Journal: bioRxiv

Article Title: Noncoding function of super enhancer derived mRNA in modulating neighboring gene expression and TAD interaction

doi: 10.1101/2023.12.05.570115

Figure Lengend Snippet: Heatmap shows the distribution of p300, H3K27ac and H3K27me3 signals around +/-5kb region of p300 only peak and p300/H3K27ac common peaks. B. Pie chart shows the distributions of annotated features around p300 only peaks in MEL cell. C. Homer motif analysis identifies significant enrichment of transcription factors binding sites at p300 only peaks. D. Binding profiles of transcription factors at p300 only peak in MEL cells. E. Distribution of different types of interaction observed at TAD boundary(blue) and chromatin loops(red). X axis represents number of different types of interactions. “Gene” represents genes that do not overlap with H3K27me3 and p300 only peak. “Intergenic” represents intergenic regions that do not overlap with H3K27me3 and p300 only peak. F and G. Aggregated peak analysis (APA) of chromatin loops anchored by p300 only peaks (F) and p300 only peak-H3K27me3 peaks (G) in G1ER late G1 cell. HiC matrix was normalized with VC_SQRT, window = 6. H. ECDF plot shows the comparison of the expression level of H3K27me3 non-overlapping and overlapping genes looped to p300 only peaks at TAD boundary or Chromatin loops. MEL cell RNA-seq processed expression quantification data was obtained from ENCODE. Wilcoxon test was used to compare the log2(TPM+1) value of H3K27me3 genes and non-H3K27me3 genes, P values ≤ 0.05 indicating a significant difference.

Article Snippet: For p300 ChIP experiment, undifferentiated MEL cells were used, p300 antibody (Santa Cruz Biotechnology, sc-48343).

Techniques: Binding Assay, Comparison, Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: Noncoding function of super enhancer derived mRNA in modulating neighboring gene expression and TAD interaction

doi: 10.1101/2023.12.05.570115

Figure Lengend Snippet: A. Location of primers used for ChIP-qPCR in C and D. B. Cpox intron 5 TFBS KO gel image. C. Cpox intron 5 TFBS KO BLAST result, MEL p300 ChIP seq data from ENCODE. D. Sanger sequencing data, black line shows the deletion site.

Article Snippet: For p300 ChIP experiment, undifferentiated MEL cells were used, p300 antibody (Santa Cruz Biotechnology, sc-48343).

Techniques: ChIP-qPCR, ChIP-sequencing, Sequencing

Journal: bioRxiv

Article Title: Noncoding function of super enhancer derived mRNA in modulating neighboring gene expression and TAD interaction

doi: 10.1101/2023.12.05.570115

Figure Lengend Snippet: UCSC genome browser tracks shows p300, H3K27ac, H3K27me3, and H3K4me1 pattern at Dcbld2 promoter and Cpox intron 5. ChIP-seq data from ENCODE. p300 only peak was highlighted in grey. B. qRT-PCR result shows the expression of Dcbld2-2 , St3gal6-2 and CpoxeRNA after Cpox intron 5 TFBS deletion in UMEL cells. Three biological replicates. Data are mean ± s.d., unpaired one-tailed t-test. C. ChIP-qPCR result shows p300 binding at the Cpox intron 5 region after Cpox knock down by shRNA in UMEL cells. Two biological replicates. Data are mean ± s.d., unpaired one-tailed t-test. D. Model of Cpox mRNA loss activate neighboring gene and enhancer. Left graph shows in normal situation, right graph shows Cpox mRNA knock down situation. Colored arrows represent the genomic locus of the protein genes and enhancer. Colored curves represent the corresponding RNA transcribed. Blue dots represent p300, orange dots represent PRC2. Rectangles on genes represent the promoters.

Article Snippet: For p300 ChIP experiment, undifferentiated MEL cells were used, p300 antibody (Santa Cruz Biotechnology, sc-48343).

Techniques: ChIP-sequencing, Quantitative RT-PCR, Expressing, One-tailed Test, ChIP-qPCR, Binding Assay, Knockdown, shRNA

Journal: Cancer Communications

Article Title: Junction plakoglobin regulates and destabilizes HIF2α to inhibit tumorigenesis of renal cell carcinoma

