Extended Data Fig. 3: The VHL E3 ubiquitin ligase complex suppresses γ-globin expression by targeting HIF1α for degradation (linked to main Fig. 1).
From: Activation of γ-globin expression by hypoxia-inducible factor 1α
a, CD34+ HSPCs were co-transfected with RNPs targeting VHL and HIF1A. %HbF measured by HPLC at day 15. (Data are presented as mean ± s.d., n = 3 independent experiments with different donor CD34+ cells). Multiplicity-adjusted P-values by one-way ANOVA with Dunnett’s MCT against control. b–d, VHL−/− HUDEP-2 clones 1 and 2 were electroporated with HIF1A sgRNA1 or sgRNA2, grown in maintenance medium, and analyzed after 5 days. b, Representative flow-cytometry plots showing %F-cells. Experiments were repeated independently 3 times with similar results. c, γ-Globin mRNA levels (left) and %γ-globin mRNA relative to γ-globin + β-globin mRNA (right). Data are presented as mean ± s.d., n = 3 independent experiments. Multiplicity-adjusted P-values by one-way ANOVA with Dunnett’s MCT against NT. d, Western blot analysis of the indicated proteins. e, Cas9-expressing VHL−/− HUDEP-2 cell clones 1 and 2 were transduced with lentiviral vectors encoding MYOM1 or NT sgRNAs, grown in maintenance medium for 7 days, then analyzed for F-cells. Indel frequencies for sgRNAs ranged from 62% to 94% (Supplementary Table 4b). MYOM1 protein was not detected in Western blots of control HUDEP-2 cells (not shown). Representative flow-cytometry plots for F-cells are shown. Experiments were repeated independently 3 times with similar results. f, %γ-globin mRNA in the cells in e. Bar charts show the mean ± s.d. from three biological replicates. g–i, CD34+ HSPCs were electroporated with RNP containing VHL sgRNA1 or NT sgRNA, then erythroid differentiation was induced. In parallel, the same untreated CD34+ HSPCs were induced to undergo erythroid differentiation in 1% O2. g, At day 13, mRNAs encoded by HIF target genes EGLN3 and BNIP3 were measured by RT-qPCR and normalized to AHSP (ERAF) mRNA. Globin mRNAs and hemoglobin proteins were measured by RT-PCR and IE-HPLC, respectively. Bar charts show average data value from two biological replicates, dots represent individual values. h, %F-cells in day 15 erythroblasts in g. mean ± s.d. are shown for two biological replicates using CD34+ cells from different donors. i, Western blot analysis of day 10 erythroblasts.
