variation of T c of pure CBX2 and RING1B systems, as well as their mixtures with varying RING1B mole fraction using our residue-resolution model (purple) and minimal model (red). For each set of data points, the T c of each system relative to the T c of the pure CBX2 system is plotted. (E) Diffusion coefficients of CBX2 measured in direct-coexistence simulations of the dense phase of mixtures with different compositions at T = 1 .

Article Snippet: Anti-RING1B mouse mAb , MBL , Cat#D139-3; RRID: AB_592650.

Techniques: Residue, Diffusion-based Assay

Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-RING1B mouse mAb , MBL , Cat#D139-3; RRID: AB_592650.

Techniques: Virus, Recombinant, Staining, Modification, Saline, Live Cell Imaging, Electroporation, Purification, Protease Inhibitor, Membrane, Stripping Membranes, Bradford Assay, Plasmid Preparation, Software, Microscopy

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: L3MBTL2 maintains MYCN-amplified neuroblastoma cell proliferation through silencing NRIP3 and BRME1 genes.

doi: 10.1111/gtc.13148

Figure Lengend Snippet: FIGURE 7 L3MBTL2-knockout reduces the level of H2AK119ub around the NRIP3 and BRME1 transcription start sites (TSSs). (a, b) Representative integrative genomics viewer (IGV) screenshots showing ChIP-seq-data-derived peaks of L3MBTL2, PCGF6, RING1B, H2AK119ub, H3K27me3, and MYCN on the NRIP1 and BRME1 loci. The blue boxes below each peak indicate peaks called using hiddenDomains with default parameters. The black boxes indicate the range used for ChIP-qPCR primer design. (c) Heatmap of PCGF6, MYCN, and H2AK119ub ChIP-seq signals in control (left) or L3MBTL2-knockout (right). PCGF6 ChIP-seq signal intensities in controls are sorted in descending order. (d) ChIP-qPCR showing changes in PCGF6, MYCN, and H2AK119ub binding around the NRIP3 and BRME1 promoter regions upon L3MBTL2 depletion. Numbers in parentheses indicate the primer sets used in Table S2. Data are presented as the mean ± SD, N = 3 (two-tailed Student's t-test).

Article Snippet: We employed the following primary antibodies: L3MBTL2 rabbit pAb (Active Motif), RING1B mouse mAb (Atsuta et al., 2004), PCGF6 rabbit pAb (Proteintech 24,103-1-AP), E2F6 rabbit pAb (LSBio LS-C352133), β-Tubulin mouse mAb (Boehringer Ingelheim, Ingelheim am Rhein, Germany), phospho-histone H2A.X (Ser139) mouse mAb (Millipore JBW301), ubiquityl-histone H2A (Lys119) rabbit mAb (CST, (D27C4) XP®, #8240), histone H3 rabbit pAb (Abcam ab1791, Bristol, United Kingdom), or DYKDDDDK Tag rabbit mAb (CST, #2368).

Techniques: Knock-Out, ChIP-sequencing, Derivative Assay, ChIP-qPCR, Control, Binding Assay, Two Tailed Test

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: L3MBTL2 maintains MYCN-amplified neuroblastoma cell proliferation through silencing NRIP3 and BRME1 genes.

doi: 10.1111/gtc.13148

Figure Lengend Snippet: FIGURE 7 L3MBTL2-knockout reduces the level of H2AK119ub around the NRIP3 and BRME1 transcription start sites (TSSs). (a, b) Representative integrative genomics viewer (IGV) screenshots showing ChIP-seq-data-derived peaks of L3MBTL2, PCGF6, RING1B, H2AK119ub, H3K27me3, and MYCN on the NRIP1 and BRME1 loci. The blue boxes below each peak indicate peaks called using hiddenDomains with default parameters. The black boxes indicate the range used for ChIP-qPCR primer design. (c) Heatmap of PCGF6, MYCN, and H2AK119ub ChIP-seq signals in control (left) or L3MBTL2-knockout (right). PCGF6 ChIP-seq signal intensities in controls are sorted in descending order. (d) ChIP-qPCR showing changes in PCGF6, MYCN, and H2AK119ub binding around the NRIP3 and BRME1 promoter regions upon L3MBTL2 depletion. Numbers in parentheses indicate the primer sets used in Table S2. Data are presented as the mean ± SD, N = 3 (two-tailed Student's t-test).

