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: 6465337
• 负责人: Susan K. Gilmour
• 金额: $ 29.17万
• 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 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6465337
• 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）是一种关键酶， 调节酶的生物合成的多胺是必不可少的 正常的细胞生长和分化。虽然ODC过表达不是 足以在正常细胞中诱导肿瘤，我们已经证明， 水平的ODC和多胺与活化的Ha-ras合作， 上皮肿瘤形成和侵袭。由于其阳离子性质， 多胺被怀疑影响整个染色体构象， 从而有助于转录调节。最近， 组蛋白乙酰转移酶（HAT）和去乙酰化酶（HDAC）活性 某些转录控制蛋白质内在靶向各种基因 启动子引起染色质的动态重塑并导致激活 或抑制转录。ODC和多胺水平的升高改变了 许多与增殖相关的基因的表达， 分化值得注意的是，我们发现它们也促进了 可以用特异性ODC抑制剂逆转的组蛋白乙酰化， α-二氟甲基鸟氨酸本项目的总体目标是确定 多胺如何介导染色质环境的变化， 在上皮肿瘤发生的背景下基因表达的后果。 因此，我们提出以下具体目标：1。建立联系 多胺诱导的组蛋白乙酰化和转录的改变 受影响的基因的活性，这是恶性转化的关键， 表皮细胞：a.识别特定的基因， 使用代表性差异分析（RDA）， 通过染色质纯化的转录活性基因组DNA 免疫沉淀（ChIP）或汞亲和层析，和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由多胺转录调节 与正常水平的ODC相比。2.确定机制， 多胺调节皮肤中HAT和HDAC的功能。使用报告 基于基因的策略，我们将：1）确定HAT/HDAC酶的作用 活性介导多胺对报告基因反式激活的影响， 抑制，和2）检查多胺水平升高对细胞增殖的影响。 染色质重塑的各种蛋白质组分之间的相互作用 参与将HAT和HDAC靶向特定基因的复合物 发起人。我们也将开始描述/确定负责的HAT 对于在来自ODC/Ras双突变的肿瘤中观察到的异常高的HAT活性， 转基因小鼠3.阐明多胺对细胞增殖的作用机制 调节特定基因启动子处的染色质重塑， 差异乙酰化，其表达相应改变， 对ODC过度表达的反应。