The transcription factor EPAS-1/hypoxia-inducible factor 2α plays an important role in vascular remodeling

We have studied the role of the basic helix–loop–helix–PAS transcription factor EPAS-1/hypoxia-inducible factor 2α in vascular development by gene targeting. In ICR/129 Sv outbred background, more than half of the mutants displayed varying degrees of vascular disorganization, typically in the yolk sac, and died in utero between embryonic day (E)9.5 and E13.5. In mutant embryos directly derived from EPAS-1−/− embryonic stem cells (hence in 129 Sv background), all embryos developed severe vascular defects both in the yolk sac and embryo proper and died between E9.5 and E12.5. Normal blood vessels were formed by vasculogenesis but they either fused improperly or failed to assemble into larger vessels later during development. Our results suggest that EPAS-1 plays an important role at postvasculogenesis stages and is required for the remodeling of the primary vascular network into a mature hierarchy pattern.

Target gene search for the metal-responsive transcription factor MTF-1

Activation of genes by heavy metals, notably zinc, cadmium and copper, depends on MTF-1, a unique zinc finger transcription factor conserved from insects to human. Knockout of MTF-1 in the mouse results in embryonic lethality due to liver decay, while knockout of its best characterized target genes, the stress-inducible metallothionein genes I and II, is viable, suggesting additional target genes of MTF-1. Here we report on a multi-pronged search for potential target genes of MTF-1, including microarray screening, SABRE selective amplification, a computer search for MREs (DNA-binding sites of MTF-1) and transfection of reporter genes driven by candidate gene promoters. Some new candidate target genes emerged, including those encoding α-fetoprotein, the liver-enriched transcription factor C/EBPα and tear lipocalin/von Ebner’s gland protein, all of which have a role in toxicity/the cell stress response. In contrast, expression of other cell stress-associated genes, such as those for superoxide dismutases, thioredoxin and heat shock proteins, do not appear to be affected by loss of MTF-1. Our experiments have also exposed some problems with target gene searches. First, finding the optimal time window for detecting MTF-1 target genes in a lethal phenotype of rapid liver decay proved problematical: 12.5-day-old mouse embryos (stage E12.5) yielded hardly any differentially expressed genes, whereas at stage 13.0 reduced expression of secretory liver proteins probably reflected the onset of liver decay, i.e. a secondary effect. Likewise, up-regulation of some proliferation-associated genes may also just reflect responses to the concomitant loss of hepatocytes. Another sobering finding concerns γ-glutamylcysteine synthetasehc (γ-GCShc), which controls synthesis of the antioxidant glutathione and which was previously suggested to be a target gene contributing to the lethal phenotype in MTF-1 knockout mice. γ-GCShc mRNA is reduced at the onset of liver decay but MTF-1 null mutant embryos manage to maintain a very high glutathione level until shortly before that stage, perhaps in an attempt to compensate for low expression of metallothioneins, which also have a role as antioxidants.

Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1.

The X chromosome-linked transcription factor GATA-1 is expressed specifically in erythroid, mast, megakaryocyte, and eosinophil lineages, as well as in hematopoietic progenitors. Prior studies revealed that gene-disrupted GATA-1- embryonic stem cells give rise to adult (or definitive) erythroid precursors arrested at the proerythroblast stage in vitro and fail to contribute to adult red blood cells in chimeric mice but did not clarify a role in embryonic (or yolk sac derived) erythroid cells. To examine the consequences of GATA-1 loss on embryonic erythropoiesis in vivo, we inactivated the GATA-1 locus in embryonic stem cells by gene targeting and transmitted the mutated allele through the mouse germ line. Male GATA-1- embryos die between embryonic day 10.5 and 11.5 (E10.5-E11.5) of gestation. At E9.5, GATA-1- embryos exhibit extreme pallor yet contain embryonic erythroid cells arrested at an early proerythroblast-like stage of their development. Embryos stain weakly with benzidine reagent, and yolk sac cells express globin RNAs, indicating globin gene activation in the absence of GATA-1. Female heterozygotes (GATA-1+/-) are born pale due to random inactivation of the X chromosome bearing the normal allele. However, these mice recover during the neonatal period, presumably as a result of in vivo selection for progenitors able to express GATA-1. Our findings conclusively establish the essential role for GATA-1 in erythropoiesis within the context of the intact developing mouse and further demonstrate that the block to cellular maturation is similar in GATA-1- embryonic and definitive erythroid precursors. Moreover, the recovery of GATA-1+/- mice from anemia seen at birth provides evidence indicating a role for GATA-1 at the hematopoietic progenitor cell level.

