The Saga Complex Member TADA2B Is a Novel Regulator of γ-Globin Production

β-hemoglobinopathies are the most common monogenic disorders in the world. They include sickle cell disease (SCD) and β-thalassemia (β-thal), which are caused by defective or inadequate production of β-globin, respectively. However, treatments for these disorders are limited in efficacy or accessibility. Reactivation of γ-globin, the β-like globin chain expressed during fetal development, reduces the clinical severity of SCD and β-thal.

While exploring a possible role for the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional regulatory complex in erythropoiesis, we discovered that deletion of multiple genes encoding members of the SAGA complex, including the transcriptional adaptor protein TADA2B, lead to γ-globin induction in HUDEP-2 cells. We next generated multiple clonal TADA2B-null HUDEP-2 cell lines. These cells exhibited an increased proportion of γ-globin expressing cells (F-cells) by flow cytometry (10-30% F-cells compared to 4% background levels).

To validate our findings, we deleted TADA2B using three unique, efficient sgRNAs in erythroid cells derived from human hematopoietic stem and progenitor cells (HSPCs) harvested from two independent donors. TADA2B deletion resulted in up to 80% F-cells by flow cytometry and 40% fetal hemoglobin (HbF) protein levels by HPLC analysis. In TADA2B depleted cells, transcripts for γ-globin were elevated 5-fold above control levels with a concurrent 2-fold reduction in β-globin transcripts. TADA2B deletion did not significantly impair cell growth or erythroid differentiation (by flow cytometry and morphologic analysis).

The SAGA complex comprises 20 unique proteins organized into distinct functional domains. TADA2B is a member of the histone acetyltransferase (HAT) domain, along with KAT2A (also known as GCN5). KAT2A deposits acetyl groups at Histone 3 Lysine 9 (H3K9), and to a lesser extent at H3K14, modifications that are associated with transcriptional activation. We pharmacologically inhibited KAT2A activity in erythroid cells derived from HSPCs obtained from two independent donors. Treatment with the KAT2A inhibitor Butyrolactone-3 resulted in an ~15% increase in F-cell formation (41% F-cells at 50uM, compared to 24% in DMSO treated cells), without impairing cell growth.

To determine whether TADA2B deletion affected the expression of known γ-globin regulators, we compared the transcriptomes of stage-matched TADA2B-edited and control erythroid cells and found that expression of the direct γ-globin repressor ZBTB7A/LRF was reduced >2-fold in TADA2B-null cells. This finding was confirmed by RT-qPCR in erythroid cells derived from a second HSPC donor. These results raise the possibility that TADA2B deletion increases γ-globin production by impairing the ability of the SAGA complex to fully induce ZBTB7A expression.

In summary, we have found that deletion of TADA2B, which encodes a member of the HAT domain of the SAGA complex, results in clinically significant elevation in γ-globin transcripts, F-cell formation, and HbF production in HSPC-derived erythroid cells. This finding may be mediated by decreased SAGA complex HAT activity, leading to reduced expression of the γ-globin repressor ZBTB7A. Ongoing studies are focused on determining the mechanism by which TADA2B deletion results in increased γ-globin production.