Extended Data Fig. 9: H3K4me3 ChIP-seq analysis in hypoxia, or following HIF or SET1B depletion.
a-c, H3K4me3 increases at HIF target gene promoters in hypoxia. a, H3K4me3 ChIP-PCR in HeLa cells incubated at 21% or 1% O2 for 6 h. CA9, PHD3, or VEGF 21% versus 1% hypoxia P ≤ 0.0001 (4 biological replicates), BAP1 (4 biological replicates); two-way ANOVA (mean ± SD, *** P < 0.0001). b, c, Total H3K4me3 levels in HeLa and A549 cells (1% O2 for indicated times). Representative of 3 biolgical replicates. d, e, H3K4me3 ChIP-seq analysis. d, Heatmaps of H3K4me3 ChIP-seq signal ± 5-kb from TSS. Genes ranked according to expression in the RNA-seq analysis (highest at top). Representative data shown for one replicate in control, HIF1β and SET1B depleted cells incubated at 21% and 1% O2 for 6 h. e, H3K4me3 signal at gene promoters. Plots showing mean H3K4me3 ChIPseq signal (counts per million – cpm) ± 5-kb from TSS of all gene promoters in control, HIF1β and SET1B depleted cells incubated at 21% and 1% O2for 6 h. f, H3K4me3 at the promoters of the top hypoxia-inducible, HIF-bound genes, defined in the RNA-seq and HIF-1α, HIF-2α, and HIF1β ChIP-seq analyses. g, Venn Diagram of high stringency canonical HIF-1 and HIF-2 binding sites (present in both HIF-1α and HIF1β replicates, and/or both HIF-2α and HIF1β replicates). 600 separate binding sites were identified, at 550 gene loci. h, Combined analysis of the 550 HIF-bound gene set and RNA-seq datasets using Gene Set Enrichment Analysis. Comparisons of HIF bound genes in 21% vs 1% O2, control HeLa vs HIF1β KO, and control HeLa vs SET1B KO shown. ES = enrichment score, NES = normalized enrichment score (P value threshold define by 1/number of permutations). i, Metagene plot of H3K4me3 ChIP-seq at the TSS and gene body of HIF1β dependent or independent genes identified in the RNA-seq analyses (Fig. 3d,e).
