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Gene-expression analysis provides evidence for dosage compensation of the X chromosome in flies, mice and worms.
Ever since the Garden of Eden, men have been fascinated by what makes them different from women. This interest has focused recent attention on the sex chromosomes X and Y. In mammals and Drosophila, males are XY and females XX. In nematodes, males are XO (as there is no Y) and hermaphrodites (or self-fertile females) are XX. The Y chromosome is relatively gene poor, so X-chromosome dosage appears to be critical for sex determination. But this set-up poses two problems: how do females deal with having a double dose of X-linked genes, and how do males deal with being aneuploid for the X chromosome? Now, in Journal of Biology , Vaijayanti Gupta and colleagues describe microarray studies to investigate how gene expression from the X chromosome or the autosomes is equilibrated in flies, worms and mice (see 'The bottom line' box for a summary of the work and the 'Background' box for definitions).
Different species have developed strikingly different strategies to deal with disparities in the dose of X chromosome between males and females: in XX female mammals, one of the two X chromosomes is randomly inactivated; XX hermaphrodite nematodes halve the expression from each X chromosome; and male Drosophila double expression from their single X chromosome in somatic cells [2,3]. These dosage compensation mechanisms serve to balance the differences between the number of copies of X-linked genes in somatic tissues of the two sexes.
"Although we now know that these species use different approaches to achieve dosage compensation, this amounts mainly to playing differently with a limited panoply of chromatin-based modifications," says Philip Avner from the Pasteur Institute in Paris, France. X inactivation in mammals requires expression of the Xist gene, which produces a large, non-coding RNA that coats the inactive X chromosome . The inactive X is characterized by DNA methylation, histone hypoacetylation, late replication and enrichment in the variant histone macroH2A. The hypertranscription of the Drosophila X chromosome in somatic cells is dependent on the 'male specific lethal' (msl) loci, which encode a histone-modifying MSL complex that acetylates histone H4 on lysine 16 (H4 K16) .
"Much less attention has been paid to the question of X/autosome dosage," says Avner. "X inactivation would be expected to lead to halved quantities of X-linked gene products compared to autosomal gene products. Haploinsufficiency for the entire X would a priori be expected to be catastrophic to the organism and lead to lethality during early embryonic development." Haploinsufficiency was the issue that Gupta and colleagues set out to address. "There was a lack of evidence for a germline dosage compensation machinery," notes Gupta, citing studies showing that in Drosophila the X chromosomes are not coated with MSL complexes or hyperacetylated on H4 K16 in male germ cells  (see the 'Behind the scenes' box for more on the rationale for the work). "Also, Parisi et al.  showed that a subset of ribosomal protein encoding genes are equally expressed in both testis and ovaries and we had seen that X;AA and XX;AA tumors showed very similar gene expression profiles," adds Gupta. "I've been thinking about this problem since I was a graduate student in the late 80s," recalls Brian Oliver who heads the research group at the National Institute of Diabetes and Digestive and Kidney Diseases in Bethesda, USA. "Until microarrays appeared, we didn't really see a good way to do a convincing experiment."
Microarray chips covering virtually the entire Drosophila genome offered Oliver's group the chance to look simultaneously at thousands of genes and their response to changing gene doses. "It would be extremely difficult to draw meaningful conclusions about the dosage compensation of an entire chromosome based on just a handful of genes," notes Gupta. They used genetic tricks to remove the influence of sex-biased expression and included many replicate hybridizations for every sample. "We ensured that the X;AA and XX;AA matched tissue samples were compared directly against each other, by hybridizing to the same array. By including the loop design, we could compare any sample in the design to any other. This increased our total number of replicates (both direct and indirect). For example, we have 7 X;AA hs-tra tumors and 17 XX;AA otu and Sxl tumors [these are the genotypes used]. There were 6 comparisons by direct hybridizations between the X;AA and XX;AA tumors. But we could additionally compute 113 X;AA versus XX;AA tumor comparisons indirectly ((17 × 7) - 6)."
The results were strikingly clear: the expression ratios between X chromosome and autosome genes were tightly centered on 1-fold, indicating that dosage compensation occurs in the germline. Gupta and colleagues performed a large number of controls using flies with deleted or duplicated autosomal segments to be sure that they could detect gene dosage affects on other chromosomes . "We were able to detect gene expression changes for our control autosomal aneuploidy corresponding to as small as 1.5-fold gene dose change, which was actually a more stringent dose difference, compared to the 2-fold dose difference in X chromosome," says Gupta.
Not content with their impressive results in flies, Oliver's group went on to reanalyze microarray data from Caenorhabditis elegans and mice. Their results led them to a similar conclusion: that the single X chromosomes of X;AA nematodes and mice were expressed at similar levels to two autosomes. Di Nguyen and Christine Disteche, at the University of Washington, Seattle, USA, recently reported a similar study of gene expression in a range of human and mouse somatic tissues . They also found that doubling of the global expression level of the X chromosome leads to dosage compensation in mammalian somatic tissues. "Interestingly, X-chromosome expression levels appear even more markedly increased in the brain," comments Avner. "Dare one suggest that the X chromosome has become specialized in cognitive function and sex? Also, although some X-linked genes are expressed in post-meiotic cells of the male and female germlines, upregulation is absent, allowing X/A ratios to be maintained in an equivalent fashion to somatic tissues."
"Taken together, these experiments suggest that, with the development of heteromorphic sex chromosomes, the driving and unifying force may have been to maintain X/autosome levels within relatively strict limits to avoid haploinsufficiency, and that mechanisms to ensure dosage compensation may well have been added in later," suggests Avner. "But these results leave us with the conundrum of how, and exactly when, upregulation of the thousand or so genes on the X chromosome is put in place during early development alongside X-inactivation. Or alternatively, when and how downregulation is achieved specifically in post-meiotic cells."
Gupta thinks that studying the germline X-chromosome gene expression in other organisms (such as mice and C. elegans) using a similar approach might bring new insights. Oliver is very keen to get a handle on the mechanism. "We'll need to probe chromatin structure in the germline by ChIP-chip [chromatin immunoprecipitation] type methods," he says. He cites recent studies investigating how many of the X-chromosome genes might be regulated by the MSL complex [8,9]. "There may be other players besides the MSLs in the soma," says Oliver. "This supports our observation of MSL-independent dosage compensation in the germline." But he is also keeping an open mind about dosage compensation. He is interested in how the apparent moderate dosage compensation on the autosomes works and what this can tell us about general properties of gene expression networks. "And what is the best reference for measuring dosage compensation?" asks Oliver provocatively. "It is still possible, as long suggested by Jim Birchler, that autosomal expression is down in X;AA individuals. 2X = AA or X = (AA)/2?" Whether it is indeed the expression on the X chromosome that goes up or expression of the autosomes that goes down, the mysteries of the X will continue to fascinate both sexes for many years to come.