Takashi Kuroha

A GATA box in the GATA-1 gene hematopoietic enhancer is a critical element in the network of GATA factors and sites that regulate this gene.

ABSTRACT A region located at kbp −3.9 to −2.6 5′ to the first hematopoietic exon of the GATA-1 gene is necessary to recapitulate gene expression in both the primitive and definitive erythroid lineages. In transfection analyses, this region activated reporter gene expression from an artificial promoter in a position- and orientation-independent manner, indicating that the region functions as the GATA-1 gene hematopoietic enhancer (G1HE). However, when analyzed in transgenic embryos in vivo, G1HE activity was orientation dependent and also required the presence of the endogenousGATA-1 gene hematopoietic promoter. To define the boundaries of G1HE, a series of deletion constructs were prepared and tested in transfection and transgenic mice analyses. We show that G1HE contains a 149-bp core region which is critical for GATA-1gene expression in both primitive and definitive erythroid cells but that expression in megakaryocytes requires the core plus additional sequences from G1HE. This core region contains one GATA, one GAT, and two E boxes. Mutational analyses revealed that only the GATA box is critical for gene-regulatory activity. Importantly, G1HE was active in SCL−/− embryos. These results thus demonstrate the presence of a critical network of GATA factors and GATA binding sites that controls the expression of this gene.

Gata3 participates in a complex transcriptional feedback network to regulate sympathoadrenal differentiation

Gata3 mutant mice expire of noradrenergic deficiency by embryonic day (E) 11 and can be rescued pharmacologically or, as shown here, by restoring Gata3 function specifically in sympathoadrenal (SA) lineages using the human DBH promoter to direct Gata3 transgenic expression. In Gata3-null embryos, there was significant impairment of SA differentiation and increased apoptosis in adrenal chromaffin cells and sympathetic neurons. Additionally, mRNA analyses of purified chromaffin cells from Gata3 mutants show that levels of Mash1, Hand2 and Phox2b (postulated upstream regulators of Gata3) as well as terminally differentiated SA lineage products (tyrosine hydroxylase, Th, and dopamineβ -hydroxylase, Dbh) are markedly altered. However, SA lineage-specific restoration of Gata3 function in the Gata3 mutant background rescues the expression phenotypes of the downstream, as well as the putative upstream genes. These data not only underscore the hypothesis that Gata3 is essential for the differentiation and survival of SA cells, but also suggest that their differentiation is controlled by mutually reinforcing feedback transcriptional interactions between Gata3, Mash1, Hand2 and Phox2b in the SA lineage.

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.

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.

An NK and T cell enhancer lies 280 kilobase pairs 3' to the Gata3 structural gene

ABSTRACT Transcription factor GATA-3 is vital for multiple stages of T cell and natural killer (NK) cell development, and yet the factors that directly regulate Gata3 transcription during hematopoiesis are only marginally defined. Here, we show that neither of the Gata3 promoters, previously implicated in its tissue-specific regulation, is alone capable of directing Gata3 transcription in T lymphocytes. In contrast, by surveying large swaths of DNA surrounding the Gata3 locus, we located a cis element that can recapitulate aspects of the Gata3-dependent T cell regulatory program in vivo. This element, located 280 kbp 3′ to the structural gene, directs both T cell- and NK cell-specific transcription in vivo but harbors no other tissue activity. This novel, distant element regulates multiple major developmental stages that require GATA-3 activity.

The Glasgow Prognostic Score as a pre-transplant risk assessment for allogeneic hematopoietic cell transplantation

Evaluation methods, such as scoring systems for predicting complications in advance, are necessary for determining the adaptation of allogeneic hematopoietic cell transplantation (HCT) and selecting appropriate conditioning regimens. The Hematopoietic Cell Transplantation‐specific Comorbidity Index (HCT‐CI), which is based on functions of main organs, is a useful tool for pre‐transplant risk assessments and has been widely applied in determining treatment strategies for patients with hematological diseases. However, as allogeneic HCT is performed on patients with diverse backgrounds, another factor, which reinforces the HCT‐CI, is required to evaluate pre‐transplant risk assessments. The Glasgow Prognostic Score (GPS), which assesses the combined C‐reactive protein and albumin, was reported to predict survival of patients with solid‐organ malignancies independently of receiving chemo/radiotherapy and stages of cancer. In this study, we applied the GPS for pre‐transplant risk assessments for allogeneic HCT. The GPS successfully stratified the patients into three risk groups of overall survival (OS) and non‐relapse mortality (NRM). Moreover, the GPS could predict outcomes independently of the HCT‐CI for OS and NRM in multivariate analysis. The GPS is considered to be a useful tool and reinforces the HCT‐CI for determining adaptation of allogeneic HCT for patients with hematopoietic neoplasms.

Refinement of the Glasgow Prognostic Score as a pre-transplant risk assessment for allogeneic hematopoietic cell transplantation

The Hematopoietic Cell Transplantation-specific Comorbidity Index (HCT-CI) is a widely used tool for pre-transplant risk assessment. Allogeneic hematopoietic cell transplantation (HCT) is performed on patients with diverse backgrounds, highlighting the need for other predictors to complement the HCT-CI and support bedside decision-making. There is a strong body of evidence supporting the use of pre-transplant serum ferritin (SF) in risk assessments of allogeneic HCT. We additionally found that the Glasgow Prognostic Score (GPS), which assesses inflammatory biomarkers and predicts survival of patients with solid organ malignancies, is a useful predictive marker for overall survival (OS) and non-relapse mortality (NRM) in allogeneic HCT, independent of HCT-CI and SF. In this study, we refined the GPS by adding pre-transplant SF to improve its prognostic ability and enable better stratification; we call this revised index the HCT-specific revised Glasgow Prognostic Score (HCT-GPS). We observed that the HCT-GPS more accurately predicted NRM and early-term OS than the GPS. Moreover, the HCT-GPS provides an independent prognostic factor adjusted for the HCT-CI and disease status, and stratifies patients into four risk groups by OS and NRM. Thus, the HCT-GPS is a useful index for predicting early-term complications after allogeneic HCT in patients with hematopoietic diseases.