Transcription factor EGR-1 suppresses the growth and transformation of human HT-1080 fibrosarcoma cells by induction of transforming growth factor beta 1.

The early growth response 1 (EGR-1) gene product is a transcription factor with role in differentiation and growth. We have previously shown that expression of exogenous EGR-1 in various human tumor cells unexpectedly and markedly reduces growth and tumorigenicity and, conversely, that suppression of endogenous Egr-1 expression by antisense RNA eliminates protein expression, enhances growth, and promotes phenotypic transformation. However, the mechanism of these effects remained unknown. The promoter of human transforming growth factor beta 1 (TGF-beta 1) contains two GC-rich EGR-1 binding sites. We show that expression of EGR-1 in human HT-1080 fibrosarcoma cells uses increased secretion of biologically active TGF-beta 1 in direct proportion (rPearson = 0.96) to the amount of EGR-1 expressed and addition of recombinant human TGF-beta 1 is strongly growth-suppressive for these cells. Addition of monoclonal anti-TGF-beta 1 antibodies to EGR-1-expressing HT-1080 cells completely reverses the growth inhibitory effects of EGR-1. Reporter constructs bearing the EGR-1 binding segment of the TGF-beta 1 promoter was activated 4- to 6-fold relative to a control reporter in either HT-1080 cells that stably expressed or parental cells cotransfected with an EGR-1 expression vector. Expression of delta EGR-1, a mutant that cannot interact with the corepressors, nerve growth factor-activated factor binding proteins NAB1 and NAB2, due to deletion of the repressor domain, exhibited enhanced transactivation of 2- to 3.5-fold over that of wild-type EGR-1 showing that the reporter construct reflected the appropriate in vivo regulatory context. The EGR-1-stimulated transactivation was inhibited by expression of the Wilms tumor suppressor, a known specific DNA-binding competitor. These results indicate that EGR-1 suppresses growth of human HT-1080 fibrosarcoma cells by induction of TGF-beta 1.

Selective constraints on the activation domain of transcription factor Pit-1.

The POU transcription factor Pit-1 activates members of the prolactin/growth hormone gene family in specific endocrine cell types of the pituitary gland. Although Pit-1 is structurally conserved among vertebrate species, evolutionary changes in the pattern of Pit-1 RNA splicing have led to a notable "contraction" of the transactivation domain in the mammalian lineage, relative to Pit-1 in salmonid fish. By site-directed mutagenesis we demonstrate that two splice insertions in salmon Pit-1, called beta (29 aa) and gamma (33 aa), are critical for cooperative activation of the salmon prolactin gene. Paradoxically, Pit-1-dependent activation of the prolactin gene in rat is enhanced in the absence of the homologous beta-insert sequence. This apparent divergence in the mechanism of activation of prolactin genes by Pit-1 is target gene specific, as activation of rat and salmon growth hormone genes by Pit-1 splice variants is entirely conserved. Our data suggest that efficient activation of the prolactin gene in the vertebrate pituitary has significantly constrained the pattern of splicing within the Pit-1 transactivation domain. Rapid evolutionary divergence of prolactin gene function may have demanded changes in Pit-1/protein interactions to accommodate new patterns of transcriptional control by developmental or physiological factors.

Transcription factor GATA-1 permits survival and maturation of erythroid precursors by preventing apoptosis.

