Journal: bioRxiv

Article Title: Epigenetic regulation of p63 blocks squamous-to-neuroendocrine transdifferentiation in esophageal development and malignancy

doi: 10.1101/2023.09.09.556982

Figure Lengend Snippet: (A) p63 and INSM1 staining in human esophageal small cell carcinoma specimens. Scale bars: 20 µm. (B) Schematics showing that TYUC-1 esophageal neuroendocrine cancer cells were treated with inhibitors of epigenetic regulators and gene expression was analyzed by qRT-PCR. (C) The transcript levels of TAp63 and ΔNp63 in TYUC-1 esophageal neuroendocrine cancer cells treated with various epigenetic inhibitors. Note that the EZH2 inhibitor EPZ-6438 increased the expression of TAp63 and ΔNp63 to the highest levels compared to other inhibitors. (D) Transcript levels of ΔNp63 in TYUC-1 cells treated with EPZ-6438 alone or in combination with JQ1 or A485. (E-F) Immunofluorescence staining and quantification of p63 cells in EPZ-6438 treated TYUC-1 cells. Note that p63 is expressed in the EPZ-6438-treated group but not the DMSO-treated group. Scale bars: 20 µm. (G) Genome browser view at the TP63 locus showing esophagus epithelium H3K27ac ChIP-seq (ENCODE SRX3205374) and TYUC-1 cell H3K27me3, H3K27ac, and BRD4 CUT&Tag upon treatment with EPZ-6438 for 3 and 6 days. Noted are putative enhancer elements near the ΔNp63 isoform TSS where H3K27 methylation is lost, and acetylation is restored.

Article Snippet: For H3K27me3 and H3K27ac, 2 μl of SNAP-ChIP K-MetStat panel and K-AcylStat panel nucleosomes (EpiCypher) respectively, were added as spike-in control at the primary antibody incubation step.

Techniques: Staining, Gene Expression, Quantitative RT-PCR, Expressing, Immunofluorescence, ChIP-sequencing, Methylation

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: bioRxiv

Article Title: Exogenous and endogenous HDAC inhibitor effects in Rubinstein-Taybi syndrome models

doi: 10.1101/2020.03.31.015800

Figure Lengend Snippet: H3K27 acetylation levels normalized on H3K4 unmodified, assessed by AlphaLISA ® ; levels of acetylation upon HDACi are expressed as ratio between the treatment and respective vehicle; on Y-axis H3K27 acetylation levels normalized and expressed as HDACi/vehicle, on X-axis lists of epigenetic treatments or untreated/treated single LCL or LCLs means A) Means of values of H3K27 acetylation in healthy donors (HD, in blue) and patients LCLs (RSTS, in pale brown) untreated and exposed to four different HDACi (TSA 2μM, SAHA 2μM, VPA 2mM and NaB 5mM). B) H3K27 acetylation in eight RSTS LCLs ( CREBBP LCLs in shades of red, EP300 LCLs in shades of pink) after exposure with the four different HDACi, compared to treated HD and RSTS means. C) Insight on single-patients’ response (RSTS 114) to the four compounds compared to untreated RSTS means and RSTS 114. Groups were compared using Student’s t-test as statistical method (*p<0.05; **p<0.01; ***p<0.001).

Article Snippet: Replicates were tested with both AlphaLISA Acetylated-Histone H3 Lysine 27 (H3K27ac) Cellular Detection Kit (AL720, PerkinElmer) and AlphaLISA unmodified Histone H3 Lysine 4 (H3K4) Cellular Detection Kit (AL719, PerkinElmer) for normalization.

Techniques:

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