Getting signals crossed in C. elegans.

The induction of an appropriate cellular response to a stimulus often depends on the intricate interplay between multiple signaling pathways. Recent work utilizing Caenorhabditis elegans has enabled the identification of points of convergence between signaling pathways and permitted the elucidation of how multiple signals work in concert to ensure a proper response.

sem-4 promotes vulval cell-fate determination in Caenorhabditis elegans through regulation of lin-39 Hox.

Vulval cell-fate determination in Caenorhabditis elegans requires the action of numerous gene products, including components of the Ras/Raf/MAPK signaling cascade and the hox gene lin-39. sem-4 encodes a zinc finger protein with previously characterized roles in fate specification of sex myoblasts, coelomocytes, and multiple neuronal lineages in C. elegans (M. Basson and R. Horvitz, 1996, Genes Dev. 10, 1953-1965). By characterizing three new alleles of sem-4 that we identified in a screen for vulval-defective mutants, we determined that loss of sem-4 activity results in abnormal specification of the secondary vulval cell lineages. We analyzed sem-4 interactions with other genes involved in vulval differentiation and determined that sem-4 does not function directly in the Ras-mediated signal transduction pathway but acts in close association with and upstream of lin-39 to promote vulval cell fate. We demonstrate that sem-4 regulates lin-39 expression and propose that sem-4 is a regulator of lin-39 in the vulval cell-fate determination pathway that may act to link lin-39 to incoming signals.

COG-2, a sox domain protein necessary for establishing a functional vulval-uterine connection in Caenorhabditis elegans.

In screens for mutants defective in vulval morphogenesis, multiple mutants were isolated in which the uterus and the vulva fail to make a proper connection. We describe five alleles that define the gene cog-2, for connection of gonad defective. To form a functional connection between the vulva and the uterus, the anchor cell must fuse with the multinucleate uterine seam cell, derived from uterine cells that adopt a (pi) lineage. In cog-2 mutants, the anchor cell does not fuse to the uterine seam cell and, instead, remains at the apex of the vulva, blocking the connection between the vulval and uterine lumens, resulting in an egg-laying defective phenotype. According to lineage analysis and expression assays for two (pi)-cell-specific markers, induction of the (pi) fate occurs normally in cog-2 mutants. We have cloned cog-2 and shown that it encodes a Sox family transcription factor that is expressed in the (pi) lineage. Thus, it appears that COG-2 is a transcription factor that regulates a late-stage aspect of uterine seam cell differentiation that specifically affects anchor cell-uterine seam cell fusion.

Two evolutionarily conserved repression domains in the Drosophila Kruppel protein differ in activator specificity.

To identify biologically functional regions in the product of the Drosophila melanogaster gene Kruppel, we cloned the Kruppel homolog from Drosophila virilis. Both the previously identified amino (N)-terminal repression region and the DNA-binding region of the D. virilis Kruppel protein are greater than 96% identical to those of the D. melanogaster Kruppel protein, demonstrating a selective pressure to maintain the integrity of each region during 60 million to 80 million years of evolution. An additional region in the carboxyl (C) terminus of Kruppel that was most highly conserved was examined further. A 42-amino-acid stretch within the conserved C-terminal region also encoded a transferable repression domain. The short, C-terminal repression region is a composite of three subregions of distinct amino acid composition, each containing a high proportion of either basic, proline, or acidic residues. Mutagenesis experiments demonstrated, unexpectedly, that the acidic residues contribute to repression function. Both the N-terminal and C-terminal repression regions were tested for the ability to affect transcription mediated by a variety of activator proteins. The N-terminal repression region was able to inhibit transcription in the presence of multiple activators. However, the C-terminal repression region inhibited transcription by only a subset of the activator proteins. The different activator specificities of the two regions suggest that they repress transcription by different mechanisms and may play distinct biological roles during Drosophila development.

Active repression mechanisms of eukaryotic transcription repressors.

Regulators of transcription and, in particular, transcriptional repressors, play central roles in vital biological processes, such as development and the regulation of cell growth. A major class of transcriptional repressors consists of DNA-binding proteins that interact with specific promoter elements and repress transcription via small, portable repression 'domains'. Such transcriptional inhibition, first identified only five years ago, has been termed active repression, because it is not mediated simply by steric hindrance mechanisms. It is unknown how interaction(s) between such a repressor and the RNA polymerase II basal or regulatory transcription machinery can derail the formation or competency of a transcription complex at a promoter. However, the recent progress toward identification of molecular targets suggests several specific mechanisms for achieving active repression.

Mapping and mutagenesis of the amino-terminal transcriptional repression domain of the Drosophila Krüppel protein.

We previously demonstrated that the Drosophila Krüppel protein is a transcriptional repressor with separable DNA-binding and transcriptional repression activities. In this study, the minimal amino (N)-terminal repression region of the Krüppel protein was defined by transferring regions of the Krüppel protein to a heterologous DNA-binding protein, the lacI protein. Fusion of a predicted alpha-helical region from amino acids 62 to 92 in the N terminus of the Krüppel protein was sufficient to transfer repression activity. This putative alpha-helix has several hydrophobic surfaces, as well as a glutamine-rich surface. Mutants containing multiple amino acid substitutions of the glutamine residues demonstrated that this putative alpha-helical region is essential for repression activity of a Krüppel protein containing the entire N-terminal and DNA-binding regions. Furthermore, one point mutant with only a single glutamine on this surface altered to lysine abolished the ability of the Krüppel protein to repress, indicating the importance of the amino acid at residue 86 for repression. The N terminus also contained an adjacent activation region localized between amino acids 86 and 117. Finally, in accordance with predictions from primary amino acid sequence similarity, a repression region from the Drosophila even-skipped protein, which was six times more potent than that of the Krüppel protein in the mammalian cells, was characterized. This segment included a hydrophobic stretch of 11 consecutive alanine residues and a proline-rich region.