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Like expression of other cell-type-specific genes, expression of the insulin gene depends on the actions of a unique set of nuclear activators. These activators cooperate synergistically in building a transcriptional activation complex that binds to the regulatory domains of the gene and activates the basal RNA polymerase machinery (reviewed in reference 7). The complexity and specificity of the interactions among these activators limit the cell types capable of building a functional activation complex. Dissection of these interactions provides insight into the mechanism by which insulin expression is limited to the correct cell type.In adult mammals, activation of the insulin gene is tightly restricted to the ␤ cells in the pancreatic islets of Langerhans, where it is expressed at high levels. This specificity is reflected in the restricted function of the insulin promoter, the proximal few hundred base pairs of which can replicate the specificity of the intact gene (19,50). Because of the complexity of the intact promoter (9, 13, 24), a short portion of the rat insulin I promoter between bp Ϫ247 and Ϫ197 upstream from the transcription initiation site has been used as a model of the types of synergistic interactions that combine to give the characteristic activity of the full promoter (15). This 50-bp fragment contains at least three distinct DNA-binding sites named E2, A3, and A4 (13). The E and A elements synergize: neither has significant activity on its own, but in combination E and A elements produce ␤-cell specific transcriptional activation (15,23).The E2 element functions as a recognition site for dimers of basic helix-loop-helix (bHLH) proteins, including a heterodimer of the ubiquitous bHLH protein E47/Pan1 and the neuroendocrine specific bHLH protein BETA2/NeuroD1 (35). The A elements each contain the sequence TAAT and have been shown to bind to several homeodomain proteins found in ␤-cell nuclei (12,16,22, 32,37 (16,38,40).The LIM homeodomain protein Lmx1.1 contains two LIM domains that form zinc-binding structures in the amino end of the molecule. The second of these two LIM domains (LIM2) directly binds to the bHLH domain of E47/Pan1 and mediates the synergy between Lmx1.1 and E47/Pan1. This interaction is specific, since analogous domains from other LIM proteins and bHLH proteins cannot substitute for the LIM2 domain of Lmx1.1 or the bHLH domain of E47/Pan1, respectively (20).PDX-1 plays an important role both in the development of the pancreas and in maintaining ␤-cell function. Mice with a targeted disruption of the pdx1 gene selectively lack a pancreas (2, 21, 36). Similar pancreatic agenesis has been found in a human patient with a single nucleotide deletion in the pdx1 gene (46). If the pancreas is allowed to develop with an intact pdx1 gene, and the pdx1 gene is disrupted only in mature ␤ cells, diabetes ensues due to impaired ␤-cell function (3). This

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 ®nger deletion of GATA-1 (DNT, DNF or DCF, 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 DNF and DCF transgenes failed to rescue the GATA-1.05/Y pups. However, transgenic mice expressing DNT, but not the DNF protein, were able to rescue de®nitive hematopoiesis. In embryos, while neither the DCF protein nor a mutant missing both N-terminal domains (DNTNF) was able to support primitive erythropoiesis, the two independent DNT and DNF mutants could support primitive erythropoiesis. Thus, lineage-speci®c transgenic rescue of the GATA-1 mutant mouse revealed novel properties that are conferred by speci®c domains of GATA-1 during primitive and de®nitive erythropoiesis, and demonstrate that the NT and NF moieties lend complementary, but distinguishable properties to the function of GATA-1.

Hypoxia-inducible factors (HIFs) are crucial for oxygen homeostasis during both embryonic development and postnatal life. Here we show that a novel HIF family basic helix-loop-helix (bHLH) PAS (Per-Arnt-Sim) protein, which is expressed predominantly during embryonic and neonatal stages and thereby designated NEPAS (neonatal and embryonic PAS), acts as a negative regulator of HIF-mediated gene expression. NEPAS mRNA is derived from the HIF-3␣ gene by alternative splicing, replacing the first exon of HIF-3␣ with that of inhibitory PAS. NEPAS can dimerize with Arnt and exhibits only low levels of transcriptional activity, similar to that of HIF-3␣. NEPAS suppressed reporter gene expression driven by HIF-1␣ and HIF-2␣. By generating mice with a targeted disruption of the NEPAS/HIF-3␣ locus, we found that homozygous mutant mice (NEPAS/ HIF-3␣ ؊/؊ ) were viable but displayed enlargement of the right ventricle and impaired lung remodeling. The expression of endothelin 1 and platelet-derived growth factor ␤ was increased in the lung endothelial cells of NEPAS/HIF-3␣-null mice. These results demonstrate a novel regulatory mechanism in which the activities of HIF-1␣ and HIF-2␣ are negatively regulated by NEPAS in endothelial cells, which is pertinent to lung and heart development during the embryonic and neonatal stages.Hypoxia-inducible factors (HIFs) are crucial for oxygen homeostasis during both embryonic development and postnatal life. HIFs are heterodimeric transcription factors consisting of ␣ and ␤ subunits. To date, three ␣ subunits (HIF-1␣, HIF-2␣, and HIF-3␣) and one ␤ subunit (HIF-1␤, also called Arnt [aryl hydrocarbon receptor nuclear translocator]) have been identified (10, 34, 37). Oxygen-dependent activity of HIFs is mainly regulated through the stability of their ␣ subunits. Under the normoxic condition, HIF-␣ protein is rapidly degraded through the ubiquitin-proteasomal pathway. During this process, HIF-␣ is hydroxylated by proline hydroxylases and specifically interacts with the von Hippel-Lindau (VHL) tumor suppressor protein (8, 21), which acts as a component of E3 ubiquitin ligase and targets HIF-␣ molecules for ubiquitination and subsequent degradation (12). Under low oxygen tension, hydroxylation of HIF-␣ is significantly reduced because the activity of proline hydroxylases is repressed by hypoxia. Since VHL can recognize exclusively the hydroxylated HIF-␣ molecules, in the hypoxic condition HIF-␣ is stabilized and activates transcription of target genes with Arnt in the nucleus.Although it is indisputable that this ubiquitin-proteasomal pathway plays a central role in determining HIF activity, an additional regulatory mechanism should be considered under certain conditions. For instance, the availability of oxygen is limited in utero and embryos are continuously exposed to hypoxia (17). Under such conditions, it is likely that HIF-␣ proteins are no longer degraded and accumulate into the nucleus. Given the fact that both HIF-1␣ and HIF-2␣ are required for early embryonic development (13,25,33), HIF...