Article Snippet: We employed the following primary antibodies: L3MBTL2 rabbit pAb (Active Motif), RING1B mouse mAb (Atsuta et al., 2004), PCGF6 rabbit pAb (Proteintech 24,103-1-AP), E2F6 rabbit pAb (LSBio LS-C352133), β-Tubulin mouse mAb (Boehringer Ingelheim, Ingelheim am Rhein, Germany), phospho-histone H2A.X (Ser139) mouse mAb (Millipore JBW301), ubiquityl-histone H2A (Lys119) rabbit mAb (CST, (D27C4) XP®, #8240), histone H3 rabbit pAb (Abcam ab1791, Bristol, United Kingdom), or DYKDDDDK Tag rabbit mAb (CST, #2368).

Techniques: Knock-Out, ChIP-sequencing, Derivative Assay, ChIP-qPCR, Control, Binding Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Distinct roles for CDK-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1101/2021.11.04.467119

Figure Lengend Snippet: (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 CDK-MED KO ESCs. (C) Heatmaps showing CDK8 ChIPseq signal at Polycomb domains (n=2097) in WT and CDK-MED KO ESCs, sorted by decreasing RING1B signal. (D) Western blot analysis of nuclear extracts from WT and CDK-MED KO ESCs probed with the indicated antibodies. TBP and HDAC1 are used as loading controls.

Article Snippet: Antibodies used for Western blot analysis were rabbit polyclonal anti-MED13L (A302-420A, Bethyl laboratories), rabbit polyclonal anti-MED13 (GTX129674, Genetex), rabbit monoclonal anti-CDK8 (ab229192, Abcam), rabbit polyclonal anti-CCNC (A301-989A, Bethyl laboratories), rabbit polyclonal anti-MED1 (A300-793A, Bethyl laboratories), rabbit polyclonal anti-MED15 (A302-422A, Bethyl laboratories), rabbit polyclonal anti-MED14 (A301-044A-T, Bethyl laboratories), rabbit monoclonal anti-RING1B (5694, Cell Signaling), rabbit monoclonal anti-SUZ12 (3737, Cell Signaling), rabbit polyclonal anti-PCGF2 (sc-10744, Santa Cruz), rabbit monoclonal anti-T7-Tag (D9E1X) (13246, Cell Signaling), mouse monoclonal anti-TBP (ab818, Abcam), rabbit monoclonal anti-HDAC1 (ab109411, Abcam), and mouse monoclonal anti-Flag (F1804, Sigma).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Distinct roles for CDK-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1101/2021.11.04.467119

Figure Lengend Snippet: (A) Western blot analysis 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. Error bars show standard deviation of three biological replicates. (C) ChIP-qPCR analysis of binding of the CDK-MED complex to the TetO array in the TetR-CDK8 and TetR-GFP lines. Error bars show standard deviation of three biological replicates. (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. (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) Western blot analysis of nuclear extracts 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 ChUPseq 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 with other Polycomb-domains (left) or non-Polycomb gene promoters with active sites (H3K27ac, right). Number of promoters (P) and interactions (int) is shown.

Article Snippet: Antibodies used for Western blot analysis were rabbit polyclonal anti-MED13L (A302-420A, Bethyl laboratories), rabbit polyclonal anti-MED13 (GTX129674, Genetex), rabbit monoclonal anti-CDK8 (ab229192, Abcam), rabbit polyclonal anti-CCNC (A301-989A, Bethyl laboratories), rabbit polyclonal anti-MED1 (A300-793A, Bethyl laboratories), rabbit polyclonal anti-MED15 (A302-422A, Bethyl laboratories), rabbit polyclonal anti-MED14 (A301-044A-T, Bethyl laboratories), rabbit monoclonal anti-RING1B (5694, Cell Signaling), rabbit monoclonal anti-SUZ12 (3737, Cell Signaling), rabbit polyclonal anti-PCGF2 (sc-10744, Santa Cruz), rabbit monoclonal anti-T7-Tag (D9E1X) (13246, Cell Signaling), mouse monoclonal anti-TBP (ab818, Abcam), rabbit monoclonal anti-HDAC1 (ab109411, Abcam), and mouse monoclonal anti-Flag (F1804, Sigma).

