POLYAMINE-MODULATED HISTONE ACETYLATION IN TUMORIGENESIS

肿瘤发生中多胺调节的组蛋白乙酰化

• 批准号: 7030345
• 负责人: Susan K. Gilmour
• 金额: $ 29.33万
• 依托单位: LANKENAU INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7030345
• 关键词: acetylation; acyltransferase; affinity chromatography; amidohydrolases; chemical carcinogenesis; chromatin; gene induction /repression; genetic transcription; genetically modified animals; histones; immunoprecipitation; laboratory mouse; neoplasm /cancer genetics; ornithine decarboxylase; polyamines; representational difference analysis; skin neoplasms

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.

描述：（由申请人提供）鸟氨酸脱羧酶（ODC）是关键 多胺生物合成中的调节酶，对于生物合成至关重要 正常的细胞生长和分化。尽管 ODC 过度表达并不 足以在正常细胞中诱导肿瘤，我们已经证明升高 ODC 和多胺的水平与激活的 Har-ras 配合，促进 上皮肿瘤的形成和侵袭。由于其阳离子性质， 多胺被怀疑会影响整体染色体构象， 从而有助于转录调控。最近已经显示了 组蛋白乙酰转移酶 (HAT) 和脱乙酰酶 (HDAC) 活性 一些转录控制蛋白固有的靶向不同基因 启动子引起染色质动态重塑并导致激活 或转录抑制。 ODC 和多胺水平升高会改变 一些与增殖相关的基因的表达 差异化。值得注意的是，我们发现它们还促进了以下方面的变化： 组蛋白乙酰化可以用特定的 ODC 抑制剂逆转， α-二氟甲基鸟氨酸。该项目的总体目标是确定 多胺如何介导染色质环境的变化，以及 上皮肿瘤发生背景下基因表达的后果。 因此，我们提出以下具体目标： 1. 在不同领域之间建立联系 多胺诱导组蛋白乙酰化和转录的改变 受影响基因的活性，这是恶性转化的关键 表皮细胞由： A.识别转录的特定基因 使用代表性差异分析 (RDA) 受多胺调节 通过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，以及 b．验证 与基因启动子相关的核小体乙酰化改变 在高表达的细胞或组织中受多胺转录调节 与 ODC 的正常水平相比。 2. 确定机制 多胺调节皮肤中 HAT 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 介导多胺对报告基因反式激活作用的活性或 抑制，2）检查多胺水平升高对 染色质重塑的各种蛋白质成分之间的相互作用 参与将 HAT 和 HDAC 靶向特定基因的复合物 发起人。我们还将开始描述/识别负责的 HAT 用于在 ODC/Ras 双肿瘤中观察到的异常高 HAT 活性 转基因小鼠。 3.阐明多胺作用的具体机制 调节特定基因启动子处染色质的重塑 差异乙酰化，其表达相应改变 对 ODC 过度表达的反应。