In C. elegans, the processes of sex determination and X-chromosome dosage compensation are coordinately controlled through
sdc-1,
sdc-2, and
sdc-3 . The SDC proteins function together in a complex that associates with X chromosomes of hermaphrodites to repress X-linked gene expression by half. The complex also binds to the promoter of the male sex-determination gene
her-1 to repress its transcription 20-fold, thereby promoting hermaphrodite development. To better understand the role of
sdc-1 in these processes, suppressors of the nonsense mutation
sdc-1(
n485) were identified (1). The semi-dominant
sdc-1 suppressor
y63 differentially suppresses the dosage compensation defects but not the sex determination defects. Suppression by
y63 is gene-specific and allele-non-specific. Our studies have shown that
y63 is a loss-of-function mutation and the dominance is due to haplo-insufficiency, suggesting that
y63 may bypass the need for
sdc-1 . Supporting this hypothesis, our western blot analysis revealed that SDC-1 protein is not present in embryonic extracts of
sdc-1(
n485) or
y63 sdc-1(
n485) embryos. In immunofluorescence experiments, no staining was detected in
sdc-1(
n485) embryos. However, slight nuclear staining that colocalized with the dosage compensation complex on the X chromosome was seen in
y63 sdc-1(
n485) embryos. This result suggested that
sdc-1(
n485) may not be a true null allele and that
y63 may function by increasing levels of SDC-1 protein. To prove that
y63 is an actual bypass suppressor, we isolated the
sdc-1 deletion allele
y415 . SDC-1 protein is absent in
sdc-1(
y415) and
y63 sdc-1(
y415) mutants, indicating that the
y63 mutation bypasses the need for SDC-1 in dosage compensation. Using single nucleotide polymorphisms,
y63 was finely mapped to a 40 kb region of the X chromosome that encompasses 11 open reading frames (ORFs). RNAi and genomic fragment rescue experiments revealed that the ORF Y62H9A.9 is defective in
y63 mutants. Y62H9A.9 was renamed
box-1 for b ypass o f X expression defect. The
y63 mutation is a G to A transition in the splice donor site of the 6 th intron. Interestingly,
y63 suppression of
sdc-1 mutants can be reversed by
sup-6(
st19) , a mutant allele of a U1 snRNA gene.
sup-6(
st19) has a substitution in the 5' end that allows proper splicing of G to A splice donor site mutations, like the one in
y63 . Since splice donor site mutations are often partial loss-of-function mutations, we isolated a deletion allele
y417 of
box-1 that removes the start codon, part of the first exon and 220 bases upstream of the ATG, in order to establish the null phenotype. Like
y63 ,
y417 does not have a phenotype on its own and is a strong semi-dominant suppressor of multiple
sdc-1 mutant alleles.
box-1 has no recognizable motifs or domains that provide a clue to its wild type function. The behavior of
box-1 suppression suggests that it may be modulating the activity of the dosage compensation complex. In order to further our understanding of the mechanism by which
box-1 mutants suppress
sdc-1 dosage compensation defects, we are developing antibodies against this gene product to examine its subcellular localization and to perform co-immunoprecipitation experiments to identify potential interacting factors. Elucidating the function of this suppressor may explain how one protein complex, the SDC protein complex, functions to repress transcription by 2-fold along the entire X chromosome compared to the 20-fold repression of
her-1 . 1. Villeneuve, A.M. and B.J. Meyer, (1990) Genetics 124 : 91-114