In vitro and in vivo characterization of Caenorhabditis elegans PHA-4/FoxA response elements.

Caenorhabditis elegans PHA-4 is a member of the FoxA group of transcription factors. PHA-4 is critical for development of the C. elegans pharynx and directly regulates most or all pharyngeal genes. The consensus binding site of PHA-4 has not been identified, with previous analysis of PHA-4 targets relying on the mammalian FoxA consensus. Here, we use in vitro and in vivo analyses to demonstrate three features of PHA-4 response elements. First, the PHA-4 consensus matches that of other FoxA proteins, but only a subset of possible sites is active in an in vivo assay. Second, sequence flanking the core PHA-4 site can influence the strength of reporter expression in vivo, as seen for other Fox proteins. Third, in the context of some pharyngeal promoters, PHA-4 response elements are flanked by distinct cis-regulatory elements that modulate response to PHA-4, generating gene expression in specific pharyngeal cell types.

ELT-2 is the predominant transcription factor controlling differentiation and function of the C. elegans intestine, from embryo to adult.

Starting with SAGE-libraries prepared from C. elegans FAC-sorted embryonic intestine cells (8E-16E cell stage), from total embryos and from purified oocytes, and taking advantage of the NextDB in situ hybridization data base, we define sets of genes highly expressed from the zygotic genome, and expressed either exclusively or preferentially in the embryonic intestine or in the intestine of newly hatched larvae; we had previously defined a similarly expressed set of genes from the adult intestine. We show that an extended TGATAA-like sequence is essentially the only candidate for a cis-acting regulatory motif common to intestine genes expressed at all stages. This sequence is a strong ELT-2 binding site and matches the sequence of GATA-like sites found to be important for the expression of every intestinal gene so far analyzed experimentally. We show that the majority of these three sets of highly expressed intestinal-specific/intestinal-enriched genes respond strongly to ectopic expression of ELT-2 within the embryo. By flow-sorting elt-2(null) larvae from elt-2(+) larvae and then preparing Solexa/Illumina-SAGE libraries, we show that the majority of these genes also respond strongly to loss-of-function of ELT-2. To test the consequences of loss of other transcription factors identified in the embryonic intestine, we develop a strain of worms that is RNAi-sensitive only in the intestine; however, we are unable (with one possible exception) to identify any other transcription factor whose intestinal loss-of-function causes a phenotype of comparable severity to the phenotype caused by loss of ELT-2. Overall, our results support a model in which ELT-2 is the predominant transcription factor in the post-specification C. elegans intestine and participates directly in the transcriptional regulation of the majority (>80%) of intestinal genes. We present evidence that ELT-2 plays a central role in most aspects of C. elegans intestinal physiology: establishing the structure of the enterocyte, regulating enzymes and transporters involved in digestion and nutrition, responding to environmental toxins and pathogenic infections, and regulating the downstream intestinal components of the daf-2/daf-16 pathway influencing aging and longevity.

Lifespan extension and increased pumping rate accompany pharyngeal muscle-specific expression of nfi-1 in C. elegans.

Caenorhabditis elegans nfi-1 belongs to the Nuclear Factor I (NFI) family of transcription factors known to regulate metazoan gene expression and development. We showed previously that loss of nfi-1 in worms results in multiple behavioral defects; slower pharyngeal pumping rate, impaired egg laying, defective motility, and a shortened life span. Here, we generated cell-type specific transgenic worms to determine the cells in which nfi-1 must be expressed to rescue the pharyngeal pumping defect. Expression of nfi-1 from the pharyngeal muscle-specific myo-2 promoter, but not from the F25B3.3 or myo-3 promoters, rescued the pharyngeal pumping defect of nfi-1 worms. Surprisingly, myo-2-driven nfi-1 expression also rescued the shortened lifespan of nfi-1 worms, demonstrating a possible cell-autonomous role of nfi-1 in pharyngeal muscle for both phenotypes. We propose some relationships between the pharyngeal pumping and lifespan phenotypes and potential mechanisms of nfi-1 function.

nfi-I affects behavior and life-span in C. elegans but is not essential for DNA replication or survival.