doi: 10.1002/cac2.12142

Figure Lengend Snippet: Recruitment of HDAC1/2 to HIF2α by JUP decreases H3K27ac levels at HIF2α‐binding sites in RCC cells. A, Endogenous HIF2α and JUP co‐immunoprecipitated with Flag‐HDAC1/2 in 786‐O cells. 786‐O cells stably expressing Flag‐HDAC1 or Flag‐HDAC2 were lysed and immunoprecipitated with anti‐Flag antibody. B, GST pull‐down assay detecting the regions of JUP that bind to HDAC1. Flag‐HDAC1 and GST‐JUP deletion mutants were ectopically expressed in HEK293T cells, followed by Western blotting analysis of Flag‐HDAC1 pulled down by GST or GST‐JUP deletion mutants. Inputs are shown in the bottom panels. C, JUP promoted both HDAC1 and HDAC2 binding to HIF2α. 786‐O cells were co‐transfected with Flag‐HDAC1 or Flag‐HDAC2 (0.6 μg) and HA‐JUP (0 or 1 μg) plasmids. Endogenous HIF2α co‐immunoprecipitated with Flag antibody. D, GST pull‐down assay detecting JUP‐HDAC1/HDAC2 interaction. E, Schematic diagram of regulatory regions in VEGFA promoter and ZNF395 enhancer shows the HIF DNA‐binding sites. Numbers indicate nucleotides with the transcription start site indicated by +1. F and G, ChIP analysis with anti‐JUP or anti‐HDAC1 antibody in WT‐ or HIF2α‐specific depleted 786‐O cells. Precipitated DNAs were quantified by qPCR of fragments around the HIF‐binding site at the VEGFA promoter (F) and ZNF395 enhancer (G). H and I, ChIP analysis with anti‐HIF2α antibody in WT‐, JUP‐, or HDAC1‐specific depleted 786‐O cells. Precipitated DNAs were quantified by qPCR of fragments around the HIF‐binding site at the VEGFA promoter (H) and ZNF395 enhancer (I). J and K, ChIP‐qPCR validated the recruitment of HDAC1 by JUP at the VEGFA promoter (J) and ZNF395 enhancer (K) in 786‐O cells. L and M, ChIP‐qPCR of p300 binding and H3K27ac enrichment at the VEGFA promoter (L) and ZNF395 enhancer (M) in WT‐ or JUP‐specific depleted 786‐O cells. * P < 0.05, ** P < 0.01, and *** P < 0.001. Abbreviations: ZNF395, zinc finger protein 395; H3K27ac, histone H3K27 acetylation; ChIP, chromatin immunoprecipitation; JUP, junction plakoglobin; RCC, renal cell carcinoma

Article Snippet: The following antibodies were used in the experiments: mouse anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (G8795, 1:10,000) and anti‐Flag (F3165, 1:2000) from Sigma‐Aldrich (St. Louis, MO, USA); mouse anti‐Myc (11667149001, 1:2000) and mouse anti‐hemagglutinin (HA) antibody (11583816001, 1:2000) from Roche Applied Science (Penzberg, Germany); rabbit anti‐Myc (562, 1:2000), rabbit anti‐HA antibody (561, 1:2000), and anti‐Strep‐tag II (M211‐3, 1:3000) from MBL Life Science (MBL, Nagoya, Japan); anti‐HIF2α (NB100‐122, 1:1000) from Novus Biologicals (Centennial, CO, USA); anti‐histone H3 (acetyl K27) (H3K27ac, 1:100 for ChIP) (ab4729) from Abcam (Cambridge, MA, USA); anti‐HDAC1 (GTX100513) from GeneTex (Irvine, CA, USA); rabbit anti‐junction plakoglobin (JUP) (#75550, 1:1000) and anti‐p300 (#86377, 1:100 for ChIP) from Cell Signaling Technology (Danvers, MA, USA); mouse anti‐JUP (#MA5‐15905, 1:1000), goat anti‐mouse IgG secondary antibody (31430, 1:20,000), and goat anti‐rabbit IgG‐horseradish peroxidase (HRP) secondary antibody (31460, 1:20,000) from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Binding Assay, Immunoprecipitation, Stable Transfection, Expressing, Pull Down Assay, Western Blot, Transfection, ChIP-qPCR, Chromatin Immunoprecipitation

Journal: Frontiers in Oncology

Article Title: Histone Modifications Drive Aberrant Notch3 Expression/Activity and Growth in T-ALL

doi: 10.3389/fonc.2019.00198

Figure Lengend Snippet: Notch3 preserves active H3K27 modification status on its gene locus. Cross-linked protein-DNA complexes from TALL-1 cells treated with 10 μM DAPT or DMSO for 48 h were subjected to immunoprecipitation with antibodies against: (A) H3K27ac, H3K27me3, p300, JMJD3, (B) p300, JMJD3, H3K4me3, and analyzed by qPCR. Immunoprecipitations against relative isotypes (IgG) were performed in parallel. Schematics position of the amplicons is shown up the charts. ChIP results are presented as the percentage of the input DNA. Data represent mean values of two biological replicates ± Standard Error of the Mean (S.E.M.); ( n = 2). (C) ChIP-seq tracks of Notch3 and H3K27ac dynamic peaks visualized by IGV surrounding the NOTCH3 locus derived from previously described ChIP-seq data of human TALL-1 cells treated with gamma-secretase inhibitor (Notch OFF) or with vehicle alone (Notch ON) (GSE104262) .

