Characterization of SET-3 and its role in DNA methylation

描述(由申请人提供)：基因表达的表观遗传控制是正常细胞发育和维持所需的基本过程。表观遗传过程的异常调节导致组蛋白异常修饰和/或DNA甲基化，是许多人类疾病的共同潜在因素，包括神经和自身免疫性疾病以及癌症。赖氨酸甲基转移酶的SET结构域蛋白超家族包括参与组蛋白和DNA甲基化的关键基因表达调节物。异常的SET蛋白活性被证明有助于恶性转化，并且在各种癌症中发现了反复出现的SET蛋白突变。由于SET-蛋白在调节染色质紧凑、DNA甲基化和恶性转化中的重要作用，我们的实验室试图鉴定和表征模式生物粗糙脉孢霉的SET-蛋白。脉孢子虫作为揭示组蛋白调控和DNA甲基化之间联系的实验系统，其独特的特性使其成为继续发现表观遗传编程调控因子的理想系统。我们实验室的初步研究表明，靶向干扰野生型SET-3功能会导致DNA超甲基化和异常甲基化扩散。我们建议确定SET-3‘S在DNA甲基化调控中的作用。我们的具体目标是：(1)表征SET-3的基因组相互作用；(2)表征SET-3的赖氨酸甲基转移酶活性；以及(3)阐明 SET-3在体内的作用。具体地说，我们将使用高通量测序技术和Selker实验室成熟的实验技术来绘制SET-3的基因组相互作用图，并表征SET-3调节的转录组。我们将通过在SET-3受损的菌株中绘制整个基因组的DNA甲基化来补充这一点。然后，我们将通过使用纯化的催化结构域在体外测试SET-3的组蛋白赖氨酸活性，以及通过体内DNA和组蛋白甲基化模式的分析来探索SET-3的生化活性。最后，我们将使用SET-3的免疫沉淀和质谱分析来鉴定SET-3-相互作用。我们将把体外活性研究与In 活体表型特征和蛋白质-蛋白质相互作用的研究，并将它们置于SET-3‘的背景下，S绘制了基因组相互作用图，得出了SET-3功能的全球特征。这些研究将有助于我们理解真核生物中的基因表达调控，并将有助于我们理解DNA超甲基化和甲基化在癌症中的扩散。我们研究的长期目标是将我们所学到的应用于人类SET-蛋白质同源基因，从而为新的靶向癌症疗法的发展提供信息。

DESCRIPTION (provided by applicant): Epigenetic control of gene expression is a fundamental process required for normal cellular development and maintenance. Abnormal regulation of epigenetic processes, resulting in aberrant histone modification and/or DNA methylation, is a common underlying contributor to many human diseases, including neurological and autoimmune disorders and cancer. The SET-domain protein superfamily of lysine methyltransferases includes critical modulators of gene expression that participate in both histone and DNA methylation. Abnormal SET- protein activity has been shown to contribute to malignant transformation, and recurrent SET-protein mutations have been identified in a wide variety of cancers. Because of the importance of SET-proteins in the regulation of chromatin compaction, DNA methylation, and malignant transformation, our lab has sought to identify and characterize the SET-proteins of the model organism Neurospora crassa. Neurospora has been instrumental as an experimental system for uncovering the connection between histone regulation and DNA methylation, and unique characteristics of Neurospora make it the ideal system for continued discovery of regulators of epigenetic programming. Preliminary studies in our lab indicate that targeted disruption of wild type SET-3 function results in DNA hypermethylation and aberrant methylation spreading. We propose to define SET-3's role in DNA methylation regulation. Our specific aims are: (1) To characterize the genomic interactions of SET- 3; (2) to characterize the lysine methyltransferase activity of SET-3; and (3) to elucidate the function of SET-3 in vivo. Specifically, we will use high-throughput sequencing technology and experimental techniques well established in the Selker lab to map the genomic interaction of SET-3 and characterize the SET-3 regulated transcriptome. We will complement this by mapping DNA methylation across the whole genome in a SET-3- compromised strain. We will then explore the biochemical activity of SET-3 by testing its histone lysine activity in vitro usin a purified catalytic domain construct and by in vivo analyses of DNA and histone methylation patterns. Finally, we will use immunoprecipitation of SET-3 followed by mass spectrometric analysis to identify SET-3-interactors. We will integrate the in vitro activity studies with the in vivo phenotype characterization and protein-protein interaction studies, and place them in the context of SET-3's mapped genomic interactions to derive a global characterization of SET-3 function. These studies should contribute to our understanding of gene expression regulation in eukaryotes, and will aid in our understanding of DNA hypermethylation and methylation spreading in cancer. The long-term goal of our studies is to apply what we have learned to human SET-protein orthologues to inform the development of new targeted cancer therapeutics.