Stage Selective Requirement For The N-Terminus Of GATA1 During Erythropoiesis

The X-linked blood transcription factor GATA1 is required for the survival and maturation of erythroid cells. Loss of Gata1 causes profound anemia and related mid-gestation lethality in mouse models and human GATA1 mutations are associated with congenital dyserythropoietic anemia, congenital thrombocytopenia, porphyria, Diamond-Blackfan anemia (DBA) and acute megakaryoblastic leukemia in children with Down syndrome (DS-AMKL). In DBA, DS-AMKL, and a subset of congenital anemia, GATA1 mutations cause the exclusive production of a shorter GATA1 isoform, termed GATA1s. The GATA1s isoform retains both zinc fingers and binds DNA and cofactors accordingly, but lacks the N-terminal 83 residues of full-length GATA1. Intriguingly, exclusive production of GATA1s promotes AMKL in the context of Down syndrome (trisomy 21), while disomic individuals harboring the same type of mutations suffer from anemia and severe red cell defects. A mutation causing exclusive production of Gata1s in mice does not appear to affect adult hematopoiesis, but has been shown to cause a marked expansion of megakaryocytes in the fetal liver. The effects of the replacement of Gata1 with Gata1s during fetal liver erythropoiesis, however, remain uncharacterized. To investigate the effects of exclusive Gata1s production during fetal hematopoiesis, we performed comprehensive phenotypic and mechanistic studies using Gata1s knock-in embryos. We found significant changes in myelo-erythroid differentiation beyond the known expansion of megakaryocytes. Flow cytometric analysis revealed altered erythroid differentiation at embryonic days 12.5 and 14.5, evidenced by decreased Ter119 and increased CD71 expression, characteristic of delayed erythroid maturation. Changes in expression of myelo-erythroid progenitor commitment markers were also discovered. Specifically, we observed marked decreases in pre-CFU-E and CFU-E committed progenitors and an increase in pre-GM and pre-MegE populations. Our findings are consistent with a bias towards megakaryopoiesis at the expense of erythroid commitment caused by the expression of Gata1s in place of full-length Gata1. A shift in differentiation was also observed in the embryonic granulocyte/ macrophage lineage, with an increased generation of macrophages with fewer developing granulocytes. Mechanistically, we found that expression of erythroid -specific Gata1 target genes such as Alas2 , Slc4a1 and Klf1 are markedly reduced in the Gata1 knock-in erythroid cells, indicating that Gata1s is a less effective transcriptional activator than full-length Gata1. In particular, given that Klf1 functions to promote erythroid specification downstream of the megakaryocyte-erythrocyte progenitor, our results suggest that Gata1s may promote megakaryocyte differentiation at the expense of erythroid differentiation, in part, by failing to activate Klf1. Taken together, our studies demonstrate a stage-specific requirement for full-length Gata1 during embryonic erythropoiesis. Furthermore, erythroid defects associated with exclusive production of Gata1s in humans may result from an incomplete activation of the erythroid transcriptional program. Disclosures: Crispino: Sanofi: Research Funding.