We developed an aqueous spreading procedure that permits simultaneous analysis of human chromosomes by Q-banding and indirect immunofluorescence. Using this methodology and anticentromere antibodies from an autoimmune patient we compared the active and inactive centromeres of an isodicentric X chromosome. We show that a family of structurally related human centromere proteins (CENP-A, CENP-B, and CENP-C) is detectable only at the active centromere. These antigens therefore may be regarded both as morphological and functional markers for active centromeres.

To explore the role of DNA methylation in maintaining dosage compensation of X chromosome-linked genes and in regulating the transcriptional activity of "housekeeping" genes, we characterized DNA methylation of active, inactive, and derepressed alleles at the locus for hypoxanthine phosphoribosyltransferase (HPRT) on the human X chromosome. The methylation of Hpa II and Hha I sites in HPRT alleles on the active X chromosome was the same in all tissues. The consensus pattern includes hypomethylation of 5' clustered sites and extensive methylation of the 3' sequence. The striking feature of methylation of inactive X-chromosome alleles is nonuniformity and less extensive hypomethylation of the 5' cluster. Analysis of HPRT alleles reactivated in response to 5-azacytidine showed at least partial restoration of the consensus pattern. These observations indicate that methylation of housekeeping genes on the X chromosome is the same as that of autosomal ones and that the overall pattern and methylation of multiple sites within a cluster may cooperate to facilitate transcription. Furthermore, the fidelity of methylation of the active allele and the extensive drift in methylation of the inactive allele suggest that mechanisms involved in X-chromosome dosage compensation may be directed at the active rather than inactive X chromosome.DNA methylation not only has been implicated as an important determinant of gene activity (1) but also has been considered to have a primary role in compensating for sex differences in the dosage of X chromosome-linked (X-linked) genes (2, 3). Evidence suggests that DNA methylation is involved in regulation of the hypoxanthine phosphoribosyltransferase (HPRT) locus. The cytosine analog 5-azacytidine (5-azaC) induces the localized derepression of inactive HPRT alleles (4-6), presumably by inducing demethylation at sites within or near the locus. DNA purified from these derepressed alleles or "reactivants" is competent to transfer HPRT activity to recipient cells, whereas DNA from the inactive alleles is not (6)(7)(8).Yet this evidence does not show any direct role for DNA methylation in dosage compensation-i.e., patterns of methylation exclusively concerned with maintaining inactivity of the silent X chromosome. Loci on the X chromosome, like those on other chromosomes, represent an array of developmentally regulated, hormone-responsive, and constitutively expressed ("housekeeping") genes (9). If there are special features of the X chromosome relevant to dosage compensation, then they must be superimposed on regulatory features common to all chromosomes. Therefore, demethylation may induce reexpression of HPRT by directly affecting transcription of the locus, rather than by reversing some developmental program specifically associated with dosage compensation. The HPRT locus is extraordinarily useful for exploring the role of DNA methylation in maintaining the silence of the inactive X chromosome and in transcriptional regulation. The inactive X chromosome provides the means to examine t...

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Sex-specific manifestations of disease are most often attributed to differences in the reproductive apparatus or in life experiences. However, a good deal of sex differences in health issues have their origins in the genes on the sex chromosomes themselves and in X inactivation-the developmental program that equalizes their expression in males and females. Most females are mosaics, having a mixture of cells expressing either their mother's or father's X-linked genes. Often, cell mosaicism is advantageous, ameliorating the deleterious effects of X-linked mutations and contributing to physiological diversity. As a consequence, most X-linked mutations produce male-only diseases. Yet, in some cases the dynamic interactions between cells in mosaic females lead to female-specific disease manifestations.