BACKGROUND: The Nuclear Factor I (one) (NFI) family of transcription/replication factors plays essential roles in mammalian gene expression and development and in adenovirus DNA replication. Because of its role in viral DNA replication NFI has long been suspected to function in host DNA synthesis. Determining the requirement for NFI proteins in mammalian DNA replication is complicated by the presence of 4 NFI genes in mice and humans. Loss of individual NFI genes in mice cause defects in brain, lung and tooth development, but the presence of 4 homologous NFI genes raises the issue of redundant roles for NFI genes in DNA replication. No NFI genes are present in bacteria, fungi or plants. However single NFI genes are present in several simple animals including Drosophila and C. elegans, making it possible to test for a requirement for NFI in multicellular eukaryotic DNA replication and development. Here we assess the functions of the single nfi-1 gene in C. elegans. RESULTS: C. elegans NFI protein (CeNFI) binds specifically to the same NFI-binding site recognized by vertebrate NFIs. nfi-1 encodes alternatively-spliced, maternally-inherited transcripts that are expressed at the single cell stage, during embryogenesis, and in adult muscles, neurons and gut cells. Worms lacking nfi-1 survive but have defects in movement, pharyngeal pumping and egg-laying and have a reduced life-span. Expression of the muscle gene Ce titin is decreased in nfi-1 mutant worms. CONCLUSION: NFI gene function is not needed for survival in C. elegans and thus NFI is likely not essential for DNA replication in multi-cellular eukaryotes. The multiple defects in motility, egg-laying, pharyngeal pumping, and reduced lifespan indicate that NFI is important for these processes. Reduction in Ce titin expression could affect muscle function in multiple tissues. The phenotype of nfi-1 null worms indicates that NFI functions in multiple developmental and behavioral systems in C. elegans, likely regulating genes that function in motility, egg-laying, pharyngeal pumping and lifespan maintenance.

Interference between the PHA-4 and PEB-1 transcription factors in formation of the Caenorhabditis elegans pharynx.

PHA-4 is a forkhead/winged helix transcription factor that acts as an organ identity factor in the development of the Caenorhabditis elegans pharynx. PEB-1 is a novel DNA-binding protein also involved in pharyngeal morphogenesis. PHA-4 and PEB-1 bind at overlapping sites on the C183 sequence element that controls pharynx-specific expression of the C. elegans myo-2 gene. It has been suggested that PHA-4 and PEB-1 act cooperatively on the C183 sequence. In this study, we test this model and assess the C183-dependent transcriptional activity of PHA-4 and PEB-1, both individually and in combination. We show that PHA-4 and PEB-1 are both modest transcriptional activators in yeast but that co-expression of the two factors does not result in significantly increased expression of a C183-regulated reporter gene. Electrophoretic mobility-shift assays provide no evidence for the formation of a PHA-4/PEB-1 complex in vitro but rather show that PHA-4 and PEB-1 cannot bind C183 simultaneously. As we have reported previously, ectopic expression of PHA-4 in C. elegans causes ectopic expression of a C183-regulated reporter gene. We show that ectopic expression of PEB-1 cannot cause ectopic expression of the same reporter but rather ectopic PEB-1 inhibits reporter gene activation by PHA-4. Overall, our results do not support a model in which PHA-4 and PEB-1 synergize in vivo but rather support a model in which PEB-1 may negatively modulate PHA-4's ability to activate transcription through C183 during formation of the C. elegans pharynx.

The Drosophila melanogaster seminal fluid protein Acp62F is a protease inhibitor that is toxic upon ectopic expression.

Drosophila melanogaster seminal fluid proteins stimulate sperm storage and egg laying in the mated female but also cause a reduction in her life span. We report here that of eight Drosophila seminal fluid proteins (Acps) and one non-Acp tested, only Acp62F is toxic when ectopically expressed. Toxicity to preadult male or female Drosophila occurs upon one exposure, whereas multiple exposures are needed for toxicity to adult female flies. Of the Acp62F received by females during mating, approximately 10% enters the circulatory system while approximately 90% remains in the reproductive tract. We show that in the reproductive tract, Acp62F localizes to the lumen of the uterus and the female's sperm storage organs. Analysis of Acp62F's sequence, and biochemical assays, reveals that it encodes a trypsin inhibitor with sequence and structural similarities to extracellular serine protease inhibitors from the nematode Ascaris. In light of previous results demonstrating entry of Acp62F into the mated female's hemolymph, we propose that Acp62F is a candidate for a molecule to contribute to the Acp-dependent decrease in female life span. We propose that Acp62F's protease inhibitor activity exerts positive protective functions in the mated female's reproductive tract but that entry of a small amount of this protein into the female's hemolymph could contribute to the cost of mating.