Genome-wide measurement of histone replacement rates: a new dimension in epigenom

描述（由申请人提供）：真核生物的表观遗传系统可以定义为一组基于染色质的过程，其建立全局基因表达状态并促进此类状态的长期维持和/或遗传性。该领域的大多数现代工作集中在组蛋白翻译后修饰（PTM）和赋予和去除这些修饰的酶，作为理解基因表达状态如何被施加和随后传播的手段。然而，最近的研究结果表明，核小体组装，驱逐和替换的动态过程是表观遗传的关键。为了在高分辨率下探索核小体动力学，我们建议开发一种新的通用技术，用于在以前难以处理的时间尺度上全基因组测量组蛋白置换动力学。该技术，我们称之为共价附着标签以捕获组蛋白和识别转换（CATCH-IT），将利用甲硫氨酸替代物叠氮基高丙氨酸（AHA）共翻译掺入蛋白质中，然后分离染色质并将生物素部分体外连接到携带AHA的核小体。这种代谢标记策略之后将是含有新合成的组蛋白的标记核小体的链霉亲和素亲和下拉，允许测量整个基因组的组蛋白置换率以及分析新合成的组蛋白上的PTM。CATCH-IT方法将在果蝇S2细胞系中开发，但鉴于其组分技术的通用性以及不需要转基因或抗体的事实，可以应用于任何细胞类型。虽然目前的ChIP芯片方法仅提供基于染色质的过程的相对静态视图，但CATCH-IT方法将允许在整个基因组中测量组蛋白周转率，为表观基因组学研究增加时间维度。 公共卫生相关性：我们建议开发一种新的技术来测量基于染色质的过程的动力学，并将其应用于关键的表观遗传签名。这将提供一个时间轴，用于使用通常适用于不同生物体、细胞类型和组织的程序动态调节染色质。因此，我们的技术有可能彻底改变表观基因组学研究，包括旨在诊断和治疗癌症和其他表观遗传疾病的努力。

DESCRIPTION (provided by applicant): The epigenetic system of eukaryotic organisms can be defined as the set of chromatin-based processes that establish global gene expression states and promote the long-term maintenance and/or heritability of such states. Most modern work in the field has focused on histone post- translational modifications (PTMs) and the enzymes that impart and remove these modifications as a means of understanding how gene expression states can be imposed and subsequently propagated. However, recent results indicate that the dynamic processes of nucleosome assembly, eviction and replacement are key to epigenetic inheritance. To explore nucleosome dynamics at high resolution, we propose to develop a novel and general technology for the genome-wide measurement of histone replacement kinetics on timescales that have been previously intractable. This technology, which we refer to as Covalent Attachment of Tags to Capture Histones and Identify Turnover (CATCH-IT), will utilize co-translational incorporation of the methionine surrogate azidohomoalanine (AHA) into proteins, followed by isolation of chromatin and in vitro ligation of a biotin moiety to AHA-bearing nucleosomes. This metabolic labeling strategy will be followed by streptavidin affinity pulldown of tagged nucleosomes containing newly synthesized histones, allowing measurement of histone replacement rates across the genome as well as analysis of PTMs on newly synthesized histones. The CATCH-IT method will be developed in the Drosophila S2 cell line but could be applied to any cell type given the generic nature of its component techniques and the fact that no transgenes or antibodies are required. Whereas current ChIP-chip methods offer only a relatively static view of chromatin-based processes, the CATCH-IT method will allow the rates of histone turnover to be measured throughout the genome, adding a temporal dimension to epigenomics research. PUBLIC HEALTH RELEVANCE: We propose to develop a novel technology for measuring the kinetics of chromatin-based processes and to apply it to key epigenetic signatures. This will provide a timeline for dynamic regulation of chromatin using a procedure that is generally applicable to different organisms, cell types and tissues. As such, our technology has the potential of revolutionizing epigenomics research, including efforts that are aimed at diagnosis and treatment of cancer and other epigenetic disorders.