A Tetramer of BCL11A is Required for Stable Protein Production and Fetal Hemoglobin Silencing

Down-regulation of BCL11A protein reverses the fetal (HbF, a2g2) to adult (HbA, a2β2) hemoglobin switch and is exploited in gene-based therapy for hemoglobin disorders. Due to reliance on ex vivo cell manipulation and marrow transplant, such therapies cannot lessen disease burden. To develop novel small molecule approaches, we interrogated the state of BCL11A protein in erythroid cells. We report that tetramer formation mediated by a single zinc-finger (ZnF0) is required for production of steady-state protein. Beyond its role in protein stability, the tetramer state is necessary for g-globin gene repression, as an engineered monomer fails to engage a critical corepressor complex. These aspects of BCL11A protein production identify tetramer formation as a vulnerability for HbF silencing, and provide opportunities for drug discovery. ATAC-seq experiments were performed in HUDEP-2 cells expressing wild-type BCL11A (WT), BCL11A with biallelic deletions of ZnF1 (DZnF1), BCL11A with biallelic deletions of both ZnF0 and ZnF1 (DZnF01), BCL11A knock-out (KO), or BCL11A with biallelic deletions of the NuRD-binding motif (DN) to assess their chromatin accessibility at various differentiation days. To assess DNA binding of WT and DZnF01 BCL11A, CUT&RUN experiment was performed using BCL11A antibody in WT and DZnF01 HUDEP-2 cells on day 7 of differentiation. Occupancy of NF-Y was analyzed in WT, DZnF0, DZnF1, and DZnF01 HUDEP-2 cells by CUT&RUN using NF-Y antibody on day 6 of differentiation. Occupancy of MTA2 was analyzed in WT, DN, DZnF0, DZnF1, DZnF01 HUDEP-2 cells by CUT&RUN using MTA2 antibody on day 7 of differentiation. RNAseq: We compared the gene expression profiles in WT, DZnF1, and DZnF01 HUDEP-2 cells by RNA-Seq on days 0 and 7 of differentiation. We compared the gene expression profiles in WT and DN HUDEP-2 cells by RNA-Seq on days 0 and 4 of differentiation.