In order to understand cell fate specification during vulval development, we are conducting a structure/function analysis of LIN-31, a member of the winged-helix family of transcription factors, which is required for the proper specification of vulval cell fates in C. elegans (Miller et al. , Genes and Dev., 7:933, 1993). The LIN-31 protein contains a DNA-binding domain, an acidic region, four MAP kinase consensus phosphorylation sites, and a small region of homology conserved among other winged-helix proteins. We are using site-directed mutagenesis to create plasmids carrying specific mutations in the MAP kinase consensus phosphorylation sites, acidic domain, or the homology region of the LIN-31 protein. These plasmids are then injected into an animal with no functional LIN-31 protein to test for their ability to provide LIN-31 function. LIN-31 is phosphorylated by the MAP kinase MPK-1 in response to an inductive signaling event (Tan et al. , Cell 93: 569, 1998). Tan et al . have already shown that removing all four MAP kinase consensus phosphorylation sites inactivates one of LIN-31's functions, but the individual contribution of each site is not known. Four individual clones are being created, with each one containing a different disrupted MAP kinase consensus phosphorylation site. In addition, it is appealing to imagine that the small acidic domain (six consecutive aspartic acid residues) adjacent to the DNA-binding domain is functioning as a transcriptional activator, as is the case with acidic regions in some other transcription factors. There is no proof, however, that this rather small acidic domain in LIN-31 is even required for function. Five mutant clones, in which different portions of the acidic domain have been removed or replaced will address this question. Finally, the carboxy-terminus of LIN-31 contains a small region of homology that shows similarity to a subset of winged-helix proteins. The function of this homology region is also unknown and is being explored in this project. This structure/function study is especially appealing since the current model (Miller et al. , 1993; Tan et al. , 1998; and Miller et al. , Genetics 156: 1595, 2000) proposes that LIN-31 has two functions: 1) to activate vulval cell fates in P5.p, P6.p, and P7.p and 2) to repress vulval cell fates in P3.p, P4.p, and P8.p. Microinjection of these clones into
lin-31(null) animals will allow us to test if each specific mutation disrupts one, both, or none of LIN-31's functions. Thus, this approach will allow us to identify the roles of specific domains or sites in the LIN-31 protein.