Cbr-TRA-2 interacts with TRA-1 to prevent spermatogenesis

2020-04-20T22:43:27Z (GMT) by Yongquan Shen Ronald E Ellis
We are studying C. briggsae TRA-1, to learn how this Gli transcription factor regulates sexual identity in worms. To begin, we generated cbr-tra-1(v455), which has an OLLAS tag inserted near the N-terminus, and cbr-tra-1(v424), which has one near the C-terminus. Both alleles develop normally, so neither tag affects function. On western blots we see that Cbr-TRA-1 is cleaved to form a product that is slightly larger than its C. elegans counterpart. This product is predicted to be a repressor that blocks the transcription of male genes. Finally, sequence alignments reveal a small conserved domain near the putative cleavage site. To analyze TRA-1 function, we used gene editing to make the following mutations: (1) cbr-tra-1(v197) is a frameshift upstream of the cleavage site. When its mRNA is stabilized, the truncated product makes a functional TRA-1 repressor, since XX animals develop normal hermaphrodite bodies. However, they make only oocytes and no sperm. (2) cbr-tra-1(v405) and cbr-tra-1(v406) are frameshifts located downstream of the cleavage site. they develop normal hermaphrodite bodies, so TRA-1 repressor is functioning. However, these mutations also cause animals to make oocytes instead of sperm, which implies that full-length TRA-1 normally promotes spermatogenesis. (3) cbr-tra-1(v197v383) alters 30 residues in the domain that binds TRA-2, and disrupts interactions between C. briggsae TRA-2 and TRA-1 in yeast two-hybrid assays. These XX mutants make significantly more sperm than the wild type. Furthermore, this mutation restores spermatogenesis to many she-1(v35) XX animals. We conclude that TRA-2 normally binds TRA-1 to block spermatogenesis. Analyses of tra-2(null); fem-3(null); she-1(null) animals support this model. This result is surprising, since C. elegans tra-2 mutations that block the interaction, cause oogenesis. (4) We are now making mutants which alter the putative cleavage domain without affecting upstream or downstream regions to see if full-length Cbr-TRA-1 activates male genes like Gli/Ci in humans and flies. (5) we and others in our lab found that mutations in several chromatin regulators alter the sperm/oocyte decision. Taken all the results together, we propose that full-length TRA-1 promotes spermatogenesis by working with chromatin regulatory factors, whereas the cleaved form of TRA-1 represses spermatogenesis. In C. briggsae, TRA-2 blocks the function of full-length TRA-1.