Journal: bioRxiv

Article Title: Angioplasty-induced epigenomic remodeling entails BRD4 and EZH2 hierarchical regulations

doi: 10.1101/2020.03.12.989640

Figure Lengend Snippet: Balloon-injured rat left common carotid arteries and contralateral arteries (uninjured control) were collected at day 7 post angioplasty and snap frozen until use for ChIPseq. A . Heatmaps of ChIPseq peak density for BRD4, H3K27ac, H3K27me3, and H3K4me1. ChIPseq signal anchors 10 kb center region with 5 kb flanking on either side of the transcription start site (TSS) of over 24000 genes. Three clusters show the main pattern of co-localization of the ChIP-seq signal and non-overlap between H3K27ac and H3K27me3. Note increased (injured- vs -uninjured) H3K27me3 ChIPseq signal mainly in Cluster-1. B . Distribution of transcript abundance of the genes associated with BRD4 and the three histone marks. C . ChIPseq intensity changes in injured ( vs uninjured) arteries. Red, increased intensity; blue, decreased intensity; a cutoff of 2-fold change of read intensity was used. Note the prevailing H3K27me3 ChIPseq signal increase after injury.

Article Snippet: Artery tissues from 40 rats were pooled for ChIPseq analysis at Active Motif per company standard procedures.

Techniques: Control, ChIP-sequencing

Journal: bioRxiv

Article Title: Angioplasty-induced epigenomic remodeling entails BRD4 and EZH2 hierarchical regulations

doi: 10.1101/2020.03.12.989640

Figure Lengend Snippet: A and B . Comparison of ChIPseq binding density (near Ezh2 ) between injured (+, light color) and uninjured (-, dark color) arteries. The profiles for Nrp2 , which is known as upregulated in balloon-injured rat carotid arteries , are presented for positive control to validate the ChIP methodology and data. Non-specific input indicates very low background noise. C and D . Effect of BRD4 silencing on EZH2 expression. BRD2, BRD3, or BRD4 was silenced with their specific siRNAs (validated in our recent reports) , . Rat aortic SMCs were cultured, starved for 6h, then transduced with BRD2,3,4 siRNA overnight, recovered for 24h and 48h before RNA and protein extraction.EZH2 protein and mRNA were measured with Western blot and qRT-PCR (normalized by ΔΔCT-log2) assays. Quantification: Mean ± SEM; n =3 independent experiments; one-way ANOVA with Bonferroni test, *P<0.05 compared to the scrambled-sequence siRNA control. E . BRD4 ChIPseq binding density focusing on Ezh2 . Red and blue bars mark enhancers. Box highlights an enhancer region where ChIPseq intensity increased in injured vs uninjured arteries. F . Effect of CRISPR-mediated enhancer region deletion on EZH2 expression. sg, short guide RNA. Quantification: Mean ± SEM; n =3 independent experiments; one-way ANOVA with Bonferroni test, *P<0.05.

Article Snippet: Artery tissues from 40 rats were pooled for ChIPseq analysis at Active Motif per company standard procedures.

Techniques: Comparison, Binding Assay, Positive Control, Expressing, Cell Culture, Transduction, Protein Extraction, Western Blot, Quantitative RT-PCR, Sequencing, Control, CRISPR

Journal: bioRxiv

Article Title: Angioplasty-induced epigenomic remodeling entails BRD4 and EZH2 hierarchical regulations

doi: 10.1101/2020.03.12.989640

Figure Lengend Snippet: MOVAS cells were cultured, starved, pre-treated with vehicle (DMSO) or the pan-EZH1/2 inhibitor UNC1999 (5 µM) for 2h, and then stimulated with PDGF-BB (final 20 ng/ml). For lentivirus-mediated overexpression or silencing, cells were transduced with lentivirus overnight, recovered for 24h, and then starved for 6h prior to adding PDGF-BB. Cells were harvested 24h or 48h after stimulation with PDGF-BB, for qRT-PCR and Western blot assay, respectively. Quantification: Mean ± SEM; n =3 independent experiments; one-way ANOVA with Bonferroni test, *P<0.05. A . H3K27me3 ChIPseq binding density at Cdkn1c (P57) and Ccnd1 (cyclin-D1) in balloon-injured (red) and uninjured (gray) artery tissues. B and C . Effect of pan-EZH1/2 inhibition on P57 and cyclin-D1 expression. D and E . Effect of EZH1 or EZH2 silencing on P57 and cyclin-D1 expression. F and G . Effect of EZH1 or EZH2 overexpression on P57 and cyclin-D1 expression.

Article Snippet: Artery tissues from 40 rats were pooled for ChIPseq analysis at Active Motif per company standard procedures.

