Skn-1 is a maternally-expressed cell linage determinant in C. elegans, which directs one of the four earliest embryonic blastomeres to form pharynx and muscle cells, presumably by activating expression of downstream genes. Its mst intriguing feature is its bipartite monomeric DNA binding motif, in which it appears that an """"""""arm"""""""" like that of the homeodomain (HD) protein antennapedia (Ant) recognizes bases in the minor groove, and a basic region (BR) similar to that of basic """"""""leucine zipper"""""""" (bZIP) proteins binds in the major groove. Consistent with the notion that Skn-1 regulates gene expression, it also contains a transcription activation domain. The first two specific aims of the proposed research are to investigate how Skn-1 interacts with DNA and how its binding specificity is determined, and to elucidate how its previously-undescribed DNA binding motif forms a structure that allows it to make contact with DNA.
These aims will be addressed by a combination of mutagenesis, molecular modeling, biochemical, and biophysical experiments. In a collaborative effort, the structure of the Skn-1-DNA complex will be determined by X- ray crystallography. These experiments are of significance in part because of the important biological role played in Skn-1, in that it provides and example of how cell fate can be determined in the early embryo, and because an understanding of its DNA-binding properties will be essential for gaining an understanding of how it functions. Of equal importance, these experiments will provide new general insights into the strategies by which proteins interact with DNA, because the finding that a bZIP-like BR which lacks the normally contiguous ZIP dimerization domain is incorporated into a monomeric DNA binding motifs is surprising and unprecedented. An understanding of the critical issues involved in protein-DNA interactions is essential for future design of therapies that will intervene in such molecular recognition events. This bipartite domains is of further evolutionary interest because it contains elements for two distinct known families of DNA-binding proteins, and probably defines a novel DNA-binding protein family with members that have yet to be discovered. The third specific aim is to investigate how Skn-1 functions as a transcriptional activator. These experiments include elucidation and study of its transcriptional activation domain, which appears to be different from those described previously, investigations of how Skn-1 is regulated post-transcriptionally in particular C. elegans blastomeres, and isolation of downstream genes that it controls. They will involve cell-culture assays, collaborative in vivo studies in C. elegans, genetic screen in yeast, and subtractive hybridization experiments. The significance of these experiments lies in the specific insights that they will provide into how Skn-1 functions in the cell, and in the general insights into biological regulation that will derive from investigation of this post-transcriptional regulatory mechanism.
| Carroll, A S; Gilbert, D E; Liu, X et al. (1997) SKN-1 domain folding and basic region monomer stabilization upon DNA binding. Genes Dev 11:2227-38 |
