Identification of novel histone modifications in a model eukaryote

描述(由申请人提供)： 表观遗传在调节细胞发育和分化中扮演着重要但知之甚少的角色。为了阐明表观遗传机制，已经确定组蛋白的翻译后修饰是表观遗传信息的主要转导。这些修饰对表观遗传控制的影响要么是通过染色质纤维的生物物理改变发生的，要么是通过将效应蛋白招募到修饰部位的能力发生的。近年来，基于高灵敏度和高通量的蛋白质组学技术应用于组蛋白修饰的研究，发现了大量新的修饰或修饰位点。然而，这些研究使用的是从异步化生长的酵母或哺乳动物细胞中提纯的组蛋白。因此，在细胞周期的特定阶段，通常存在于低水平的重要修饰很容易被错过，因为它们不会在纯化的组蛋白总库中显著丰富。此外，在这些早期研究中，高度不稳定的修饰，如泛素化和总酰化，很可能被遗漏，因为标准的组蛋白提取和纯化方案不能阻止酶去除这些不稳定的修饰。为了绕过这些技术问题，我们创造了一系列萌芽酵母菌株，每个组蛋白基因都有一个拷贝，基因组上标记着一个表位序列，允许变性蛋白质纯化。我们将使用这些菌株在变性条件下纯化单个组蛋白，以及两个组蛋白变体Htz1和Cse4，在细胞周期的每个阶段。为了检测在每个组蛋白上可能的PTM位点的最大覆盖率，这些纯化的组蛋白将通过使用“自上而下”和“自下而上”的基于MS的方法进行分析，以互补的方式表征在谨慎的细胞周期阶段驻留在这些组蛋白上的全部修饰。这些方法将使我们能够确定通常由于其不稳定的性质或丰度不足而无法检测到的新的修饰和/或修饰部位。因此，这项提议旨在创建一个真核“表观基因组图谱”，它将定义整个细胞周期中存在于酵母组蛋白上的组蛋白修饰的完整补充。我们的研究不仅有助于识别在表观基因组学倡议下可以在人类干细胞中进一步分析的新标记，而且还将提供有价值的信息，了解在细胞分裂过程中影响染色体生物学的组蛋白修饰的动态。因此，这项工作将为理解人类中发生的复杂的表观遗传调控以及调控失调如何促进癌症等病理过程奠定基础。公共卫生相关性：染色质组织、DNA包装及其可及性方面的缺陷是导致包括癌症在内的人类疾病的主要原因。这些拟议的研究旨在确定新的表观遗传学组蛋白标记，这无疑将被证明是基于DNA的活动的中央调节因子。这一知识将有助于解决癌症和其他公共卫生问题的根本原因。

DESCRIPTION (provided by applicant): Epigenetic inheritance plays an important but poorly understood role in regulating cell development and differentiation. Efforts to elucidate epigenetic mechanisms have identified the post-translational modification of histone proteins as a major transducer of epigenetic information. The effect these modifications have on epigenetic control occurs either through biophysical alteration of the chromatin fiber or through the ability to recruit effector proteins to the site of modification. In recent years the application of sensitive and high throughput mass spectrometry (MS)-based proteomic techniques to characterize histone modifications has revealed a large number of novel modifications or sites of modification on these proteins. However, these studies used histones that were purified from asynchronously growing yeast or mammalian cells. As a consequence, important modifications that normally exist at low levels during specific phases of the cell cycle could easily have been missed since they would not be significantly enriched in the total pool of histones purified. Furthermore, highly labile modifications such as ubiquitylation and sumoylation are highly likely to have been missed in these early studies since standard histone extraction and purification protocols do not prevent enzymatic removal of these labile modifications. To circumvent these technical issues, we have created a series of budding yeast strains that have one copy of each histone gene genomically tagged with an epitope sequence that allows denaturing protein purification. We will use these strains to purify under denaturing conditions the individual histones, and the two histone variants Htz1 and Cse4, during each phase of the cell cycle. To detect the maximum coverage of possible PTM sites at each individual histone, these purified histones will be analyzed by using both "top-down" and "bottom-up" MS-based approaches in a complimentary way to characterize the full complement of modifications residing on these histones at discreet cell-cycle phases. These approaches will allow us to identify novel modifications and/or sites of modification that normally would be undetectable due to either their labile nature or insufficient abundance. This proposal thus aims to create a eukaryotic "epigenomic atlas" that will define the full complement of histone modifications that exist on yeast histones throughout the cell cycle. Our study will not only facilitate the identification of novel marks that can be further analyzed in human stem cells under the epigenomics initiative, but it also will provide valuable information into the dynamics of histone modifications that influence chromosome biology during cell division. This work will therefore serve as a foundation for understanding the complex epigenetic regulation that occurs in humans, and how dysfunctional regulation promotes pathological processes such as cancer. PUBLIC HEALTH RELEVANCE: Defects in chromatin organization, DNA packaging and its accessibility is a major cause of human disease, including cancer. These proposed studies aim to identify novel epigenetic histone marks, which will undoubtedly turn out to be central regulators of DNA-based activities. This knowledge will help to address the underlying causes of cancer and other public health concerns.