Techniques: Western Blot, Capture-C, Expressing, Binding Assay, Standard Deviation

Journal: bioRxiv

Article Title: Distinct roles for CDK-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1101/2021.11.04.467119

Figure Lengend Snippet: (A) Western blot analysis of CDK8 immunoprecipitation from nuclear extracts from Med13/13l fl/fl (WT) and Med13/13l -/- (CDK-MED 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 CDK-MED KO ESCs. (C) Aggregate analysis of super enhancer interactions in WT and CDK-MED KO ESCs. The difference between WT and KO is shown. (D) Boxplot analysis of Capture-C interaction scores from WT and CDK-MED KO ESCs showing interactions between Polycomb gene promoters with other Polycomb-domains (left) or non-Polycomb gene promoters with active sites (H3K27ac, right). Number of promoters (P) and interactions (int) is shown.

Article Snippet: Antibodies used for Western blot analysis were rabbit polyclonal anti-MED13L (A302-420A, Bethyl laboratories), rabbit polyclonal anti-MED13 (GTX129674, Genetex), rabbit monoclonal anti-CDK8 (ab229192, Abcam), rabbit polyclonal anti-CCNC (A301-989A, Bethyl laboratories), rabbit polyclonal anti-MED1 (A300-793A, Bethyl laboratories), rabbit polyclonal anti-MED15 (A302-422A, Bethyl laboratories), rabbit polyclonal anti-MED14 (A301-044A-T, Bethyl laboratories), rabbit monoclonal anti-RING1B (5694, Cell Signaling), rabbit monoclonal anti-SUZ12 (3737, Cell Signaling), rabbit polyclonal anti-PCGF2 (sc-10744, Santa Cruz), rabbit monoclonal anti-T7-Tag (D9E1X) (13246, Cell Signaling), mouse monoclonal anti-TBP (ab818, Abcam), rabbit monoclonal anti-HDAC1 (ab109411, Abcam), and mouse monoclonal anti-Flag (F1804, Sigma).

Techniques: Western Blot, Immunoprecipitation, Hi-C, Capture-C

Journal: bioRxiv

Article Title: Distinct roles for CDK-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1101/2021.11.04.467119

Figure Lengend Snippet: (A) Heatmaps showing RING1B (PRC1) and CDK8 ChIPseq signal at Polycomb domains (n=2097), sorted by decreasing RING1B signal. (B) A genomic snapshot of a Polycomb-bound locus, showing CDK8, RING1B, PCGF2 and H3K27me3 ChIPseq signal in WT (+) and CDK-MED KO (-) ESCs. (C) Heatmaps showing RING1B, PCGF2 and H3K27me3 ChIPseq signal at Polycomb domains (n=2097) in WT (+) and CDK-MED KO (-) ESCs, sorted by decreasing RING1B signal. (D) Metaplot analysis of RING1B, PCGF2 and H3K27me3 enrichment at Polycomb domains (n=2097) in WT and CDK-MED KO ESCs.

Article Snippet: Antibodies used for Western blot analysis were rabbit polyclonal anti-MED13L (A302-420A, Bethyl laboratories), rabbit polyclonal anti-MED13 (GTX129674, Genetex), rabbit monoclonal anti-CDK8 (ab229192, Abcam), rabbit polyclonal anti-CCNC (A301-989A, Bethyl laboratories), rabbit polyclonal anti-MED1 (A300-793A, Bethyl laboratories), rabbit polyclonal anti-MED15 (A302-422A, Bethyl laboratories), rabbit polyclonal anti-MED14 (A301-044A-T, Bethyl laboratories), rabbit monoclonal anti-RING1B (5694, Cell Signaling), rabbit monoclonal anti-SUZ12 (3737, Cell Signaling), rabbit polyclonal anti-PCGF2 (sc-10744, Santa Cruz), rabbit monoclonal anti-T7-Tag (D9E1X) (13246, Cell Signaling), mouse monoclonal anti-TBP (ab818, Abcam), rabbit monoclonal anti-HDAC1 (ab109411, Abcam), and mouse monoclonal anti-Flag (F1804, Sigma).

