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. 2021 Sep 29;10:e70557. doi: 10.7554/eLife.70557

Figure 2. The induction of HbF in Δ3′HS1 cells is BCL11A independent.

(A) Volcano plot of differentially expressed genes in two Δ3′HS1 clones (B6 and D3) vs. two wild-type HUDEP-2 biological duplicates. Differentially expressed globin and olfactory receptor genes are labeled. (B) The volcano plot of differentially expressed genes in two 3′HS1 inversion clones (A2 and G3) vs. two wild-type HUDEP-2 biological duplicates. Differentially expressed globin and olfactory receptor genes are labeled. (C) Expression level of β-globin genes in Δ3′HS1 clones, 3′HS1 inversion clones, and wild-type HUDEP-2 cells. (D) Expression level of known fetal hemoglobin repressor genes in Δ3′HS1 clones, 3′HS1 inversion clones, and wild-type HUDEP-2 cells. (E) Western blot shows the level of BCL11A and ZBTB7A (LRF) in Δ3′HS1 clones, 3′HS1 inversion clones, Δ3′HS-5 clones, and wild-type HUDEP-2 cells. Refer to Figure 2—source data 1 for original blot picture. (F) The composition of β-like hemoglobin genes in the WT HDUEP-2 cells with BCL11A + 58 enhancer deleted with CRISPR/Cas9 and Δ3′HS1 HDUEP-2 cells with BCL11A + 58 enhancer deleted with CRISPR/Cas9.

Figure 2—source data 1. The immunoblot data of BCL11A, ZBTB7A, β-actin, β-globin, and γ-globin of clones displayed in Figure 2.

Figure 2.

Figure 2—figure supplement 1. BCL11A loss further promotes fetal hemoglobin induction in Δ3′HS-1 background.

Figure 2—figure supplement 1.

(A) Integrated Genome Viewer (IGV) track view of ATAC-seq and H3K27ac ChIP-seq at β-globin gene locus of Δ3′HS1 HDUEP-2 cell clones, 3′HS1 inversion HDUEP-2 cells, and wild-type HUDEP-2 cells. GATA1, CTCF, and BCL11A CUT&RUN data is shown below the track. Regions highlighted in orange are paralogous HBG1/2 promoter. (B) Sanger sequencing of BCL11A +58 enhancer-disrupted clones. (C) qPCR quantification of BCL11A gene expression in +58 enhancer-deleted clones. (D) Flow cytometry measurement of HbF in WT and B6 clone after the deletion of BCL11A + 58 enhancer by CRISPR/Cas9. (E) Western blot quantification of BCL11A protein in BCL11A + 58 enhancer-deleted clones in WT and Δ3′HS-1 background. (F) The differentiation stage of BCL11A + 58 enhancer-disrupted HUDEP-2 cell clones profiled by flow cytometry of CD71 and CD235a.
Figure 2—figure supplement 1—source data 1. The immunoblot data of BCL11A, ZBTB7A, β-actin, β-globin, and γ-globin of clones displayed in Figure 2—figure supplement 1.

Figure 2—figure supplement 2. Pomalidomide enhances fetal hemoglobin production induced by 3′HS-1 deletion.

Figure 2—figure supplement 2.

(A) qRT-PCR quantification of BCL11A in the pomalidomide-treated WT and 3′-HS-1-deleted HUDEP2 cell clones. (B) Composition of β-like globin by qRT-PCR in clones treated with DMSO and 1 μM pomalidomide described in panel (A). (C) The flow cytometry plot of HbF in clones treated with DMSO and 1 μM pomalidomide described in panel (A). (D) Western blot of BCL11A, ZBTB7A, β-globin, and γ-globin clones treated with DMSO and 1 μM pomalidomide described in panel (A). (E) The differentiation stage of HUDEP-2 cell clones used in panel (A) profiled by flow cytometry of CD71 and CD235a.
Figure 2—figure supplement 2—source data 1. The immunoblot data of BCL11A, ZBTB7A, β-actin, β-globin, and γ-globin of clones displayed in Figure 2—figure supplement 2.