Journal: Molecular Cancer

Article Title: Exclusion of HDAC1/2 complexes by oncogenic nuclear condensates

doi: 10.1186/s12943-024-02002-1

Figure Lengend Snippet: Onco-condensate remodels H3K27ac. a Representative image of immunofluorescence for H3K27ac in the BJ fibroblasts with lentiviral expression of SS18-EGFP or SS18-SSX1-EGFP. Scale bars, 5 μm. b Violin plot shows the pixel intensity distribution of immunofluorescence for H3K27ac in ( a ). Outside and inside groups indicate the distribution of random pixels’ fluorescence intensity of H3K27ac normalized by Z score from outside and inside of SS18 or SS18-SSX1 condensates, respectively. Two-sided Wilcoxon test adjusted for multiple comparisons. n = 30 pixels, from 3 nuclei. **** p < 0.0001. c Representative image of immunofluorescence for H3K27ac and H2AK119ub in the BJ fibroblasts with lentiviral expression of SS18-SSX1. Scale bars, 5 μm. d Heatmap shows the intensity changes of H3K27ac occupancy at SS18-SSX1 binding sites upon expression of the onco-fusion protein. e Jointly analysis of Hi-C, CUT&Tag of histone modification, SS18-SSX1 and cohesin-CTCF at three representative loci in BJ fibroblasts with the expression of SS18-SSX or condensate-deficient SS18(YA)-SSX as control. The green boxes indicate the newly formed long-range interaction. The blue and black dotted lines indicate the intrinsic chromatin domains. f Venn plot shows the intersection of cohesin component RAD21 binding sites in BJ cells with the overexpression of SS18-SSX1 and SS18(YA)-SSX as control. SS, SS18-SSX1. g Venn plot shows the intersection of cohesin component SMC3 binding sites in BJ cells with the overexpression of SS18-SSX1 and SS18(YA)-SSX as control. SS, SS18-SSX1. h Venn plot shows the intersection of CTCF binding sites in BJ cells with the overexpression of SS18-SSX1 and SS18(YA)-SSX as control. SS, SS18-SSX1. i H3K27ac histone modification sites were divided into three groups by Venn plot, i.e., control specific sites, SS18-SSX1 specific sites and the overlapped sites. j The colocalization analysis between the overlapped H3K27ac sites in ( i ) with cohesin component RAD21 binding sites in BJ cells with the overexpression of SS18-SSX1 and SS18(YA)-SSX as control, respectively. SS, SS18-SSX1. k The colocalization analysis between the specific H3K27ac sites in ( i ) with cohesin component RAD21 binding sites in BJ cells with the overexpression of SS18-SSX1 and SS18(YA)-SSX as control, respectively. SS, SS18-SSX1

Article Snippet: We used PRT4165 (TargetMol T3110) and A485 (MCE HY-107455) to block the H2AK119ub and H3K27ac modification, respectively.

Techniques: Immunofluorescence, Expressing, Fluorescence, Binding Assay, Hi-C, Modification, Control, Over Expression

Journal: Molecular Cancer

Article Title: Exclusion of HDAC1/2 complexes by oncogenic nuclear condensates

doi: 10.1186/s12943-024-02002-1

Figure Lengend Snippet: H3K27ac deposition can be targeted pharmacologically. a The expression of representative genes in BJ fibroblasts expressing control or SS18-SSX1 treated with DMSO or 1 μM A485 inhibitor. Data are mean ± s.d., two-sided, unpaired t-test; n = 3 independent experiments. ** p < 0.01, *** p < 0.001, **** p < 0.0001. b Venn plots showing the intersection between upregulated genes and A485 treatment downregulated genes in BJ fibroblasts expressing SS18-SSX1. c Representative image of EdU assay in synovial sarcoma cell line SW982 with lentiviral expression of EGFP or SS18-SSX1 treated by DMSO or 1 μM A485. Scale bars, 200 μm. d Histogram shows the ratio of EdU positive cells in ( c ). Data are mean ± s.d., two-sided, unpaired t-test, n = 4, from 2 independent experiments. e Tumor growth curve of SW982 cell overexpressing SS18-SSX1-EGFP xenografts established in NCG mice. Mice were treated intraperitoneally with vehicle control or A-485 at 100 mg/kg/dose, twice daily for 12 days (BID × 12). Data are mean ± s.d., two-sided, unpaired t test of n = 7 mice per group from two biological replicates, ** p < 0.01. f A Model for condensate remodeling as a driver for synovial sarcoma. SS18 WT protein forms innate phase separation for its normal physiological function (left panel). In contrast, SS18-SSX1 has enhanced condensates formation tendency mediated by ICBS mechanism via H2AK119ub histone modification and remodels 3D genome structure (right panel). The enhanced onco-fusion condensates remodel H3K27ac by excluding HDACs complexes, which resulting in aberrant activation of embryonic developmental genes

