Ritsuko Shimizu

Essential and Instructive Roles of GATA Factors in Eosinophil Development

GATA transcription factors are major regulators of hematopoietic and immune system. Among GATA factors, GATA-1, GATA-2, and GATA-3 play crucial roles in the development of erythroid cells, hematopoietic stem, and progenitor cells, and T helper type 2 (Th2) cells, respectively. A high level of GATA-1 and GATA-2 expression has been observed in eosinophils, but their roles in eosinophil development remain uncertain both in vitro and in vivo. Here we show that enforced expression of GATA-1 in human primary myeloid progenitor cells completely switches myeloid cell fate into eosinophils. Expression of GATA-1 exclusively promotes development and terminal maturation of eosinophils. Functional domain analyses revealed that the COOH-terminal finger is essential for this capacity while the other domains are dispensable. Importantly, GATA-1–deficient mice failed to develop eosinophil progenitors in the fetal liver. On the other hand, GATA-2 also showed instructive capacity comparable to GATA-1 in vitro and efficiently compensated for GATA-1 deficiency in terms of eosinophil development in vivo, indicating that proper accumulation of GATA factors is critical for eosinophil development. Taken together, our findings establish essential and instructive roles of GATA factors in eosinophil development. GATA-1 and GATA-2 could be novel molecular targets for therapeutic approaches to allergic inflammation.

In vivo requirements for GATA-1 functional domains during primitive and definitive erythropoiesis

GATA‐1 is a transcription factor essential for erythroid/megakaryocytic cell differentiation. To investigate the contribution of individual domains of GATA‐1 to its activity, transgenic mice expressing either an N‐terminus, or an N‐ or C‐terminal zinc finger deletion of GATA‐1 (ΔNT, ΔNF or ΔCF, respectively) were generated and crossed to GATA‐1 germline mutant (GATA‐1.05) mice. Since the GATA‐1 gene is located on the X‐chromosome, male GATA‐1 mutants die by embryonic day 12.5. Both ΔNF and ΔCF transgenes failed to rescue the GATA‐1.05/Y pups. However, transgenic mice expressing ΔNT, but not the ΔNF protein, were able to rescue definitive hematopoiesis. In embryos, while neither the ΔCF protein nor a mutant missing both N‐terminal domains (ΔNTNF) was able to support primitive erythropoiesis, the two independent ΔNT and ΔNF mutants could support primitive erythropoiesis. Thus, lineage‐specific transgenic rescue of the GATA‐1 mutant mouse revealed novel properties that are conferred by specific domains of GATA‐1 during primitive and definitive erythropoiesis, and demonstrate that the NT and NF moieties lend complementary, but distinguishable properties to the function of GATA‐1.

A Remote GATA2 Hematopoietic Enhancer Drives Leukemogenesis in inv(3)(q21;q26) by Activating EVI1 Expression

Chromosomal inversion between 3q21 and 3q26 results in high-risk acute myeloid leukemia (AML). In this study, we identified a mechanism whereby a GATA2 distal hematopoietic enhancer (G2DHE or -77-kb enhancer) is brought into close proximity to the EVI1 gene in inv(3)(q21;q26) inversions, leading to leukemogenesis. We examined the contribution of G2DHE to leukemogenesis by creating a bacterial artificial chromosome (BAC) transgenic model that recapitulates the inv(3)(q21;q26) allele. Transgenic mice harboring a linked BAC developed leukemia accompanied by EVI1 overexpression-neoplasia that was not detected in mice bearing the same transgene but that was missing the GATA2 enhancer. These results establish the mechanistic basis underlying the pathogenesis of a severe form of leukemia through aberrant expression of the EVI1 proto-oncogene.

A Gata2 intronic enhancer confers its pan-endothelia-specific regulation.

GATA-2, a transcription factor that has been shown to play important roles in multiple organ systems during embryogenesis, has been ascribed the property of regulating the expression of numerous endothelium-specific genes. However, the transcriptional regulatory hierarchy governing Gata2 activation in endothelial cells has not been fully explored. Here, we document GATA-2 endothelial expression during embryogenesis by following GFP expression in Gata2-GFP knock-in embryos. Using founder transgenic analyses, we identified a Gata2 endothelium enhancer in the fourth intron and found that Gata2 regulation by this enhancer is restricted to the endocardial, lymphatic and vascular endothelium. Whereas disruption of three ETS-binding motifs within the enhancer diminished its activity, the ablation of its single E box extinguished endothelial enhancer-directed expression in transgenic mice. Development of the endothelium is known to require SCL (TAL1), and an SCL-E12 (SCL-Tcfe2a) heterodimer can bind the crucial E box in the enhancer in vitro. Thus, GATA-2 is expressed early in lymphatic, cardiac and blood vascular endothelial cells, and the pan-endothelium-specific expression of Gata2 is controlled by a discrete intronic enhancer.