Techniques:

Journal: bioRxiv

Article Title: Distinct roles for CDK-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1101/2021.11.04.467119

Figure Lengend Snippet: (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 CDK-MED KO ESCs. (C) Heatmaps showing CDK8 ChIPseq signal at Polycomb domains (n=2097) in WT and CDK-MED KO ESCs, sorted by decreasing RING1B signal. (D) Western blot analysis of nuclear extracts from WT and CDK-MED KO ESCs probed with the indicated antibodies. TBP and HDAC1 are used as loading controls.

Article Snippet: Antibodies used for Western blot analysis were rabbit polyclonal anti-MED13L (A302-420A, Bethyl laboratories), rabbit polyclonal anti-MED13 (GTX129674, Genetex), rabbit monoclonal anti-CDK8 (ab229192, Abcam), rabbit polyclonal anti-CCNC (A301-989A, Bethyl laboratories), rabbit polyclonal anti-MED1 (A300-793A, Bethyl laboratories), rabbit polyclonal anti-MED15 (A302-422A, Bethyl laboratories), rabbit polyclonal anti-MED14 (A301-044A-T, Bethyl laboratories), rabbit monoclonal anti-RING1B (5694, Cell Signaling), rabbit monoclonal anti-SUZ12 (3737, Cell Signaling), rabbit polyclonal anti-PCGF2 (sc-10744, Santa Cruz), rabbit monoclonal anti-T7-Tag (D9E1X) (13246, Cell Signaling), mouse monoclonal anti-TBP (ab818, Abcam), rabbit monoclonal anti-HDAC1 (ab109411, Abcam), and mouse monoclonal anti-Flag (F1804, Sigma).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Distinct roles for CDK-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1101/2021.11.04.467119

Figure Lengend Snippet: (A) A schematic illustration 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) ChIPseq 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 illustration of the cPRC1 ( Pcgf4 -/- Pcgf2 fl/fl ) conditional knock-out 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 ChIPseq) is shown as a reference. (E) Boxplot analysis of normalised read counts from WT and cPRC1 KO ESCs showing interactions between Polycomb gene promoters with other Polycomb-domains (left) or non-Polycomb gene promoters with active sites (H3K27ac, right). Number of promoters (P) and interactions (int) is shown.

Article Snippet: Antibodies used for Western blot analysis were rabbit polyclonal anti-MED13L (A302-420A, Bethyl laboratories), rabbit polyclonal anti-MED13 (GTX129674, Genetex), rabbit monoclonal anti-CDK8 (ab229192, Abcam), rabbit polyclonal anti-CCNC (A301-989A, Bethyl laboratories), rabbit polyclonal anti-MED1 (A300-793A, Bethyl laboratories), rabbit polyclonal anti-MED15 (A302-422A, Bethyl laboratories), rabbit polyclonal anti-MED14 (A301-044A-T, Bethyl laboratories), rabbit monoclonal anti-RING1B (5694, Cell Signaling), rabbit monoclonal anti-SUZ12 (3737, Cell Signaling), rabbit polyclonal anti-PCGF2 (sc-10744, Santa Cruz), rabbit monoclonal anti-T7-Tag (D9E1X) (13246, Cell Signaling), mouse monoclonal anti-TBP (ab818, Abcam), rabbit monoclonal anti-HDAC1 (ab109411, Abcam), and mouse monoclonal anti-Flag (F1804, Sigma).

