Tracking how molecular machines propagate epigenetic information in time and space

摘要 异染色质是一种抑制基因的核超微结构，是基因组正常构型所必需的。 进入活跃和非活跃区域，以保持结构完整性，推动和维持发展命运。 虽然异染色质组装是由DNA序列局部成核的，但大多数图案化过程 需要它沿着染色质模板扩散。异染色质的一种主要形式，与这种图案有关 组蛋白3(H3)的赖氨酸9(K9)处的甲基化(Me)是信号。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.如何 异染色质分布在化学、结构和稳定机制截然不同的基因座上？3.你的生活怎么样？ 在发育过程中，反应调节为扩张或收缩，以稳定细胞命运开关？在过去的四年里， 我的实验室已经制定了解决这些问题的策略。我们已经开发出了单细胞传感器 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)，常色特征如何塑造传播反应(Greenstein 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 Al，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。 染色质链和整体生化方法，以探索H3K9me“写入机”的功能。完毕 在接下来的五年里，我们将部署这些实验系统来充分阐明异染色质的扩散 过程从三个角度进行：1.编写机：我们将使用单分子和生化测序 解开酶复合体“写”的机制和分子轨迹的方法 H3K9me沿着染色质模板。2.底物：异染色质从根本上扩散 不同的染色质景观，不能适合“一刀切”的模式。我们将使用单细胞异染色质 在裂解酵母中传播传感器以检测不同活性状态或相同基因座的染色质 不同的历史影响了反应。此外，我们将定义使能和调谐的遗传电路 在不同的染色质环境中传播。3.观点形式发展：我们关注发展中的 关键的H3K9甲基酶G9a/GLP，并将区分关于发育如何触发的不同假说， 依赖于G9a/GLP的异染色质扩张和收缩是在哺乳动物干细胞中实现的。 此外，由于G9a/GLP的H3K9甲基化和沉默之间的关系难以捉摸，我们将定义 H3K9甲基化后基因抑制必须发生的步骤。这套项目结合在一起，将 异染色质向稳定期和发育期扩散的内在生化特征 这种独特的超微结构具有动态的基因组分割功能，这是基因组和细胞命运稳定的基础。

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.