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.

How is the mec-3 Gene Regulated?

Ding Xue, Marty Chalfie, Mike Finney and Gary Ruvkun

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 
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