C. elegans equalizes X-linked gene expression between XX hermaphrodites and XO males by halving transcript levels from each of the hermaphrodite X chromosomes. Although the mechanism of dosage compensation is not known, molecular analysis of
dpy-27 has yielded significant insight. DPY-27 is a member of the evolutionarily conserved SMC family of proteins, which are involved in chromosome condensation and segregation. Moreover, DPY-27 is specifically localized to the X chromosome in XX animals. The similarity of DPY-27 to SMC proteins and its X localization suggest that dosage compensation is achieved through changes in X chromosome structure. DPY-27, however, does not act alone.
sdc-1,
sdc-2,
sdc-3,
dpy-21,
dpy-26,
dpy-27,
dpy-28, and
dpy-30 are also required for the proper execution of dosage compensation. To further our understanding of dosage compensation we undertook a molecular analysis of
dpy-26. Homozygous mutant daughters of
dpy-26 heterozygotes are dumpy and produce 4% male progeny. The XX progeny of these homozygous mutants die as embryos or L1s, whereas XO animals develop into normal males. The eight alleles of
dpy-26 have indistinguishable phenotypes. XX lethality is caused by the inability of these animals to implement the hermaphrodite mode of dosage compensation, leading to the overexpression of X-linked genes. Our cloning and DNA sequence analysis has revealed that the predicted
dpy-26 gene product is an acidic 1263 aa (142.5 kD) protein with no similarity to any known motif, protein, or predicted protein sequence. The DNA change in
dpy-26(
n199) is a C to T transition that results in a premature termination of translation at codon 525.
dpy-26 is trans-spliced to both SL1 and SL2 leaders. Preliminary analysis suggests that
dpy-26 is the third gene in a three gene operon. Rescue of
dpy-26 XX lethality is achieved with a subclone extending only 625 bp 5 of the first codon, indicating that the upstream portion of the operon is not required for rescue. Mouse polyclonal antibodies were raised against two DPY-26 polypeptides (aa 127-738 and aa 739-1263). Staining with either antibody revealed that DPY-26 co-localizes precisely with DPY-27 in older (>50 cells) wild-type XX embryos, indicating that DPY-26 is associated with the X chromosome. DPY-26 colocalizes with DPY-27 in all other embryos studied as well. DPY-26 is not localized to the X chromosome in XO male embryos or young (<50 cell) XX embryos, but is distributed diffusely throughout the nucleus. This diffuse nuclear staining is also observed in animals carrying either an
sdc-2 or an
sdc-3 mutation, revealing that these genes are required for the proper localization of DPY-26 to X. DPY-26 is mislocalized to the X chromosome of
xol-1 XO embryos, which have inappropriately activated the XX mode of dosage compensation. DPY-26 X chromosome localization is unaffected by mutations in
sdc-1 or
dpy-21.
dpy-26 and
dpy-28 are necessary for the synthesis or stability of DPY-27, since there is abundant
dpy-27 mRNA but no DPY-27 protein in
dpy-26 or
dpy-28 mutants. We find that DPY-27 and DPY-28 are probably required for the stability of DPY-26, since there is no detectable DPY-26 in either a
dpy-27 or a
dpy-28 mutant. Northern analysis to determine
dpy-26 transcript levels in these mutants is underway. These data support the speculation that DPY-26, DPY-27, and DPY-28 are part of a large dosage compensation protein complex associated specifically with the X chromosome. There is one noteworthy difference between the distribution of DPY-26 and DPY-27. In wild-type XX adults, DPY-27 is excluded from the germline. As predicted by the Him phenotype of
dpy-26 mutants, DPY-26 is expressed in the germline. We will continue our study of
dpy-26-induced dosage compensation defects and meiotic non-disjunction in search of a molecular explanation of these phenotypes.