Germ cell fate determination: Cloning and characterization of <italic>fog-3</italic> and its homologs in <italic>Caenorhabditis</italic>.

Abstract

In <italic>C. elegans</italic>, the choice of sex is determined by a cascade of sex determination genes. Genetic studies indicate that <italic> fog-3</italic> acts at the end of this cascade, and that <italic>fog-3</italic> might directly determine male sexual fate in the germ line. Studies of <italic> fog-3</italic> in this thesis show that FOG-3 is a nematode homolog of the Tob family of proteins, which have been suggested to suppress proliferation or promote differentiation. The domain that BTG1 and BTG2 share with FOG-3 interacts with a transcriptional regulatory complex that has been conserved in all eukaryotes. Finally, analysis of mutations shows that this domain is essential for <italic>fog-3</italic> to function. Thus, one possibility is that FOG-3 controls transcription of genes required for germ cells to initiate spermatogenesis rather than oogenesis. How is FOG-3 activity regulated? Several lines of evidence suggest <italic>fog-3</italic> is regulated by <italic> tra-1</italic>. First, Northern analyses and RT-PCR experiments indicate that expression of <italic>fog-3</italic> mRNA is controlled by <italic>tra-1</italic>. Second, studies of <italic>fem;tra-1</italic> double mutants show that this control could be direct. Third, the <italic>fog-3</italic> promoter contains multiple sites that bind TRA-1A in gel shift assays, and mutations in these sites alter the activity of <italic>fog-3 in vivo</italic>. These results establish <italic>fog-3</italic> as one of the first known targets of transcriptional regulation by TRA-1A. Furthermore, we show that <italic>tra-1</italic> controls a terminal regulator of sexual fate in germ cells, just as it is thought to do in the soma. To learn if the function of <italic>fog-3</italic> has been conserved during evolution, we cloned its homologs from <italic>C. briggsae </italic> and <italic>C. remanei</italic>. Both BTF and TF are highly conserved in <italic>Cb</italic>-FOG-3 and <italic>Cr</italic>-FOG-3, suggesting that these domains are important for <italic>fog-3</italic> to function. Furthermore, one additional region that is unique to the FOG-3 proteins is also highly conserved. The phenotypes of <italic>Cb-fog-3</italic> and <italic>Cr-fog-3 </italic> RNAi animals suggest that <italic>fog-3</italic> is required to promote male germ cell fates in all three nematode species. Finally, there are TRA-1A binding sites upstream of the coding regions of <italic>Cb-fog-3 </italic> and <italic>Cr-fog-3</italic>. This suggests that the regulation of <italic>fog-3</italic> by TRA-1A has also been conserved during evolution.