Skin fibroblasts of human males affected with adrenoleukodystrophy (ALD) have previously been shown to be abnormal with respect to C26 fatty acid content. Skin fibroblast clones from heterozygotes in three families segregating this mutation have been analyzed and are oftwo types: clones with normal ratios of C26 to C22 fatty acids and clones with an excess ofC26 fatty acids similar to that found in cells of affected males. This indicates not only that the locus is X linked but also that it is subject to inactivation. In most of the heterozygotes there were significantly more clones of abnormal type than those expressing the normal allele, indicating a proliferative advantage in vitro for skin fibroblasts of mutant type. The increased levels offatty acids in plasma in most heterozygotes and the phenotype of blood cells of women heterozygous for both ALD and glucose-6-phosphate dehydrogenase (G6PD) in one family are evidence that selection favoring the mutant allele may occur in vivo as well as in vitro and may explain why many heterozygotes manifest clinical symptoms of the disease. These studies have also revealed the close linkage between ALD and G6PD loci, because there are no recombinants among 18 informative offspring of doubly heterozygous mothers. Therefore, the ALD locus can be mapped on the human X chromosome near the G6PD locus at Xq28. Adrenoleukodystrophy (ALD), a lipid storage disease, is characterized by adrenal insufficiency and progressive demyelination of the cerebral white matter (1). The onset of symptoms is usually between 4 and 8 years of age, with death in 1-4 years. ALD is believed to be an X-linked mutation, on the basis ofthe pattern of inheritance; usually males are affected and no maleto-male transmission has been reported. ALD has many features in common with a more indolent neurological disorder, adrenomyeloneuropathy (AMN), which affects females as well as males. Characteristic cytoplasmic inclusions and the accumulation of very long chain fatty acids are found in both disorders (2). Stronger evidence for a relationship between the two disorders is that they cosegregate in the same family (3), and skin fibroblasts from individuals affected with either ALD or AMN are abnormal with regard to the quantity ofC26 long-chain fatty acids (4-6).Because the ALD phenotype is demonstrable in cultured fibroblasts, we initiated studies of heterozygous females to determine if this locus is indeed X linked, ifit is subject to X chromosome inactivation (7), and to explore the relationship with AMN. Our strategy was to analyze skin fibroblast clones from heterozygotes to determine if some clones were normal and others expressed the mutant gene, as expected for X-chromosomal loci at which inactivation occurs (8). Studies of families segregating not only the ALD mutation but also electrophoretic variants of glucose-6-phosphate dehydrogenase (G6PD) revealed that the ALD mutation is X linked and that the locus is subject to inactivation and is closely linked to G6PD. These studies also suggest ...

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The role of X-inactivation is often ignored as a prime cause of sex differences in disease. Yet, the way males and females express their X-linked genes has a major role in the dissimilar phenotypes that underlie many rare and common disorders, such as intellectual deficiency, epilepsy, congenital abnormalities, and diseases of the heart, blood, skin, muscle, and bones. Summarized here are many examples of the different presentations in males and females. Other data include reasons why women are often protected from the deleterious variants carried on their X chromosome, and the factors that render women susceptible in some instances.

Transcriptional silencing of the human inactive X chromosome is induced by the XIST gene within the human X-inactivation center. The XIST allele must be turned off on one X chromosome to maintain its activity in cells of both sexes. In the mouse placenta, where X inactivation is imprinted (the paternal X chromosome is always inactive), the maternal Xist allele is repressed by a cis-acting antisense transcript, encoded by the Tsix gene. However, it remains to be seen whether this antisense transcript protects the future active X chromosome during random inactivation in the embryo proper. We recently identified the human TSIX gene and showed that it lacks key regulatory elements needed for the imprinting function of murine Tsix. Now, using RNA FISH for cellular localization of transcripts in human fetal cells, we show that human TSIX antisense transcripts are unable to repress XIST. In fact, TSIX is transcribed only from the inactive X chromosome and is coexpressed with XIST. Also, TSIX is not maternally imprinted in placental tissues, and its transcription persists in placental and fetal tissues, throughout embryogenesis. Therefore, the repression of Xist by mouse Tsix has no counterpart in humans, and TSIX is not the gene that protects the active X chromosome from random inactivation. Because human TSIX cannot imprint X inactivation in the placenta, it serves as a mutant for mouse Tsix, providing insights into features responsible for antisense activity in imprinted X inactivation.