HLH-29, like other members of the REF-1 family of transcription factor,s is a basic helix-loop-helix (bHLH) protein that contains two fully intact bHLH domains. Previously we demonstrated that HLH-29 affects development of the somatic gonad and the vulva in C. elegans and is expressed in the spermatheca of adult hermaphrodites. More recently, our results from yeast-two-hybrid screens suggested that HLH-29 interacts with the sexual identity factor, FKH-6. Our objectives are to demonstrate a genetic and molecular interaction between HLH-29 and FKH-6 in vitro and in vivo. First, we show by Far-western blotting and by pull-down assays that HLH-29 and FKH-6 interact via the N-terminal most bHLH domain, Domain A. Second, we show that
hlh-29 animals have normal brood sizes, but also lay significantly more unfertilized oocytes than wild-type animals. We show that this phenotype is due at least in part to defective ovulation cycles that worsen as the animals age and that vary according to gonad arm. Third, we show that the interactions between FKH-6 and HLH-29 are likely to affect development and morphology of the gonad arm, in addition to ovulation, as HLH-29;FKH-6 (+/-) animals have more severe phenotypes than single mutant animals. Finally, our preliminary RT-qPCR data show that expression of the IP3 signaling pathway genes
ipp-5 and
ppk-1 is regulated by HLH-29 alone, while the expression of the genes C37H5.6,
dis-3, and
emb-9 is regulated by HLH-29 together with FKH-6.