Session: 101. Red Cells and Erythropoiesis, Excluding Iron: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, hematopoiesis, Biological Processes
BCL11A is a master regulator of γ-globin gene silencing through association with the NuRD complex and blocking of NFYA recruitment to γ-globin gene promoters. The role of BCL11A in regulating gene expression during erythroid differentiation is not well studied. HDAC1 mediated GATA-1 and BCL11A association was validated by pull down and immunoprecipitation assay. Next, the BCL11A and HDAC1 interacting site was determined. The N-terminal region of BCL11A is found to be important for HDAC1 interaction by pull down assay. Through analysis of the secondary structure of BCL11A N-terminal, L12/K14 are predicted to be important for protein-protein interaction. Indeed, L12A/K14A mutation significantly reduced HDAC1 interaction as well as HDAC1 mediated GATA-1 and NuRD complex association. To assess the effects of the L12A/K14A mutation of BCL11A on gene expression, we performed RNA-seq analysis using BCL11A knock down (KD) or L12A/K14A mutant overexpressed in HUDEP-2 cells. The result shows that the L12A/K14A mutation de-repressed γ-globin expression and positively affects genes for erythrocyte development that is similar to the expression profile of BCL11A KD. Therefore, L12/K14 residues of BCL11A might have an important role in γ-globin silencing and erythropoiesis by mediating HDAC1 associated GATA-1 and NuRD complex. The Cut&Run assay was performed to study the recruitment of BCL11A, GATA-1, HDAC1, NuRD complex, and NFYA in WT, BCL11A KD, and Flag-BCL11A L12A/K14A mutant cells. There are over 7000 GATA-1 and BCL11A co-binding peaks detected in wild type cells. Within these peaks, about 2000 peaks are at BCL11A binding motif site. GATA-1, HDAC1 and NuRD binding were abolished at these sites in BCL11A KD or L12A/K14A mutant cells, while BCL11A binding was not affected. There are about 3000 peaks at GATA-1 binding motif sites. While GATA-1 binding is not affected at these sites in BCL11A KD or L12A/K14A mutant cells, BCL11A, HDAC1 and NuRD complex binding were significantly reduced. This study demonstrates that there are two modes of BCL11A and GATA-1 co-recruitment: BCL11A can recruit GATA-1 and GATA-1 can recruit BCL11A to their specific binding motifs, respectively. Interestingly, these two modes regulate differential gene targets. BCL11A recruits GATA-1 at genes mainly involved in chromatin organization and cell cycle, while GATA-1 recruits BCL11A to genes mainly involved in transcription, erythrocyte and megakaryocyte differentiation. At the β-globin locus, BCL11A KD or L12A/K14A mutation reduced GATA-1 and NuRD binding and enhanced NFYA binding at the γ-globin promoter, suggesting that BCL11A cooperates with GATA-1, HDAC1, and NuRD complex and prevents NFYA binding at the distal binding region to suppress γ-globin expression. At LCR hypersensitive site 2 (HS2) region, we also observed a reduction of GATA-1 and NuRD recruitment with BCL11A KD or L12A/K14A mutation while GATA-1 recruitment at other promoters or LCR regions were not changed, suggesting that the HS2 region may be involved in reactivating γ-globin in BCL11A KD or L12A/K14A mutation cells. Future study will continue to analyze how BCL11A KD or mutation affects BCL11A and GATA-1 co-binding and target gene expression, and NFYA or other factors recruitment. In summary, our study shows that BCL11A interacts with GATA-1 in a HDAC1 dependent manner and this interaction is important in regulating fetal globin expression and erythroid development.
Disclosures: No relevant conflicts of interest to declare.
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