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