The Caenorhabditis elegans peb-1 gene encodes a novel DNA-binding protein involved in morphogenesis of the pharynx, vulva, and hindgut.

Gene expression in the Caenorhabditis elegans pharynx is regulated in part by organ-specific signals, which in the myo-2 gene target a regulatory sequence called the C sub-element. C sub-element activity requires the organ specification factor PHA-4, a winged-helix transcription factor expressed in all pharyngeal cells. To identify additional factors involved in pharyngeal organogenesis, we performed a yeast one-hybrid screen for C sub-element binding proteins. Here we describe the novel factor PEB-1, which is coexpressed with PHA-4 in many pharyngeal cell types, including muscles, epithelial cells, marginal cells, and glands, but is undetectable in the pharyngeal nervous system. PEB-1 is also detected outside the pharynx in cells surrounding the rectum and vulva, as well as in the germ line. Reduction of peb-1 function using RNAi results in morphological defects in the somatic tissues in which peb-1 is expressed. We have mapped the PEB-1 DNA-binding domain to a 158-residue region, which is unrelated to known DNA-binding proteins but shares some sequence similarity to the Drosophila Mod(mdg4) proteins. PEB-1 specifically recognizes a site in the C subelement that partially overlaps the PHA-4 binding site. Both the PEB-1 and the PHA-4 binding sites are necessary for strong C sub-element enhancer activity in some cells in which these factors are coexpressed. In contrast the PEB-1 site is dispensable for C sub-element activity in pharyngeal neurons. We propose that PEB-1 functions with PHA-4 to activate target gene expression in cells in which they are coexpressed.

The DAF-3 Smad binds DNA and represses gene expression in the Caenorhabditis elegans pharynx.

Gene expression in the pharyngeal muscles of Caenorhabditis elegans is controlled in part by organ-specific signals, which in the myo-2 gene target a short DNA sequence termed the C subelement. To identify genes contributing to these signals, we performed a yeast one-hybrid screen for cDNAs encoding factors that bind the C subelement. One clone recovered was from daf-3, which encodes a Smad most closely related to vertebrate Smad4. We demonstrated that DAF-3 binds C subelement DNA directly and specifically using gel mobility shift and DNase1 protection assays. Mutation of any base in the sequence GTCTG interfered with binding in the gel mobility shift assay, demonstrating that this pentanucleotide is a core recognition sequence for DAF-3 binding. daf-3 is known to promote formation of dauer larvae and this activity is negatively regulated by TGFbeta-like signaling. To determine how daf-3 affects C subelement enhancer activity in vivo, we examined expression a gfp reporter controlled by a concatenated C subelement oligonucleotide in daf-3 mutants and other mutants affecting the TGFbeta-like signaling pathway controlling dauer formation. Our results demonstrate that wild-type daf-3 can repress C subelement enhancer activity during larval development and, like its dauer-promoting activity, daf-3's repressor activity is negatively regulated by TGFbeta-like signaling. We have examined expression of this gfp reporter in dauer larvae and have observed no daf-3-dependent repression of C activity. These results suggest daf-3 directly regulates pharyngeal gene expression during non-dauer development.

Induction of glyoxylate cycle expression in Caenorhabditis elegans: a fasting response throughout larval development.

The mRNA and the bifunctional protein for the two glyoxylate cycle (GC) enzymes, isocitrate lyase and malate synthase, are expressed in a tissue- and stage-specific pattern in Caenorhabditis elegans. Since expression of the two enzymes for the carbon-conserving glyoxylate cycle is regulated by the availability of carbon sources in microorganisms, we have studied the bifunctional GCP gene expression under fasting conditions and in certain mutants of C. elegans in order to understand possible mechanisms regulating its expression during nematode development. The GCP mRNA and protein levels were elevated in early larvae which were never fed, a result consistent with previous enzyme activity measurements (Khan, F.R., & McFadden, B.A. (1982) Exp. Parasitol. 54, 48-54]. However, larvae of later stages also expressed higher levels of GCP mRNA and protein when they were shifted from normal to fasting growing conditions. The GCP expression appeared to be regulated primarily at the transcriptional level throughout development. Although the expression of both the GCP gene and lin-14 peaks at about the same time during development and are induced by fasting, the regulation of the GCP gene is independent of the heterochronic lin-14 control mechanism of postembryonic lineages, as demonstrated by the fact that there was no significant change of the GCP at both mRNA and protein levels in the heterochronic lin-4 (lf) and lin-14 (gf) mutants compared to the wild type.