Techniques: Cell Culture, Over Expression, Transduction, Quantitative RT-PCR, Western Blot, Binding Assay, Inhibition, Expressing

Journal: bioRxiv

Article Title: Angioplasty-induced epigenomic remodeling entails BRD4 and EZH2 hierarchical regulations

doi: 10.1101/2020.03.12.989640

Figure Lengend Snippet: MOVAS cells were cultured, starved, pre-treated with UNC1999 or transduced with lentivirus, stimulated with PDGF-BB, and assayed, as described for . Quantification: Mean ± SEM; n =3 independent experiments; one-way ANOVA with Bonferroni test, *P<0.05. A . H3K27me3 ChIPseq binding density at Uhrf1 in balloon-injured (red) and uninjured (gray) artery tissues. B . Effect of pan-EZH1/2 inhibition on UHRF1 expression. C . Effect of EZH1 or EZH2 silencing on UHRF1 expression. D . Effect of EZH1 or EZH2 overexpression on UHRF1 expression.

Article Snippet: Artery tissues from 40 rats were pooled for ChIPseq analysis at Active Motif per company standard procedures.

Techniques: Cell Culture, Transduction, Binding Assay, Inhibition, Expressing, Over Expression

Journal: Cancers

Article Title: SMYD3 Promotes Cell Cycle Progression by Inducing Cyclin D3 Transcription and Stabilizing the Cyclin D1 Protein in Medulloblastoma

doi: 10.3390/cancers14071673

Figure Lengend Snippet: Chromatin immunoprecipitation (ChIP) analysis of transcriptomic and genome-wide SMYD3 binding profiles in MB. ( A ) Heat map showing the distribution and peak intensity of SMYD3 genomic occupancy from 5 kb downstream to 5 kb upstream. ( B ) Schematic representation of peaks into promoter peaks, upstream peaks, intron peaks, exon peaks, and intergenic peaks; pie diagram showing the distribution of peaks. ( C ) Graph depicting the ChIP peak at the global TSS (green) vs. Input peak (orange). ( D ) Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showing SMYD3 enrichment scores. ( E ) List of genes and their representative pathways whose promoters were bound by SMYD3, as predicted by KEGG pathway analysis.

Article Snippet: The anti-SMYD3 antibody ChIP-enriched DNA was sent for ChIP seq analysis (Arraystar Inc., Rockville, MD, USA).

Techniques: Chromatin Immunoprecipitation, Genome Wide, Binding Assay

Journal: Cancers

Article Title: SMYD3 Promotes Cell Cycle Progression by Inducing Cyclin D3 Transcription and Stabilizing the Cyclin D1 Protein in Medulloblastoma

doi: 10.3390/cancers14071673

Figure Lengend Snippet: SMYD3 binds to the Cyclin D3 promoter. ( A ) Representation of the cyclin D3 (CCND3) gene promoter sequence; (red circles) the EPD software detected four SMYD3 consensus binding sites (−1166, −890, −247, −121 bps) on the cyclin D3 gene promoter. (Blue box) Region −311 to +367 was identified by SMYD3_ChIP-seq analysis. ( B ) Agarose gel showing the PCR amplification of RI (ChIP identified sequence), RII (SMYD3 binding sites within RI), and RIII regions (negative control). ( C ) Scheme depicting the RI, RII, and RIII regions cloned into the pXPG reporter vector. ( D ) Sequence peaks showing the SMYD3 binding sites on the cloned CCND3 promoter regions within the pXPG plasmid. ( E , F ) Effect of SMYD3 on Cyclin D3 (RI region) promoter activity by luciferase reporter assay in D458 (top) and MB002 (bottom) cells. D458 and MB002 cells were transfected with shRNA or overexpression vectors targeting SMYD3 for 48 h or treated with BCI-121 (80 μM) for 24 h. The statistical significance * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The anti-SMYD3 antibody ChIP-enriched DNA was sent for ChIP seq analysis (Arraystar Inc., Rockville, MD, USA).

Techniques: Sequencing, Software, Binding Assay, ChIP-sequencing, Agarose Gel Electrophoresis, Amplification, Negative Control, Clone Assay, Plasmid Preparation, Activity Assay, Luciferase, Reporter Assay, Transfection, shRNA, Over Expression

Journal:

Article Title: In vitro analysis of DNA-protein interactions by proximity ligation

doi: 10.1073/pnas.0611229104

Figure Lengend Snippet: Fig. 4.

Article Snippet: The HNF-4α antibodies were purchased from Active Motif and from Santa Cruz Biotechnology (Santa Cruz, CA) (catalog no. C19).

Techniques: