scholarly journals Differential transcriptional regulation of the human thrombin receptor gene by the Sp family of transcription factors in human endothelial cells

1998 ◽  
Vol 330 (3) ◽  
pp. 1469-1474 ◽  
Author(s):  
Yaxu WU ◽  
Johannes RUEF ◽  
N. Gadiparthi RAO ◽  
Cam PATTERSON ◽  
S. Marschall RUNGE
Keyword(s):  
Endothelial Cells ◽  
Promoter Activity ◽  
Receptor Gene ◽  
Thrombin Receptor ◽  
Wild Type ◽  
Mobility Shift ◽  
Human Thrombin ◽  
Gc Box

The mitogenic effects of thrombin are mediated by a G-protein-coupled receptor. Because the effects of thrombin are strongly influenced by the expression of its receptor, an understanding of its regulatory mechanisms is essential. To identify mechanisms of human thrombin receptor (HTR) gene regulation, a series of HTR-promoter-luciferase constructs were made and transfected into human microvascular endothelial cells for analysis. Deletion from bp -303 to -164 abolished reporter gene expression. Dimethyl sulphate treatment in vivo and DNase I footprinting in vitro demonstrated that a cluster of three GC box consensus sites was occupied, and electrophoretic mobility-shift assays established that Sp1 and Sp3 both bind to this 3ʹ GC box cluster. We mutated each of the three GC boxes individually and all three collectively within this 3ʹ cluster. Basal promoter activity was decreased to 46%, 78% and 29% of control for each of the GC boxes mutated individually, and to 6% when the three were mutated collectively. To test the individual abilities of Sp1 and Sp3 to activate or repress HTR transcription, we conducted co-transfection experiments with wild-type or mutated HTR-promoter-luciferase constructs. Co-transfection with Sp1 significantly augmented wild-type HTR promoter activity. Sp3 alone did not affect activity, and inhibited Sp1-mediated activation. Competition for shared binding sites by Sp1 and Sp3 might differentially regulate HTR expression in vascular endothelial cells.

scholarly journals Estrogen Up-Regulates Hepatic Expression of Suppressors of Cytokine Signaling-2 and -3 in Vivo and in Vitro

Endocrinology ◽  
2004 ◽  
Vol 145 (12) ◽  
pp. 5525-5531 ◽  
Author(s):  
Gary M. Leong ◽  
Sofia Moverare ◽  
Jesena Brce ◽  
Nathan Doyle ◽  
Klara Sjögren ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Hepatoma Cells ◽  
Cytokine Signaling ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Wild Type ◽  
Hepatic Expression ◽  
Suppressors Of Cytokine Signaling

Abstract Suppressors of cytokine signaling (SOCS) are important negative regulators of cytokine action. We recently reported that estrogen stimulates SOCS-2 expression and inhibits GH signaling in kidney cells. The effects of estrogen on SOCS expression in other tissues are unclear. The aim of this study was to investigate in vivo and in vitro whether estrogen affected SOCS expression in the liver, a major target organ of GH. The in vivo hepatic effects of estrogen on ovariectomized mice lacking estrogen receptor (ER)-α, ERβ, or both and their wild-type littermates were examined by DNA microarray analysis. In vitro, the effects of estrogen on SOCS expression in human hepatoma cells were examined by reverse transcription quantitative PCR. Long-term (3 wk) estrogen treatment induced a 2- to 3-fold increase in hepatic expression of SOCS-2 and -3 in wild-type and ERβ knockout mice but not in those lacking ERα or both ER subtypes. Short-term treatment (at 24 h) increased the mRNA level of SOCS-3 but not SOCS-2. In cultured hepatoma cells, estrogen increased SOCS-2 and -3 mRNA levels by 2-fold in a time- and dose-dependent manner (P < 0.05). Estrogen induced murine SOCS-3 promoter activity by 2-fold (P < 0.05) in constructs containing a region between nucleotides −1862 and −855. Moreover, estrogen and GH had additive effects on the SOCS-3 promoter activity. In summary, estrogen, via ERα, up-regulated hepatic expression of SOCS-2 and -3, probably through transcriptional activation. This indicates a novel mechanism of estrogen regulation of cytokine action.

scholarly journals Genetic regulation, biochemical properties and physiological importance of arginase from Sinorhizobium meliloti

Microbiology ◽  
2020 ◽  
Vol 166 (5) ◽  
pp. 484-497 ◽  
Author(s):  
Alejandra Arteaga Ide ◽  
Victor M. Hernández ◽  
Liliana Medina-Aparicio ◽  
Edson Carcamo-Noriega ◽  
Lourdes Girard ◽  
...  
Keyword(s):  
Type Species ◽  
Sinorhizobium Meliloti ◽  
Arginase Activity ◽  
Wild Type ◽  
Mobility Shift ◽  
Content Type ◽  
Link Type ◽  
Arginine Catabolism

In bacteria, l-arginine is a precursor of various metabolites and can serve as a source of carbon and/or nitrogen. Arginine catabolism by arginase, which hydrolyzes arginine to l-ornithine and urea, is common in nature but has not been studied in symbiotic nitrogen-fixing rhizobia. The genome of the alfalfa microsymbiont Sinorhizobium meliloti 1021 has two genes annotated as arginases, argI1 (smc03091) and argI2 (sma1711). Biochemical assays with purified ArgI1 and ArgI2 (as 6His-Sumo-tagged proteins) showed that only ArgI1 had detectable arginase activity. A 1021 argI1 null mutant lacked arginase activity and grew at a drastically reduced rate with arginine as sole nitrogen source. Wild-type growth and arginase activity were restored in the argI1 mutant genetically complemented with a genomically integrated argI1 gene. In the wild-type, arginase activity and argI1 transcription were induced several fold by exogenous arginine. ArgI1 purified as a 6His-Sumo-tagged protein had its highest in vitro enzymatic activity at pH 7.5 with Ni2+ as cofactor. The enzyme was also active with Mn2+ and Co2+, both of which gave the enzyme the highest activities at a more alkaline pH. The 6His-Sumo-ArgI1 comprised three identical subunits based on the migration of the urea-dissociated protein in a native polyacrylamide gel. A Lrp-like regulator (smc03092) divergently transcribed from argI1 was required for arginase induction by arginine or ornithine. This regulator was designated ArgIR. Electrophoretic mobility shift assays showed that purified ArgIR bound to the argI1 promoter in a region preceding the predicted argI1 transcriptional start. Our results indicate that ArgI1 is the sole arginase in S. meliloti , that it contributes substantially to arginine catabolism in vivo and that argI1 induction by arginine is dependent on ArgIR.

scholarly journals Developmental aspects of adipose tissue in GH receptor and prolactin receptor gene disrupted mice: site-specific effects upon proliferation, differentiation and hormone sensitivity

2006 ◽  
Vol 191 (1) ◽  
pp. 101-111 ◽  
Author(s):  
David J Flint ◽  
Nadine Binart ◽  
Stephanie Boumard ◽  
John J Kopchick ◽  
Paul Kelly
Keyword(s):  
Adipose Tissue ◽  
Receptor Gene ◽  
Metabolic Effects ◽  
Wild Type ◽  
Extrinsic Factors ◽  
Differentiation Potential ◽  
Site Specific ◽  
Proliferation And Differentiation

Direct metabolic effects of GH on adipose tissue are well established, but effects of prolactin (PRL) have been more controversial. Recent studies have demonstrated PRL receptors on adipocytes and effects of PRL on adipose tissue in vitro. The role of GH in adipocyte proliferation and differentiation is also controversial, since GH stimulates adipocyte differentiation in cell lines, whereas it stimulates proliferation but inhibits differentiation of adipocytes in primary cell culture. Using female gene disrupted (ko) mice, we showed that absence of PRL receptors (PRLRko) impaired development of both internal and s.c. adipose tissue, due to reduced numbers of adipocytes, an effect differing from that of reduced food intake, where cell volume is decreased. In contrast, GHRko mice exhibited major decreases in the number of internal adipocytes, whereas s.c. adipocyte numbers were increased, even though body weight was decreased by 40–50%. The changes in adipose tissue in PRLRko mice appeared to be entirely due to extrinsic factors since preadipocytes proliferated and differentiated in similar fashion to wild-type animals in vitro and their response to insulin and isoproterenol was similar to wild-type animals. This contrasted with GHRko mice, where s.c. adipocytes proliferated, differentiated, and responded to hormones in identical fashion to controls, whereas parametrial adipocytes exhibited markedly depressed proliferation and differentiation potential and failed to respond to insulin or noradrenaline. Our results provide in vivo evidence that both GH and PRL stimulate differentiation of adipocytes but that the effects of GH are site specific and induce intrinsic changes in the precursor population, which are retained in vitro.

Abstract 5566: Recovery of Erk Signaling Restores Defective Angiogenesis and Arteriogenesis in Synectin-Deficient Animals

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Bin Ren ◽  
Arpita Mukhopadhyay* ◽  
Anthony A Lanahan ◽  
Zhen W Zhuang ◽  
Karen L Moodie ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Branching Morphogenesis ◽  
Erk Signaling ◽  
Dependent Manner ◽  
Wild Type ◽  
Erk Activation ◽  
Arterial Development ◽  
Constitutively Active

Background : Arterial morphogenesis is an important and poorly understood process. We have previously demonstrated that disruption of synectin gene expression in mice and zebrafish results in impaired arterial development and branching morphogenesis. Synectin null endothelial cells demonstrate reduced VEGF responsiveness in terms of migration, proliferation and differentiation and ERK-1/2 activation (Chittenden et al, Dev Cell 2006). Since ERK has been established as major participants in the regulation of cell growth and differentiation and Erk activation has been previously linked to arterial morphogenesis, we evaluated whether activation of Erk signaling in synectin disrupted mice and zebrafish as well as synectin KO arterial endothelial cells (ECs) would restore defective migration, arterial differentiation, angiogenesis and arteriogenesis. To stimulate ERK signaling we used partial inhibition of PI3-K activity to reduce Akt-dependent suppression of Raf1 activation or introduction of constitutively active ERK construct. Methods : In vitro studies were conducted with primary arterial ECs isolated from synectin wild type (WT) and knock out (KO) mice. In vivo studies were carried out in WT and synectin deficient mice and synectin knockdown zebrafish embryos. Results: Exposure of synectin −/− arterial EC to two selective PI3K inhibitors GS4898 or LY294002 in vitro restored ERK activation in a dose-dependent manner and returned cell migration and in vitro branching morphogenesis to wild type levels. Transduction of a constitutively active ERK construct in vitro or in a Matrigel model in vivo had similar effect. Systemic treatment of synectin −/− mice with GS4898 fully restored impaired angiogenesis and arterial morphogenesis in adult animals in the setting of hindlimb ischemia. Similar treatment nearly completely restored arterial development defects in zebrafish treated with a synectin morpholino. Conclusions: ERK activation plays a key role in arteriogenesis both in adult tissues and during embryonic development. Activation of compromised ERK-1/2 signaling may be a novel therapeutic intervention to stimulate arteriogenesis.

Factor VIIa induces extracellular vesicles from the endothelium: A potential mechanism for its hemostatic effect

Blood ◽  
2021 ◽  
Author(s):  
Kaushik Das ◽  
Shiva Keshava ◽  
Shabbir A Ansari ◽  
Vijay Kumar Reddy Kondreddy ◽  
Charles Esmon ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Factor Viia ◽  
Mutant Mice ◽  
In Vivo Studies ◽  
Cell Protein ◽  
Wild Type ◽  
Hemostatic Effect

