Expression of cis-regulatory mutations of the white locus in metafemales of Drosophila melanogaster.

At the white eye colour locus, there are a number of alleles that have altered expression between males and females. To test these regulatory mutations of the white eye colour locus for their phenotypic expression in metafemales (3X; 2A) compared to diploid females and males, eleven alleles or transduced copies of white were analysed. Two alleles that exhibit dosage compensation between males and females (apricot, blood) also exhibit dosage compensation in metafemales. White-ivory and white-eosin, which fail to dosage compensate in males compared to females, but that are distinct physical lesions, also show a dosage effect in metafemales. Two alleles with greater expression in males than females (spotted, spotted-55) exhibit even lower expression in metafemales. Lastly, five transduced copies of white carrying three different lengths of the white promoter, but that all exhibit higher expression in males, show reduced expression in metafemales, exhibiting an inverse correlation between the level of expression and the dosage of the X chromosome. Because these alleles of white respond to dosage compensation in metafemales as a continuum of the male and female responses, it is concluded that the same basic mechanism of dosage compensation is involved and that the dosage of the X chromosome conditions the sexually dimorphic expression.     

Dosage Regulation of the Active X Chromosome in Human Triploid Cells

In mammals, dosage compensation is achieved by doubling expression of X-linked genes in both sexes, together with X inactivation in females. Up-regulation of the active X chromosome may be controlled by DNA sequence–based and/or epigenetic mechanisms that double the X output potentially in response to autosomal factor(s). To determine whether X expression is adjusted depending on ploidy, we used expression arrays to compare X-linked and autosomal gene expression in human triploid cells. While the average X:autosome expression ratio was about 1 in normal diploid cells, this ratio was lower (0.81–0.84) in triploid cells with one active X and higher (1.32–1.4) in triploid cells with two active X''s. Thus, overall X-linked gene expression in triploid cells does not strictly respond to an autosomal factor, nor is it adjusted to achieve a perfect balance. The unbalanced X:autosome expression ratios that we observed could contribute to the abnormal phenotypes associated with triploidy. Absolute autosomal expression levels per gene copy were similar in triploid versus diploid cells, indicating no apparent global effect on autosomal expression. In triploid cells with two active X''s our data support a basic doubling of X-linked gene expression. However, in triploid cells with a single active X, X-linked gene expression is adjusted upward presumably by an epigenetic mechanism that senses the ratio between the number of active X chromosomes and autosomal sets. Such a mechanism may act on a subset of genes whose expression dosage in relation to autosomal expression may be critical. Indeed, we found that there was a range of individual X-linked gene expression in relation to ploidy and that a small subset (∼7%) of genes had expression levels apparently proportional to the number of autosomal sets.     

Analysis of Autosomal Dosage Compensation Involving the Alcohol Dehydrogenase Locus in Drosophila Melanogaster

An example of autosomal dosage compensation involving the expression of the alcohol dehydrogenase (Adh) locus is described. Flies trisomic for a quarter of the length of the left arm of chromosome two, including Adh, have diploid levels of enzyme activity and alcohol dehydrogenase messenger RNA. Subdivision of the compensating trisomic into smaller ones revealed a region that exerts an inverse regulatory effect on alcohol dehydrogenase activity and messenger RNA levels and a smaller region surrounding the structural gene that exhibits a direct gene dosage response. The two opposing effects are of sufficient magnitude that they cancel when simultaneously present resulting in the observed compensation in the larger aneuploid. An Adh promoter-white structural gene fusion construct is affected by the inverse regulatory region indicating that the effect is mediated through the Adh promoter sequences. The role of autosomal dosage compensation in understanding aneuploid syndromes and karyotype evolution in Drosophila species is discussed.     

