Worm Breeder's Gazette 13(3): 39 (June 1, 1994)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
C. elegans compensates for the difference in X-linked gene dosage between males and hermaphrodites by equalizing X-specific transcript levels. The genes dpy-21 , dpy-26 , dpy-27 , dpy-28 ,and dpy-30 are essential components of the dosage compensation process in XX animals. Genetic analysis of these genes suggests that they might act together to regulate X chromosome expression. However, the mechanism by which they achieve this regulation is unknown.
In order to understand the mechanism of dosage compensation, we have taken a molecular and biochemical approach to study the function of the dpy-27 gene. We have cloned and sequenced dpy-27 .The complete dpy-27 cDNA contains one open reading frame of 4407 bp, which encodes a protein of 1469 aa. Database searches revealed sequence similarity between dpy-27 and the yeast chromosome segregation protein SMC1 .This similarity suggests a possible role of chromatin structure in dosage compensation. Both Dpy-27 and SMC1 contain a potential ATP-binding motif at their N-termini. Purified recombinant Dpy-27 protein exhibits ATPase activity in vitro. In addition, site-directed mutagenic changes of the conserved lysine in the ATP-binding motif to either isoleucine or glutamate abolish the rescuing activity of dpy-27 .These results suggest that the ATP-binding motif is important for dosage compensation in vivo.
To further understand the function of dpy-27 in dosage compensation, we generated antibodies against Dpy-27 and performed in situ antibody staining. Antibodies against Dpy-27 stain diffusely throughout the whole nucleus in oocytes and early embryos. However, in older XX embryos staining is restricted to part of the nucleus, a pattern which is very similar to that of fluorescent in situ hybridization with YACs spanning an entire chromosome (chromosome paint). Double staining experiments with Dpy-27 antibody and X chromosome paint indicated that Dpy-27 protein is localized to the X chromosome in XX embryos. In control experiments, Dpy-27 staining does not colocalize with chromosome III paint. The X chromosome localization is also seen in larval and adult XX animals. In contrast, Dpy-27 protein is not localized to the X chromosome in XO embryos. These results provide support for the model that Dpy-27 down regulates X chromosome transcript levels in XX animals by direct association with the X chromosome, presumably when the dosage compensation process becomes active.
To investigate the involvement of other gene products in the production and localization of Dpy-27 to the X chromosome, we stained mutants defective in one of several genes in the sex determination and dosage compensation pathways. Our results show that Sdc-3 and Dpy-30 are required for the proper localization of Dpy-27 to the X chromosome in XX embryos, since the Dpy-27 staining remains diffuse throughout the nucleus. Sdc-2 , Dpy-26 and Dpy-28 are required for the production or stability of Dpy-27 in XX embryos, since staining is severely reduced in these mutant backgrounds. The functional significance of direct association of Dpy-27 with the X chromosome in down regulating X-specific transcripts is further demonstrated by examining xol-1 XO embryos. These embryos die due to the execution of the hermaphrodite mode of dosage compensation. Consistent with this, Dpy-27 is inappropriately localized to the X chromosome in xol-1 XO embryos.
We are trying to use the Dpy-27 antibodies to identify the other components with which Dpy-27 might directly interact to further elucidate the biochemical mechanism of dosage compensation.