Escapees on the X chromosome.

I many organisms, differentiation of the sex chromosome complement resulted in the coordinated regulation of genes on whole chromosomes to equalize gene expression between the sexes. In mammals, X inactivation evolved to restore equal expression of X-linked genes in males and females (1). Although X inactivation consists in the general repression of most genes on the X, some genes escape inactivation (reviewed in ref. 2). Recent advances in the Human Genome Project now allow the inactivation status of many Xlinked genes to be systematically studied. In this issue of PNAS, Carrel et al. (3) report such a systematic analysis. Their data are so extensive that they are summarized in the paper but are fully accessible only as a file on the World Wide Web (www.pnas.orgysupplementary.shtml). According to Carrel et al. (3), ‘‘escapees’’ (genes that escape X inactivation) are not rare: conservatively, they number as much as 19%, or about one-fifth, of the genes on the human X. Another striking finding of Carrel et al. (3) is that the distribution of escapees and of genes subject to X inactivation is nonrandom. Many more escapees are located on the short arm of the human X (21%) than on the long arm (3%). The origin of these patterns of expression and organization can be tied to the evolutionary history of the sex chromosomes. The existence of these patterns also has implications for the regulation and role of X chromosomal genes in human disease. It is useful to begin by placing Carrel et al.’s findings in the context of our understanding of sex chromosome evolution. Ohno (4) proposed that the sex chromosomes derived from a homomorphic autosome-like pair of chromosomes. Determination of the male sex by a gene (SRY) on the Y resulted in inhibition of recombination between the sex chromosomes. Decay of the Y and accumulation of genes advantageous to males close to the male determinant resulted in dramatic differences in size and gene content between the sex chromosomes (5, 6). Extensive gene loss from the Y provided a strong impetus for X inactivation (Fig. 1). Expression from a single allele on the active X in both sexes would, however, have led to an expression imbalance relative to biallelic autosomal genes. Hence, upregulation of genes on the active X likely evolved together with—or before—X inactivation (Fig. 1) (4, 7). The ancestral sex chromosomes of mammals were probably smaller than the present-day eutherian X chromosomes. Graves (8) has suggested that most of the short arm of the human X is a recent addition to an ancestral chromosome. This ancestral region [X-conserved-region (XCR)] is also present in marsupials and monotremes, whereas the added region [X-added-region (XAR)] is present only in eutherians (Fig. 2). An interesting new study by Lahn and Page (9) defines four evolutionary strata on the human X chromosome on the basis of sequence divergence between XyY genes. Thus at least four events (likely inversions of the Y) must have shaped the human sex chromosomes. The oldest strata, 1 and 2, correspond to the XCR, and the two newest strata, 3 and 4, to the XAR. The three most recent strata are all on the X short arm, precisely where the majority of escapees are located (Fig. 2) The process of incorporation of genes into the X inactivationyup-regulation system within each stratum was likely progressive. In fact, it may have occurred on a gene-by-gene basis, with the Y gene being first lost or differentiated to acquire a male-specific function. Indeed, there is no known example of a gene that is subject to X inactivation but has not lost its functionally equivalent Y partner. Thus it seems that degeneration of the Y took place before the acquisition of X inactivation (9, 10). Studies in marsupials provide some information about acquisition of X inactivation. Marsupial X inactivation is incomplete and unstable, perhaps because of the absence of locking mechanisms (e.g., methylation), whereas genes on eutherian inactive Xs (except for escapees) are usually stably inactivated and methylated at their CpG islands (reviewed in refs. 11, 12). Histone deacetylation appears to be a more ancestral mechanism found in both eutherian and marsupial X inactivation. Although the marsupial Xinactive-specific-transcript gene (XIST) has not been identified, its hypothetical location lies in the XCR, in stratum 1. Thus it appears that some primitive form of X inactivation may have evolved before the divergence of eutherians and marsupials, and the XAR may have been translocated to an ancestral chromosome already regulated by X inactivation. The escapees identified by Carrel et al. (3) in the short arm of the human X are likely remnants of incomplete incorporation of individual genes into the X inactivation system but could also result in part from incomplete inactivation spreading into regions added to an ancestral X. Whether X inactivation spreading occurred or not in the XAR, it is interesting to compare the results of Carrel et al. (3) with findings in Xyautosome translocations. In such translocations, spreading is often incomplete, and many of the autosomal genes attached to the inactive X escape in a discontinuous fashion (13). Spreading, in cases of unbalanced Xyautosome translocations, provides partial correction of the trisomic state and thus would be favorable. Yet, as in the study of Carrel et al. (3), gene inactivation is discontinuous and many genes escape. Thus a selection for corrected gene dosage may not operate on all genes, either because dosage is unimportant for some genes or because they cannot be stably inactivated. One would expect that the high density of escapees on the X short arm would be accompanied by a high density of remaining XyY gene pairs in this region. In Fig. 2, we have indicated the position of XyY genes among the escapees, and indeed the density of XyY gene pairs is higher on the short arm, which includes the three newest strata defined by Lahn and Page (9). The most distal short arm region is the pseudoautosomal region 1 (PAR1) where pairing occurs between the sex chromosomes. All genes that could be assayed in the PAR1 escape X inactivation, as expected for genes equally expressed from the X and Y. Another small pseudoautosomal region 2, located at the extremity of the long arms of the human sex chromosomes, represents a recent addition that contains one