Extended Data Fig. 6: HIF1-induced transcription factor recruitment and epigenetic changes at BGLT3 (linked to main Fig. 3).
From: Activation of γ-globin expression by hypoxia-inducible factor 1α
a, Normal adult donor CD34+ cells were transfected with RNPs containing Cas9 + VHL-targeting sgRNA2 or NT sgRNA, maintained in culture with erythroid cytokines, and analyzed after 11 days. The Genome Browser screenshot shows transcription factor occupancy and histone modifications determined by CUT&RUN analysis and open chromatin regions identified by ATAC-seq. b, Zoom-in on ATAC-seq and CUT&RUN analyses of the LCR in VHL-disrupted erythroblasts generated from CD34+ cells. Data show the average signals from two biological replicate experiments performed using VHL sgRNA1 (main Fig. 3a) or sgRNA2 (panel a of this figure). Note that the scale of the low-magnitude HIF1α peaks are expanded relative to that in panel a. Arrows and dashed lines indicate the positions of forward and reverse HRE motifs in the region. c, CD34+ HSPCs were co-transfected with RNPs targeting VHL and HIF1A, after which erythroid differentiation was induced. The bar chart shows BGLT3 mRNA expression normalized to α-globin mRNA as determined by RT-qPCR analysis at 13 days. Data are presented as mean ± s.d., n = 3 independent experiments with different donor CD34+ cells. Multiplicity-adjusted P-values by ordinary one-way ANOVA with Šídák’s MCT between selected groups. d, CUT&RUN and ATAC-seq analysis of WT HUDEP-2 cells and VHL−/− clones 1 and 2.