Techniques: Capture-C, Knock-Out, Binding Assay

Journal: bioRxiv

Article Title: Distinct roles for CDK-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1101/2021.11.04.467119

Figure Lengend Snippet: (A) Western blot analysis 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. Error bars show standard deviation of three biological replicates. (C) ChIP-qPCR analysis of binding of the CDK-MED complex to the TetO array in the TetR-CDK8 and TetR-GFP lines. Error bars show standard deviation of three biological replicates. (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. (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) Western blot analysis of nuclear extracts 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 ChUPseq 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 with other Polycomb-domains (left) or non-Polycomb gene promoters with active sites (H3K27ac, right). Number of promoters (P) and interactions (int) is shown.

Article Snippet: Antibodies used for Western blot analysis were rabbit polyclonal anti-MED13L (A302-420A, Bethyl laboratories), rabbit polyclonal anti-MED13 (GTX129674, Genetex), rabbit monoclonal anti-CDK8 (ab229192, Abcam), rabbit polyclonal anti-CCNC (A301-989A, Bethyl laboratories), rabbit polyclonal anti-MED1 (A300-793A, Bethyl laboratories), rabbit polyclonal anti-MED15 (A302-422A, Bethyl laboratories), rabbit polyclonal anti-MED14 (A301-044A-T, Bethyl laboratories), rabbit monoclonal anti-RING1B (5694, Cell Signaling), rabbit monoclonal anti-SUZ12 (3737, Cell Signaling), rabbit polyclonal anti-PCGF2 (sc-10744, Santa Cruz), rabbit monoclonal anti-T7-Tag (D9E1X) (13246, Cell Signaling), mouse monoclonal anti-TBP (ab818, Abcam), rabbit monoclonal anti-HDAC1 (ab109411, Abcam), and mouse monoclonal anti-Flag (F1804, Sigma).

Techniques: Western Blot, Capture-C, Expressing, Binding Assay, Standard Deviation

Journal: bioRxiv

Article Title: Distinct roles for CDK-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1101/2021.11.04.467119

Figure Lengend Snippet: (A) A schematic illustration of the differentiation of WT and CDK-MED KO ESCs for cnRNAseq. (B) Boxplot analysis of the expression of CDK-MED-dependent genes (n=631) in WT ESCs and following RA-induction (WT and CDK-MED KO). (C) A schematic illustration of the differentiation of WT and cPRC1 KO ESCs for cnRNAseq; (D) As in (B) for cPRC1 cKO cells. (E) A screen-shot showing the expression of genes within the HoxB cluster following RA induction of CDK-MED cKO or cPRC1 KO cells. Forward strand is shown on top and reverse strand is shown at the bottom of each track. ChIPseq tracks for CDK8 and cPRC1 (PCGF2) enrichment are shown. (F) Boxplot analysis of the expression of RA-induced genes from the Polycomb (PcG) network (top) and CDK-MED-dependent genes from the PcG network (bottom) following RA induction of CDK-MED cKO or cPRC1 KO cells.

Article Snippet: Antibodies used for Western blot analysis were rabbit polyclonal anti-MED13L (A302-420A, Bethyl laboratories), rabbit polyclonal anti-MED13 (GTX129674, Genetex), rabbit monoclonal anti-CDK8 (ab229192, Abcam), rabbit polyclonal anti-CCNC (A301-989A, Bethyl laboratories), rabbit polyclonal anti-MED1 (A300-793A, Bethyl laboratories), rabbit polyclonal anti-MED15 (A302-422A, Bethyl laboratories), rabbit polyclonal anti-MED14 (A301-044A-T, Bethyl laboratories), rabbit monoclonal anti-RING1B (5694, Cell Signaling), rabbit monoclonal anti-SUZ12 (3737, Cell Signaling), rabbit polyclonal anti-PCGF2 (sc-10744, Santa Cruz), rabbit monoclonal anti-T7-Tag (D9E1X) (13246, Cell Signaling), mouse monoclonal anti-TBP (ab818, Abcam), rabbit monoclonal anti-HDAC1 (ab109411, Abcam), and mouse monoclonal anti-Flag (F1804, Sigma).