Histone acetylation and gene expression analysis of sex lethal mutants in Drosophila

The evolution of sex determination mechanisms is often accompanied by reduction in dosage of genes on a whole chromosome. Under these circumstances, negatively acting regulatory genes would tend to double the expression of the genome, which produces compensation of the single-sex chromosome and increases autosomal gene expression. Previous work has suggested that to reduce the autosomal expression to the female level, these dosage effects are modified by a chromatin complex specific to males, which sequesters a histone acetylase to the X. The reduced autosomal histone 4 lysine 16 (H4Lys16) acetylation results in lowered autosomal expression, while the higher acetylation on the X is mitigated by the male-specific lethal complex, preventing overexpression. In this report, we examine how mutations in the principal sex determination gene, Sex lethal (Sxl), impact the H4 acetylation and gene expression on both the X and autosomes. When Sxl expression is missing in females, we find that the sequestration occurs concordantly with reductions in autosomal H4Lys16 acetylation and gene expression on the whole. When Sxl is ectopically expressed in Sxl(M) mutant males, the sequestration is disrupted, leading to an increase in autosomal H4Lys16 acetylation and overall gene expression. In both cases we find relatively little effect upon X chromosomal gene expression.     

Implications of the gene balance hypothesis for dosage compensation

Dosage compensation refers to the equal expression between the sexes despite the fact that the dosage of the X chromosome is different in males and females. In Drosophila there is a twofold upregulation of the single male X. In triple X metafemales, there is also dosage compensation, which occurs by a two-thirds downregulation. There is a concomitant reduction in expression of many autosomal genes in metafemales. The male specific lethal (MSL) complex is present on the male X chromosome. Evidence is discussed showing that the MSL complex sequesters a histone acetyltransferase to the X chromosome to mute an otherwise increased expression by diminishing the histone acetylation on the autosomes. Several lines of evidence indicate that a constraining activity occurs from the MSL complex to prevent overcompensation on the X that might otherwise occur from the high level of acetylation present. Together, the evidence suggests that dosage compensation is a modification of a regulatory inverse dosage effect that is a reflection of intrinsic gene regulatory mechanisms and that the MSL complex has evolved in reaction in order to equalize the expression on both the X and autosomes of males and females.     

The Influence of Whole-Arm Trisomy on Gene Expression in Drosophila

The biochemical consequences of extensive aneuploidy in Drosophila have been examined by measuring the levels of specific proteins in larvae trisomic for entire chromosome arms. By far the most common effect is a reduction in gene product levels (per gene template) by one-third from the diploid quantity, consistent with the model that concentration-dependent repressors of these loci reside on the duplicated chromosome arms. Most loci appear sensitive to such repression in one or more of the trisomies examined, suggesting that such regulatory loci might be quite common. Repression of gene-product levels in trisomies may significantly contribute to their inviability. Few loci are activated in trisomies implying that most factors necessary for gene expression are in excess. While autosomal trisomies can repress the expression of both X-linked and autosomal loci, X-chromosomal trisomies have little effect on most autosomal genes. A family of genes coding for larval serum proteins do not respond similarly in trisomies, suggesting that regulation operates on a process which is not common to their coordinate regulation. Finally, Adh genes transposed to new chromosomal positions maintain their ability to be repressed in 3L trisomies suggesting that this response to regulation involves a closely linked cis-acting regulatory element.     

Dosage Compensation of X-Linked Muller Element F Genes but Not X-Linked Transgenes in the Australian Sheep Blowfly

