Capturing the dynamic epigenome using single molecule and single cell approaches

项目摘要 在早期发育和多细胞期间保留先前存在的组蛋白修饰模式 分化对于维持细胞特性是必不可少的。组蛋白H3赖氨酸9甲基化(H3K9me)是 与转录沉默的特殊染色质结构域的形成有关，也称为 异染色质。异染色质的建立是正常着丝粒和端粒功能所必需的， 沉默转座子和重复的DNA元件并保留基因的谱系特异性模式 表情。H3K9甲基化缺失与基因组不稳定和非整倍体有关，这些都是 被广泛认为是与癌症有关的最常见的异常。异染色质的建立 是一个动态过程，涉及组蛋白修饰物和它们之间的弱而瞬时的相互作用 同源核小体底物。与直觉相反的是，这些弱的蛋白质-蛋白质相互作用产生了表观遗传 在世代时间尺度上保持可遗传的状态。这些动态属性与 表观基因组暴露了重大的概念和方法差距，本提案将主要解决这些差距： 1)由组蛋白修饰读取器、写入器和擦除器组成的动态表观遗传复合体是如何 在体外和活细胞中组装？2)组蛋白修饰物如何穿过复杂的染色质景观 定位它们的作用部位？3)表观遗传记忆是如何存储和传递的 世世代代？目前的观点是，组蛋白修饰起着惰性支架的作用，被动地 促进组蛋白修饰物在基因组中不同位置的定位。根据我们最近的研究，我们 提出一种对这一范式的颠倒。我们的结果显示，H3K9甲基化在 催化异染色质调节复合体的协同组装。我们的研究强调了 H3K9甲基化依赖的表观遗传复合体组装限制蛋白质-蛋白质 与特定染色质环境的相互作用。在这项提案中，我们将使用体外生物化学来重建 异染色质调节复合体，单分子成像以确定组装的顺序和时间 这些复合体在体外和天然染色质背景下以及高通量微流控平台内 定义表观遗传记忆是如何在个体谱系中传播的。实时视觉化 高空间和时间分辨率的异染色质组装将阐明瞬时分子 相互作用可以协同作用，建立稳定和可遗传的基因表达模式。

Project Summary Retaining pre-existing patterns of histone modifications during early development and multicellular differentiation is essential for the maintenance of cellular identity. Histone H3 lysine 9 methylation (H3K9me) is associated with the formation of transcriptionally silent, specialized chromatin domains also referred to as heterochromatin. Heterochromatin establishment is essential for normal centromere and telomere function, silencing of transposons and repetitive DNA elements and preserving lineage-specific patterns of gene expression. The loss of H3K9 methylation is associated with genome instability and aneuploidy which are widely recognized as the most common abnormalities associated with cancer. Heterochromatin establishment is a dynamic process which involves weak and transient interactions between histone modifiers and their cognate nucleosome substrates. Counterintuitively, these weak protein-protein interactions produce epigenetic states that remain heritable across generational timescales. These dynamic properties associated with the epigenome exposes significant conceptual and methodological gaps that this proposal will principally address: 1) How do dynamic epigenetic complexes consisting of histone modification readers, writers and erasers assemble in vitro and in living cells? 2) How do histone modifiers traverse a complex chromatin landscape to locate their sites of action? 3) How is epigenetic memory stored and transmitted across subsequent generations? The current belief is that histone modifications function as inert scaffolds which passively promote the localization of histone modifiers to distinct sites in the genome. Based on our recent studies, we propose an inversion of this paradigm. Our results reveal that H3K9 methylation has an active role in catalyzing the cooperative assembly of a heterochromatin regulatory complex. Our studies underscore how the assembly of epigenetic complexes in an H3K9 methylation dependent manner restricts protein-protein interactions to specific chromatin contexts. In this proposal, we will use in vitro biochemistry to reconstitute heterochromatin regulatory complexes, single molecule imaging to define the order and timing of assembly of these complexes in vitro and within a native chromatin context and a high-throughput microfluidic platform to define how epigenetic memory is propagated within individual lineages. The real-time visualization of heterochromatin assembly at high spatial and temporal resolution will illuminate how transient molecular interactions can synergize to establish stable and heritable patterns of gene expression.