Incorrect regulation of gene expression is observed during mammalian aging and may contribute to the pathology of senescence. One class of genetic misregulation is the reactivation of previously repressed genes. The proposed experiments will examine the aging-related re-expression of genes from inactive Xchromosomes. Inactivated X chromosomes of female mammals are transcriptionally repressed and maintained as highly-condensed, late-replicating heterochromatic DNA. The mechanism for maintenance of X-inactivation is uncharacterized, as are the DNA sequences which contribute to the inactive state. In addition, X-inactivation may share common features with the maintenance of other forms of heterochromatin and the programmed inactivation of autosomal genes. Two genetic loci from the mouse X chromosome, sparsefur and Mottled, have previously been shown to undergo aging-dependent reactivation. However, it has not been demonstrated that reactivation is a general phenomenon of X-linked genes. For this proposal, transcriptional reactivation will be examined from a defined set of X-chromosome genes during normal aging. In the female mouse, alleles from each X-chromosome homologue can be assayed independently. Allele-specific expression assays are based on naturally-occurring RNA sequence polymorphisms between interbreeding mouse species. Interspecific hybrid (F1) females maintain one X-chromosome from each parental species. Consequently, these animals are heterozygous for alleles at virtually every genetic locus. For each locus tested, RNA polymorphism assays will be used to detect the relative contribution from each allele in mRNA samples prepared from aged F1 female mice. Aging-dependent reactivation will be detected as increased mRNA expression from the initially repressed allele. The proposed experiments will focus on the reactivation time-course of genes within the X-chromosomal region adjacent to the centromere. The X-pericentromere is a unique genomic location where inactive-X heterochromatin and centromeric heterochromatin may contribute overlapping effects to expression. In addition, the approximate 3 cM centromere-proximal region contains several characterized genetic and molecular loci, including the known reactivated gene ornithine transcarbamylase (Otc=sparsefur). The high density of genomic mapping resources for the region will expedite assembly of a comprehensive physical map. Such a map will be essential for assessing relationships between gene location and aging-dependent reactivation.
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