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: Nature Communications

Article Title: PHC1 maintains pluripotency by organizing genome-wide chromatin interactions of the Nanog locus

doi: 10.1038/s41467-021-22871-0

Figure Lengend Snippet: a PRC1 and PRC2 gene expression was examined by RT-qPCR in HFFs, hESCs, and hiPSCs. ACTIN was used as the house keeping gene and expression was normalized to HFFs. n = 4 independent experiments for PRC1 genes; n = 3 independent experiments for PRC2 genes including EZH1 , EZH2 , EED , and SUZ12 . Error bars represent the s.e.m. Two-tailed unpaired t -tests were used ( p = 0.0256 for CBX8 , p = 0.0201 for PCGF2 , p = 0.0498 for PHC1 in HFFs vs. ESCs, p = 0.0104 for PHC1 in HFFs vs. iPSCs, ** p = 0.0022 for EZH2 , *** p = 0.0003 for EZH2 , ** p = 0.0034 for SUZ12 , * p = 0.0111 for SUZ12 ). b Expressions of PRC1 and PRC2 genes and key pluripotency factors POU5F1 and NANOG during embryoid body differentiation (D0–D7) of hPSCs were examined by RT-qPCR. n = 3 independent experiments for CBX2, CBX7 , and EZH2 ; n = 4 independent experiments for the other genes. Error bars represent the s.e.m. c WB analysis of PHC1 and NANOG protein expression in hPSCs and HFFs. d Analysis of the published single-cell RNA-seq data of early human embryos (E5–7) showing expression of NANOG and PHC1 in epiblast (Epi), primitive endoderm (PE), and trophectoderm (TE) . e Co-immunostaining of Phc1 with Nanog and Sox2, or Gata6 and Sox2 in mouse E4.5 embryos. Scale bars, 31 μm. Source data are provided as a Source Data file.

Article Snippet: The primary antibodies NANOG (Cell Signaling Technology, 4903), OCT4 (Cell Signaling Technology, 2750), SOX2 (Cell Signaling Technology, 23064), RING1B (Cell Signaling Technology, 5694), RING1B (Abcam, ab181140), and PHC1 (Active motif, 39723) were all used at the concentration of 1:100.

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Two Tailed Test, RNA Sequencing, Immunostaining

Journal: Nature Communications

Article Title: PHC1 maintains pluripotency by organizing genome-wide chromatin interactions of the Nanog locus

doi: 10.1038/s41467-021-22871-0

Figure Lengend Snippet: a , b Morphology and colony-forming capacity of sh Scr -, sh PHC1 .1-, and sh PHC1 .2-infected hESCs. Scale bars, 600 μm. Cells were stained for alkaline phosphatase activity 14 or 18 days after plating. Bar plot shows mean colony-formation efficiencies normalized to the sh Scr . Mean ± s.d. of n = 3 independent experiments. Two-tailed unpaired t -tests were used ( ** p = 0.0022, * p = 0.0458). c Comparison of tumor sizes about 1 month after injection of sh Scr -, and sh PHC1 -infected hESCs into NOD/SCID mice. Data are presented as mean ± s.e.m. In each group, 4 NOD/SCID mice were injected. Two-tailed unpaired t -tests were used ( * p = 0.0211). d WB analysis of PHC1, NANOG, OCT4, SOX2, RING1B, H2AK119ub1, and ACTIN protein levels in sh Scr and sh PHC1 -infected hESCs. e WB analysis of PHC1 and NANOG protein levels in hESCs infected with control and CRISPR-CAS9 vector targeting human PHC1 gene. f Immunofluorescent co-staining of PHC1 with NANOG, OCT4, or SOX2 in sh Scr - and sh PHC1 -infected hESCs. Arrowheads showed cells with low PHC1 and NANOG signals. Scale bars, 20 μm. g The ratios of cells exhibiting PHC1 high NANOG low and PHC low NANOG low signals to the total number of stained cells in ( f ) were quantified. Mean ± s.d. of n = 3 independent counts for sh PHC1 .1 and sh PHC1 .2. Two-tailed unpaired t -tests were used ( p = 0.0065 for sh PHC1 .1, p = 0.0048 for sh PHC1 .2). Source data are provided as a Source Data file.

Article Snippet: The primary antibodies NANOG (Cell Signaling Technology, 4903), OCT4 (Cell Signaling Technology, 2750), SOX2 (Cell Signaling Technology, 23064), RING1B (Cell Signaling Technology, 5694), RING1B (Abcam, ab181140), and PHC1 (Active motif, 39723) were all used at the concentration of 1:100.

Techniques: Infection, Staining, Activity Assay, Two Tailed Test, Comparison, Injection, Control, CRISPR, Plasmid Preparation

Journal: Nature Communications

Article Title: PHC1 maintains pluripotency by organizing genome-wide chromatin interactions of the Nanog locus

doi: 10.1038/s41467-021-22871-0

Figure Lengend Snippet: a Designing sgRNAs targeting the 2nd exon and 3rd intron of mouse Phc1 , and the morphology of the Phc1 +/+ and Phc1 −/− mESCs. Scale bars, 300 μm. b WB analysis of Phc1, Nanog, Oct4, Sox2, and Actin protein levels in the Phc1 +/+ and Phc1 −/− mESCs. c qPCR analysis of transcript levels of Pou5f1 , Sox2 Nanog , and its known direct target genes including Klf4 and Esrrb . Mean ± s.d. of n = 4 independent experiments. Two-tailed t -tests were used ( p < 0.0001 for Nanog ; p = 0.0023 for Pou5f1 ; p = 0.0216 for Sox2 ; p < 0.0001 for Klf4 ; p < 0.0001 for Esrrb ). d Flow cytometry analysis quantifying GFP signal after Phc1 knockdown in a mESC line carrying the Nanog -GFP reporter. Nanog suppression was used as the positive control. Mean ± s.d. of n = 3 independent experiments. e Morphology of Phc1 +/+ and Phc1 −/− mESCs transfected with an empty or Flag-Nanog vector. Scale bars, 600 μm. f Immunoblotting of Flag, Nanog, and Tubulin in extracts of mESCs in ( e ). g Alkaline phosphatase staining of mESCs in ( e ). h Quantification of colony-forming efficiencies of mESCs relative to Phc1 +/+ + vector as the control in ( e ). Mean ± s.e.m. of n = 4 independ e nt experiments. One-way ANOVA test with Bonferroni’s multiple comparison was used ( ** p = 0.0024, *** p = 0.0003, * p = 0.0319). Source data are provided as a Source Data file.

Article Snippet: The primary antibodies NANOG (Cell Signaling Technology, 4903), OCT4 (Cell Signaling Technology, 2750), SOX2 (Cell Signaling Technology, 23064), RING1B (Cell Signaling Technology, 5694), RING1B (Abcam, ab181140), and PHC1 (Active motif, 39723) were all used at the concentration of 1:100.

Techniques: Two Tailed Test, Flow Cytometry, Knockdown, Positive Control, Transfection, Plasmid Preparation, Western Blot, Staining, Control, Comparison