CREB BINDING PROTEIN AND GENE REGULATION

CREB ​​结合蛋白和基因调控

• 批准号: 6634728
• 负责人: WILLIAM H LUDLAM
• 金额: $ 13.21万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6634728
• 关键词: Adenoviridae; Drosophilidae; acetyl coA; acyltransferase; binding sites; biological signal transduction; cAMP response element binding protein; chromatin; enzyme activity; gene deletion mutation; genetic regulation; histones; point mutation; protein sequence; virus protein

The cAMP mediated second messenger pathway is a well established model of gene regulation. Within a given cell, many transcription factors are known to interact with and work through the protein kinase A/CREB pathway making integration of multiple signals through common regulatory factors an exceedingly complex phenomenon. The mechanism by which CBP (a CREB co-activator) influences gene regulation is not fully understood, but it is likely that this factor contributes to transcriptional signal integration. There is increasing evidence that this activity may be, in part, due to histone acetylation (HAT) of chromatin that exposes DNA for transcription. The main thrust of this project is to characterize the intrinsic HAT activity of CBP/p300. Four Specific Aims are proposed: (1) CBP/p300 proteins mutated in the putative intrinsic HAT domain (between the bromodomain and the glutamine-rich region of CBP/p300) will be developed to determine and test whether they retain their intrinsic HAT activity. This will also be helpful in mapping the HAT domain. (2) To test whether the intrinsic HAT activity of CBP/p300 for nucleosomes and polynucleosomes is blocked by E1A, since the mechanism by which adenovirus 12S E1A blocks the CBP/p300 co-activator is unknown. (3) To determine whether mutants lose their ability to activate reconstituted chromatin (DNA bound to histones), yet maintain their ability to activate transcription in DNA not bound to histones. (4) To study CBP HAT mutants in developing flies. This lab has previously shown that dCBP rescues CBP-null flies. The inability of CBP HAT mutants to rescue CBP-null flies would be strong evidence that intrinsic HAT activity of CBP (and p300) is critical for in vivo function, and that histone acetylation plays a central role in their gene regulatory function. Gaining insight to the mechanisms of gene activation and silencing has far reaching relevance in the treatment of many diseases including cancers and endocrinopathies. Through a mentored career development plan, the Principal Investigator will gain an enhanced capacity to be productive in the field of molecular biology with a focus on gene regulation. This will be accomplished by conducting the above described research, attending and presenting at local and national seminars and meetings, and participating in courses teaching molecular biology techniques. The proposed mentoring laboratory and institution are rich in intellectual and physical facility resources that provide an excellent environment for the Principal Investigator to transition to independence in the design and implementation of research in the field of gene regulation.

CAMP介导的第二信使途径是一个成熟的基因调控模型。在一个给定的细胞内，已知许多转录因子与蛋白激酶A/CREB途径相互作用并通过其发挥作用，使得通过共同调控因子整合多种信号成为一种极其复杂的现象。CBP(一种CREB共激活因子)影响基因调控的机制尚不完全清楚，但这一因素很可能有助于转录信号整合。越来越多的证据表明，这种活性可能部分归因于染色质的组蛋白乙酰化(HAT)，它暴露了DNA进行转录。该项目的主旨是表征CBP/p300的固有HAT活性。提出了四个具体的目标：(1)将开发在CBP/p300的固有HAT结构域(位于CBP/p300的溴区和富含谷氨酰胺的区域之间)突变的CBP/p300蛋白，以确定和测试它们是否保持其固有的HAT活性。这也将有助于映射HAT域。(2)由于腺病毒12S E1a阻断CBP/p300共激活子的机制尚不清楚，因此检测E1a是否阻断CBP/p300对核小体和多核小体的固有HAT活性。(3)确定突变体是否失去了激活重组染色质(与组蛋白结合的DNA)的能力，但仍保持激活未与组蛋白结合的DNA的转录的能力。(4)研究CBP HAT突变体在发育过程中的作用。这个实验室之前已经证明，dCBP可以拯救CBP零值的苍蝇。CBP HAT突变体不能拯救CBP缺失型果蝇，这将是CBP(和p300)固有的HAT活性对体内功能至关重要的有力证据，并且组蛋白乙酰化在其基因调控功能中发挥核心作用。深入了解基因激活和沉默的机制对包括癌症和内分泌疾病在内的许多疾病的治疗具有深远的意义。通过有指导的职业发展计划，首席调查员将获得在分子生物学领域提高生产力的能力，重点是基因调控。这将通过开展上述研究、出席和在地方和国家研讨会和会议上发言以及参加教授分子生物学技术的课程来实现。拟议的辅导实验室和机构拥有丰富的智力和物质设施资源，为首席研究人员在基因调控领域的研究设计和实施方面向独立过渡提供了良好的环境。