TR4 and BCL11A Repress gamma-globin Transcription via Independent Mechanisms

Nuclear receptor TR4 was previously shown to bind to the -117 position of the gamma-globin gene promoters in vitro, which overlaps with the BCL11A binding site recently described. The role of TR4 in human gamma-globin gene repression has not been extensively characterized in vivo, and the relationship between TR4 and BCL11A binding to the gamma-globin promoters remained elusive. We showed in vitro that TR4 and BCL11A competitively bind to overlapping but distinct consensus sequences which both include the -117 position of the gamma-globin promoter. We show here that TR4 represses gamma-globin transcription and HbF accumulation in a BCL11A-independent manner. We characterized the chromatin occupancy of TR4 within the beta-globin locus in comparison to BCL11A and found that both bind to the hypersensitive sites avidly but only BCL11A binds to the gamma-globin promoters at significant levels, suggesting that BCL11A is the predominant repressor acting through the -117 gamma-globin promoter sequence in vivo. These data resolve an important discrepancy in the literature, and thus helps to clarify possible approaches to treatments for sickle cell disease or beta-thalassaemia. To elucidate how TR4 might repress γ-globin transcription, we performed TR4 CNR in HUDEP-2 cells and compared its binding to BCL11A. HUDEP-2 clones were generated by genome editing bearing either of 2 modifications: TR4 C-terminally tagged with 3FLAG+3MYC epitopes (cell line T) and BCL11A tagged with 3HA+3MYC epitopes (cell line B). In addition, a third clone was created bearing both BCL11A::3HA3MYC and TR4::3FLAG3MYC modifications (cell line TB). These epitope-tagged clones not only improved the recovery of the epitopes, but also facilitated comparison of TR4 and BCL11A chromatin occupancy by immune-precipitation of the common MYC epitope using CUT&RUN.