Post-translational protein modifications are systems designed to expand restricted genomic information through functional conversion of target molecules. Ubiquitin-like post-translational modifiers regulate numerous cellular events through their covalent linkages to target protein(s) by an enzymatic cascade analogous to ubiquitylation consisting of E1 (activating), E2 (conjugating) and E3 (ligating) enzymes. In this study, we report the essential role of Uba5, a specific activating enzyme for the ubiquitin-like modifier, Ufm1, in erythroid development. Mice lacking Uba5 exhibited severe anaemia, followed by death in utero. Although Uba5 was dispensable for the production of erythropoietin, its genetic loss led to impaired development of megakaryocyte and erythroid progenitors from common myeloid progenitors. Intriguingly, transgenic expression of Uba5 in the erythroid lineage rescued the Uba5-deficient embryos from anaemia and prolonged their survival, demonstrating the importance of Uba5 in cell-autonomous erythroid differentiation. Our results suggest that one of the ubiquitin-like protein modification systems, the Ufm1 system, is involved in the regulation of haematopoiesis.

The transcription factor Gata1 is expressed in several hematopoietic lineages and plays essential roles in normal hematopoietic development during embryonic stages. The lethality of Gata1-null embryos has precluded determination of its role in adult erythropoiesis. Here we have examined the effects of Gata1 loss in adult erythropoiesis using conditional Gata1 knockout mice expressing either interferon-or tamoxifen-inducible Cre recombinase (Mx-Cre and Tx-Cre, respectively). Mx-Cre-mediated Gata1 recombination, although incomplete, resulted in maturation arrest of Gata1-null erythroid cells at the proerythroblast stage, thrombocytopenia, and excessive proliferation of megakaryocytes in the spleen. Tx-Cremediated Gata1 recombination resulted in depletion of the erythroid compartment in bone marrow and spleen. Formation of the early and late erythroid progenitors in bone marrow was significantly reduced in the absence of Gata1. Furthermore, on treatment with a hemolytic agent, these mice failed to activate a stress erythropoietic response, despite the rising erythropoietin levels. These results indicate that, in addition to the requirement of Gata1 in adult megakaryopoiesis, Gata1 is necessary for steady-state erythropoiesis and for erythroid expansion in response to anemia. Thus, ablation of Gata1 in adult mice results in a condition resembling aplastic crisis in human. IntroductionProduction of mature blood cells from the pluripotent hematopoietic stem cells is continuous throughout life. This process is controlled by combinatorial functions of lineage-specific and ubiquitous transcription factors. The zinc finger transcription factor Gata1 is a prototype of lineage-specific transcription factors, with restricted expression in several myeloid lineages. 1,2 Gene ablation studies of Gata1 in mice revealed its essential role in erythroid cell development. Embryos lacking Gata1 die at approximately embryonic day 11.5 because of arrested maturation of primitive erythroid cells. 3 A similar phenotype was observed in embryos bearing a Gata1 knockdown mutation (Gata1.05) that causes 95% reduction of Gata1 mRNA level in erythroid cells. 4 A critical role for Gata1 in megakaryocyte maturation and platelet formation has been shown in mice with megakaryocyte-specific Gata1 deficiency. 5,6 A remarkable phenotype of these mutant mice is that defective maturation of megakaryocytes is associated with excessive proliferation, suggesting that Gata1 inhibits cell proliferation during megakaryocytic maturation.Although roles for Gata1 in postnatal megakaryopoiesis have been thoroughly investigated, the embryonic lethality caused by Gata1 deficiency in erythroid precursors has precluded studies of its role in postnatal erythropoiesis. In adults, the rate of red cell production in the steady-state condition is much lower than that in the fetal and neonatal stages. However, in response to erythropoietic stress, the rate of erythropoiesis is rapidly increased. 7 The plasma levels of erythropoietin (Epo), the central regulator of erythr...

Transcription factor GATA-1 is essential for erythroid cell differentiation. GATA-binding motifs have been found in the regulatory regions of various erythroid-specific genes, suggesting that GATA-1 contributes to gene regulation during the entire process of erythropoiesis. A GATA-1 germ-line mutation results in embryonic lethality due to defective primitive erythropoiesis and GATA-1-null embryonic stem cells fails to differentiate beyond the proerythroblast stage. Therefore, the precise roles of GATA-1 in the later stages of erythropoiesis could not be clarified. Under the control of a GATA-1 gene hematopoietic regulatory domain, a GATA-1 mutant lacking the N-finger domain (∆ ∆ ∆ ∆ NF mutant) was over-expressed in mice. These mice exhibited abnormal morphology in peripheral red blood cells (RBCs), reticulocytosis, splenomegaly, and erythroid hyperplasia, indicating compensated hemolysis. These mice were extremely sensitive to phenylhydrazine (PHZ), an agent that induces hemolysis, and their RBCs were osmotically fragile. Importantly, the hemolytic response to PHZ was partially restored by the simultaneous expression of wild-type GATA-1 with the ∆ ∆ ∆ ∆ NF mutant, supporting our contention that ∆ ∆ ∆ ∆ NF protein competitively inhibits the function of endogenous GATA-1. These data provide the first in vivo evidence that the NF domain contributes to the gene regulation that is critical for differentiation and survival of mature RBCs in postnatal erythropoiesis.