Capturing the dynamic epigenome using single molecule and single cell approaches

项目摘要 在早期发育和多细胞过程中保留预先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（H3 K9 me）是 与转录沉默的特化染色质结构域的形成相关， 异染色质异染色质的建立对于正常的着丝粒和端粒功能是必不可少的， 转座子和重复DNA元件的沉默以及基因的谱系特异性模式的保持 表情H3 K9甲基化的丧失与基因组不稳定性和非整倍性相关， 被广泛认为是与癌症相关的最常见的异常。异染色质建立 是一个动态的过程，涉及组蛋白修饰剂和它们之间的微弱和短暂的相互作用。 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 这些状态在几代人的时间尺度上仍然是可遗传的。这些与 表观基因组暴露出重大的概念和方法上的差距，本提案将主要解决这些差距： 1)如何动态表观遗传复合体组成的组蛋白修饰读者，作家和橡皮擦 在体外和活细胞中组装？2)组蛋白修饰剂如何穿越复杂的染色质景观， 定位他们的行动地点3)表观遗传记忆是如何在随后的遗传过程中储存和传递的？ 世代？目前认为组蛋白修饰作为惰性支架， 促进组蛋白修饰剂定位于基因组中的不同位点。根据我们最近的研究，我们 提出了一个颠覆这种模式的方案。我们的研究结果表明，H3 K9甲基化在细胞凋亡中起着积极的作用。 催化异染色质调节复合物的协同组装。我们的研究强调了 表观遗传复合物以H3 K9甲基化依赖方式的组装限制了蛋白质-蛋白质 与特定染色质环境的相互作用。在这个建议中，我们将使用体外生物化学来重建 异染色质调节复合物，单分子成像，以确定组装的顺序和时间， 这些复合物在体外和天然染色质环境中以及高通量微流体平台中， 定义了表观遗传记忆是如何在个体谱系中传播的。的实时可视化 高空间和时间分辨率异染色质组装将阐明瞬时分子 相互作用可以协同作用以建立稳定的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.