Fig 2: The role of BGLT3 in γ-globin induction by BCL11A and ZBTB7A.a Validation of shRNA-mediated knockdown efficiency of BGLT3 in BCL11A- or ZBTB7A-knockout HUDEP-2 cells. Data are mean ± SD from three independent experiments. b Relative expression of γ-globin in BCL11A- or ZBTB7A-knockout HUDEP-2 cells with or without BGLT3 knockdown. Data are mean ± SD from three independent experiments. c Relative expression of BCL11A and ZBTB7A in BGLT3-overexpressing HUDEP-2 cells. Data are mean ± SD from three independent experiments. P-value was calculated by the one-way ANOVA with Tukey’s post hoc test.

Fig 3: Regulation of occupancies of BCL11A and ZBTB7A at the BGLT3 gene locus by G9a.a, b ChIP-qPCR analyses using antibodies against BCL11A (a) and ZBTB7A (b) in HUDEP-2 cells treated with 1 μM RK-701 or DMSO for 4 days. Data are mean ± SD from three independent experiments. c Peptide pull-down assay with biotinylated histone H3 peptides from HUDEP-2 cell lysates. A Representative image of two independent experiments is shown. d ChIP-qPCR analyses using antibodies against CHD3 or CHD4 in HUDEP-2 cells treated with 1 μM RK-701 or DMSO for 4 days. Data are mean ± SD from three independent experiments. e, f Interaction between CHD4 and BCL11A (e) or ZBTB7A (f) in cells. HEK293T cells were transiently transfected with plasmids for expression of His-tagged CHD4 and FLAG-tagged BCL11A or ZBTB7A. Proteins immunoprecipitated with the anti-FLAG antibody were analyzed by immunoblotting using the indicated antibodies. A representative image of two independent experiments is shown. P-value was calculated by two-tailed t-test.

Fig 4: A schematic model of the molecular mechanism by which G9a represses fetal globin gene expression.Two major HbF repressors, BCL11A and ZBTB7A, recruit G9a onto the BGLT3 gene locus, where G9a facilities H3K9me2. G9a-mediated H3K9me2 recruits BCL11A and ZBTB7A onto the BGLT3 gene locus via CHD4, which may further enhance locus-specific H3K9me2. This positive feedback loop of enhancement of H3K9me2 at the BGLT3 gene locus ensures to repress BGLT3 lncRNA and subsequent fetal globin expression. RK-701 reactivates fetal globin by disrupting this G9a–BCL11A/ZBTB7A system by inhibiting the enzymatic activity of G9a.

Fig 5: Regulation of G9a-induced H3K9me2 at the BGLT3 gene locus by BCL11A and ZBTB7A.a Effect of BCL11A or ZBTB7A knockout on RK-701–induced expression of BGLT3. Relative expression of BGLT3 in parental, BCL11A-, or ZBTB7A-knockout HUDEP-2 cells treated with 1 μM RK-701 for 4 days was analyzed by RT-qPCR. Data are mean ± SD from three independent experiments. b ChIP-qPCR analyses using an antibody against H3K9me2 in parental, BCL11A-, or ZBTB7A-knockout HUDEP-2 cells. Data are mean ± SD from three independent experiments. c, d Interaction between G9a and BCL11A (c) orZBTB7A (d) in cells. HEK293T cells were transiently transfected with plasmids for expression of FLAG-tagged BCL11A or ZBTB7A. Proteins immunoprecipitated with the anti-FLAG antibody were analyzed by immunoblotting using the indicated antibodies. A representative image of three independent experiments is shown. P-value was calculated by the one-way ANOVA with Tukey’s post hoc test (a) or the two-tailed t-test (b).