From critters to cancers: bridging comparative and clinical research on oxygen sensing, HIF signaling, and adaptations towards hypoxia.

The objective of this symposium at the First International Congress of Respiratory Biology (ICRB) was to enhance communication between comparative biologists and cancer researchers working on O(2) sensing via the HIF pathway. Representatives from both camps came together on August 13-16, 2006, in Bonn, Germany, to discuss molecular adaptations that occur after cells have been challenged by a reduced (hypoxia) or completely absent (anoxia) supply of oxygen. This brief "critters-to-cancer" survey discusses current projects and new directions aimed at improving understanding of hypoxic signaling and developing therapeutic interventions.

Protein tyrosine and serine-threonine phosphatases in the sea urchin, Strongylocentrotus purpuratus: identification and potential functions.

Protein phosphatases, in coordination with protein kinases, play crucial roles in regulation of signaling pathways. To identify protein tyrosine phosphatases (PTPs) and serine-threonine (ser-thr) phosphatases in the Strongylocentrotus purpuratus genome, 179 annotated sequences were studied (122 PTPs, 57 ser-thr phosphatases). Sequence analysis identified 91 phosphatases (33 conventional PTPs, 31 dual specificity phosphatases, 1 Class III Cysteine-based PTP, 1 Asp-based PTP, and 25 ser-thr phosphatases). Using catalytic sites, levels of conservation and constraint in amino acid sequence were examined. Nine of 25 receptor PTPs (RPTPs) corresponded to human, nematode, or fly homologues. Domain structure revealed that sea urchin-specific RPTPs including two, PTPRLec and PTPRscav, may act in immune defense. Embryonic transcription of each phosphatase was recorded from a high-density oligonucleotide tiling microarray experiment. Most RPTPs are expressed at very low levels, whereas nonreceptor PTPs (NRPTPs) are generally expressed at moderate levels. High expression was detected in MAP kinase phosphatases (MKPs) and numerous ser-thr phosphatases. For several expressed NRPTPs, MKPs, and ser-thr phosphatases, morpholino antisense-mediated knockdowns were performed and phenotypes obtained. Finally, to assess roles of annotated phosphatases in endomesoderm formation, a literature review of phosphatase functions in model organisms was superimposed on sea urchin developmental pathways to predict areas of functional activity.

The Caenorhabditis elegans hif-1 gene encodes a bHLH-PAS protein that is required for adaptation to hypoxia.

Hypoxia-inducible factor, a heterodimeric transcription complex, regulates cellular and systemic responses to low oxygen levels (hypoxia) during normal mammalian development or tumor progression. Here, we present evidence that a similar complex mediates response to hypoxia in Caenorhabditis elegans. This complex consists of HIF-1 and AHA-1, which are encoded by C. elegans homologs of the hypoxia-inducible factor (HIF) alpha and beta subunits, respectively. hif-1 mutants exhibit no severe defects under standard laboratory conditions, but they are unable to adapt to hypoxia. Although wild-type animals can survive and reproduce in 1% oxygen, the majority of hif-1-defective animals die in these conditions. We show that the expression of an HIF-1:green fluorescent protein fusion protein is induced by hypoxia and is subsequently reduced upon reoxygenation. Both hif-1 and aha-1 are expressed in most cell types, and the gene products can be coimmunoprecipitated. We conclude that the mechanisms of hypoxia signaling are likely conserved among metazoans. Additionally, we find that nuclear localization of AHA-1 is disrupted in an hif-1 mutant. This finding suggests that heterodimerization may be a prerequisite for efficient nuclear translocation of AHA-1.

Oral-aboral axis specification in the sea urchin embryo. I. Axis entrainment by respiratory asymmetry.

