CREB BINDING PROTEIN AND GENE REGULATION

cAMP介导的第二信使途径是基因调控的良好建立的模型。 在给定的细胞内，已知许多转录因子与蛋白激酶A/CREB途径相互作用并通过蛋白激酶A/CREB途径起作用，使得通过共同调节因子的多个信号的整合成为极其复杂的现象。 CBP（CREB共激活因子）影响基因调控的机制尚不完全清楚，但该因子可能有助于转录信号整合。 越来越多的证据表明，这种活性可能部分是由于染色质的组蛋白乙酰化（HAT）暴露DNA进行转录。 该项目的主要目的是表征CBP/p300的内在HAT活性。 提出了四个具体的目的：（1）CBP/p300蛋白质的突变在推定的内在HAT结构域（之间的溴结构域和谷氨酰胺丰富的区域CBP/p300）将被开发，以确定和测试它们是否保留其内在的HAT活性。 这也将有助于映射HAT域。 (2)由于腺病毒12 S E1 A阻断CBP/p300共激活因子的机制尚不清楚，因此测试CBP/p300对核小体和多核小体的固有HAT活性是否被E1 A阻断。 (3)确定突变体是否失去激活重组染色质（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转录的能力。 (4)研究发育中果蝇的CBP HAT突变体。该实验室先前已经表明，dCBP拯救CBP无效苍蝇。CBP HAT突变体不能拯救CBP-无效的苍蝇将是CBP（和p300）的内在HAT活性对体内功能至关重要的有力证据，组蛋白乙酰化在其基因调控功能中起着核心作用。 深入了解基因激活和沉默的机制在治疗包括癌症和内分泌疾病在内的许多疾病中具有深远的意义。 通过指导的职业发展计划，首席研究员将获得增强的能力，在分子生物学领域的生产力，重点是基因调控。 这将通过进行上述研究，参加并在地方和国家研讨会和会议上发言，并参加分子生物学技术教学课程来实现。 拟议的指导实验室和机构拥有丰富的智力和物理设施资源，为首席研究员在基因调控领域的研究设计和实施中过渡到独立提供了良好的环境。

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.