The transcription factors GATA-1 and GATA-2 play key roles in gene regulation during erythropoiesis. Gene ablation studies in mouse revealed that GATA-2 is crucial for the maintenance and proliferation of immature hematopoietic progenitors, whereas GATA-1 is essential for the survival of erythroid progenitors as well as the terminal differentiation of erythroid cells. Both GATA-1 and GATA-2 are regulated in a cell-type-specific manner, their expression being strictly controlled during the development and differentiation of erythroid cells. Closer examination revealed a cross-regulatory mechanism by which GATA-1 can control the expression of GATA-2 and vice versa, possibly via essential GATA binding sites in their cis-acting elements. In addition, recent studies identified several human inherited hematopoietic disorders that are caused by mutations in cis-acting GATA binding motifs or mutations in GATA-1 itself.

Umbilical cord blood (UCB) has been used as a potential source of various kinds of stem cells, including hematopoietic stem cells, mesenchymal stem cells, and endothelial progenitor cells (EPCs), for a variety of cell therapies. Recently, EPCs were introduced for restoring vascularization in ischemic tissues. An appropriate procedure for isolating EPCs from UCB is a key issue for improving therapeutic efficacy and eliminating the unexpected expansion of nonessential cells. Here we report a novel method for isolating EPCs from UCB by a combination of negative immunoselection and cell culture techniques. In addition, we divided EPCs into 2 subpopulations according to the aldehyde dehydrogenase (ALDH) activity. We found that EPCs with low ALDH activity (Alde-Low) possess a greater ability to proliferate and migrate compared to those with high ALDH activity (Alde- High IntroductionEndothelial progenitor cells (EPCs) were originally identified as a population of stem cells in human peripheral blood (PB) and characterized by the expression of CD34, KDR (VEGFR-2), and CD133 markers. 1-3 Subsequently, EPCs have been isolated from other sources, such as bone marrow (BM), fetal liver, and umbilical cord blood (UCB). [4][5][6] Recent studies have shown that EPCs are a potential tool for therapeutic angiogenesis in the treatment of patients suffering from severe limb ischemia or myocardial infarction. 7 EPCs have been identified as contributors to vessel development in both normal physiological processes such as wound healing and pathological processes such as cancer. 8 The definition of an EPC has been controversial and hence the method to isolate EPCs has been variable among investigators. 9 Several studies have demonstrated that there are 2 distinct types of EPCs, the so-called early and late EPCs, which appear sequentially. 10 Early EPCs, likely originating from monocytic/dendritic cells, are characterized by the expression of CD45 and CD14, together with some endothelial cell (EC) markers, and have a short lifespan of 3 to 4 weeks. On the other hand, late EPCs rapidly grow out from mononuclear cells with a cobblestone-like morphology and are characterized by EC markers such as CD31, CD34, VEGFR2, and VE-cadherin, but are negative for myeloid markers. 10 However, Yoder et al have recently demonstrated that progeny of the CD45 ϩ CD14 ϩ cells that coexpress EC markers are not endothelial progenitor cells but hematopoietic-derived myeloid progenitor cells. 11 Alternatively, Ingram et al divided ECs into several subpopulations according to their clonogenic and proliferative potential. 12 They identified a population of highly proliferative endothelial potential-colony-forming cells (HPP-ECFCs), which form secondary and ternary colonies in human UCB. Given the therapeutic usefulness of EPCs, the effective isolation of highly proliferative EPCs is centrally important for the generation of reliable and safe cell-based therapy.Aldehyde dehydrogenase (ALDH) is an enzyme responsible for oxidizing intercellular aldehydes. 13 This ...

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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.