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. Author manuscript; available in PMC: 2021 Dec 21.
Published in final edited form as: Nat Genet. 2021 Jun 21;53(7):1022–1035. doi: 10.1038/s41588-021-00887-y

Fig. 3. SET1B selectively drives mRNA expression of HIF target genes.

Fig. 3

(a) RNA-seq: HeLa cells were transduced with sgRNA targeting HIF1β or SET1B, and control or mixed KO populations were treated at 21% or 1% O2 for 12 h before RNA was extracted and sequenced using HiSeq (n = 2 biologically independent samples). (b, c) Volcano plots of differential mRNA expression in control vs. HIF1β (b) or SET1B (c) at 21% O2. Significantly up or downregulated genes were selected based on a log2(fold-change) of >0.5 or <-0.5. Genes significantly upregulated (red) and downregulated (blue) are shown. (d) Venn diagram highlighting hypoxia inducible genes (purple), HIF1β-dependent (blue) and SET1B-dependent (green) genes. (e) RNA-seq HIF1β- and/or SET1B-dependent mRNA expression in 1% oxygen. Volcano plot of log2(fold-change) against –log10 P value for all genes in 21% and 1% O2. Hypoxia inducible (purple), HIF1β-dependent genes (blue), SET1B-dependent genes (light green) and combined HIF1β- and SET1B-dependent genes (dark green) are shown. (f) Heat map displaying log2(fold-change) in mRNA expression in control, HIF1β or SET1B mixed KO cells of validated HIF target genes. (g) Quantitative PCR (qPCR) of HIF targets (CA9, PHD3, VEGF and GLUT1) and a non-HIF target (BAP1) in HeLa and A549 cells incubation in 1% or 21% oxygen for 24 h (n = 3 biologically independent samples, mean ± SD, *** P ≤ 0.0001, two-way ANOVA). (h) Gene expression levels of all hypoxia induced transcripts ranked from high expression to low by Fragments Per Kilobase of transcript per Million mapped reads (FPKM). SET1B dependent (green) and independent (grey) genes are shown. (i, j) VEGF ELISA from control, HIF1β- or SET1B-depleted A549 (i) or 786-0 cells (j) (n = 3 biologically independent samples). Cells were grown in 21% or 1% O2 before supernatants were collected. (i) Control vs. HIF1β P ≤ 0.0001, control vs. SET1B P ≤ 0.0001; two-way ANOVA. (j) Control vs. HIF1β P <0.0002, control vs. SET1B P = 0.003; two-way ANOVA. Ct = control. Graphs show mean ± SD, ** P < 0.01, *** P < 0.001, **** P < 0.0001.