Omithine decarboxylase (ODC) is a key regulatory enzyme in the biosynthesis of polyamines which are essential for normal cell growth and differentiation. Although ODC overexpression is not sufficient to induce tumors in normal cells, we have demonstrated that elevated levels of ODC and polyamines cooperate with activated Ha-ras to promote epithelial tumor formation and invasion. Due to their cationic nature, polyamines have been suspected to affect overall chromosome conformation, thereby contributing to transcriptional regulation. Recently it has been shown that histone acetyltransferase (HAT) and deacetylase (HDAC) activities intrinsic to some transcriptional control proteins are targeted to various gene promoters causing dynamic remodeling of chromatin and leading to the activation or repression of transcription. Elevated levels of ODC and polyamines alter the expression of a number of genes that are associated with proliferation and differentiation. Significantly, we have found that they also promote changes in histone acetylation which can be reversed with the specific ODC inhibitor, alpha-difluoromethylomithine. The overall goal of this project is to determine how polyamines mediate changes in the chromatin environment, and the consequences for gene expression in the context of epithelial tumorgenesis. Thus, we propose the following specific aims: 1. To establish a link between polyamine induced alterations in histone acetylation and transcriptional activity of impacted genes which are key to malignant transformation of epidermal cells by: a. identifying specific genes that are transcriptionally regulated by polyamines using representative difference analysis (RDA) of transcriptionally competent genomic DNA purified by chromatin immunoprecipitation (ChIP) or mercury affinity chromatography, and b. verifying altered acetylation of nucleosomes associated with promoters of genes transcriptionally regulated by polyamines in cells or tissue expressing high versus normal levels of ODC. 2. To identify the mechanism(s) by which polyamines modulate the function of HATs and HDACs in the skin. Using reporter gene-based strategies, we will: 1) determine the role of HAT/HDAC enzymatic activities in mediating polyamine effects on reporter gene transactivation or repression, and 2) examine the effect of elevated levels of polyamines on the interactions between various protein components of chromatin remodeling complexes that are involved in targeting HATs and HDACs to specific gene promoters. We will also begin to characterize/identify the HAT(s) responsible for the aberrantly high HAT activity observed in tumors from ODC/Ras double transgenic mice. 3.To elucidate the individual mechanism(s) by which polyamines modulate the remodeling of chromatin at promoters of specific genes that are differentially acetylated and whose expression is correspondingly altered in response to ODC overexpression.
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