MATERIALS AND METHODS Cell Lines, DNA, and RNA Preparations. We conclude that the inactivation status of at least some X-linked genes varies among different females and may reflect chromatin differences among different X chromosomes that determine whether a given gene escapes from or is subject to X inactivation. Surprisingly, expression of one of these genes, REP1, was heterogeneous monoallelic expression, consistent with X inactivation, was detected in some lines, whereas biallelic expression, indicating escape from X inactivation, was detected in others. To demonstrate the utility of this approach, seven X-linked genes were tested by using this system. To develop a complementary approach to the study of X inactivation directly in human diploid cells and to avoid the potentially confounding aspects of the somatic cell hybrid system, we describe here an assay system that tests the expression of X-linked genes by using transcribed polymorphisms to distinguish Xa and Xi expression in an extensive panel of primary human cell lines from females with nonrandom X inactivation caused by the presence of a structurally abnormal X. These findings suggest that Xi gene expression is under a previously unsuspected level of genetic or epigenetic control, likely involving local or regional changes in chromatin organization that determine whether a gene escapes or is subject to X inactivation. These data indicate that REP1 is expressed from the Xi in all cells, but that the level of expression relative to Xa levels is reduced. The cellular basis of Xi expression was examined by expression assays in single cells. Furthermore, levels of Xi expression varied among cell lines that expressed REP1. However, a novel pattern of expression was observed for another gene, REP1 monoallelic expression, indicating inactivation, was detected in some lines, whereas biallelic expression, indicating escape from inactivation, was detected in others. Six X-linked genes used to document this assay system showed monoallelic expression in all informative cell lines, consistent with X inactivation. In this report, we test the expression of human X-linked genes in primary cell lines from females with complete nonrandom X inactivation, by using transcribed polymorphisms to distinguish Xa and Xi expression. Whereas it is well established that some X-linked genes “escape” X inactivation and are expressed from both active (Xa) and inactive (Xi) X chromosomes, most models for the chromosomal control of X-linked gene expression assume that the X inactivation status of a given gene is constant among different females within a population. In mammalian females, most genes on one X chromosome are transcriptionally silenced as a result of X chromosome inactivation.
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