A common TBP-binding site mutation elevates γ-globin levels by competitive globin switching change in β-thalassemia

• A large-scale targeted NGS on 1142 β-thalassemia patients identified common genetic variants associated with the expression of HbF

• HBG: c.-78A>G reactivates Hb F expression and ameliorates β-thalassemia severity by disrupting the TBP-binding site in HBB promoters

β-thalassemia is a common monogenic disorder caused by genetic defects in β-globin genes (HBB), resulting in imbalanced synthesis of α-/β-globin and ineffective erythropoiesis. It has been well documented that β-thalassemia patients, or even carriers, mostly experience re-activation of fetal hemoglobin (Hb F), but its underlying mechanisms are incompletely understood. We took advantage of a previously established cohort of 1142 β-thalassemia patients with diverse thalassemic mutations subjected to targeted next-generation sequencing. Genotype-phenotype association studies demonstrated that the HBB: c. -78A>G showed a remarkable effect on the elevation of Hb F levels compared to other β-thalassemic mutations. To experimentally validate the conclusion above, the RNP transfection complex through homology-directed repair (HDR) by electroporation was performed, from which we observed a consistent increase of Hb F expression in both HUDEP-2 and primary CD34+ cell lines. Furthermore, ChIP-qPCR, Dual-luciferase reporter assay, and circular chromosome conformation capture (4C) assays validated a decreased occupancy of the HBB TATA-Box by TBP, leading to boosted expression of γ-globin genes by enhanced interaction between locus control regions (LCRs) and γ-globin gene promoters. The patient-based investigation and experimental validations presented in this study might lead to a better understanding of stage-specific globin-gene expression mediated by competitive binding of distal enhancers (LCRs).