TADA1 plays a regulatory role in erythropoiesis and the expression of HBG. Free

Sickle cell anemia and β-thalassemia are caused by mutations in the adult β-globin gene. Reactivation of fetal γ-globin expression can compensate for the defective β-globin and restore hemoglobin's oxygen carrying capacity. Therefore, identifying therapeutic targets or drugs that promote fetal hemoglobin (HbF) reactivation is of great significance for the treatment of these diseases.In this study, we performed a genome-wide CRISPR-Cas9 screen and identified TADA1 as a novel regulator of HBG expression. This finding was validated in both HUDEP2 cells and primary CD34+ hematopoietic stem cells using Western blotting and qPCR. TADA1 knockout significantly increased the proportion of HbF-positive cells and upregulated γ-globin expression. It also promoted erythroid differentiation.Analysis of adult bone marrow single-cell transcriptomic data together with erythroid differentiation transcriptomes from peripheral blood and cord blood revealed that TADA1 is highly expressed in early erythroid progenitors. Importantly, TADA1 expression is largely restricted to early erythroblasts within the bone marrow, with minimal expression in other cell types. These observations suggest that TADA1 may represent a naturally specific and promising target for modulating HbF levels.TADA1 is a core component of the SAGA complex (Spt-Ada-Gcn5 acetyltransferase complex), which plays a broad role in transcriptional regulation. The SAGA complex consists of four functional modules, and TADA1 resides in the central core module where it promotes transcription initiation by enhancing the binding of TBP to the TATA box in gene promoters.To explore the roles of different SAGA subunits in γ-globin activation, we conducted a targeted screen across the complex and found that TADA1 exhibited the strongest effect on HBG induction. In contrast, other components such as KAT2A and TADA2B from the acetyltransferase module and USP22 from the deubiquitination module showed only minor effects. This suggests that histone-modifying activities may not be essential for HBG reactivation.We further investigated the molecular mechanism underlying TADA1-mediated repression of γ-globin and found that TADA1 knockout did not significantly alter the mRNA levels of key HBG repressors such as BCL11A, ZBTB7A, EIF2AK1, and WIZ. However, we observed a marked reduction in BCL11A protein levels upon TADA1 deletion. This decrease appeared to result from impaired translational capacity, as TADA1 deficiency led to downregulation of genes involved in ribosome biogenesis and translation. We confirmed this mechanism through ribo-seq and Western blotting. More intriguingly, we discovered that TADA1 protein levels are regulated by autophagy, which also explains the mechanism by which rapamycin enhances γ-globin expression.In addition, our results showed that TADA1 deficiency downregulated c-MYC and KIT expression, thereby promoting erythroid differentiation.Taken together, our findings reveal that TADA1 regulates erythroid differentiation and γ-globin expression through dual mechanisms involving the regulation of key transcription factors and ribosomal translation related proteins. These insights not only advance our understanding of the regulatory network governing erythropoiesis but also highlight TADA1 as a promising therapeutic target for sickle cell disease and β-thalassemia.