The transcription factor TRA-1 is the terminal global regulator of the sex determination pathway in C. elegans. Hermaphrodites require TRA-1 activity to repress the transcription of genes that specify male cell fates. In the absence of TRA-1 activity, males develop. We have observed that while both sexes accumulate low levels of TRA-1A (Mr ≈ 125kD), hermaphrodites but not males accumulate higher levels of a series of TRA-1 phosphoisoforms of Mr ≈ 90-100kD. We have evidence that these isoforms, which we believe to result from proteolytic cleavage of TRA-1A, are sufficient to repress several known TRA-1 target genes. The transcription factor Cubitus interruptus (Ci) is the Drosophila homologue of TRA-1 and both proteins bind similar DNA sequences. Protein kinase A (PKA) phosphorylates Ci to stimulate its proteolytic processing to produce a repressor isoform. We hypothesize that PKA may similarly regulate the production of hermaphrodite-specific cleaved isoforms of TRA-1. This leads us to predict that increased PKA activity would feminize male animals. To test whether PKA may play a role in sex determination, a heat-inducible transgene was used to overexpress the catalytic subunit (PKAc) in C. elegans. A vitellogenin gene reporter (vit-2::gfp) provides a marker of feminization of the intestine. Expression of PKAc induced vit-2::gfp expression in the intestinal cells of male worms. Worms lacking tra-1, however, did not express vit-2::gfp in response to PKAc overexpression. These results suggest that PKAc has TRA-1-dependent feminizing activity in the intestine. Western blot analysis will be carried out to determine whether PKAc overexpression results in accumulation of truncated phosphoisoforms of TRA-1. As a further test of the involvement of PKA in TRA-1 regulation, we have deleted sequences encoding a cluster of potential PKA target sites from a rescuing tra-1 transgene that includes the entire tra-1 locus (see abstract by Cooperstock, Sassi, et al.). Preliminary analysis has revealed that the deletion construct has reduced feminizing activity in tra-1(o) animals. We are now testing whether this reduced ability to rescue hermaphrodite development correlates with a change in the accumulation of the hermaphrodite-specific phosphoisoforms. To test whether truncation of TRA-1A is necessary for the greater accumulation of TRA-1 isoforms in hermaphrodites and thus for the feminizing activity of tra-1, we are producing a derivative of our rescuing tra-1 transgene that is resistant to cleavage. We will present a comparison of its activity to that of the wild-type transgene.

Affiliation:
- Dept Molec & Medical Genetics, Univ Toronto, Toronto, ON, Canada