Article Snippet: We used PRT4165 (TargetMol T3110) and A485 (MCE HY-107455) to block the H2AK119ub and H3K27ac modification, respectively.

Techniques: Expressing, Control, EdU Assay, Modification, Activation Assay

Journal: Nature Cardiovascular Research

Article Title: Nuclear ATP-citrate lyase regulates chromatin-dependent activation and maintenance of the myofibroblast gene program

doi: 10.1038/s44161-024-00502-3

Figure Lengend Snippet: a , ChIP–qPCR with H3K27ac antibody (ab4729) regulatory regions of Acta2 and Postn . Normalized to Ct values of rabbit IgG. One-way ANOVA with Dunnett’s HSD with TGFβ DMSO as positive control, n = 3 biological replicates. Data are depicted as mean ± s.e.m. b , Principal component analysis showing two principal components (PC1 and PC2) for H3K27ac-occupied regions for control (con; n = 4), con + ACLYi ( n = 4), TGFβ + veh ( n = 4) and TGFβ + ACLYi ( n = 3). Cohorts are color coded, and each sample is represented by an individual point. The open circle represents a sample removed from downstream analysis. c , CUT&RUN-seq H3K27ac MA plots comparing TGFβ + veh versus con and TGFβ + ACLY versus con. Pink dots indicate regions with significant change in occupancy (FDR < 0.05). d , Venn diagram of H3K27ac-occupied regions increased in TGFβ versus con (orange) and regions increased in TGFβ + ACLYi versus con (green). The 1,829 regions that were increased only between TGFβ and con were examined for further study. e , Genome browser viewing tracks of ACLYi-sensitive regions outlined by dashed lines. Representative genes thrombospondin 1 ( Thbs1 ) and hexokinase 2 ( Hk2 ). f , DAVID gene ontology enrichment analysis of the 253 genes corresponding to the 1,829 ACLYi-sensitive regions, representing cellular components. The bottom x -axis represents the fold enrichment of genes in red bars of interest over a mouse genome background. The numbers in parentheses are the number of genes aligned to the GO term. The top x -axis represents −log 10 (FDR) by dot plot of genes of interest over a mouse genome background. g , DAVID gene ontology enrichment analysis of the 253 genes corresponding to the 1,826 ACLYi-sensitive regions representing biological processes. BMP, bone morphogenetic protein; VEGF, vascular endothelial growth factor. h , Co-immunoprecipitation of immortalized CF lysate treated with and without TGFβ for 12 h. Full-length western blots are available as source data.

Article Snippet: Modifications are described as follows: CUT&RUN wash buffer was modified by additionally preparing it with 0.05% SDS, 1% Triton X-100 and 1 μM Trichostatin A. Antibodies used were Histone H3K27ac Antibody, SNAP-ChIP Certified (EpiCypher, 13-0045) and CUTANA IgG Negative Control Antibody for CUT&RUN and CUT&Tag (EpiCypher, 13-0042).

