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Variable X inactivation of the human TIMP1 gene Anderson, Catherine Linda


X inactivation silences most of the genes on one of the two X chromosomes in mammalian females, presumably to achieve dosage compensation of X-linked gene products. The human X chromosome preserves its activation status when isolated in rodent/human somatic cell hybrids. Surprisingly, the human tissue inhibitor of metalloproteinases-1 gene (TIMP1) is expressed in some but not all inactive Xcontaining hybrids, suggesting that this gene is either prone to reactivation or variable in its inactivation. Although many genes that escape X inactivation are clustered along the X chromosome, genes flanking TIMP1 (ARAF1, ELK1, ZNF41, and Z/VF757) were expressed only from the active X (Xa) chromosome, demonstrating that the factors allowing TIMP1 expression from the inactive X (Xi) are specific to the TIMP1 gene. This variable X inactivation of TIMP1 is not limited to the hybrid cell environment because TIMP1 expression from the Xi was demonstrated in female cells. As TIMP1 and its target metalloproteinases are involved in many biological processes, women with elevated TIMP1 expression may exhibit different disease susceptibilities, TIMP1 expression levels were analyzed. The range of TIMP1 RNA levels from the Xa precluded analysis of the contribution of the Xi to total TIMP1 RNA levels in females, so I examined expression in Xi hybrids. TIMP1 expression levels varied more widely from the Xi than the Xa, suggesting variable retention of the epigenetic silencing mechanisms. I examined methylation and expression patterns and found that TIMP1 was generally unmethylated when expressed. Methylation was associated with unstable expression because only 58% of clones derived from a methylated TIMP1+ culture retained expression. One of the 33 TIMP1- subclones reactivated TIMP1, suggesting another epigenetic feature differs in the Xi hybrids expressing TIMP1. The acetylation status of histone H3 was examined and intriguingly, in the clones from Xi hybrids with TIMP1 expression, the TIMP1 promoter was always hyperacetylated regardless of current expression status. This was not a reflection of chromatin configuration because the promoter remained nuclease insensitive in all silent clones, similar to the methylation results. These results establish a hierarchy to the epigenetic silencing of TIMP1, with histone acetylation preceding expression whereas methylation and chromatin structure are concordant with expression.

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