In most animals that have X and Y sex chromosomes, chromosome-wide mechanisms are used to balance X-linked gene expression in males and females. In the fly Drosophila melanogaster, the dosage compensation mechanism also generally extends to X-linked transgenes. Over 70 transgenic lines of the Australian sheep blowfly Lucilia cuprina have been made as part of an effort to develop male-only strains for a genetic control program of this major pest of sheep. All lines carry a constitutively expressed fluorescent protein marker gene. In all 12 X-linked lines, female larvae show brighter fluorescence than male larvae, suggesting the marker gene is not dosage compensated. This has been confirmed by quantitative RT-PCR for selected lines. To determine if endogenous X-linked genes are dosage compensated, we isolated 8 genes that are orthologs of genes that are on the fourth chromosome in D. melanogaster. Recent evidence suggests that the D. melanogaster fourth chromosome, or Muller element F, is the ancestral X chromosome in Diptera that has reverted to an autosome in Drosophila species. We show by quantitative PCR of male and female DNA that 6 of the 8 linkage group F genes reside on the X chromosome in L. cuprina. The other two Muller element F genes were found to be autosomal in L. cuprina, whereas two Muller element B genes were found on the same region of the X chromosome as the L. cuprina orthologs of the D. melanogaster Ephrin and gawky genes. We find that the L. cuprina X chromosome genes are equally expressed in males and females (i.e., fully dosage compensated). Thus, unlike in Drosophila, it appears that the Lucilia dosage compensation system is specific for genes endogenous to the X chromosome and cannot be co-opted by recently arrived transgenes.     

Genetic Analysis of X-Chromosome Dosage Compensation in Caenorhabditis elegans

We have shown that the phenotypes resulting from hypomorphic mutations (causing reduction but not complete loss of function) in two X-linked genes can be used as a genetic assay for X-chromosome dosage compensation in Caenorhabditis elegans between males (XO) and hermaphrodites (XX). In addition we show that recessive mutations in two autosomal genes, dpy-21 V and dpy-26 IV, suppress the phenotypes resulting from the X-linked hypomorphic mutations, but not the phenotypes resulting from comparable autosomal hypomorphic mutations. This result strongly suggests that the dpy-21 and dpy-26 mutations cause increased X expression, implying that the normal function of these genes may be to lower the expression of X-linked genes. Recessive mutations in two other dpy genes, dpy-22 X and dpy-23 X, increase the severity of phenotypes resulting from some X-linked hypomorphic mutations, although dpy-23 may affect the phenotypes resulting from the autosomal hypomorphs as well. The mutations in all four of the dpy genes show their effects in both XO and XX animals, although to different degrees. Mutations in 18 other dpy genes do not show these effects.     

Sex chromosome evolution: life,death and repetitive DNA

Dimorphic sex chromosomes create problems. Males of many species, including Drosophila, are heterogametic, with dissimilar X and Y chromosomes. The essential process of dosage compensation modulates the expression of X-linked genes in one sex to maintain a constant ratio of X to autosomal expression. This involves the regulation of hundreds of dissimilar genes whose only shared property is chromosomal address. Drosophila males dosage compensate by up regulating X-linked genes 2 fold. This is achieved by the Male Specific Lethal (MSL) complex, which is recruited to genes on the X chromosome and modifies chromatin to increase expression. How the MSL complex is restricted to X-linked genes remains unknown. Recent studies of sex chromosome evolution have identified a central role for 2 types of repetitive elements in X recognition. Helitrons carrying sites that recruit the MSL complex have expanded across the X chromosome in at least one Drosophila species.¹ Our laboratory found that siRNA from an X-linked satellite repeat promotes X recognition by a yet unknown mechanism.² The recurring adoption of repetitive elements as X-identify elements suggests that the large and mysterious fraction of the genome called “junk” DNA is actually instrumental in the evolution of sex chromosomes.     

A liveborn 69,XXX triploid. Origin, X chromosome activity and gene dosage

A 69,XXX female liveborn triploid survived 45 days. The phenotype was consistent with the average clinical picture of liveborn triploids. Autopsy revealed slight atrophy of cerebral cortex and corpus callosum and severe adrenal hypoplasia. Chromosome polymorphisms indicated that the origin of this triploid was dispermy. Replication studies of the X chromosome performed on lymphocytes and fibroblasts showed that the majority of cells had two late replicating X chromosomes. X chromosome inactivation in spontaneous abortuses and liveborn triploids is discussed. Nine enzymes encoded by autosomal genes were tested, five had normal, three increased, and one reduced levels of activity. The reduced activity of alpha-galactosidase, an X-linked enzyme, is in agreement with cytogenetic findings and demonstrated a gene dosage effect.