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（由申请方提供）：组蛋白脱乙酰酶（HDAC）抑制剂曲马斯他汀A（TSA）发挥强效抗肿瘤作用。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HDAC抑制剂TSA下调NF-κ B和NF-κ B控制的靶基因表达的分子机制。该建议的具体假设是TSA诱导的p100加工和p52乙酰化在调节NF-κ B活性和NF-κ B控制的细胞周期蛋白D1基因转录中起因果作用。我们的假设基于以下观察结果：1）TSA促进NF-κ B 2/p100加工，2）TSA增强p52水平和乙酰化，3）乙酰化p52的积累与NF-κ B DNA结合的破坏和细胞周期蛋白D1的下调一致。根据这些意见，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-κ B2/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需要在某些位点乙酰化的假设。 2.鉴定p52上的乙酰化位点，并确定它们在TSA处理后调节NF-κ B DNA结合中的因果作用。来自用p52乙酰化突变体或野生型p52转染的细胞的核蛋白的电泳迁移率变动测定（EMSA）应当揭示TSA破坏NF-κ B 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将不能下调细胞周期蛋白D1 RNA和蛋白质的表达。此外，我们将使用EMSA和染色质免疫沉淀（ChIP）来确定p52乙酰化是否影响转录因子Sp-1、AP-1、CREB与cyclin D1启动子的结合。