To equalize X-linked gene expression between males (XO) and hermaphrodites (XX), Caenorhabditis elegans hermaphrodites reduce transcript levels from each of their two X chromosomes. In situ localization and biochemical experiments show that DPY-26 is an essential member of a dosage compensation protein complex that superimposes this chromosome-wide repression upon the spatial and temporal regulation of individual genes. Antibodies raised against DPY-26 reveal that it is concentrated on all mitotic chromosomes in young (<30 cells) XX and XO embryos. When dosage compensation is activated (>30 cells), DPY-26 becomes localized specifically to the X chromosomes of XX, but not XO embryos. How is DPY-26 directed to the X chromosomes of hermaphrodites but not males? Genes that control the hermaphrodite modes of both sex determination and dosage compensation (
sdc-2 and
sdc-3), or dosage compensation alone (
dpy-27 and
dpy-30) are required for the X localization of DPY-26. DPY-26 remains associated with mitotic chromosomes but is diffusely distributed in the interphase nuclei of embryos mutant in these genes. In addition, DPY-26 staining is abolished in
dpy-28 mutants and reduced in
dpy-27 mutants, suggesting that these two genes are required for the stability of DPY-26 in somatic nuclei and that all three proteins physically interact. In males,
xol-1 represses the sdc genes, thereby preventing DPY-26 from localizing to X. Dying
xol-1 mutant XO animals are forced into the hermaphrodite mode of dosage compensation and consequently misdirect DPY-26 to their single X. Immunoprecipitations from C. elegans nuclear extract reveal that DPY-26, DPY-27, and two proteins of ~150 and ~160 kD physically interact. This protein complex is stable in 400 mM salt and its members co-fractionate through ion-exchange chromatography and sucrose gradients. Preliminary data suggests that the 150 kD member of the protein complex is a C. elegans SMC2 (Structural Maintenance of Chromosomes) homolog. The SMC family of proteins has been conserved from bacteria to man and is known to be involved in several aspects of chromosome dynamics, including chromosome condensation and segregation. Previous molecular analysis of
dpy-27 showed that DPY-27 is specifically localized to the X chromosomes of XX animals and is an SMC1 homolog. In situ staining with antibodies raised against the C. elegans SMC2 homolog show that it is associated with all mitotic chromosomes and the X chromosomes of interphase nuclei in XX animals. It fails to localize to the X chromosomes in XO animals or XX animals mutant for
sdc-2,
sdc-3, or
dpy-30. Therefore, an essential part of X-chromosome dosage compensation may be the recruitment of general mitotic chromosome condensation factors to the X chromosome during interphase. Consistent with the 4% meiotic non-disjunction phenotype found in
dpy-26 mutants, DPY-26 is found in germline nuclei of both XO and XX adults. This distribution is unaffected by mutations in
sdc-2,
sdc-3,
dpy-27, or
dpy-30, indicating that the function of DPY-26 in meiotic chromosome segregation is independent of its role in dosage compensation. In contrast, mutations in
dpy-28 abolish
dpy-26 germline staining, suggesting that DPY-26 and DPY-28 physically interact. The absence of DPY-27 from the germline may allow a germline-specific complex of DPY-26 and DPY-28 to function in the proper segregation of meiotic chromosomes. Together, our data indicate that DPY-26 acts in a complex with at least DPY-27, DPY-28, and a C. elegans SMC2 homolog to specifically reduce somatic X-linked gene expression through modifications of X chromosome structure. Additionally, DPY-26 is likely to form a germline-specific complex with DPY-28 that is required for the proper segregation of meiotic chromosomes.