Epigenetic regulation by PWWP domain proteins

描述(申请人提供)：组蛋白的共价修饰，如乙酰化、甲基化、磷酸化和泛素化，是染色质结构和功能的重要调节因子。这些修饰的调节缺陷在许多发育障碍和疾病中起到了因果作用。然而，调控组蛋白修饰活动的机制还不是很清楚。此外，除了几个众所周知的例子外，目前还不清楚细胞机械如何解释这些修改。PWWP结构域类似于Chromo、Tudor和MBT结构域，这些结构域以识别甲基化的组蛋白来调节不同的细胞过程而闻名。然而，PWWP结构域的功能仍然是一个谜。我们的初步研究表明，PWWP结构域蛋白直接与组蛋白相互作用，形成具有组蛋白修饰活性的复合体。因此，我假设PWWP结构域蛋白可能识别修饰的染色质成分来调节相关的酶。我将对分裂酵母中PWWP结构域蛋白的功能进行表征，以获得对它们在调节染色质结构和功能中的作用的机械性见解。具体目的是：(A)通过下拉试验和染色质免疫沉淀(CHIP)分析确定与每个PWWP结构域蛋白相互作用的组蛋白修饰；(B)通过亲和纯化标记的PWWP结构域蛋白来鉴定PWWP结构域蛋白质复合体，以阐明每个蛋白质对这些复合体功能的贡献；(C)分析PWWP结构域蛋白在基于染色质的过程中的细胞功能，目的是直接将表型与特定的组蛋白修饰联系起来。许多含有PWWP结构域的蛋白质的突变与人类疾病密切相关。例如，Dnmt3b的PWWP区域的单个氨基酸改变导致ICF(免疫缺陷、着丝粒不稳定和面部畸形)综合征。更好地了解PWWP结构域蛋白的功能可能会导致治疗这些疾病的新方法。

DESCRIPTION (provided by applicant): Covalent modification of histones, such as acetylation, methylation, phosphorylation, and ubiquitylation, are essential regulators of chromatin structure and function. Defects in the regulation of these modifications have causal roles in numerous developmental disorders and diseases. However, the mechanisms that regulate histone-modifying activities are not well understood. In addition, apart from a few well-known examples, it is not clear how the cellular machinery interprets these modifications. The PWWP domain is similar to Chromo, Tudor, and MBT domains, which are well known for their ability to recognize methylated histones to regulate diverse cellular processes. However, the function of the PWWP domain is still a mystery. Our preliminary studies demonstrated that PWWP domain proteins directly interact with histones and form complexes with histone-modifying activities. I therefore hypothesize that PWWP domain proteins might recognize modified chromatin components to regulate associated enzymes. I will characterize the functions of PWWP domain proteins in fission yeast to gain mechanistic insights into their roles in regulating chromatin structure and function. The specific aims are designed to (a) determine the histone modification that interacts with each PWWP domain protein with pull-down assays and chromatin immunoprecipitation (ChIP) analysis; (b) characterize PWWP domain protein complexes identified by affinity purification of epitope-tagged PWWP domain proteins to elucidate the contribution of each protein to the function of these complexes; (c) analyze the cellular functions of PWWP domain proteins in chromatin based processes, with the goal of directly linking phenotypes to specific histone modifications. Mutations in many PWWP domain-containing proteins are intimately linked to human diseases. For example, a single amino acid alteration in the PWWP domain of Dnmt3b is responsible for ICF (immunodeficiency, centromeric instability, and facial anomalies) syndrome. A better understanding of the function of PWWP domain proteins may lead to novel therapeutic approaches to treat these diseases.