Identifying Co-Factors That Drive TRA-1 Activator Function

Gli proteins are involved in cell fate determination, proliferation, and patterning in many species and are major effectors of Hedgehog (Hh) signaling. There are three Gli proteins in humans, and mutations or errors in their regulation lead to a variety of developmental disorders or cancer. However, the mechanisms by which they interact with co-factors are poorly understood. We are analyzing co-factors of Gli proteins using TRA-1 in Caenorhabditis nematodes. The TRA-1 zinc fingers are structurally like those of other Gli proteins, and TRA-1 can be cleaved like other Gli proteins to form a repressor. However, its function has changed during evolution — in nematodes, TRA-1 controls sexual fates and plays a central role in self-fertility, which makes it easy to assay mutant phenotypes. Furthermore, worms lack classical Hedgehog signaling, so study of nematode TRA-1 should reveal other types of regulation. Our lab has shown that full-length TRA-1 can work as an activator and promote spermatogenesis, and that the mutation cbr-tra-1(v48) disrupts this process and prevents spermatogenesis. We suspect that regulation of TRA-1 activator plays a major role in the evolution of hermaphrodite spermatogenesis in nematodes. Because v48 was isolated in a classical EMS mutagenesis, we recently made other activator mutations to confirm that all of its phenotypes were due solely to the alteration of TRA-1. Since TRA-1 activator is likely to interact with a diverse set of co-factors, whose activities might help determine whether specific targets are activated or repressed, we identified sites in TRA-1 where an OLLAS tag does not inactivate the protein, and used anti-OLLAS antibodies to isolate TRA-1 complexes. We are now characterizing the products, and preparing to analyze them with mass spectrometry. While doing so, we will look for important modifications to TRA-1 itself, as well as the precise site of cleavage that forms the repressor. In addition, we hope to identify TRA-1 co-factors and learn how they regulate Gli activity. Finally, we will see if any of these co-factors has a novel role in species that produce self-fertile hermaphrodites