T. Keith Blackwell
Department of Genetics
Joslin Diabetes Center
Pathology, Rm. 655B
One Joslin Place
Boston, MA 02115
1995 Searle Scholar
Molecular Mechanisms of SKN-1 and PIE-1 FunctionIn the nematode C. elegans, maternally-expressed SKN-1 and PIE-1 proteins are required for proper cell lineage specification during the earliest embryonic stages. The first embryonic cell division produces an anterior (AB) and a posterior (P1) daughter cell. SKN-1 expression is detectable in the nuclei of P1 and its descendants through the twelve-cell stage, and is required for specification of pharyngeal and gut precursors. In embryos produced by skn-1 mutants, these precursors instead give rise to excess hypodermal (skin) cells. Although we have determined that SKN-1 is a DNA-binding transcription factor, its target genes have yet to be identified. The PIE-1 protein is required for specification of germ cell precursors, which are derived from the P1 cell, and it is expressed specifically in these cells throughout embryogenesis. In pie-1 (pharyngeal and intestinal excess) mutants, germ cell precursors form pharyngeal, gut, and muscle cells by a skn-1-dependent pathway. Not only does PIE-1 inhibit SKN-1 from functioning in these cells; recent evidence suggests that its role may be to prevent any transcription of zygotic genes from occurring in them. The PIE-1 amino acid sequence contains two Cys(3)-His zinc finger motifs, which are of unknown structure and function. These motifs are related to those present in the TIS11 genes, which are found in species ranging from humans to yeast. TIS11 genes were first identified as immediate early genes induced by treatment of mammalian cells with growth factors, but their functions remain unknown.
Our laboratory is interested in understanding how SKN-1 and PIE-1 proteins function as transcriptional regulators. In a distinct but related effort, we are investigating the SKN-1 DNA binding domain, which is unusual in that is composed of sub-domains from bZIP and homeodomain proteins. To date, otherwise we have focused primarily on studying the PIE-1 protein, and have employed mammalian cell culture assays to evaluate the effects of PIE-1 expression on transcription. Our preliminary findings indicate that in transient transfections, expression of PIE-1 can repress transcription driven by SKN-1 and a variety of other activators, and even by viral enhancers. For example, PIE-1 expression reduces dramatically transcription from the SV40 enhancer. Fusion of portions of PIE-1 to the yeast GAL4 DNA binding domain have demonstrated that it contains residues that can function as a powerful repressor domain, when thus tethered to a promoter. Based on this last finding, and on the apparent wide range of PIE-1 function, we hypothesize that PIE-1 may act on an evolutionarily conserved component of the transcriptional regulatory machinery, and bring to it residues that are capable of effecting repression.
In the coming months, we plan to test this hypothesis by evaluating further the capabilities of PIE-1 to repress transcription. We are determining whether PIE-1 can repress transcription from different types of promoters, and we are evaluating how particular PIE-1 residues mediate its repressive effect. We are also employing various approaches to look for factors that interact with PIE-1, to identify the step(s) in transcriptional activation at which it might function, and to search for its vertebrate homologs.
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