Article Snippet: For Chip experiments following antibodies were used: Notch3 (2889; Cell Signaling Technology), Notch1 (3608; Cell Signaling Technology), H3K27me3 (07-473; Merck Millipore, Darmstadt, Germany), H3K27ac (4353; Cell Signaling Technology), H3K4me3 (07-473; Merck Millipore), JMJD3 (ab38113; Abcam, Cambridge, UK), p300 (sc-584, Santa Cruz Biotechnology, Santa Cruz, CA, USA), normal rabbit IgG (sc-2027; Santa Cruz Biotechnology) and normal mouse IgG (sc-2025; Santa Cruz Biotechnology).

Techniques: Modification, Immunoprecipitation, ChIP-sequencing, Derivative Assay

Journal: Frontiers in Oncology

Article Title: Histone Modifications Drive Aberrant Notch3 Expression/Activity and Growth in T-ALL

doi: 10.3389/fonc.2019.00198

Figure Lengend Snippet: Notch1 sustains active H3K27 modification status on NOTCH3 gene locus. Cross-linked protein-DNA complexes from MOLT3 cells treated with 10 μM DAPT or DMSO for 48 h were subjected to immunoprecipitation with antibodies against: (A) the C-terminal domain of Notch1 (N1ICD), (B) H3K27ac, H3K27me3, p300, and JMJD3, (C) p300, JMJD3 and H3K4me3, and analyzed by qPCR. Immunoprecipitation against relative isotypes (IgG) were performed in parallel. Schematics positions of the amplicons are shown up the charts. ChIP results are presented as the percentage of the input DNA. Data represent mean values of two biological replicates ± Standard Error of the Mean (S.E.M.); ( n = 2).

Article Snippet: For Chip experiments following antibodies were used: Notch3 (2889; Cell Signaling Technology), Notch1 (3608; Cell Signaling Technology), H3K27me3 (07-473; Merck Millipore, Darmstadt, Germany), H3K27ac (4353; Cell Signaling Technology), H3K4me3 (07-473; Merck Millipore), JMJD3 (ab38113; Abcam, Cambridge, UK), p300 (sc-584, Santa Cruz Biotechnology, Santa Cruz, CA, USA), normal rabbit IgG (sc-2027; Santa Cruz Biotechnology) and normal mouse IgG (sc-2025; Santa Cruz Biotechnology).

Techniques: Modification, Immunoprecipitation

Journal: Frontiers in Oncology

Article Title: Histone Modifications Drive Aberrant Notch3 Expression/Activity and Growth in T-ALL

doi: 10.3389/fonc.2019.00198

Figure Lengend Snippet: The histone modifiers JMJD3 and p300 sustain Notch signaling and cell viability in T-ALL . (A) Notch signaling trans-activation is primed by the binding between a Notch receptor (N3R or N1R) and a ligand expressed on the surface of two neighboring cells. Ligand interaction allows two consecutive proteolytic cleavages of the receptor by ADAM metalloproteases and by the gamma-secretase complex that releases the active Notch intracellular domain (N3ICD or N1ICD) from the cell membrane leading to its nuclear translocation. In the nucleus, N3ICD or N1ICD associates with the DNA binding protein CBF1–Suppressor of Hairless(SuH)–LAG1 (CSL) and assembles a multifactorial transcriptional complex including MAML1 complex (MAM) and p300 to promote Notch target gene expression. By our study, we demonstrated that the binding of N3ICD, as well as N1ICD, to the regulatory region of NOTCH3 , is strictly associated with high levels of accumulation of the active chromatin marks H3K27ac and H3K4me3 on the NOTCH3 gene locus and combines with the co-recruitment of the histone modifiers p300 and JMJD3. Moreover, we found that the interplay between Notch and these histone modifiers is required to sustain NOTCH3 expression in T-ALL cells. (B) Indeed, pharmacological inhibition of the last step of Notch signaling activation, by the gamma-secretase inhibitor DAPT, restrains the binding of JMJD3 and p300 to the NOTCH3 gene, allows H3K27me3 accumulation and counteracts NOTCH3 expression. (C) Therefore, JMJD3 and p300 sustain the expression and the oncogenic transcriptional activity of Notch3 and Notch1 receptors in T-ALL cells. (D) Accordingly, the pharmacological inhibition of their enzymatic activity by treatment with GSKJ4 and A-485, respectively produced the following outcomes: (i) reduced NOTCH3 and NOTCH1 levels; (ii) decreased the expression of Notch target genes DELTEX1 and c-MYC; (iii) promotes the accumulation of the cell cycle-regulator factors p27 Kip1 and of the apoptosis-related cleaved form of PARP (C PARP); (ìv) impairs viability in several T-ALL cell lines.