In embryos of indirectly developing echinoids, the secondary (oral-aboral) larval axis is established after fertilization by an as yet undiscovered process. One of the earliest manifestations of this axis is an asymmetry in mitochondrial respiration, with the prospective oral side of the embryo exhibiting a higher rate of respiration than the prospective aboral side. We show here that respiratory asymmetry can be experimentally induced within embryos by immobilizing them in tight clusters of four ("rosettes"). Within such clusters a redox gradient is established from the inside to the outside of the rosette. Vital staining of clustered embryos demonstrates that the side of the embryo facing the outside of the rosette (i.e., the most oxidizing) tends to become the oral side, while the side facing the inside tends to become the aboral side. Effective entrainment of the oral-aboral axis requires that the embryos remain immobilized in rosettes until the hatching blastula stage. To begin to investigate the molecular mechanisms underlying this effect we made use of P3A2, a transcriptional regulatory protein whose activity is spatially modulated along the oral-aboral axis. When synthetic mRNA encoding P3A2 fused to the VP16 activation domain is injected into eggs, it activates embryonic expression of a green fluorescent protein reporter gene containing a basal promoter and a single strong P3A2 target site. In embryo rosettes, such activation occurs predominantly on the outside of the rosette, suggesting that the activity of the P3A2 protein is spatially regulated by the respiratory asymmetry established by clustering the embryos. These findings are discussed with reference to earlier work on both oral-aboral axis specification and P3A2 and used to develop a testable model of the mechanism of oral-aboral axis specification in the sea urchin embryo.

Saccharomyces cerevisiae GATA sequences function as TATA elements during nitrogen catabolite repression and when Gln3p is excluded from the nucleus by overproduction of Ure2p.

Saccharomyces cerevisiae selectively uses good nitrogen sources (glutamine) in preference to poor ones (proline) by repressing GATA factor-dependent transcription of the genes needed to transport and catabolize poor nitrogen sources, a physiological process designated nitrogen catabolite repression (NCR). We show that some NCR-sensitive genes (CAN1, DAL5, DUR1,2, and DUR3) produce two transcripts of slightly different sizes. Synthesis of the shorter transcript is NCR-sensitive and that of the longer transcript is not. The longer transcript also predominates in gln3Delta mutants irrespective of the nitrogen source provided. We demonstrate that the longer mRNA species arises through the use of an alternative transcription start site generated by Gln3p-binding sites (GATAAs) being able to act as surrogate TATA elements. The ability of GATAAs to serve as surrogate TATAs, i.e. when synthesis of the shorter, NCR-sensitive transcripts are inhibited, correlates with sequestration of enhanced green fluorescent protein (EGFP)-Gln3p in the cytoplasm in a way that is indistinguishable from that seen with EGFP-Ure2p. However, when the shorter, NCR-sensitive DAL5 transcript predominates, EGFP-Gln3p is nuclear. These data suggest that the mechanism underlying NCR involves the cytoplasmic association of Ure2p with Gln3p, an interaction that prevents Gln3p from reaching it is binding sites upstream of NCR-sensitive genes.

Cellular localization of huntingtin in striatal and cortical neurons in rats: lack of correlation with neuronal vulnerability in Huntington's disease.

Immunohistochemistry and single-cell RT-PCR were used to characterize the localization of huntingtin and/or its mRNA in the major types of striatal neurons and in corticostriatal projection neurons in rats. Single-label immunohistochemical studies revealed that striatum contains scattered large neurons rich in huntingtin and more numerous medium-sized neurons moderate in huntingtin. Double-label immunohistochemical studies showed that the large huntingtin-rich striatal neurons include nearly all cholinergic interneurons and some parvalbuminergic interneurons. Somatostatinergic striatal interneurons, which are medium in size, rarely contained huntingtin. Calbindin immunolabeling showed that the vast majority of the medium-sized striatal neurons that contain huntingtin are projection neurons, but only approximately 65% of calbindin-labeled projection neurons (localized to the matrix compartment of striatum) were labeled for huntingtin. Calbindin-containing projection neurons of the matrix compartment and calbindin-negative projection neurons of the striatal patch compartment contained huntingtin with comparable frequency. Single-cell RT-PCR confirmed that striatal cholinergic interneurons contain huntingtin, but only approximately 65% of projection neurons contained detectable huntingtin message. The finding that huntingtin is not consistently found in striatal projection neurons [which die in Huntington's disease (HD)] but is abundant in striatal cholinergic interneurons (which survive in Huntington's disease) suggests that the mutation in huntingtin that causes HD may not directly kill neurons. In contrast to the heterogeneous expression of huntingtin in the different striatal neuron types, we found all corticostriatal neurons to be rich in huntingtin protein and mRNA. One possibility raised by our findings is that the HD mutation may render corticostriatal neurons destructive rather than render striatal neurons vulnerable.

Caenorhabditis elegans orthologs of the aryl hydrocarbon receptor and its heterodimerization partner the aryl hydrocarbon receptor nuclear translocator.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, until now described only in vertebrates, that mediates many of the carcinogenic and teratogenic effects of certain environmental pollutants. Here, we describe orthologs of AHR and its dimerization partner AHR nuclear translocator (ARNT) in the nematode Caenorhabditis elegans, encoded by the genes ahr-1 and aha-1, respectively. The corresponding proteins, AHR-1 and AHA-1, share biochemical properties with their mammalian cognates. Specifically, AHR-1 forms a tight association with HSP90, and AHR-1 and AHA-1 interact to bind DNA fragments containing the mammalian xenobiotic response element with sequence specificity. Yeast expression studies indicate that C. elegans AHR-1, like vertebrate AHR, requires some form of post-translational activation. Moreover, this requirement depends on the presence of the domains predicted to mediate binding of HSP90 and ligand. Preliminary experiments suggest that if AHR-1 is ligand-activated, its spectrum of ligands is different from that of the mammalian receptor: C. elegans AHR-1 is not photoaffinity labeled by a dioxin analog, and it is not activated by beta-naphthoflavone in the yeast system. The discovery of these genes in a simple, genetically tractable invertebrate should allow elucidation of AHR-1 function and identification of its endogenous regulators.

Covalent variation is a general property of transcription factors in the sea urchin embryo.

Nine different transcription factors of the Strongylocentrotus purpuratus embryo, belonging to diverse structural families, were examined by high-resolution, two-dimensional gel electrophoresis. These factors had all been cloned previously, and antibodies against the recombinant proteins were available. The factors were visualized immunologically in nuclear extracts from 24-hour blastula-stage embryos. Remarkably, every one of the nine factors displayed multiple charge variants. Three factors, SpZ12-1, SpP3A2, and SpRunt-1, were studied at different stages of embryonic development. The prevalence and distribution of the variant isoforms of all three factors differed at each stage examined; and in all cases the complexity of the variants was greatest in the 24-hour blastula-stage extracts. The most complex set of variants was observed for SpP3A2, and phosphatase treatment demonstrated that some but not all, of the covalent modifications defining these variants are phosphorylations. As the transcription factors were chosen for this study merely on the basis of the availability of antibodies, we conclude that deployment of transcription factors in sea urchin embryos generally involves their covalent modification.

Nitrogen GATA factors participate in transcriptional regulation of vacuolar protease genes in Saccharomyces cerevisiae.

The expression of most nitrogen catabolic genes in Saccharomyces cerevisiae is regulated at the level of transcription in response to the quality of nitrogen source available. This regulation is accomplished through four GATA-family transcription factors: two positively acting factors capable of transcriptional activation (Gln3p and Gat1p) and two negatively acting factors capable of down-regulating Gln3p- and/or Gat1p-dependent transcription (Dal80p and Deh1p). Current understanding of nitrogen-responsive transcriptional regulation is the result of extensive analysis of genes required for the catabolism of small molecules, e.g., amino acids, allantoin, or ammonia. However, cells contain another, equally important source of nitrogen, intracellular protein, which undergoes rapid turnover during special circumstances such as entry into stationary phase, and during sporulation. Here we show that the expression of some (CPS1, PEP4, PRB1, and LAP4) but not all (PRC1) vacuolar protease genes is nitrogen catabolite repression sensitive and is regulated by the GATA-family proteins Gln3p, Gat1p, and Dal80p. These observations extend the global participation of GATA-family transcription factors to include not only well-studied genes associated with the catabolism of small nitrogenous compounds but also genes whose products are responsible for the turnover of intracellular macromolecules. They also point to the usefulness of considering control of the nitrogen-responsive GATA factors when studying the regulation of the protein turnover machinery.

Cross regulation of four GATA factors that control nitrogen catabolic gene expression in Saccharomyces cerevisiae.

Nitrogen catabolic gene expression in Saccharomyces cerevisiae has been reported to be regulated by three GATA family proteins, the positive regulators Gln3p and Gat1p/Nil1p and the negative regulator Dal80p/Uga43p. We show here that a fourth member of the yeast GATA family, the Dal80p homolog Deh1p, also negatively regulates expression of some, but not all, nitrogen catabolic genes, i.e., GAP1, DAL80, and UGA4 expression increases in a deh1 delta mutant. Consistent with Deh1p regulation of these genes is the observation that Deh1p forms specific DNA-protein complexes with GATAA-containing UGA4 and GAP1 promoter fragments in electrophoretic mobility shift assays. Deh1p function is demonstrable, however, only when a repressive nitrogen source such as glutamine is present; deh1 delta mutants exhibit no detectable phenotype with a poor nitrogen source such as proline. Our experiments also demonstrate that GATA factor gene expression is highly regulated by the GATA factors themselves in an interdependent manner. DAL80 expression is Gln3p and Gat1p dependent and Dal80p regulated. Moreover, Gln3p and Dal80p bind to DAL80 promoter fragments. In turn, GAT1 expression is Gln3p dependent and Dal80p regulated but is not autogenously regulated like DAL80. DEH1 expression is largely Gln3p independent, modestly Gat1p dependent, and most highly regulated by Dal80p. Paradoxically, the high-level DEH1 expression observed in a dal80::hisG disruption mutant is highly sensitive to nitrogen catabolite repression.

Extensive maternal representation of DNA-binding proteins that interact with regulatory target sites of the Strongylocentrotus purpuratus CyIIIa gene.

Nine different embryonic transcription factors interact at specific target sites in the cis-regulatory domain of the Strongylocentrotus purpuratus CyIIIa gene. We tested eight of these site sequences and show here that for every one a DNA-binding protein was present in unfertilized egg cytoplasm. The concentrations of active DNA-binding proteins per egg were estimated. We also present a new and convenient method for preparation of a material cytoplasmic fraction that retains these factors in active form.

SpMyb functions as an intramodular repressor to regulate spatial expression of CyIIIa in sea urchin embryos.

The CyIIIa actin gene of Strongylocentrotus purpuratus is transcribed exclusively in the embryonic aboral ectoderm, under the control of 2.3 kb cis-regulatory domain that contains a proximal module that controls expression in early embryogenesis, and a middle module that controls expression in later embryogenesis. Previous studies demonstrated that the SpRunt-1 target site within the middle module is required for the sharp increase in CyIIIa transcription which accompanies differentiation of the aboral ectoderm, and that a negative regulatory region near the SpRunt-1 target site is required to prevent ectopic transcription in the oral ectoderm and skeletogenic mesenchyme. This negative regulatory region contains a consensus binding site for the myb family of transcription factors. In vitro DNA-binding experiments reveal that a protein in blastula-stage nuclei interacts specifically with the myb target site. Gene transfer experiments utilizing CyIIIa reporter constructs containing oligonucleotide substitutions indicate that this site is both necessary and sufficient to prevent ectopic expression of CyIIIa. Synthetic oligonucleotides containing the myb target site were used to purify a protein from sea urchin embryo nuclear extracts by affinity chromatography. This protein is immunoprecipitated by antibodies specific to the evolutionarily conserved myb domain, and amino acid sequences obtained from the purified protein were found to be identical to sequences within the myb domain. Sequence information was used to obtain cDNA clones of SpMyb, the S. purpuratus member of the myb family of transcription factors. Through interactions within the middle module, SpMyb functions to repress activation of CyIIIa in the oral ectoderm and skeletogenic mesenchyme.

Combinatorial regulation of the Saccharomyces cerevisiae CAR1 (arginase) promoter in response to multiple environmental signals.

CAR1 (arginase) gene expression responds to multiple environmental signals; expression is induced in response to the intracellular accumulation of arginine and repressed when readily transported and catabolized nitrogen sources are available in the environment. Up to 14 cis-acting sites and 9 trans-acting factors have been implicated in regulated CAR1 transcription. In all but one case, the sites are redundant. To test whether these sites actually participate in CAR1 expression, each class of sites was inactivated by substitution mutations that retained the native spacing of the CAR1 cis-acting elements. Three types of sites function independently of the nitrogen source: two clusters of Abflp- and Rap1p-binding sites, and a GC-rich sequence. Two different sets of nitrogen source-dependent sites are also required: the first consists of two GATAA-containing UASNTR sites that mediate nitrogen catabolite repression-sensitive transcription, and the second is arginine dependent and consists of three UAS1 elements that activate transcription only when arginine is present. A single URS1 site mediates repression of CAR1 arginine-independent upstream activator site (UAS) activity in the absence of arginine and the presence of a poor nitrogen source (a condition under which the inducer-independent Gln3p can function in association with the UASNTR sites). When arginine is present, the combined activity of the UAS elements overcomes the negative effects mediated by URS1. Mutation of the classes of sites either singly or in combination markedly alters CAR1 promoter operation and control, supporting the idea that they function synergistically to regulate expression of the gene.

Onset of C. elegans gastrulation is blocked by inhibition of embryonic transcription with an RNA polymerase antisense RNA.

Cleavage and gastrulation initiation in Caenorhabditis elegans embryos are characterized by an invariant temporal and spatial pattern of cell divisions and cell movements. Although bulk embryonic transcription does not begin until gastrulation onset, some transcription can be detected as early as the 4-cell stage. To determine whether any early transcripts are required for normal cleavage-stage patterning, we blocked transcription in embryos by injecting hermaphrodite parental gonads with RNA antisense to the ama-1 gene, which encodes the large subunit of RNA polymerase II. This treatment prevented the expression of a reporter gene driven by an early embryonic promoter but did not detectably perturb the maternally controlled segregation of the germ line P granules or the pattern of cell division through the first four cleavages. In the fifth cell cycle, however, the two endodermal precursor (E) cells divided early and abnormally and failed to initiate gastrulation. The embryos arrested between the sixth and seventh cell cycles with less than 100 cells. These results indicate that embryonically transcribed gene products are required for gastrulation initiation. They also demonstrate the efficacy of a method for blocking embryonic transcription that may be useful in other organisms.

Gat1p, a GATA family protein whose production is sensitive to nitrogen catabolite repression, participates in transcriptional activation of nitrogen-catabolic genes in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells selectively use nitrogen sources in their environment. Nitrogen catabolite repression (NCR) is the basis of this selectivity. Until recently NCR was thought to be accomplished exclusively through the negative regulation of Gln3p function by Ure2p. The demonstration that NCR-sensitive expression of multiple nitrogen-catabolic genes occurs in a gln3 delta ure2 delta dal80::hisG triple mutant indicated that the prevailing view of the nitrogen regulatory circuit was in need of revision; additional components clearly existed. Here we demonstrate that another positive regulator, designated Gat1p, participates in the transcription of NCR-sensitive genes and is able to weakly activate transcription when tethered upstream of a reporter gene devoid of upstream activation sequence elements. Expression of GAT1 is shown to be NCR sensitive, partially Gln3p dependent, and Dal80p regulated. In agreement with this pattern of regulation, we also demonstrate the existence of Gln3p and Dal80p binding sites upstream of GAT1.

SpRunt-1, a new member of the runt domain family of transcription factors, is a positive regulator of the aboral ectoderm-specific CyIIIA gene in sea urchin embryos.

In this paper we present a structural and functional characterization of a new sea urchin embryo transcription factor, SpRunt-1. This factor was isolated by means of its specific interaction with a cis-regulatory target site of the CyIIIa gene. Here we show that this target site, the P7I site, is required for normal embryonic activation of CyIIIa x CAT reporter gene constructs. An oligonucleotide affinity column bearing the P7I target site purifies a 21-kDa polypeptide from blastula-stage nuclear extracts, and the amino acid sequence obtained from this polypeptide was used to generate a nucleic acid probe with which the corresponding cDNA was cloned. The cDNA encodes an approximately 60-kDa protein, SpRunt-1, which includes a "runt domain" that is closely homologous to those of Drosophila and mammalian runt domain transcription factors. RNA and genomic blots show that SpRunt-1 is represented by a single embryonic transcript, encoded by one of possibly two runt-domain-containing genes. By RNA probe protection we found that transcripts of SpRunt-1 increase in concentration dramatically after the blastula stage of development, suggesting that the up-regulation of CyIIIa that occurs after blastula stage is a function of zygotically transcribed SpRunt-1. These results are discussed with reference to known features of the runt domain family of transcription factors.

Genetic evidence for Gln3p-independent, nitrogen catabolite repression-sensitive gene expression in Saccharomyces cerevisiae.

The expression of many nitrogen catabolic genes decreases to low levels when readily used nitrogen sources (e.g., asparagine and glutamine) are provided in the growth medium; this physiological response is termed nitrogen catabolite repression (NCR). Transcriptional activation of these genes is mediated by the cis-acting element UASNTR and the trans-acting factor Gln3p. A second protein encoded by URE2 possesses the genetic characteristics of a negative regulator of nitrogen catabolic gene expression. A third locus, DAL80, encodes a repressor that binds to sequences required for Gln3p-dependent transcription and may compete with Gln3p for binding to them. These observations are consistent with an NCR regulatory pathway with the structure environmental signal-->Ure2p-->(Gln3p/Dal80p)-->UASNTR operation-->NCR-sensitive gene expression. If NCR-sensitive gene expression occurs exclusively by this pathway, as has been thought to be the case, then the NCR sensitivity of a gene's expression should be abolished by a ure2 delta mutation. This expectation was not realized experimentally; the responses of highly NCR-sensitive genes to ure2 delta mutations varied widely. This suggested that NCR was not mediated exclusively through Ure2p and Gln3p. We tested this idea by assaying GAP1, CAN1, DAL5, PUT1, UGA1, and GLN1 expression in single, double, and triple mutants lacking Gln3p, Dal80p, and/or Ure2p. All of these genes were expressed in the triple mutant, and this expression was NCR sensitive for four of the six genes. These results indicate that the NCR regulatory network consists of multiple branches, with the Ure2p-Gln3p-UASNTR pathway representing only one of them.

SpGCF1, a sea urchin embryo DNA-binding protein, exists as five nested variants encoded by a single mRNA.

Several Strongylocentrotus purpuratus gene cis-regulatory regions contain asymmetric C4 sequences which are core elements of target sites for a specific DNA-protein interaction. Blastula stage nuclear extract contains five proteins which specifically bind to these target sites, resulting in a characteristic pattern of complexes in gel mobility shift assays. We used automated affinity chromatography to purify a protein which binds to these sites and have isolated the corresponding cDNA. This protein, SpGCF1, is a novel sea urchin DNA-binding protein with no overall homology to proteins reported in the databases currently available. The DNA-binding domain of this protein was identified by a deletion analysis. As demonstrated both for protein translated in vitro and for bacterial protein expressed from a cDNA clone, a single SpGCF1 mRNA serves as a template for the synthesis of five DNA-binding polypeptides. We show that these five polypeptides are most likely produced by differential usage of a nested set of AUG start codons in the SpGCF1 cDNA and thus contain variable amounts of a proline-rich N-terminal domain. Since proline-rich regions often serve as transcriptional activation domains, the five SpGCF1 proteins apparently possess different "activation potentials."

The URE2 protein regulates nitrogen catabolic gene expression through the GATAA-containing UASNTR element in Saccharomyces cerevisiae.

Many of the gene products that participate in nitrogen metabolism are sensitive to nitrogen catabolite repression (NCR), i.e., their expression is decreased to low levels when readily used nitrogen sources such as asparagine are provided. Previous work has shown this NCR sensitivity requires the cis-acting UASNTR element and trans-acting GLN3. Here, we extend the analysis to include the response of their expression to deletion of the URE2 locus. The expression of these nitrogen catabolic genes becomes, to various degrees, NCR insensitive in the ure2 deletion. This response is shown to be mediated through the GATAA-containing UASNTR element and supports the current idea that the NCR regulatory circuit involves the following steps: environmental signal-->URE2-->GLN3-->UASNTR operation-->NCR-sensitive gene expression. The various responses of the nitrogen catabolic genes' expression to deletion of the URE2 locus also indicate that not all NCR is mediated through URE2.