Recombinant FVIIa (rFVIIa) is used as a hemostatic agent to treat bleeding disorders in hemophilia patients with inhibitors and other groups of patients. Our recent studies showed that FVIIa binds endothelial cell protein C receptor (EPCR) and induces protease-activated receptor 1 (PAR1)-mediated biased signaling. The importance of FVIIa-EPCR-PAR1-mediated signaling in hemostasis is unknown. In the present study, we show that FVIIa induces the release of extracellular vesicles (EVs) from endothelial cells both in vitro and in vivo. Silencing of EPCR or PAR1 in endothelial cells blocked the FVIIa-induced generation of EVs. Consistent with these data, FVIIa treatment enhanced the release of EVs from murine brain endothelial cells isolated from wild-type, EPCR overexpressors, and PAR1-R46Q mutant mice, but not EPCR-deficient or PAR1-R41Q mutant mice. In vivo studies revealed that administration of FVIIa to wild-type, EPCR overexpressors, and PAR1-R46Q mutant mice, but not EPCR-deficient or PAR1-R41Q mutant mice, increase the number of circulating EVs. EVs released in response to FVIIa treatment exhibit enhanced procoagulant activity. Infusion of FVIIa-generated EVs and not control EVs to platelet-depleted mice increased thrombin generation at the site of injury and reduced blood loss. Administration of FVIIa-generated EVs or generation of EVs endogenously by administering FVIIa augmented the hemostatic effect of FVIIa. Overall, our data reveal that FVIIa treatment, through FVIIa-EPCR-PAR1 signaling, releases EVs from the endothelium into the circulation, and these EVs contribute to the hemostatic effect of FVIIa.

Exogenous clustered neuropilin 1 enhances vasculogenesis and angiogenesis

Blood ◽  
2001 ◽  
Vol 97 (6) ◽  
pp. 1671-1678 ◽  
Author(s):  
Yoshihiro Yamada ◽  
Nobuyuki Takakura ◽  
Hirofumi Yasue ◽  
Hisao Ogawa ◽  
Hajime Fujisawa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Low Dose ◽  
Vascular Development ◽  
Vegf Receptor ◽  
Wild Type ◽  
Flag Epitope ◽  
Neuropilin 1 ◽  
Vasculogenesis And Angiogenesis

Neuropilin 1 (NP-1) is a receptor for vascular endothelial growth factor (VEGF) 165 (VEGF165) and acts as a coreceptor that enhances VEGF165 function through tyrosine kinase VEGF receptor 2 (VEGFR-2). Transgenic overexpression of np-1results in an excess of capillaries and blood vessels and a malformed heart. Thus, NP-1 may have a key role in vascular development. However, how NP-1 regulates vascular development is not well understood. This study demonstrates how NP-1 can regulate vasculogenesis and angiogenesis in vitro and in vivo. In homozygous np-1mutant (np-1−/−) murine embryos, vascular sprouting was impaired in the central nervous system and pericardium. Para-aortic splanchnopleural mesoderm (P-Sp) explants fromnp-1−/− mice also had vascular defects in vitro. A monomer of soluble NP-1 (NP-1 tagged with Flag epitope) inhibited vascular development in cultured wild-type P-Sp explants by sequestering VEGF165. In contrast, a dimer of soluble NP-1 (NP-1 fused with the Fc part of human IgG) enhanced vascular development in cultured wild-type P-Sp explants. Moreover, the NP-1–Fc rescued the defective vascular development in culturednp-1−/− P-Sp explants. A low dose of VEGF alone did not promote phosphorylation of VEGFR-2 on endothelial cells from np-1−/− embryos, but simultaneous addition of a low dose of VEGF and NP-1–Fc phosphorylated VEGFR-2 significantly. Moreover, NP-1–Fc rescued the defective vascularity of np-1−/− embryos in vivo. These results suggest that a dimer form of soluble NP-1 delivers VEGF165 to VEGFR-2–positive endothelial cells and promotes angiogenesis.