Leukemogenesis Caused by Incapacitated GATA-1 Function

ABSTRACT GATA-1 is essential for the development of erythroid and megakaryocytic lineages. We found that GATA-1 gene knockdown female (GATA-1.05/X) mice frequently develop a hematopoietic disorder resembling myelodysplastic syndrome that is characterized by the accumulation of progenitors expressing low levels of GATA-1. In this study, we demonstrate that GATA-1.05/X mice suffer from two distinct types of acute leukemia, an early-onset c-Kit-positive nonlymphoid leukemia and a late-onset B-lymphocytic leukemia. Since GATA-1 is an X chromosome gene, two types of hematopoietic cells reside within heterozygous GATA-1 knockdown mice, bearing either an active wild-type GATA-1 allele or an active mutant GATA-1.05 allele. In the hematopoietic progenitors with the latter allele, low-level GATA-1 expression is sufficient to support survival and proliferation but not differentiation, leading to the accumulation of progenitors that are easily targeted by oncogenic stimuli. Since such leukemia has not been observed in GATA-1-null/X mutant mice, we conclude that the residual GATA-1 activity in the knockdown mice contributes to the development of the malignancy. This de novo model recapitulates the acute crisis found in preleukemic conditions in humans.

GATA1-related leukaemias

GATA1 is a prototypical lineage-restricted transcription factor that is central to the correct differentiation, proliferation and apoptosis of erythroid and megakaryocytic cells. Mutations in GATA1 can generate a truncated protein, which contributes to the genesis of transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukaemia (AMKL) in infants with Down syndrome. Similarly, Gata1 knockdown to 5% of its wild-type level causes high incidence of erythroid leukaemia in mice. The GATA1-related leukaemias in both human and mouse could provide important insights into the mechanism of multi-step leukaemogenesis. Efforts are afoot to produce mouse models that are reflective of TMD and AMKL.

A minigene containing four discrete cis elements recapitulates GATA-1 gene expression in vivo

Background:  The GATA‐1haematopoietic enhancer (G1HE), located between 3.9 and 2.6 kb 5′ to the haematopoietic first exon, is essential for GATA‐1 gene transcription in erythroid cells. However, G1HE is not sufficient to confer tissue specificity on the GATA‐1 gene in vivo, indicating that additional regulatory sequences are necessary.

GATA factor switching during erythroid differentiation.

Purpose of reviewEfforts have been made to understand how erythroid differentiation is regulated, and recent discoveries have clarified that lineage-specific transcription factor networks are essential for proper differentiation of erythroid cells. The transcription factors GATA1 and GATA2 are involved in such networks that regulate erythroid gene expression. Importantly, expression of Gata1 and Gata2 genes is also under the control of such regulatory networks. The present review is focused on the mechanism of Gata1 and Gata2 gene regulation during erythropoiesis and the physiological significance of their dynamic regulation. Recent findingsGata1 and Gata2 genes are regulated by multiple transcription factors, including their own products GATA1 and GATA2. GATA1 and GATA2 recognize specific regulatory GATA motifs, and their expression levels change dynamically during erythroid differentiation, leading to diversified gene expression during erythropoiesis. SummaryStrict regulations of the Gata1 and Gata2 genes are critical for proper lineage commitment and development of erythroid cells. It has been shown in transgenic mouse analyses that cis-acting GATA binding motifs are critical for the expression of Gata1 and Gata2 genes. Furthermore, expression of Gata1 and Gata2 genes along with a set of erythroid genes appeared to be regulated by GATA factor switching.

The Ufm1-activating enzyme Uba5 is indispensable for erythroid differentiation in mice

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.

Characterization of GATA‐1+ hemangioblastic cells in the mouse embryo

Hemangioblasts are thought to be one of the sources of hematopoietic progenitors, yet little is known about their localization and fate in the mouse embryo. We show here that a subset of cells co‐expressing the hematopoietic marker GATA‐1 and the endothelial marker VE‐cadherin localize on the yolk sac blood islands at embryonic day 7.5. Clonal analysis demonstrated that GATA‐1+ cells isolated from E7.0–7.5 embryos include a common precursor for hematopoietic and endothelial cells. Moreover, this precursor possesses primitive and definitive hematopoietic bipotential. By using a transgenic complementation rescue approach, GATA‐1+ cell‐derived progenitors were selectively restored in Runx1‐deficient mice. In the rescued mice, definitive erythropoiesis was recovered but the rescued progenitors did not display multilineage hematopoiesis or intra‐aortic hematopoietic clusters. These results provide evidence of the presence of GATA‐1+ hemangioblastic cells in the extra‐embryonic region and also their functional contribution to hematopoiesis in the embryo.