Tracking how molecular machines propagate epigenetic information in time and space

摘要 异染色质是一种基因抑制性的细胞核超微结构，是基因组正常形成所必需的 分为活跃区和非活跃区，以保持结构完整性并驱动和维持发展命运。 虽然异染色质组装是由DNA序列局部成核的，但大部分的图案化过程都是由DNA序列局部成核的。 需要它沿着染色质模板扩散。异染色质的一种主要形式参与这种模式 通过组蛋白3（H3）的赖氨酸9（K9）处的甲基化（me）发出信号。主要问题仍未得到解答 关于异染色质扩散，这限制了我们有效操纵这一过程的能力， 再生医学或合成生物学：1。它背后的生化机制是什么？2.怎么能 异染色质分布在化学、结构和稳定性截然不同的位点上？和3.怎么样 在发育过程中调整反应以扩大或收缩以稳定细胞命运开关？在过去的四年里， 我的实验室设计了解决这些问题的策略。我们已经开发了单细胞传感器， 异染色质扩散使我们能够实时记录反应的内在行为 （Al-Sady等人，2016; Greenstein等人，2018），常染色质特征如何塑造扩散反应（Greenstein et al 2019），并定义了在不同染色质环境中实现这一过程的基因（Greenstein & Ng et 等人，2020）。此外，我们还开发了单分子系统来研究个体的组蛋白甲基化， 染色质链和大量的生物化学方法来探测H3 K9 me“书写机器”的功能。超过 在接下来的五年里，我们将部署这些实验系统，以充分阐明异染色质扩散 过程从三个角度：1.作者机器：我们将使用单分子和生化测序 揭示酶复合物“书写”的机制和分子轨迹的方法 H3 K9沿着染色质模板。2.底物：异染色质扩散发生在 不同的染色质景观，不能适合“一刀切”的模型。我们将使用单细胞异染色质 在分裂酵母中散布传感器，以检查不同活性状态或相同位点的染色质位点如何 不同的历史影响反应。此外，我们将定义基因电路， 在不同的染色质环境中传播。3.发展观：我们专注于发展 关键的H3 K9甲基化酶G9 a/GLP，并将区分不同的假设如何发展触发， G9 a/GLP依赖性异染色质扩张和收缩在哺乳动物干细胞中实现。 此外，由于G9 a/GLP引起的H3 K9甲基化与沉默之间的关系是难以捉摸的，我们将定义 H3 K9甲基化后基因抑制必须发生的步骤。这套项目将 异染色质扩散的内在生化特征，以稳定状态和发育 这种独特的超微结构的动态基因组分配功能，是基因组和细胞命运稳定性的基础。

ABSTRACT Heterochromatin, a gene-repressive nuclear ultrastructure, is required for the normal patterning of the genome into active and inactive regions, to preserve structural integrity and drive and maintain developmental fates. While heterochromatin assembly is locally nucleated by DNA-sequences, the majority of the patterning process requires it to spread along the chromatin template. A major form of heterochromatin involved in this patterning is signaled by methylation (me) at Lysine 9 (K9) of histone 3 (H3). Major questions have remained unanswered about heterochromatin spreading, which has limited our ability to effectively manipulate this process for regenerative medicine or synthetic biology: 1. What are the biochemical mechanisms underlying it? 2. How can heterochromatin spread over loci of vastly different chemical, structural and stability regimes? And 3. How is the reaction tuned to expand or contract during development to stabilize cell fate switches? Over the last four years, my laboratory has devised strategies to tackle these questions. We have developed single-cell sensors of heterochromatin spreading that have enabled us to document the intrinsic behavior of the reaction in real-time (Al-Sady et al, 2016; Greenstein et al, 2018), how euchromatic features sculpt the spreading reaction (Greenstein et al 2019) and defined genes that enable this process in different chromatin environments (Greenstein & Ng et al, 2020). Additionally, we have developed single molecule systems to study histone methylation on individual chromatin strands and bulk biochemical methods to probe the function of the H3K9me “writer machines”. Over the next five years, we will deploy these experimental systems to fully illuminate the heterochromatin spreading process from three angles: 1. The writer machine: We will use single molecule and biochemical sequencing approaches to unravel the mechanisms and molecular trajectories by which the enzyme complexes “write” H3K9me along the chromatin template. 2. The substrate: Heterochromatin spreading occurs over radically different chromatin landscapes and cannot fit a “one-size-fits-all” model. We will use single-cell heterochromatin spreading sensors in fission yeast to examine how chromatin loci of different activity states or the same locus with different histories impact the reaction. Further, we will define the genetic circuitry that enables and tunes spreading in different chromatin environments. 3. The view form development: We focus on the developmentally crucial H3K9 methylase G9a/GLP and will distinguish different hypotheses on how developmentally triggered, G9a/GLP-dependent heterochromatin expansions and contractions are implemented in mammalian stem cells. Further, since the relationship between H3K9 methylation by G9a/GLP and silencing is elusive, we will define the steps that must occur for gene repression after H3K9 methylation. Together, this suite of projects connects the intrinsic biochemical features of heterochromatin spreading, to the steady-state and developmentally dynamic genome partitioning function of this unique ultrastructure, which underlies genome and cell fate stability.