The
her-1gene is required for male somatic development in C. elegans.
her-1 produces two male-specific transcripts from two tandem promoters: P1 and P2. The larger 1.2 kb transcript,
her-1a, is the functional regulator of male development and is predicted to encode a 20 kDa novel secreted protein. Two gain-of-function (gf) mutations that map two bp upstream of the transcriptional start site result in the inappropriate expression of
her-1 in XX animals and their subsequent masculinization. These two mutations may define a binding site for a negative regulator(s) of
her-1. Four loss-of-function (lf) mutations in the
her-1 promoter have been isolated; they occur within nine bp, 45 bp upstream of the transcriptional start site and cause variable feminization of XO animals. We hypothesize that this cluster of mutations defines a site required for
her-1 expression in XO animals. To identify trans-acting factors that regulate
her-1, we performed a yeast one-hybrid screen using two short oligonucleotides from the lf and gf sites in the P1 promoter. F13G3.1, a putative general transcription factor containing three zinc fingers, was identified in the screen using the lf site oligonucleotide. This interaction has been confirmed by gel mobility shift assays. The gf site binds specifically to C26C6.5, the worm homologue of
p66, a subunit of Mi-2 that is the central component of the nucleosome remodeling and histone deacetylase complex. These data suggest that a chromatin remodeling complex is involved in the negative regulation of
her-1. Consistent with this hypothesis, RNAi with C26C6.5 in a
her-1 gf strain caused a substantial increase in the frequency of strongly masculinized animals. To assess the biological relevance of the
her-1 promoter interactions, we are currently carrying out several experiments. We have constructed transcriptional GFP reporter constructs of both genes to look at their expression patterns. We are also creating transgenic lines in which F13G3.1 and C26C6.5 are overexpressed. We predict that overexpression of F13G3.1 will masculinize XX animals and overexpression of C26C6.5 will feminize XO animals. In addition we are injecting the lf and gf promoter sites into worms and looking for the effects of titrating F13G3.1 and C26C6.5. Finally we would like to determine whether F13G3.1 and C26C6.5 colocalize to the
her-1 promoter in vivo.