Fig. 4.

HIF-3 binding is associated with the canonical HRE in the promoter regions of its target genes. a HIF-3α2 ChIP-seq enrichment signal associates with the canonical HRE sequence with preference for A at position R. b, c HIF-3α2 enrichment is observed on the EPO and ANGPTL4 genes near the promoter-TSS, co-localizing with sites that contain six canonical HRE sequences (5′-RCGTG-3′, included HREs underlined) on forward and reverse strands as denoted by blue and red lines, respectively. Interestingly, no HIF-3α2 enrichment is detected on the LIE immediately 3′ to EPO. HIF-3α2 enrichment (first track) is shown relative to input (lower track). Samples were prepared in triplicate and pooled for ChIP. d–i Validation of a subset of HIF-3α2 chromatin-binding enrichment sites by ChIP-qPCR. ChIP-qPCR by HIF3A and normal rabbit IgG antibodies shows amplification in the HIF-3α2 enrichment site on five genes, namely EPO (d), EPOR (f), EIF5A (g), PSMD5 (h), and ANGPTL4 (i), and no amplification with a control primer set designed to target regions of EPO where no enrichment is observed (e), n = 3. The ChIP-qPCR results are normalized with respect to input. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student’s t test