Shen YS and Ellis RE.pdf (2.58 MB)
Cbr-TRA-2 interacts with TRA-1 to prevent spermatogenesis
poster
posted on 2020-04-20, 22:43 authored by Yongquan Shen, Ronald E EllisWe
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.