The transcription factor GATA-1 recognizes a consensus motif present in regulatory regions of numerous erythroid-expressed genes. Mouse embryonic stem cells lacking GATA-1 cannot form mature red blood cells in vivo. In vitro differentiation of GATA-1- embryonic stem cells gives rise to a population of committed erythroid precursors that exhibit developmental arrest and death. We show here that the demise of GATA-1- erythroid cells is accompanied by several features characteristics of apoptosis. This process occurs despite normal expression of all known GATA target genes examined, including the erythropoietin receptor, and independent of detectable accumulation of the tumor suppressor protein p53. Thus, in addition to its established role in regulating genes that define the erythroid phenotype, GATA-1 also supports the viability of red cell precursors by suppressing apoptosis. These results illustrate the multifunctional nature of GATA-1 and suggest a mechanism by which other hematopoietic transcription factors may ensure the development of specific lineages.

Reduction of insulin gene transcription in HIT-T15 beta cells chronically exposed to a supraphysiologic glucose concentration is associated with loss of STF-1 transcription factor expression.

Chronic exposure of HIT-T15 beta cells to elevated glucose concentrations leads to decreased insulin gene transcription. The reduction in expression is accompanied by diminished binding of a glucose-sensitive transcription factor (termed GSTF) that interacts with two (A+T)-rich elements within the 5' flanking control region of the insulin gene. In this study we examined whether GSTF corresponds to the recently cloned insulin gene transcription factor STF-1, a homeodomain protein whose expression is restricted to the nucleus of endodermal cells of the duodenum and pancreas. We found that an affinity-purified antibody recognizing STF-1 supershifted the GSTF activator complex formed from HIT-T15 extracts. In addition, we demonstrated a reduction in STF-1 mRNA and protein levels that closely correlated with the change in GSTF binding in HIT-T15 cells chronically cultured under supraphysiologic glucose concentrations. The reduction in STF-1 expression in these cells could be accounted for by a change in the rate of STF-1 gene transcription, suggesting a posttranscriptional control mechanism. In support of this hypothesis, no STF-1 mRNA accumulated in HIT-T15 cells passaged in 11.1 mM glucose. The only RNA species detected was a 6.4-kb STF-1 RNA species that hybridized with 5' and 3' STF-1-specific cDNA probes. We suggest that the 6.4-kb RNA represents an STF-1 mRNA precursor and that splicing of this RNA is defective in these cells. Overall, this study suggests that reduced expression of a key transcriptional regulatory factor, STF-1, contributes to the decrease in insulin gene transcription in HIT-T15 cells chronically cultured in supraphysiologic glucose concentration.

Complexity of the erythroid transcription factor NF-E2 as revealed by gene targeting of the mouse p18 NF-E2 locus.

High-level globin expression in erythroid precursor cells depends on the integrity of NF-E2 recognition sites, transcription factor AP-1-like protein-binding motifs, located in the upstream regulatory regions of the alpha- and beta-globin loci. The NF-E2 transcription factor, which recognizes these sites, is a heterodimer consisting of (i) p45 NF-E2 (the larger subunit), a hematopoietic-restricted basic leucine zipper protein, and (ii) a widely expressed basic leucine zipper factor, p18 NF-E2, the smaller subunit. p18 NF-E2 protein shares extensive homology with the maf protooncogene family. To determine an in vivo role for p18 NF-E2 protein we disrupted the p18 NF-E2-encoding gene by homologous recombination in murine embryonic stem cells and generated p18 NF-E2-/- mice. These mice are indistinguishable from littermates throughout all phases of development and remain healthy in adulthood. Despite the absence of expressed p18 NF-E2, DNA-binding activity with the properties of the NF-E2 heterodimer is present in fetal liver erythroid cells of p18 NF-E2-/- mice. We speculate that another member of the maf basic leucine zipper family substitutes for the p18 subunit in a complex with p45 NF-E2. Thus, p18 NF-E2 per se appears to be dispensable in vivo.

