Functional analysis of the WT1-BASP1 transcriptional repressor complex

描述(由申请人提供)： Wilms‘s Tumor 1蛋白是一种发育转录因子，可以激活或抑制与生长、凋亡和分化有关的基因。WT1的这些不同的活动是如何调控的尚不清楚，但在确定其在多种癌症中的作用方面具有重要的意义。我们的长期目标是了解WT1在发育和疾病中调节转录的机制。BASP1是WT1的关键调控因子，其功能由转录激活因子转换为抑制因子，并通过与靶基因控制区的WT1结合发挥作用。我们的新发现是，BASP1的N端肉豆蔻酸化和与磷脂酰肌醇4，5-二磷酸(PIP2)相互作用的能力是其转录共抑制功能的关键。此外，我们最近的数据表明，BASP1是一个大型多蛋白复合体的一部分，该复合体包含转录抑制因子禁止素和组蛋白脱乙酰酶活性。当前提案的目标是为WT1-BASP1参与转录调控的机制和PIP2的新角色提供关键的新见解。其具体目的是：1.阐明BASP1-PIP2相互作用调控WT1转录功能的机制。PIP2在WT1-BASP1通过染色质重塑和RNA聚合酶II活性进行转录调控中的作用将使用芯片、转录分析和共定位方法进行研究。这些研究将为基因特异性调控转录抑制的新机制提供新的见解。2.分离BASP1复合体的组成成分，研究其在WT1调控中的作用。我们最近已经确定转录抑制因子Prohibitin和组蛋白脱乙酰酶I是BASP1复合体的组成部分，我们将进一步分析它们的作用机制。将确定该复合体的其他成分，并阐明它们在BASP1介导的转录抑制中的作用。3.确定BASP1复合体和PIP2在WT1调控分化中的作用。依赖WT1和BASP1活性的足细胞分化细胞模型将被用来确定BASP1复合体的关键成分，并揭示PIP2在驱动分化过程的WT1依赖的基因表达中的作用。 公共卫生相关性： Wilms‘s Tumor 1蛋白WT1在儿童肾癌中作为肿瘤抑制基因，但在成人恶性肿瘤中作为癌基因。这项提案将研究WT1辅因子BASP1如何调节WT1的活动。这些结果将为WT1是如何被控制的提供洞察力，并有助于设计WT1依赖的肿瘤的新治疗方法。

DESCRIPTION (provided by applicant): The Wilms' tumor 1 protein is a developmental transcription factor that can either activate or repress genes involved in growth, apoptosis and differentiation. How these disparate activities of WT1 are regulated is not clear, but have important implications in determining its role in multiple cancers. Our long-term goal is to understand the mechanisms by which WT1 regulates transcription in development and disease. BASP1 is a critical regulator of WT1 that switches its function from a transcriptional activator to a repressor, and acts by binding to WT1 at the control regions of target genes. We have made the novel finding that the N-terminal myristoylation of BASP1 and the capacity of BASP1 to interact with phosphatidyinositol 4,5-bisphosphate (PIP2) is critical for its transcriptional cosuppressor function. Moreover, our recent data demonstrate that BASP1 is part of a large multiprotein complex that contains the transcriptional repressor prohibitin and histone deacetylase activity. The goal of the current proposal is to provide critical new insights into the mechanisms involved in the regulation of transcription by WT1-BASP1 and the novel role of PIP2. The specific aims are to; 1. Elucidate the mechanisms by which BASP1-PIP2 interactions control the transcription function of WT1. The role of PIP2 in transcriptional regulation by WT1-BASP1 through chromatin remodeling and RNA polymerase II activity will be investigated using ChIP, transcription analysis, and colocalization approaches. These studies will provide new insights into a novel mechanism of transcriptional repression by gene-specific regulators. 2. Isolate the components of the BASP1 complex and study their function in WT1 regulation. We have recently identified the transcriptional repressor prohibitin and histone deacetylase I as components of the BASP1 complex, which will be analyzed further to determine their mechanism of action. The other components of the complex will be identified and their role in BASP1-mediated transcriptional repression elucidated. 3. Determine the role of the BASP1 complex and PIP2 in the control of differentiation by WT1. A cell line model of podocyte differentiation that is dependent on the activities of WT1 and BASP1 will be employed to identify the critical components of the BASP1 complex, and uncover the role of PIP2 in WT1-dependent gene expression that drives the differentiation process. PUBLIC HEALTH RELEVANCE: The Wilms' tumor 1 protein WT1 acts as a tumor suppressor in childhood kidney cancer, but as an oncogene in adult malignancies. This proposal will study how the WT1 cofactor BASP1 regulates the activities of WT1. The results will provide insights into how WT1 is controlled and aid in the design of new treatments for WT1-dependent tumors.