In C. elegans , sex is determined by a chromosome counting mechanism that senses the ratio of X chromosomes to sets of autosomes. The low X:A ratio in XO diploid worms permits high expression of the sex switch gene
xol-1 and hence male development. Conversely, a high X:A ratio in XX animals results in low
xol-1 activity and hermaphrodite development. Although the autosomal component of the sex signal is uncharacterized, the X portion is comprised of several X-signal elements (XSE) that act cumulatively to repress
xol-1 function in a dose-dependent manner. A mere two-fold difference in the dose of XSEs is translated to a greater than ten-fold difference in
xol-1 expression between the sexes. The molecular identities of two XSEs have been determined. SEX-1 is a nuclear hormone receptor that represses
xol-1 transcriptionally, and FOX-1 is an RNA-binding protein that represses
xol-1 post-transcriptionally. We report the molecular mechanism by which FOX-1 represses
xol-1 and the identification of two new putative XSEs. Additionally, we report the initial characterization of the autosomal sex signal. Our transgenic studies have shown that FOX-1 regulates
xol-1 expression through
xol-1 ’s sixth intron, which is both necessary and sufficient for repression. Overexpression of FOX-1 prevents removal of the sixth intron in vivo . The sixth intron can confer FOX-1-mediated repression of a reporter gene. Recombinant FOX-1 binds directly to the sixth intron in vitro . The region required for FOX-1 binding has been localized to two separate 37-nucleotide regions with extensive sequence overlap. The contribution of these binding sites to
xol-1 regulation is being analyzed in vivo . Together, these data indicate that FOX-1 represses
xol-1 directly by preventing proper splicing of its sixth intron. Two new putative XSEs have also been identified. An XSE duplication strain that is 100% XO-lethal was screened by feeding double-stranded RNA from half of the ~140 ORFs in a region of X known to contain at least one XSE. RNAi of
ceh-39 , a member of the evolutionarily conserved ONECUT homeodomain family, suppresses the male lethality of this duplication and strongly enhances the sex determination and dosage compensation phenotypes of
fox-1 and
sex-1 . A genetic screen of the XSE duplication strain for suppressors of XO lethality identified three suppressors,
y323 ,
y324 , and
y326 . These alleles have synergistic sex determination and dosage compensation defects in combination with
fox-1 or
sex-1 and map to a small region of X not previously known to contain an XSE. The sex signal also consists of an autosomal component whose precise nature is unknown but presumably serves to increase the activity of
xol-1 . Discrete autosomal signal elements (ASE) that are analogous to, but act in opposition to, the XSEs may exist. If true, ASE loss-of-function mutations should suppress the complete XX lethality caused by mutating the two XSEs
fox-1 and
sex-1 . To test this hypothesis, we screened 4500 mutagenized haploid genomes for suppressors of the hermaphrodite lethality of
fox-1 sex-1 double mutants. Of the 47 mutations isolated, including dominant and recessive X-linked and autosomal suppressors, seven autosomal alleles with strong suppression phenotypes are under analysis to determine their role in chromosome counting.