Formation, Structure and Function in Heterochromatin

描述（由申请人提供）：DNA在分化细胞中可被稳定地包装成正染色质和异染色质形式，是表观遗传的重要形式。异染色质系统的故障可导致基因表达的错误调节，以及染色体不稳定，并且是许多健康问题的一个促成因素，包括癌症和基于遗传的发育障碍。异染色质包装可以通过一组相互作用的结构蛋白得以延续和传播，其中异染色质蛋白1 （HP1a）在识别关键的组蛋白修饰标记（H3K9的甲基化）和与组蛋白甲基转移酶（HMT）相互作用中发挥关键作用。我们的长期目标是了解为什么以及如何选择基因组的某些区域进行异染色质组装，并确定所需的染色体蛋白质及其相互作用。所有的工作都将在果蝇中进行，这是一个允许遗传学、细胞学和生化方法的模型系统；用位置效应色差（PEV）作为异染色质包装的指标。我们对4号染色体（通过几种测量完全异色）的研究表明，1360 DNA转座子的残余物是异染色质形成的靶标，但稳定的异染色质需要高重复密度。在第一个特定目标中，将使用具有不同类型和重复元件拷贝数的报告P元件来测试基因组组织的作用和特定靶标的身份。在我们的第二个目标中，结合研究和结构分析将探讨HP1a如何区分其几个合作伙伴以组装沉默结构。将鉴定4号染色体的HMT特异性，探索其相互作用的模式，并考虑对传播的影响。其他异染色质成分将通过筛选酵母双杂交系统中与HP2相互作用的蛋白质来鉴定，利用最近恢复的HP2突变作为对照。在第三个长期目标中，我们将询问是否某些第四染色体基因在异色环境中发挥最佳功能，并探索这种适应。综上所述，这些研究将阐明异染色质形成是如何开始和持续的，并将在结构域水平上为基因调控提供新的见解。

DESCRIPTION (provided by applicant): The alternative packaging of DNA into euchromatic and heterochromatic forms can be stably maintained in differentiated cells, and represents an important form of epigenetic inheritance. Malfunction in the heterochromatin system can lead to misregulation of gene expression, as well as chromosome instability, and is a contributing factor in many health problems, including cancer and genetically-based developmental disabilities. Heterochromatin packaging can be perpetuated and spread by virtue of an interacting set of structural proteins, with Heterochromatin Protein 1 (HP1a) playing a key role in both recognizing a critical histone modification mark (methylation of H3K9) and interacting with the histone methyl transferase (HMT). Our long term goal is to understand why and how certain regions of the genome are selected for heterochromatin assembly, and to identify the required chromosomal proteins and their interactions. All work will be carried out in Drosophila, a model system that allows genetic, cytological, and biochemical approaches; position effect variegation (PEV) is used as an indicator of heterochromatin packaging. Our studies of chromosome four (entirely heterochromatic by several measures) indicate that remnants of the 1360 DNA transposon serve as targets for heterochromatin formation, but that stable heterochromatin requires a high repeat density. In the first specific aim, the role of genome organization and identity of specific targets will be tested using reporter P elements with different types and copy number of repetitious elements. In our second aim, binding studies and structural analysis will explore how HP1a discriminates among its several partners to assemble a silencing structure. The HMT specific for chromosome four will be identified, its pattern of interactions explored, and the impact on spreading considered. Additional heterochromatin components will be identified by screening for proteins interacting with HP2 in a yeast two hybrid system exploiting recently recovered HP2 mutations as controls. In a third long-term aim, we will ask whether some fourth chromosome genes function optimally in a heterochromatic environment, and explore that adaptation. Taken together these studies will illuminate how heterochromatin formation is initiated and sustained, and will provide new insights into gene regulation at the domain level.