Prostaglandin E Synthase Is Upregulated By Gas6 during Cancer-Induced Venous Thromboembolism

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 111-111
Author(s):  
Meghedi N Aghourian ◽  
Catherine A Lemarie ◽  
Francois-Rene Bertin ◽  
Mark D Blostein
Keyword(s):  
Endothelial Cells ◽  
Venous Thromboembolism ◽  
Microarray Analysis ◽  
Conflicts Of Interest ◽  
Prostaglandin E ◽  
Wild Type ◽  
Prostaglandin E Synthase ◽  
Murine Lung

Abstract Venous thromboembolism (VTE) is the most common morbid complication related to cancer and its treatments. Although malignancies are characterized by a hypercoagulable state leading to VTE, the pathophysiology of this state has not been well studied. Growth arrest specific 6 (Gas6) is a protein that has pro-coagulant properties. Gas6 deficient mice develop smaller venous thrombi as compared to wild type mice, and express less tissue factor in the endothelium when challenged with thrombotic stimuli. We hypothesize that Gas6 may be involved in cancer-induced venous thrombosis. In order to test this hypothesis, venous thrombi were induced in wild type (WT) and Gas6 null (-/-) mice injected with M27 murine lung cancer cell lines. Thrombus size was measured using ultrasonography, thrombus weight and histology. We observed that WT mice with cancer developed larger thrombi than their healthy counterparts (p<0.05). However, these larger thrombi induced by cancer were not seen in Gas6-/- mice, suggesting that Gas6 has a pathophysiologic role in promoting malignancy associated VTE. Whole genome microarray analysis was then used to identify differential gene expression in WT and Gas6-/- endothelial cells co-cultured with M27 murine lung carcinoma cells. Microarray analysis revealed that prostaglandin E synthase (PTGES) was increased in WT endothelial cells but not in Gas6-/- cells co-cultured with M27. These results were confirmed using real-time PCR and immunofluorescence staining (p<0.05). In WT endothelial cells, PTGES expression was regulated through ERK1/2 phosphorylation. We also show that co-culture of WT endothelial cells with M27 augments the secretion of PGE2, the enzymatic product of PTGES. PGE2 activates platelets in vitro after binding to its receptor, EP3. In vivo, EP3 receptor antagonism reversed the effect of cancer-induced thrombosis in WT mice. These results show that Gas6, through upregulation of PGE2, contributes to cancer-induced VTE. Disclosures No relevant conflicts of interest to declare.

The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences

1989 ◽  
Vol 9 (11) ◽  
pp. 4706-4712
Author(s):  
A H Siddiqui ◽  
M C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Complex Formation ◽  
Binding Activity ◽  
Lacz Gene ◽  
Wild Type ◽  
Mobility Shift ◽  
Proline Utilization

The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction.

Protease-activated receptors 1 and 4 mediate thrombin signaling in endothelial cells

Blood ◽  
2003 ◽  
Vol 102 (9) ◽  
pp. 3224-3231 ◽  
Author(s):  
Hiroshi Kataoka ◽  
Justin R. Hamilton ◽  
David D. McKemy ◽  
Eric Camerer ◽  
Yao-Wu Zheng ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Wild Type ◽  
Protease Activated Receptors ◽  
Erk Phosphorylation ◽  
Low Concentrations ◽  
Extracellular Signal ◽  
High Concentrations ◽  
Mouse Aorta

AbstractDefining the relative importance of protease-activated receptors (PARs) for thrombin signaling in mouse endothelial cells is critical for a basic understanding of thrombin signaling in these cells and for the rational use of knockout mice to probe the roles of thrombin's actions on endothelial cells in vivo. We examined thrombin- and PAR agonist–induced increases in cytoplasmic calcium, phosphoinositide hydrolysis, extracellular signal-regulated kinase (ERK) phosphorylation, and gene expression in endothelial cells from wild-type and PAR-deficient mice. PAR1 and PAR4 agonists triggered responses in wild-type but not in Par1–/– and Par4–/– endothelial cells, respectively. Calcium imaging confirmed that a substantial fraction of individual endothelial cells responded to both agonists. Compared with wild-type cells, Par1–/– endothelial cells showed markedly decreased responses to low concentrations of thrombin, and cells that lacked both PAR1 and PAR4 showed no responses to even high concentrations of thrombin. Similar results were obtained when endothelial-dependent vasorelaxation of freshly isolated mouse aorta was used as an index of signaling in native endothelial cells. Thus PAR1 is the major thrombin receptor in mouse endothelial cells, but PAR4 also contributes. These receptors serve at least partially redundant roles in endothelial cells in vitro and in vivo and together are necessary for the thrombin responses measured.

