Regulation of NF-kappaB2 by TSA: Role of Acetylation

TSA 对 NF-kappaB2 的调节：乙酰化的作用

• 批准号: 6851620
• 负责人: JING HU
• 金额: $ 15.53万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-13 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6851620
• 关键词: DNA binding protein; acetylation; active sites; amidohydrolases; cAMP response element binding protein; chromatin immunoprecipitation; enzyme inhibitors; gel mobility shift assay; gene expression; gene induction /repression; genetic transcription; molecular cloning; nuclear factor kappa beta; site directed mutagenesis

DESCRIPTION (provided by applicant): Histone deacetylase (HDAC) inhibitor trichostatin A (TSA) exerts potent anti-tumor effect. Our findings suggest that Nuclear Factor (B (NF-(B)is a primary transcription factor target of TSA. NF-kappaB is important in many aspects of tumor development and treatment. Our long-term goal is to elucidate the molecular mechanisms by which HDAC inhibitor TSA down-regulates NF-kappaB and NF-kappaB controlled target gene expression. The specific hypothesis of this proposal is that TSA-induced p100 processing and p52 acetylation plays a causal role in the regulation of NF-kappaB activity and NF-kappaB controlled cyclin D1 gene transcription. We base that hypothesis on the observations that 1) TSA promotes NF-kappaB2/p100 processing, 2) TSA enhances p52 level and acetylation, 3) Accumulation of acetylated p52 coincides with disruption of NF-kappaB DNA binding and cyclin D1 downregulation. Based on these observations, our specific aims are to: 1. Determine the role of p100 acetylation in HDAC inhibitor TSA induced NF-kappaB2/p100 processing. We will use p100 acetylation mutants to test the hypothesis that p100 processing into p52 requires acetylation at certain sites. 2. Identify the acetylation sites on p52 and ascertain their causal role in regulating NF-kappaB DNA binding upon TSA treatment. The Electrophoretic-Mobility Shift Assay (EMSA) of nuclear protein from cells transfected with p52 acetylation mutants or wild type p52 should reveal which acetylation sites are required for TSA disruption of NF-kappaB DNA binding. 3. Determine the possible causal role of p52 acetylation in TSA down regulated cyclin D1 gene expression. If p52 acetylation at certain sites contributes to the down regulation of cyclin D1 expression by TSA, then transfection of p52 mutated at those sites will fail to down regulate cyclin D1 RNA and protein expression. In addition, we will determine whether p52 acetylation affects the binding of transcription factors Sp-1, AP-1, CREB to cyclin D1 promoter using EMSA and chromatin immunoprecipitation (ChIP).

描述（申请人提供）：组蛋白脱乙酰酶（HDAC）抑制剂曲古抑菌素A（TSA）发挥有效的抗肿瘤作用。我们的研究结果表明，核因子 (B (NF-(B)) 是 TSA 的主要转录因子靶标。NF-kappaB 在肿瘤发展和治疗的许多方面都很重要。我们的长期目标是阐明其分子机制HDAC 抑制剂 TSA 下调 NF-kappaB 和 NF-kappaB 控制的靶基因表达。该提议的具体假设是 TSA 诱导的 p100 加工和 p52 乙酰化发挥着重要作用。我们的假设基于以下观察结果：1) TSA 促进 NF-kappaB2/p100 加工，2) TSA 增强 p52 水平和乙酰化，3)。乙酰化 p52 的积累与 NF-kappaB DNA 结合的破坏和细胞周期蛋白 D1 的下调同时发生。根据这些观察，我们的具体目标是： 1.确定p100乙酰化在HDAC抑制剂TSA诱导的NF-kappaB2/p100加工中的作用。我们将使用 p100 乙酰化突变体来检验 p100 加工成 p52 需要在某些位点进行乙酰化的假设。 2. 鉴定 p52 上的乙酰化位点并确定其在 TSA 处理后调节 NF-kappaB DNA 结合中的因果作用。对 p52 乙酰化突变体或野生型 p52 转染细胞的核蛋白进行电泳迁移率变化分析 (EMSA)，应能揭示 TSA 破坏 NF-kappaB DNA 结合所需的乙酰化位点。 3.确定p52乙酰化在TSA下调细胞周期蛋白D1基因表达中可能的因果作用。如果 p52 在某些位点的乙酰化有助于 TSA 下调细胞周期蛋白 D1 的表达，那么转染在这些位点突变的 p52 将无法下调细胞周期蛋白 D1 RNA 和蛋白的表达。此外，我们将使用 EMSA 和染色质免疫沉淀 (ChIP) 确定 p52 乙酰化是否影响转录因子 Sp-1、AP-1、CREB ​​与细胞周期蛋白 D1 启动子的结合。