Worm Breeder's Gazette 11(4): 62
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
The predicted amino acid sequence of mec-3 suggests that it is a transcription factor that regulates the expression of genes in the touch cells, the PVD and FLP cells (Way Chalfie, Cell 54: 5, 1988; Xue & Chalfie WBG 11:3). Genetic and molecular experiments suggest that several genes regulate the expression of mec-3 (Chalfie & Au, Science 243: 1027, 1988; Way & Chalfie, Genes & Dev. 3: 1823, 1989; Finney & Ruvkun, WBG 11:3). These include unc-86, which is required for (at least) the initial expression of mec-3, and mec-3 itself, which is needed for the gene's continued expression. One prediction from these observations is that the unc-86 and mec-3 gene products should bind to cis elements within the mec-3 gene if they directly control mec-3 expression. Conserved elements in the C. elegans and C. briggsae mec-3 genes ( Xue & Chalfie, WBG 11:3) hint at a direct involvement of unc-86 and mec-3. Four conserved regions (26 to 42 bp long: CS1-CS4) upstream of the putative transcription start and a fifth (CS5) located after the putative transcription start are potential cis-regulatory elements. Both CS1 and CS2 contain a sequence (AAATGCAT) that is similar to the binding sites for several POU proteins (Pit-1, Oct-1, and Oct-2), so these sites are potential unc-86 binding sites. CS3 contains an isl-1 protein binding site (isl-1 is a LIM domain homeoprotein like mec-3; Karlsson et al., Nature 344: 879, 1990), so this site may bind the mec- 3 product. To test these hypotheses we have begun DNA-protein binding studies by performing gel mobility shift analysis and DNase I footprinting using unc-86 protein generated in E. coli and gel shifts using full- length mec-3 protein produced in E. coli. Proteins from both genes bind to mec-3 DNA. In the gel-shift experiments, mec-3 protein, as predicted, retarded a CS3 oligo (it may also bind to other regions). The unc-86 protein retarded the movement of both CS2 and CS3 oligos. Although CS3 does not share sequence similarity with CS1 and CS2, it does contain sequences that have been involved in vitro POU-protein binding (Garcia-Blanco et al. Genes & Dev. 3:739, 1989). DNase I footprinting has confirmed the gel retardation data on unc-86, showing that the protein binds CS1, CS2, and CS3. We are currently doing the footprint analysis with mec-3 protein to determine whether both unc-86 and mec-3 proteins bind to the same sequence within CS3 (and, perhaps, at other sites), and we are investigating whether mec-3 and unc-86 cooperate or inhibit each other's binding. Two retarded species are seen in gel shifts of unc-86 protein with oligos for either the CS2 or CS3 regions. At low unc-86 concentrations only the more rapidly migrating species is seen. As the unc-86 concentration is increased, the more slowly migrating species appears and becomes predominant. It is likely that this latter species contains a dimer of bound unc-86 protein. We are investigating whether this binding is cooperative as has been suggested by Ingraham et al. (Cell 61: 1021, 1990) for Pit-1 binding. The above data suggest that CS1, CS2, and CS3 contain cis-regulatory elements that are responsible for the initiation of mec-3 expression ( by binding unc-86, and possibly other, protein) and that CS3 (and possibly other sites) may also be important for the maintained expression of the gene (by binding mec-3 protein and perhaps other proteins, such as mec-17). A third form of regulation, repression, has been suggested by experiments by Jeff Way (WBG 11:2) in which deletion of 90 bp of mec-3 DNA in a mec-3:lacZ fusion results in fusion expression, so this conserved sequence may be important for this regulation. Two extra mec-7-positive cells are also seen in the tails of sem-4 mutants (see Mitani & Chalfie in this issue). Although we do not yet know whether these are the same as those that Jeff sees ( we have not yet made the appropriate double), this observation may indicate that the sem-4 product negatively regulate mec-3 expression, perhaps by acting at the CS4 site. We do not know a function for the CS5, but are testing the requirements for this and the other CS sites by in vitro mutagenesis and transformation. In addition we are beginning to look for other proteins that may bind to these and other sites within the mec-3 gene and regulate its expression