Techniques: ChIP-qPCR, Positive Control, Control, Immunoprecipitation, Western Blot

Journal: Nature Cardiovascular Research

Article Title: Nuclear ATP-citrate lyase regulates chromatin-dependent activation and maintenance of the myofibroblast gene program

doi: 10.1038/s44161-024-00502-3

Figure Lengend Snippet: (a) CUT&RUN-Seq H3K27ac MA plots of all comparisons made between the four groups in Fig. . Control (Con), Vehicle (Veh). Upper left compares ACLYi+Con vs Con, upper right compares TGFβ+Veh vs Con (reproduced from Fig. ), lower left compares TGFβ+ACLYi vs Con (reproduced from Fig. ), and lower right compares TGFβ+ACLYi vs TGFβ+Veh. Pink dots indicate regions with significant change in occupancy (FDR < 0.05). (b) Venn Diagram of H3K27ac occupied regions increased in TGFβ vs Con (red) and regions decreased in TGFβ+ACLYi vs TGFβ+Veh (green). The 99 regions which overlapped in these two comparisons were examined for further study. DAVID Gene Ontology enrichment analysis of the 89 genes corresponding to the 99 overlapping regions from (b), with (c) representing Cellular Components and (d) representing Biological Processes. Bottom x-axis represents the fold enrichment of genes in red bars of interest over mouse genome background. Parentheses give the number of genes aligned to the GO term. Top x-axis represents –log 10 (FDR) by dot plot of genes of interest over mouse genome background. DAVID gene ontology enrichment analysis of the 89 genes corresponding to the 99 overlapping regions from (b), with (e) representing Molecular Function and (f) representing KEGG Pathway analysis. Bottom x-axis represents the fold enrichment of genes in grey bars of interest over mouse genome background. Top x-axis represents –log 10 (FDR) by dot plot of genes of interest over mouse genome background. (g) Transcription factor binding sites enriched in the 99 overlapping regions from (b). p values calculated by Fisher exact test using HOMER.

Article Snippet: Modifications are described as follows: CUT&RUN wash buffer was modified by additionally preparing it with 0.05% SDS, 1% Triton X-100 and 1 μM Trichostatin A. Antibodies used were Histone H3K27ac Antibody, SNAP-ChIP Certified (EpiCypher, 13-0045) and CUTANA IgG Negative Control Antibody for CUT&RUN and CUT&Tag (EpiCypher, 13-0042).

Techniques: Control, Binding Assay

Journal: Nutrients

Article Title: A Hypomethylating Ketogenic Diet in Apolipoprotein E-Deficient Mice: A Pilot Study on Vascular Effects and Specific Epigenetic Changes

doi: 10.3390/nu13103576

Figure Lengend Snippet: The effect of the experimental diets (ketogenic diet, KD; low methyl ketogenic diet, LMKD; and control) on the vascular content of specific epigenetic tags at lysine 27 on histone H3 (H2K27me3, tri-methylation; H3K27ac, acetylation) in the aortic arch and brachiocephalic artery (BCA). ( A ) Representative images of brightfield and immunofluorescence quantification of BCA cross sections. Total histone 3 (H3) in green, and histone 3 trimethylation at lysine 27 (H3K27me3) or acetylation (H3K27ac) in magenta. Scale bars = 200 μm. ( B ) Immunohistochemistry results. Data are the mean ± SD, n = 35. Bars bearing different letters were significantly different ( p < 0.05).

Article Snippet: The antibodies used were: rabbit anti-histone H3K27me3 pAb (1:200, Epigentek, Cat.# A-4039), rabbit anti-histone H3K27ac pAb (1:200, Epigentek, Cat.#A-4708), mouse anti-histone H3 mAb 1B1-B2 (1:200, BioLegend, cat.#819404), Alexa Fluor 555 goat anti-rabbit IgG (1:250, Invitrogen, Cat.# A-21428) and Alexa Fluor 488 goat anti-mouse IgG3 (1:250, Invitrogen, Cat.# A-21151).

Techniques: Control, Methylation, Immunofluorescence, Immunohistochemistry

Journal: Developmental cell

Article Title: Hedgehog signaling activates a mammalian heterochronic gene regulatory network controlling differentiation timing across lineages

doi: 10.1016/j.devcel.2022.08.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-H3K27ac , FUJIFILM Wako , Cat# 306-34849; RRID: AB_10552905; Clone MABI0309.

Techniques: Virus, Subcloning, Recombinant, Modification, Lysis, Western Blot, Bicinchoninic Acid Protein Assay, SYBR Green Assay, Reporter Assay, Control, Transgenic Assay, Software, Staining