Extended Data Fig. 2: Disruption of ELOC or CUL2 induces γ-globin and HbF expression (linked to main Fig. 1).
From: Activation of γ-globin expression by hypoxia-inducible factor 1α
Cas9-expressing HUDEP-2 cells were transduced with lentiviral vectors encoding sgRNAs targeting the genes encoding Elongin-C (ELOC, E) or Cullin 2 (CUL2, C), after which erythroid differentiation was induced for 5 days. a, The %γ-globin mRNA. The bar charts show the mean ± s.d. from 3 independent experiments. Multiplicity-adjusted P-values by ordinary one-way ANOVA with Dunnett’s MCT against NT. b, Western blot analysis. c, Representative flow cytometry plots showing %F-cells. The mean ± s.d. from 3 independent experiments are shown. d, Gene ontology (GO) analysis of mRNAs that were induced in erythroblasts derived from VHL-disrupted versus control CD34+ HSPCs (false-discovery rated–adjusted P-value < 0.05, log2 fold-change > 1). e, Western blot analysis of day 10 erythroblasts generated from CD34+ HSPCs treated with Cas9 + VHL or NT sgRNAs. f, Relative expression of prolyl hydroxylase domain (PHD) enzyme-encoding mRNAs shown as transcripts per kilobase million (TPM) in day 10 erythroblasts described in panel e. g, Relative expression (in TPM) of GATA1, BCL11A, HIF1A, and HIF2A mRNAs in day 10 erythroblasts described in panel e. h, Western blot analysis of VHL, HIF1a, and HIF2a proteins in day 10 erythroblasts described in panel e and in A549 adenocarcinoma cells treated with Cas9 + VHL or NT sgRNAs. The arrow indicates the HIF2α signal. i, j, Gene set enrichment analysis (GSEA) of mRNAs that are altered in erythroblasts generated from VHL-disrupted (sgRNA1) vs. control (NT sgRNA) CD34+ cells. Fetal-enriched and adult-enriched erythroid gene sets were derived from the work of Huang et al35. NES, normalized enrichment score. k, Relative expression (in TPM) of globin mRNAs in day 10 erythroblasts described in panel e. Bar charts show average data value from two biological replicates, dots represent individual values in f, g, k.
