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（由申请人提供）：组蛋白去乙酰化酶（HDAC）抑制剂trichostatin A （TSA）具有较强的抗肿瘤作用。我们的研究结果表明核因子（NF-(B)）是TSA的主要转录因子靶点。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-kappaB和NF-kappaB控制靶基因表达的分子机制。本提案的具体假设是tsa诱导的p100加工和p52乙酰化在NF-kappaB活性调控和NF-kappaB控制cyclin 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-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cyclin D1的下调相一致。基于这些观察，我们的具体目标是：