Article Snippet: For Chip experiments following antibodies were used: Notch3 (2889; Cell Signaling Technology), Notch1 (3608; Cell Signaling Technology), H3K27me3 (07-473; Merck Millipore, Darmstadt, Germany), H3K27ac (4353; Cell Signaling Technology), H3K4me3 (07-473; Merck Millipore), JMJD3 (ab38113; Abcam, Cambridge, UK), p300 (sc-584, Santa Cruz Biotechnology, Santa Cruz, CA, USA), normal rabbit IgG (sc-2027; Santa Cruz Biotechnology) and normal mouse IgG (sc-2025; Santa Cruz Biotechnology).

Techniques: Activation Assay, Binding Assay, Membrane, Translocation Assay, Targeted Gene Expression, Expressing, Inhibition, Activity Assay, Produced

Journal: PLoS ONE

Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages

doi: 10.1371/journal.pone.0093971

Figure Lengend Snippet: (A and B), Nucleosome occupancy at a putative enhancer 10 kb upstream of the TSS of IL1A was determined in BMDMs prior to (blue bars and lines) and upon 1.5 h (yellow) or 3 h (red) induction with 1 μg/ml LPS as described in the legend of . ConSite predicted consensus sites for PU.1, C/EBP, IRF, AP1 and NFκB are indicated. (C and D), Nucleosome occupancy at the promoter of IL1A was determined as described in panel (A) in a region surrounding the TSS of IL1A. The TSS (black bar) and a putative TATA-box (blue bar) is indicated in panel (C). (E), Kinetics of nucleosome removal at IL1A and IL12B. BMDMs were induced with LPS for 15′, 30′, 60′ and 90′, and nucleosome occupancy was analyzed as described in (A). Nucleosome removal at three locations in the distal enhancer of IL12B (red lines) and in the enhancer of IL1A (blue lines) is shown. The data is displayed as the fold change in nucleosome occupancy over the levels found before induction at each location. The absence of changes in nucleosome occupancy at the TSS of IL12B (black line) and of IL1A (gray line) is shown for comparison. (F), ChIP experiments with antibodies against H3 (dark blue bars), H2A.Z (light blue), H3K4me1 (green), H3K4me3 (yellow) and H3K27ac (red) were performed as described in the . For these experiments cross-linked chromatin was lightly digested with MNase before incubation with the respective antibodies to increase resolution of the ChIP signal and the data was normalized to a region in the ORF of RPL4. Changes upon LPS induction in histone binding and histone modifications at the enhancers and promoters of IL12B and IL1A as well as at a control region in the GAPDH pseudo gene are shown as fold over levels found before induction. For H3K27ac the changes 1.5 h after LPS induction, and for all other histone variants and modifications the changes after 3 h of induction are shown. The error bars show the SEM of at least 3 independent experiments. Statistical significance of the changes in H3K4me3 and H3K27ac upon LPS induction compared to levels found prior to induction determined by Student's T-tests is indicated (*P<0.05; **P<0.01).

Article Snippet: For all other ChIP experiments isolated chromatin was directly diluted with High Salt ChIP buffer and incubated with either 1 μg of anti-PolI antibody (sc-56767), 6 μg anti-TBP (sc-204), 4 μg anti-PU.1 (sc-352), 4 μg anti-C/EBPβ (sc-150), 6 μg anti-NFκB (sc-372), 5 μg anti-c-Jun (sc-45), 6 μg anti-p300 (sc-585) or 10 μg anti-IRF3 (sc-9082) all from Santa Cruz Biotechnologies.

Techniques: Comparison, Incubation, Binding Assay, Control

Journal: PLoS ONE

Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages

doi: 10.1371/journal.pone.0093971

Figure Lengend Snippet: (A and B), ChIP experiments were performed as described in the legend of with antibodies that detect (A) PolII or (B) TBP in BMDMs before (dark blue bars), and upon 1.5 h (yellow) or 3 h (red) LPS induction. Cross-linked chromatin was either untreated (solid bars), or lightly digested with MNase (hatched bars) as described in the . The data was normalized to a region in the KIT promoter and genomic locations are indicated. The experiment was performed twice and error bars indicating the SEM are shown.

Article Snippet: For all other ChIP experiments isolated chromatin was directly diluted with High Salt ChIP buffer and incubated with either 1 μg of anti-PolI antibody (sc-56767), 6 μg anti-TBP (sc-204), 4 μg anti-PU.1 (sc-352), 4 μg anti-C/EBPβ (sc-150), 6 μg anti-NFκB (sc-372), 5 μg anti-c-Jun (sc-45), 6 μg anti-p300 (sc-585) or 10 μg anti-IRF3 (sc-9082) all from Santa Cruz Biotechnologies.

Techniques: