Ation for the duration of angiogenesis, we measured H3K27ac chromatin occupancy genomewide in human umbilical vein endothelial cells (HUVECs) treated for 0 (unstimulated), 1, four, and 12 h with VEGFA (Supplemental Table 1). General, our ChIP-seq information correlated with H3K27ac in ENCODE data sets, with some variations probably attributable to variations in growth conditions (Supplemental Fig. 1). Inspection of your H3K27ac ChIP-seq information showed regions with substantial modifications in H3K27ac because of VEGFA stimulation (boxed regions, Fig. 1A,B). To identify regions with VEGFA-induced variation in H3K27ac, we calculated the H3K27ac signal-normalized variance (variance score) for 200-bp sliding windows across the 12-h time course. This method captured a large number of regions withA dynamic VEGFA-regulated H3K27ac signature is tightly linked to EP300 chromatin occupancyThe transcriptional coactivator EP300 acetylates histones (Ogryzko et al. 1996) and occupies tissue-specific enhancers (Visel et al. 2009). To define the partnership of EP300 chromatin occupancy to dynamic H3K27ac websites, we measured EP300 chromatin occupancy during the VEGFA stimulation time course by ChIP-seq. The EP300 ChIP-seq data have been reproducible involving independent biological duplicates (Pearson r 0.92) (Supplemental Fig. 2A). Comparison to publicly available ENCODE EP300 occupancy information for the immortalized B-cell cell line GM12878 (Reddy et al. 2012) indicated that EP300 binding was largely cell type-specific (Supplemental Fig. 2B). Subsequent, in the VEGFA time course data, we calculated the distance from EP300 web-sites to neighboring H3K27acFigure 1. VEGFA-stimulated dynamic adjustments in H3K27ac occupancy. (A,B) Browser views of H3K27ac and EP300 ChIP-seq enrichment at HLX and NR4A1 at 4 time points following VEGFA stimulation. Note regions with varying H3K27ac occupancy in response to VEGFA (boxed regions). Bracketed regions are enlarged in portions of panel C. (C ) H3K27ac variance score histogram. VEGFA-stimulated significant changes in H3K27ac regions for the duration of the 12-h time course. The plot only includes regions using a log2 variance score greater than or equal to 2. (D) Chip-qPCR validation of VEGFA-induced alterations in H3K27ac occupancy. Browser views show ChIP-seq enrichment, and reduce plots indicate H3K27ac ChIP-qPCR using numbered amplicons indicated within the browser views. Underlined numbers indicate regions exactly where ChIP-seq predicted VEGFA-stimulated H3K27ac occupancy adjust, when regions with out underline are adjacent controls.AzddMeC Nucleoside Antimetabolite/Analog Horizontal scale bars: (blue) 1 kb, (green) 0.Unesbulin manufacturer five kb.PMID:24187611 Genome Researchwww.genome.orgA dynamic H3K27ac enhancer signaturesites, stratified by their H3K27ac variance score. Interestingly, the majority of the regions together with the greatest H3K27ac variance scores occurred inside two kb of EP300 websites, when less variant H3K27ac web pages tended to become further from EP300 (Fig. 2A; Supplemental Fig. three). This outcome supported a tight relationship among EP300 and dynamic but not static H3K27ac marks. The enrichment of EP300 close to dynamic H3K27ac websites recommended that EP300 is functionally involved in deposition of H3K27ac in response to VEGFA stimulation. To test the functional requirement for EP300 in VEGFA-stimulated deposition of H3K27ac, we measured the effect of EP300 knockdown on H3K27ac chromatin occupancy. siRNA EP300 knockdown (Supplemental Fig. 4) blocked VEGFA-stimulated deposition of H3K27ac at NR4A1, HLX, and KDR (Fig. 2B; Supplemental Fig. 5A). To ascertain if EP300 histone acety.