Oncogenic Transcription Factor Evi1 Regulates Hematopoietic Stem Cell Proliferation through GATA-2 Expression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 228-228 ◽  
Author(s):  
Hiromi Yuasa ◽  
Yuichi Oike ◽  
Atsushi Iwama ◽  
Daisuke Sugiyama ◽  
Ichiro Nishikata ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Network Formation ◽  
Vascular System ◽  
Upstream Region ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Adult Bone

Abstract Chromosomal abnormalities, such as translocation, mutation or deletion, are central to the pathogenesis of human cancers. Recently, several transcription factors have been isolated as genes responsible for leukemia from the region surrounding chromosomal breakpoints, which are implicated in the regulation of normal hematopoiesis. Among on them, ecotropic viral integration site-1 (Evi1) is a transcription factor activated by retroviral integration in murine leukemias and chromosomal rearrangements in human leukemias. Evi1 is a zinc finger transcription factor and contains two separated DNA-binding domains. It was reported that Evi1−/− embryos die at approximately E10.5, exhibiting widespread hypocellularity and hemorrhaging. However, the role in normal hematopoiesis or authentic target genes of Evi1 has not been elucidated. Here, we show that Evi1 is predominantly expressed in hematopoietic stem cells (HSCs) in embryos and adult bone marrows, and Evi1−/− embryos are markedly decreased in numbers of HSC. One embryo-equivalent cells from E9.5 P-Sp of Evi1+/+, Evi1+/− and Evi1−/− embryos (Ly5.2) were transplanted into a busulfan-conditioned newborn recipient (Ly5.1). At 2 months posttransplant, donor-derived Ly5.2(+) cells could be detected in the peripheral blood of the recipients that received P-Sp cells from the Evi1+/+ and Evi1+/− but not from the Evi1−/− embryos. Thus, Evi1 is critical for the generation of HSCs in the P-Sp. Both Evi1−/− embryos and yolk sac showed marked retardation in the organization of the vascular system, particularly in vascular remodeling, compared with controls. Using an in vitro P-Sp culture analysis, we found normal in vitro differentiation of endothelial cells in Evi1−/− P-Sp cultures but defects in their in vitro network formation, which is normally promoted by Ang-1 secreted from developing HSCs in P-Sp cultures. HSCs from adult bone marrow or HSCs from E9.5 wild type embryos rescued defective angiogenesis in Evi1−/− embryos. The fine vascular network coincided with the region where HSCs formed a colony. Their round morphology confirmed that exogenous adult HSCs did not differentiate into elongated endothelial cells. We showed that recombinant Ang-1 alone restored the defective angiogenesis in Evi1−/− embryos to a wild type level. It is suggested that the defect in hematopoietic cells induced defective angiogenesis in Evi1−/− embryos mediated by Ang-1. Notably, mRNA expression of GATA-2, which is essential for proliferation of definitive HSCs, was profoundly reduced in Evi1−/− embryos. Analysis of the GATA-2 promotor revealed that Evi1 directly binds to the 5′ upstream region of the GATA−2 exon and positively regulates its promoter activity in vitro and in vivo. Restoration of GATA-2 expression dramatically rescued the defective expansion of Evi1−/− embryos HSCs in vitro. Our results reveal that GATA-2 is a critical in vivo target for Evi1 and indicate hierarchical regulation of the HSC pool by transcriptional regulators.

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