Promoter binding factors regulating cyclin B transcription in the sea urchin embryo.

Cyclin B is a key regulatory protein of the cell cycle, central to the control of the G2/M transition. In the developing sea urchin embryo, the cyclin B gene is transcriptionally regulated in concert with changing patterns of cell division. In an effort to understand the mechanism controlling cyclin B expression during development, we have conducted an analysis of the Strongylocentrotus purpuratus cyclin B gene promoter. DNase I foot-printing of the cyclin B upstream region revealed eight binding regions within 435 bp of the start of transcription; seven of these sites were within 215 bp. Found within these regions were consensus sequences for two CCAAT boxes, TATA, and E-boxes and sequences with some similarity to E2F and octamer binding motifs. Upstream sequences were functionally defined by generating cyclin B-CAT fusion genes, containing deletions and base specific mutations, and testing for relative levels of expression by gene transfer. Both CCAAT boxes were found to be essential for maximal levels of expression. A third binding site (PR7) with no recognizable consensus sequence was also found to act as a positive element. Our results suggest that protein binding to the E2F-like sequences may act to reduce expression. Protein binding was further characterized by gel mobility-shift and methylation interference. The CCAAT boxes were found to bind similar, if not identical, proteins. Sequence comparisons and methylation interference data indicate that the likely protein binding these CCAAT sequences is the characterized CCAAT-binding protein CP1. A probe containing site PR7 formed multiple gel shift complexes that, by methylation interference, appeared to be interrelated. One major complex was formed with an oligonucleotide containing the two E2F-like sequences. Protein binding to this probe was specific and required bases within the E2F-like sequences. Our results indicate that cyclin B is subject to positive and negative regulation, involving multiple factors that bind between -200 and -90 bp from the start of transcription.

Bifunctional glyoxylate cycle protein of Caenorhabditis elegans: a developmentally regulated protein of intestine and muscle.

The reaction of an abundant 106-kDa polypeptide with a specific monoclonal antibody has been localized in intestinal and muscle cells of the nematode Caenorhabditis elegans. This protein was first detected in 4-6 cells of the clonal E lineage of 100-cell embryos. This lineage is committed to the intestinal cell fate. The antigen continued to be expressed in the differentiating gut and then appeared in early differentiating body wall muscle cells of 400- to 500-cell embryos. Molecular cloning and sequencing showed that the largest cDNA clone contained 3274 bp and encoded a sequence of 1005 amino acids. The predicted polypeptide of 112,799 MW contains separate domains for the glyoxylate cycle enzymes isocitrate lyase and malate synthase. Their enzymatic activities had been shown previously to be highest in embryos and L1 larvae (Khan, F. R., and McFadden, B. A. (1980). FEBS Lett. 115, 312-314; Khan, F. R., and McFadden, B. A. (1982). Exp. Parasitol. 54, 48-54; Wadsworth, W. G., and Riddle, D. L. (1989). Dev. Biol. 132, 167-173). The domain-specific sequences were shown to be contiguous in genomic DNA and are separated by an intron of 68 bp. A single polypeptide and both enzymatic activities are precipitated by the antibody, and peptide fragments resulting from limited proteolytic digestion contained amino acid sequences which overlap the predicted junctional region. The physical localization of the gene correlates with a small region of the left arm of Linkage Group V to which multiple embryonic lethal mutations have been mapped.

The role of the CCAAT/enhancer-binding protein in the transcriptional regulation of the gene for phosphoenolpyruvate carboxykinase (GTP).

Previous studies have identified a region in the promoter of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (positions -460 to +73) containing the regulatory elements which respond to cyclic AMP, glucocorticoids, and insulin and confer the tissue- and developmental stage-specific properties to the gene. We report that CCAAT/enhancer-binding protein (C/EBP) binds to the cyclic AMP-responsive element CRE-1 as well as to two regions which have been previously shown to bind proteins enriched in liver nuclei. The DNase I footprint pattern provided by the recombinant C/EBP was identical to that produced by a 43-kDa protein purified from rat liver nuclear extracts, using a CRE oligonucleotide affinity column, which was originally thought to be the CRE-binding protein CREB. Transient contransfection experiments using a C/EBP expression vector demonstrated that C/EBP could trans activate the PEPCK promoter. The trans activation occurred through both the upstream, liver-specific protein-binding domains and the CRE. The CRE-binding protein bound only to CRE-1 and not to the upstream C/EBP-binding sites. The results of this study, along with physiological properties of C/EBP, indicate an important role for this transcription factor in providing the PEPCK gene with several of its regulatory characteristics.