GATA-1 Acetylation Negatively Regulates Erythroid Differentiation

It is well known that activity of hematopoietic factor GATA-1 is modulated through p300/CBP-mediated acetylation and FOG-1 mediated interaction with NuRD complex. Our early study established the role of HDAC1 in regulating FOG-1/NuRD activity. We recently showed that FOG-1/NuRD complex does not deacetylate GATA-1. However, HDAC1/2 can deacetylate GATA-1 through binding to a different region of GATA-1. The binding site is mapped at the linker region between two zinc fingers of GATA-1. The mutation on the interaction site (2RA mutant) constitutive GATA-1 acetylation in vitro and in vivo, and fails to induce erythroid differentiation. GATA-1^2RA knock-in (KI) mice suffer mild anemia and thrombocytopenia with accumulation of immature erythrocytes and megakaryocytes in bone marrow and spleen. Single cell RNA-seq analysis of Lin⁻ cKit⁺ (LK) cells further reveal a profound change in cell subpopulations and signature gene expression patterns in HSC, myeloid progenitors, and erythroid/megakaryocyte clusters in KI mice. Having established that blockage of HDAC1 mediated GATA-1 deacetylation negatively regulates GATA-1 function, next, we like to further dissect the role of GATA-1 acetylation per se in erythroid differentiation. Key GATA-1 acetylation sites at K245, K246, K312, K314, K315 were mutated to acetyl-mimicking glutamine (KQ), non-acetyl-mimicking arginine (KR) or mutated to nonpolar amino acid alanine (KA) in mouse erythroid progenitor G1E cells. KQ mutant blocks erythroid differentiation while KR mutant does not affect differentiation, suggesting nonacetylated form of lysine are important for GATA-1 activation, also supporting the notion that GATA-1/HDAC1 interaction that mediates GATA-1 deacetylation are important for erythropoiesis. Consistent with previous published report, KA mutation also abolishes erythroid differentiation. RNA-seq analysis shows KQ mutant affects genes important for cell cycle, gene transcription and erythrocyte development. Interestingly, KR mutant upregulates about 60% of genes that are downregulated by KQ mutant, and downregulated 36% of genes that are upregulated by KQ mutant, suggesting dynamic acetylation/deacetylation play a role to maintain optimum transcription activity of GATA-1 for erythrocyte development. To investigate whether GATA-1 acetylation affects binding to its interacting partners, we performed LC-MS/MS analysis on GATA-1 associated proteins by tandem anti-Flag and streptavidin affinity purification in MEL cell extracts. The total of 160 interacting proteins were identified with wild type GATA-1 protein. KQ mutation reduces or abolishes interaction in about 50% of these proteins. GO analysis revealed that the affected proteins are important for mRNA processing, cell transport and cell cycle control. These proteins including DEAD box polypeptide 17 (DDX17), WDR33 and PHF5A. The interaction with KLF1 is also significantly reduced by acetylation. Research are in progress in investigating the role of these proteins in GATA-1 mediated transcription. However, the mutation does not affect interaction with FOG1 and NuRD complex. The mutation also does not affect interaction with other key transcription factors, such as TAL1, RUNX-1, PU.1 and LMO2. Interestingly, KQ mutation also does not affect interaction with bromodomain proteins, including BRD3, BRD4 and BRD7, suggesting the acetylated epitope is not required for GATA-1 to interact with these bromodomain proteins. In summary, our study shows GATA-1 acetylation negatively regulates transcriptional activity that control erythroid commitment and differentiation.

No relevant conflicts of interest to declare.