© 1995 Oxford University Press Human Molecular Genetics, 1995, Vol. 4, No. 4 731-739 Three genes that escape X chromosome inactivation are clustered within a 6 Mb YAC contig and STS map inXp11.21-p11.22 Andrew P.MMIer 1 - 2 , Karen Gustashaw 1 , Daynna J.Wolff 1 , Sue H.Rider 3 , Anthony P.Monaco 3 , Brian Eble 4 , David Schlessinger 4 , Jerome L.Gorski 5 , Gert-Jan van Ommen 6 , Jean Weissenbach 7 and Huntington F.Willard 1 * department of Genetics and Center for Human Genetics, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, department of Genetics, Stanford University, Stanford, California 94306, USA, 3 ICRF Laboratories, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK, 4 Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, departments of Pediatrics and Human Genetics, University of Michigan, Ann Arbor, Ml 48109, USA, department of Genetics, Leiden University, Leiden, the Netherlands and 7 lnstitut Pasteur and Genethon, Paris, France Received January 19, 1995; Revised and Accepted February 8, 1995 GenBank accesssion nos: G02061-G02O82 (incl.; In order to study the distribution of genes that escape X chromosome inactivation, a high density yeast artificial chromosome (YAC) contig and STS map spanning approximately 6 Mb has been constructed in Xp11.21-p11.22. The contig contains 113 YACs mapped with 53 markers, including 10 genes. Four genes have been assayed for their expression status on both the active and inactive human X chromo- somes, and these data have been combined with previous results on two other genes in the contig. Three of these genes escape X inactivation and have been localized to a single YAC clone of -1075 kb. The other three genes are subject to inactivation, with two of them lying among the genes that escape inactivation. These results suggest that there are both regional control signals as well as gene-specific elements that determine the X inactivation status of genes on the proximal short arm of the human X chromosome. INTRODUCTION X chromosome inactivation is the process whereby one of the two X chromosomes of normal females is inactivated early in development in order to compensate for the dosage difference of X-linked genes between males and females (1). The mechan- ism of X inactivation is unknown; however, the isolation of a gene, XIST, which maps to the X inactivation center and is expressed exclusively from the inactive X chromosome might aid in the understanding of how this complex developmental process occurs (2). A puzzling feature of X inactivation has been the finding of certain genes that 'escape' inactivation and are expressed from both the active and inactive X chromosomes (3-7). To begin to understand why these genes escape X inactivation, we have examined the expression pattern of several genes within Xpl 1.21-pi 1.22 and have analyzed the distribution of genes within this region that escape inactivation. If a gene contains a specific sequence within the promoter or another such element that marks the locus in such a way that the gene escapes X inactivation, these genes would be expected to be randomly distributed with respect to genes that are subject to inactivation. In contrast, if a chromosomal domain or region is marked to escape inactivation, perhaps through a particular local chromatin structure, the genes that escape inactivation would be expected to be clustered within specific regions. The pseudoautosomal region and the neighboring region of X—Y homology do contain clusters of genes that escape inactivation (e.g. 5,6). The pseudoautosomal region, however, may represent a special case, in that it does not need to be dosage compensated and the neighboring region might have only recently become X-specific due to the relocation during evolution of the pseudoautosomal boundary from a position once proximal to these genes (8). Other genes that have been shown to escape X inactivation appear to be distributed widely along the X chromosome (9), but at a resolution too low to provide useful information about potential clustering. To address this question of local versus region-specific inactivation patterns, we have chosen to examine Xpl 1.21 - p i 1.22 in some detail. This region is early replicating (10-12) and appears to be a site of histone H4 acetylation (13) on the inactive X chromosome, providing cytological indications that it might contain genes that escape inactivation. Other evidence, such as the structure of bipartite Barr bodies formed from isodicentric chromosomes (14) and the location of chromosomal rearrangements in Turner syndrome (15), are consistent with this possibility. Two genes in this region have already been shown to escape X inactivation, DXS1272E (also known as XE169 and SMCX) (7,16) and DXS423E (17), while a third gene in Xpll, UBE1, mapping ~8 Mb distal to these two genes (9), also escapes inactivation (3). Localization of these genes in relation to other genes that are either subject to or escape from X inactivation should further the understanding of X chromosome inactivation. Yeast artificial chromosomes (YACs) can be used as a tool to isolate an entire chromosome region in a set of overlapping clones and, therefore, allow one to easily characterize large segments of the human genome (18-20). We describe here a *To whom correspondence should be addressed at Tufts University on December 28, 2012 http://hmg.oxfordjournals.org/ Downloaded from