Extended Data Figure 5: XBP1 and HIF1α co-occupy HIF1α targets.

a, Track view of XBP1 ChIP-seq density profile (two biological replicates) on HIF1α target genes. b, XBP1 and HIF1α co-bind to DDIT4, VEGFA and PDK1 promoters under hypoxic conditions. A ChIP assay was performed using anti-XBP1 or anti-HIF1α antibody to detect enriched fragments. GST antibody was used as mock ChIP control. c, XBP1 and HIF1α co-bind to JMJD1A and JMJD2C promoters under hypoxic conditions. Upper panel: schematic diagram of the primer locations across the JMJD2C or JMJD1A promoter. Grey boxes indicate exon. A ChIP assay was performed using anti-XBP1 polyclonal antibody or anti-HIF1α polyclonal antibody to detect enriched fragments. Fold enrichment is the relative abundance of DNA fragments at the amplified region over a control amplified region. GST antibody was used as mock ChIP control. d, XBP1s and HIF1α co-occupy JMJD1A, DDIT4, NDRG1, PDK1 and VEGFA promoters. A ChIP-re-ChIP assay was performed using the anti-XBP1s antibody first (X). The eluants were then subjected to a second ChIP assay using an anti-HIF1α antibody (XH) or a control IgG antibody (XC). All ChIP data (b–d) are shown as mean ± s.d. of technical triplicates. Results show a representative of two independent experiments. *P < 0.05; **P < 0.01. e, HIF1α, but not XBP1s, binds to a probe in Twist promoter in 293T cells. DNA pull-down assay was used to analyse the binding of XBP1s or HIF1α on a probe in Twist promoter. The nuclear extracts of 293T cells overexpressing XBP1s or Flag–HIF1α was incubated with the wild-type probe and immunoblot analysis was performed with anti-XBP1s or anti-Flag antibody. f, XBP1s binds to Twist promoter in MDA-MB-231 cells under 0.1% O₂. The nuclear extract of MDA-MB-231 cells cultured under 0.1% O₂ for 24 h was incubated with the wild-type or mutant probe (HIF1α consensus sequence was mutated) and immunoblot analysis was performed with anti-XBP1s or anti-HIF1α antibody. The lower panel shows the sequence of the probes used. g, 3×HRE reporter was co-transfected with XBP1s expression plasmid or empty vector into MDA-MB-231 cells and luciferase activity measured. Experiments were performed in triplicate and data are shown as mean ± s.d. **P < 0.01. h, RT–PCR analysis of XBP1 expression as in g. Data are shown as mean ± s.d. of technical triplicates.