Worm Breeder's Gazette 13(1): 60 (October 1, 1993)
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.
In Caenorhabditis elegans, dosage compensation is required to equalize the disparity in X chromosome dosage between males (XO) and hermaphrodites (XX). Several genetic and molecular studies have indicated that C. elegans dosage compensation is achieved by reducing transcription from each X chromosome in the 2X hermaphrodite. Additional studies have identified five autosomal genes, dpy-21 , dpy-26 , dpy-27 , dpy-28 ,and dpy-30 ,as essential components of this process. Mutations in each of these genes cause elevated levels of X-linked expression in XX animals. Furthermore, three of the dpy genes display certain secondary phenotypes or homologies that suggest that C. elegans dosage compensation is mediated through sex-specific alterations in chromatin structure of the X chromosome.
In an effort to better understand the molecular mechanisms underlying the dosage compensation process in C. elegans, we would like to determine whether sex-specific changes in chromatin structure of the X chromosome occur. There are now several lines of evidence from other organisms indicating that the alterations of chromatin structure associated with global transcriptional regulation are influenced by the acetylation states of the four core histones. Specifically, recent studies in Drosophila using anitsera that distinguished histone H4 molecules acetylated at the functionally important Iysine residues (Iysine 5, 8, 12, and 16) have shown that site-specific acetylation of histone H4 is associated with different forms of chromatin structure (Turner, et. al., Cell 69, 375-384). Particularly striking was the finding that H4 acetylated at Iysine 16 is uniquely associated with the hyperactive X chromosome in male larvae and that H4 acetylated at Iysine 12 is preferentially associated with chromocentric heterochromatin.
The availability of antisera to acetylated histone H4 isoforms enabled us to examine whether differentially acetylated H4 isoforms are associated with defined regions of the C. elegans genome. If dosage compensation (reduced X-linked transcription) in hermaphrodites is achieved through the establishment of an altered form of chromatin, it is possible that there may be a unique association or specific absence of a particular H4 isoform from the X chromosomes. Using indirect immunofluorescence, adult, larval, and embryonic N2 or dpy-27 animals were stained with antisera specific for histone H4 molecules in which one of the four Iysine residues 5, 8, 12, or 16 is acetylated. Identical staining patterns were observed for both N2 and dpy-27 strains.
H4 acetylated at Iysines 8 or 12 (H4A c8 or H4A c12 )appear to be localized to the nuclei of all cells at all developmental stages. Closer examination of individual oocyte and early embryonic nuclei showed that both H4A c8 or H4A c12 are associated in relatively equivalent amounts with each of the six pairs of chromosomes. No X chromosome-specific staining was detected. Antisera specific for H4 acetylated at lysine 5 (H4A c5 )also stains the nucleus and chromosomes of oocytes and early embryos, but again this isoform does not exhibit any differential staining between autosomes and X chromosomes. Interestingly, however, in late stage embryos, larvae and adults, H4A c5 becomes concentrated in the nucleolus. The localization of H4A c5 to the nucleolus was confirmed with a ncl-1 strain. In this mutant background the nucleolus becomes visibly enlarged with respect to the nucleus, and the immunostaining pattern of H4A c5 increases correspondingly. Lastly, H4 acetylated at Iysine 16 (H4A c16 )gives a unique pattern of staining: the H4A c16 isoform could not be detected in embryos, however in L4 hermaphrodites and adult males, H4A c16 appears to be localized to a specific region of the gonad. Further analysis of two sperm defective mutants of C. elegans ( tra-2 (gf)and fog-2 )indicate that H4A c16 is specifically associated with the region of the gonad in which sperm development occurs. Very low levels of H4A c16 could also be detected in oocyte nuclei, where it is specifically associated with chromosomes. Again, we could not detect any variable association with the X chromosome.
A more recent study of histone acetylation states in mammals has shown that the inactive X chromosome lacks histone H4 acetylation (Jeppesen and Turner, Cell 74, 281-289). Therefore, it remains possible that chromatin alterations in C. elegans will be reflected in the relative amount of histone acetylation on the X chromosome rather than association with a specific H4 isoform. However, the dosage compensation mechanism in C. elegans potentially involves only a 2 fold reduction of X-linked transcription, and a corresponding partial reduction in H4 acetylation on the X chromosome may be difficult to detect. Moreover, the dosage compensation process may not yet be active in oocytes and early embryos when individual chromosomes stain with the highest resolution.