Skip to main content
. 2021 Sep 29;10:e70557. doi: 10.7554/eLife.70557

Figure 3. The induction of HbF in Δ3′HS1 cells is modulated by HPFH enhancer.

(A) Upper panel: IGV view of ATAC-seq in primary human blood cells followed by GATA1 and CTCF ChIP-seq in HUDEP-2 cells around the β-globin locus. Lower panel: ATAC-seq of CD34+ hematopoietic stem and progenitor cell (HSPC), megakaryocyte–erythroid progenitor (MEP), and erythroblast is shown in the zoomed view for the OR52A1 region. Red-shaded area indicates the locus of OR52A1. HPFH 3' beak and δβ-thalassemia 3' break is annotated (Feingold and Forget, 1989). (B) The experimental scheme of hereditary persistence of fetal hemoglobin (HPFH) deletion in the 3'HS1 deletion background. (C) The composition of β-like globin Δ3′HS1 (clone B6) HUDEP-2 cells with GATA1 binding site and HPFH region deletion. Mean ± SD is displayed, n = 3. (D) Relative expression of HBE, HBG (probe measures both HBG1 and HBG2), and HBB in the Δ3’HS1 (clone B6) HUDEP-2 cells with GATA1 binding site and HPFH enhancer region deletion. Mean ± SD is displayed, n = 3. (E) The representative HbF flow plot of Δ3’HS1 (clone B6) HUDEP-2 cells with GATA1 binding site and HPFH enhancer region deletion. (F) Evolution conservation of OR52A1 GATA1 binding site in vertebrates. GATA1 binding motif is shown in the middle. The site in mouse and rat associated with human GATA1 binding is boxed out. (G) Chromatin landscape of mouse β-globin gene cluster in mouse erythroid cells MEL and G1-ER4. CTCF, GATA1, and TAL1 ChIP-seq is shown. Orange stripe highlights the mouse homolog of human OR52A1–Olfr68.

Figure 3.

Figure 3—figure supplement 1. HPFH enhancer in edited HUDEP-2 cells.

Figure 3—figure supplement 1.

(A) The v4C tracks generated by Juicebox from WT, Δ3′HS1 HUDEP2 clone B6, and 3′HS1 inversion clone A2. The HPFH enhancer region is highlighted in orange and viewpoint of 4C is highlighted in orange and eye symbol on HPFH enhancer and HBG2 promoter region, respectively. (B) The experimental scheme of deleting 48 kb region between HPFH enhancer and 3′HS1. The Sanger sequencing validation result of a Δ(HPFH-3’HS1) clone B5 is displayed in the lower panel. (C) HBG, HBE, and HBB expression in WT and Δ(HPFH-3’HS1) clone B5. Mean ± SD is shown. ***p<0.001, *p<0.05. t-test was performed to determine the p value. (D) The flow cytometry plot of HbF in WT and Δ(HPFH-3’HS1) clone B5.