GATA1-Mediated Megakaryocyte Differentiation and Growth Control Can Be Uncoupled and Mapped to Different Domains in GATA1.

Abstract

The zinc finger transcription factor GATA1 plays a critical non-redundant role in megakaryopoiesis in mice and human patients with both germline and acquired GATA1 mutations. Germline deletion of the mouse regulatory element, HSI (ΔneoΔHS mice), specifically reduces megakaryocyte GATA1 expression to 5% of wild-type levels. ΔneoΔHS mice are viable but have defective megakaryocyte maturation and growth control. Mutant megakaryocytes fail to shed proplatelets in vitro and the platelet count is ~5 fold lower in mutant mice. Moreover, mutant megakaryocyte precursors hyperproliferate in vivo and in vitro.Here, we show there is a specific ~10-fold increase in abnormal CD41+ FcγRlowc-kit+CD9+ megakaryocyte-erythroid progenitor cells in fetal liver. These progenitors form abnormally large colonies in vitro and are unable to form proplatelets. In contrast, earlier myeloid progenitors (the common myeloid progenitor CMP) were present at expected frequencies and differentiated to all non-megakaryocyte myeloid lineages normally. Consistent with this GATA1 expression was selectively reduced in the abnormal progenitors and immature megakaryocyte precursors.We then used these GATA1-deficient primary megakaryocyte progenitors as a cellular substrate to define the amino acid domains within GATA1 required to facilitate proplatelet release and retard immature megakaryocyte growth. Previous data suggested that the bi-functional GATA1 zinc fingers could bind both DNA and interact with protein partners. The C-terminal zinc finger is essential for binding to all GATA sites whereas the N-terminal finger helps binding to a subset of GATA sites and mediates interaction with the critical GATA1 cofactor, FOG-1. Finally, the N-terminal 84 amino acids of GATA1 are important for megakaryopoiesis. Neonates and children with Down Syndrome (DS) can develop megakaryocyte leukaemia acquire mutations in GATA1 exon 2 in fetal life that result in the production of a short form of GATA1 protein (GATA1s) lacking the N-terminal 84 amino acids. In this study, we have systematically dissected GATA1 domains required for platelet release and control of megakaryocyte growth by using retroviral-mediated gene transfer to ectopically express modified GATA1 molecules in primary GATA1-deficient fetal megakaryocyte CD41+FcγRlowc-kit+CD9+progenitors. In addition to the GATA1-FOG-1 interaction and C-terminal zinc finger, two domains within the first N-terminal 110 amino acids are required for platelet release. To restrict megakaryocyte growth, two domains are required; the C-terminal zinc finger and residues in the N-terminus between amino acids 54–110. Importantly, interaction with FOG-l is not required to restrain megakaryocyte growth. Furthermore, we showed that chicken GATA1 was able to rescue both, platelet release and growth control. As only a few residues are conserved in the N-terminus between mouse and chicken GATA1, this potentially pinpoints critical functional residues in the N-terminus. In summary, distinct GATA1 domains regulate terminal megakaryocyte gene expression leading to platelet release and restrain megakaryocyte growth. Analysis of distinct mutations in the N-terminus and a modified version of GATA1 that is crippled in its interaction with FOG-1 shows platelet production and megakaryocyte growth can be uncoupled. Finally, these results have implications for how GATA1s may function in megakaryocytic leukemia in DS.