POLYAMINE-MODULATED HISTONE ACETYLATION IN TUMORIGENESIS

描述：(申请人提供)欧米氨酸脱羧酶(ODC)是一个关键 调节酶在多胺的生物合成中是必不可少的 正常的细胞生长和分化。尽管ODC过度表达不是 足以在正常细胞中诱发肿瘤，我们已经证明 ODC和多胺水平与激活的Ha-ras协同促进 上皮性肿瘤的形成和侵袭。由于它们的阳离子性质， 多胺被怀疑会影响染色体的整体构象， 从而有助于转录调控。最近，它被展示出来了 组蛋白乙酰转移酶(HAT)和脱乙酰酶(HDAC)活性 一些转录控制蛋白的固有作用于不同的基因。 导致染色质动态重塑并导致激活的启动子 或对转录的抑制。ODC和多胺水平的升高会改变 一些与增殖相关的基因的表达和 差异化。值得注意的是，我们发现它们还促进了 组蛋白乙酰化，可以用特定的ODC抑制剂逆转， α-二氟甲亚胺。这个项目的总体目标是确定 多胺如何调节染色质环境的变化，以及 上皮性肿瘤发生背景下基因表达的后果。 为此，我们提出了以下具体目标：1.建立 多胺诱导组蛋白乙酰化和转录的改变 受影响基因的活性，这些基因是恶性转化的关键 通过：A.鉴定转录水平上的特定基因 多胺调控的代表性差异分析(RDA) 染色质纯化具有转录活性的基因组DNA 免疫沉淀(CHIP)或汞亲和层析；B.验证 与基因启动子相关的核小体乙酰化改变 在高表达细胞或组织中受多胺的转录调控 与正常的ODC水平相比。2.明确机制(S) 多胺调节皮肤中HATS和HDAC的功能。使用记者 基于基因的策略，我们将：1)确定HAT/HDAC酶的作用 多胺对报告基因反式激活的调节作用 抑制，以及2)检查多胺水平升高对 染色质重塑不同蛋白质组分之间的相互作用 参与靶向特定基因的HATS和HDAC的复合体 推动者。我们还将开始表征/确定负有责任的帽子(S) 在ODC/RAS双重来源的肿瘤中观察到异常高的HAT活性 转基因小鼠。3.阐明多胺的个体机制(S) 在特定基因的启动子上调节染色质的重塑 差异乙酰化，其表达相应地改变在 对ODC过度表达的响应。

DESCRIPTION: (provided by applicant) 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.