Specificity in transforming growth factor β-induced transcription of the plasminogen activator inhibitor-1 gene: Interactions of promoter DNA, transcription factor μE3, and Smad proteins

Transforming growth factor β (TGF-β) regulates a broad range of biological processes, including cell growth, development, differentiation, and immunity. TGF-β signals through its cell surface receptor serine kinases that phosphorylate Smad2 or Smad3 proteins. Because Smad3 and its partner Smad4 bind to only 4-bp Smad binding elements (SBEs) in DNA, a central question is how specificity of TGF-β-induced transcription is achieved. We show that Smad3 selectively binds to two of the three SBEs in PE2.1, a TGF-β-inducible fragment of the plasminogen activator inhibitor-1 promoter, to mediate TGF-β-induced transcription; moreover, a precise 3-bp spacer between one SBE and the E-box, a binding site for transcription factor μE3 (TFE3), is essential for TGF-β-induced transcription. Whereas an isolated Smad3 MH1 domain binds to TFE3, TGF-β receptor-mediated phosphorylation of full-length Smad3 enhances its binding to TFE3. Together, these studies elucidate an important mechanism for specificity in TGF-β-induced transcription of the plasminogen activator inhibitor-1 gene.

Activation of transcription factor AP-2 mediates UVA radiation- and singlet oxygen-induced expression of the human intercellular adhesion molecule 1 gene

UVA radiation is the major component of the UV solar spectrum that reaches the earth, and the therapeutic application of UVA radiation is increasing in medicine. Analysis of the cellular effects of UVA radiation has revealed that exposure of human cells to UVA radiation at physiological doses leads to increased gene expression and that this UVA response is primarily mediated through the generation of singlet oxygen. In this study, the mechanisms by which UVA radiation induces transcriptional activation of the human intercellular adhesion molecule 1 (ICAM-1) were examined. UVA radiation was capable of inducing activation of the human ICAM-1 promoter and increasing ICAM-1 mRNA and protein expression. These UVA radiation effects were inhibited by singlet oxygen quenchers, augmented by enhancement of singlet oxygen life-time, and mimicked in unirradiated cells by a singlet oxygen-generating system. UVA radiation as well as singlet oxygen-induced ICAM-1 promoter activation required activation of the transcription factor AP-2. Accordingly, both stimuli activated AP-2, and deletion of the putative AP-2-binding site abrogated ICAM-1 promoter activation in this system. This study identified the AP-2 site as the UVA radiation- and singlet oxygen-responsive element of the human ICAM-1 gene. The capacity of UVA radiation and/or singlet oxygen to induce human gene expression through activation of AP-2 indicates a previously unrecognized role of this transcription factor in the mammalian stress response.

Fusion of the transcription factor TFE3 gene to a novel gene, PRCC, in t(X;1)(p11;q21)-positive papillary renal cell carcinomas

The (X;1)(p11;q21) translocation is a recurrent chromosomal abnormality in a subset of human papillary renal cell carcinomas, and is sometimes the sole cytogenetic abnormality present. Via positional cloning, we were able to identify the genes involved. The translocation results in a fusion of the transcription factor TFE3 gene on the X chromosome to a novel gene, designated PRCC, on chromosome 1. Through this fusion, reciprocal translocation products are formed, which are both expressed in papillary renal cell carcinomas. PRCC is ubiquitously expressed in normal adult and fetal tissues and encodes a putative protein of 491 aa with a relatively high content of prolines. No relevant homologies with known sequences at either the DNA or the protein level were found.