Techniques: Expressing

Journal: bioRxiv

Article Title: Distinct roles for CDK-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1101/2021.11.04.467119

Figure Lengend Snippet: (A) A schematic illustration of the generation of the T7-MED14 expressing Med13/13l fl/fl ESC line. (B) PCR showing amplification of homozygously-tagged T7-Med14 alleles. (C) Western blot analysis of nuclear extract from the T7-MED14 Med13/13l fl/fl ESC line, following tamoxifen (TAM) treatment. Extract from an untagged ESC line was used as a control. HDAC1 was used as a loading control. (D) Heatmaps of CDK8 and T7-MED14 ChIPseq signal at Polycomb domains (n=2097) and H3K27ac peaks (n=4037), sorted by decreasing CDK8 signal. (E) Boxplots showing gene expression change (log2FC) of CDK-MED-dependent (n=631) and CDK-MED-independent (n=2689) RA-induced genes following RA differentiation of WT ESCs. (F) Boxplots showing T7-MED14 ChIPseq signal at the TSS (1000bp) of the different classes of RA-induced gene classes as defined in (E) in ESCs and RA-induced cells (WT and CDK-MED KO).

Article Snippet: Antibodies used for Western blot analysis were rabbit polyclonal anti-MED13L (A302-420A, Bethyl laboratories), rabbit polyclonal anti-MED13 (GTX129674, Genetex), rabbit monoclonal anti-CDK8 (ab229192, Abcam), rabbit polyclonal anti-CCNC (A301-989A, Bethyl laboratories), rabbit polyclonal anti-MED1 (A300-793A, Bethyl laboratories), rabbit polyclonal anti-MED15 (A302-422A, Bethyl laboratories), rabbit polyclonal anti-MED14 (A301-044A-T, Bethyl laboratories), rabbit monoclonal anti-RING1B (5694, Cell Signaling), rabbit monoclonal anti-SUZ12 (3737, Cell Signaling), rabbit polyclonal anti-PCGF2 (sc-10744, Santa Cruz), rabbit monoclonal anti-T7-Tag (D9E1X) (13246, Cell Signaling), mouse monoclonal anti-TBP (ab818, Abcam), rabbit monoclonal anti-HDAC1 (ab109411, Abcam), and mouse monoclonal anti-Flag (F1804, Sigma).

Techniques: Expressing, Amplification, Western Blot

Journal: bioRxiv

Article Title: Distinct roles for CDK-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1101/2021.11.04.467119

Figure Lengend Snippet: (A) A genomic snapshot of two CDK-MED-dependent genes, showing CDK8 and T7-MED14 ChIPseq and cnRNAseq in WT (+) and CDK-MED KO (-) ESCs (top) and following RA-induction (bottom). (B) Heatmaps showing CDK8 and T7-MED14 ChIPseq signal at promoters (+/- 2.5kb) of CDK-MED-dependent genes in ESCs and following RA-induction (n=631). T7-MED14 signal is shown for WT and CDK-MED KO RA-induced cells. Genes are sorted by decreasing T7-MED14 signal in RA-treated cells. Metaplots showing read density are shown on top of each heatmap.

Article Snippet: Antibodies used for Western blot analysis were rabbit polyclonal anti-MED13L (A302-420A, Bethyl laboratories), rabbit polyclonal anti-MED13 (GTX129674, Genetex), rabbit monoclonal anti-CDK8 (ab229192, Abcam), rabbit polyclonal anti-CCNC (A301-989A, Bethyl laboratories), rabbit polyclonal anti-MED1 (A300-793A, Bethyl laboratories), rabbit polyclonal anti-MED15 (A302-422A, Bethyl laboratories), rabbit polyclonal anti-MED14 (A301-044A-T, Bethyl laboratories), rabbit monoclonal anti-RING1B (5694, Cell Signaling), rabbit monoclonal anti-SUZ12 (3737, Cell Signaling), rabbit polyclonal anti-PCGF2 (sc-10744, Santa Cruz), rabbit monoclonal anti-T7-Tag (D9E1X) (13246, Cell Signaling), mouse monoclonal anti-TBP (ab818, Abcam), rabbit monoclonal anti-HDAC1 (ab109411, Abcam), and mouse monoclonal anti-Flag (F1804, Sigma).

Techniques: