A GBF-binding site and a novel AT element define the minimal sequences sufficient to direct prespore-specific expression in Dictyostelium discoideum.

In order to better understand the molecular mechanisms of cellular differentiation in Dictyostelium discoideum, we have identified the minimum regulatory sequences of the prespore-specific gene SP60/cotC that are sufficient to confer cell-type-specific expression on a heterologous promoter. This region includes at least two essential cis-acting elements: a novel AT-rich element (or elements) and CAE3. The essential function of the AT element is confirmed through point mutations that decrease expression below the level of detection. CAE3 is one of three CA-rich elements (CAEs) required for the induction of SP60/cotC during development or in response to extracellular cyclic AMP. The CAEs have differential affinities for a specific developmentally induced nuclear activity (CAE1 > CAE2 >> CAE3). Here, we identify this activity as G-box-binding factor (GBF) and show that in vitro-transcribed and -translated GBF binds all three SP60/cotC CAEs in a sequence-specific manner. Previous studies have suggested that GBF mediates the induction of some prestalk genes, and these results demonstrate that it also has a specific role in prespore gene activation.

Spatial and temporal expression of the Dictyostelium discoideum G alpha protein subunit G alpha 2: expression of a dominant negative protein inhibits proper prestalk to stalk differentiation.

Previous results have shown that the G alpha protein subunit G alpha 2 is required for aggregation in Dictyostelium discoideum and is essential for coupling cell-surface cAMP receptors to downstream effectors in vivo during this stage of development. G alpha 2 expresses at least four distinct transcripts that are differentially regulated during development; two of the transcripts are expressed exclusively in the multicellular stages and their expression is restricted to prestalk cells. We partially dissected the G alpha 2 promoter and identified a component that is expressed exclusively during the multicellular stages using luciferase gene fusions. When this promoter region is coupled to lacZ, beta-gal expression is restricted to the multicellular stages and localized in prestalk cells with a pattern similar to that of the ecmA prestalk-specific promoter. We show that expression in wild-type cells of the G alpha 2 mutant protein [G alpha 2(G206T)] during the early stages of development blocks aggregation and cAMP-mediated activation of adenylyl cyclase and guanylyl cyclase, suggesting it functions as a dominant negatively active G alpha subunit. When this mutant G alpha protein is expressed from the ecmA prestalk-specific promoter, abnormal stalk differentiation during culmination is observed. Expression of the mutant G alpha 2 from the SP60 prespore promoter or wild-type G alpha 2 from either the ecmA or the SP60 promoter results in no detectable phenotype. The results suggest that G alpha 2 plays an essential role during the culmination stage in prestalk cells and may mediate cAMP receptor activation of these processes during multicellular development.

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.

Characterization of a novel Dictyostelium discoideum prespore-specific gene, PspB, reveals conserved regulatory sequences.

While Dictyostelium discoideum has been studied as a developmental system for decades, and many regulatory proteins have been cloned, the molecular mechanisms of cell-type-specific gene expression are poorly understood. In this paper we characterize a novel prespore gene, PspB, and undertake a comparative analysis of the regulatory regions in prespore-specific D. discoideum promoters. Sequence alignment of the PSPB gene product with other prespore-specific proteins identifies a conserved, repeated 12 amino acid cysteine-containing motif that may be involved in spore coat function or assembly. Analysis of the PspB promoter identifies two domains essential for developmentally induced promoter activity. The first region includes two CA-rich elements (CAEs) that we show to be functionally homologous to the cAMP-inducible elements previously identified in the SP60 (cotC) promoter. The PspB CAEs compete with the SP60 (cotC) CAEs for binding in vitro to a developmentally regulated nuclear activity. We identify this activity as G-box Binding Factor, a developmentally induced transcription factor. The PspB CAEs and adjacent nucleotides direct a very low level of prespore-enriched expression, but high levels of cell-type-specific expression requires a second promoter region: a 46-bp AT-rich sequence that does not resemble the CAEs or any other previously described late gene promoter elements. Comparison of the PspB AT element with regulatory regions of the SP60 (cotC), SP70 (cotB), and D19 (pspA) promoters reveals an extensive consensus sequence. We suggest that these AT-rich sequences may represent a common regulatory element (or elements) required for prespore gene activation.

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.

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.

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.