Herpes simplex virus 1 α regulatory protein ICP0 functionally interacts with cellular transcription factor BMAL1

The infected cell protein no. 0 (ICP0) of herpes simplex virus 1 (HSV-1) is a promiscuous transactivator shown to enhance the expression of gene introduced into cells by infection or transfection. At the molecular level, ICP0 is a 775-aa ring finger protein localized initially in the nucleus and late in infection in the cytoplasm and mediates the degradation of several proteins and stabilization of others. None of the known functions at the molecular level account for the apparent activity of ICP0 as a transactivator. Here we report that ICP0 functionally interacts with cellular transcription factor BMAL1, a member of the basic helix–loop–helix PER-ARNT-SIM (PAS) super family of transcriptional regulators. Specifically, sequences mapped to the exon II of ICP0 interacted with BMAL1 in the yeast two-hybrid system and in reciprocal pull-down experiments in vitro. Moreover, the enhancement of transcription of a luciferase reporter construct whose promoter contained multiple BMAL1-binding sites by ICP0 and BMAL1 was significantly greater than that observed by ICP0 or BMAL1 alone. Although the level of BMAL1 present in nuclei of infected cells remained unchanged between 3 and 8 h after infection, the level of cytoplasmic BMAL1 was reduced at 8 h after infection. The reduction of cytoplasmic BMAL1 was significantly greater in cells infected with the ICP0-null mutant than in the wild-type virus-infected cells, suggesting that ICP0 mediates partial stabilization of the protein. These results indicate that ICP0 interacts physically and functionally with at least one cellular transcription-regulatory factor.

Amphibian transcription factor IIIA proteins contain a sequence element functionally equivalent to the nuclear export signal of human immunodeficiency virus type 1 Rev.

The human immunodeficiency virus type 1 (HIV-1) Rev protein is required for nuclear export of late HIV-1 mRNAs. This function is dependent on the mutationally defined Rev activation domain, which also forms a potent nuclear export signal. Transcription factor IIIA (TFIIIA) binds to 5S rRNA transcripts and this interaction has been proposed to play a role in the efficient nuclear export of 5S rRNA in amphibian oocytes. Here it is reported that amphibian TFIIIA proteins contain a sequence element with homology to the Rev activation domain that effectively substitutes for this domain in inducing the nuclear export of late HIV-1 mRNAs. It is further demonstrated that this TFIIIA sequence element functions as a protein nuclear export signal in both human cells and frog oocytes. Thus, this shared protein motif may play an analogous role in mediating the nuclear export of both late HIV-1 RNAs and 5S rRNA transcripts.

Alternative translation initiation site usage results in two functionally distinct forms of the GATA-1 transcription factor.

GATA-1 is a zinc-finger transcription factor that plays a critical role in the normal development of hematopoietic cell lineages. In human and murine erythroid cells a previously undescribed 40-kDa protein is detected with GATA-1-specific antibodies. We show that the 40-kDa GATA-1 (GATA-1s) is produced by the use of an internal AUG initiation codon in the GATA-1 transcript. The GATA-1 proteins share identical binding activity and form heterodimers in erythroleukemic cells but differ in their transactivation potential and in their expression in developing mouse embryos.

The nuclear matrix protein NMP-1 is the transcription factor YY1.

NMP-1 was initially identified as a nuclear matrix-associated DNA-binding factor that exhibits sequence-specific recognition for the site IV regulatory element of a histone H4 gene. This distal promoter domain is a nuclear matrix interaction site. In the present study, we show that NMP-1 is the multifunctional transcription factor YY1. Gel-shift and Western blot analyses demonstrate that NMP-1 is immunoreactive with YY1 antibody. Furthermore, purified YY1 protein specifically recognizes site IV and reconstitutes the NMP-1 complex. Western blot and gel-shift analyses indicate that YY1 is present within the nuclear matrix. In situ immunofluorescence studies show that a significant fraction of YY1 is localized in the nuclear matrix, principally but not exclusively associated with residual nucleoli. Our results confirm that NMP-1/YY1 is a ubiquitous protein that is present in both human cells and in rat osteosarcoma ROS 17/2.8 cells. The finding that NMP-1 is identical to YY1 suggests that this transcriptional regulator may mediate gene-matrix interactions. Our results are consistent with the concept that the nuclear matrix may functionally compartmentalize the eukaryotic nucleus to support regulation of gene expression.