Genome Plasticity during ES Cell Differentiation to Neural Lineages

描述（由申请人提供）：我们的长期目标是了解 DNA 复制在细胞表观遗传状态中的作用。染色质在复制叉处组装，不同类型的染色质在 S 期的不同时间组装。此外，许多研究将复制时间的变化与不同细胞谱系和癌症中基因表达的变化联系起来，但没有一个能够解决伴随这些变化的中间状态。机理研究需要一个能够以足够的同步性和同质性引发这些变化的系统，以便进行生化和分子分析。我们在本提案中描述了这样一个系统。我们检测到单个细胞周期内复制时间的动态变化，并且与小鼠 ES 细胞分化为神经前体期间的关键细胞命运变化相一致。早期到晚期的复制变化与多能性的丧失和 ES 特异性基因的不可逆下调一致，而晚期到早期的变化与神经谱系的形成和神经特异性基因的上调一致。由于复制时间是在大染色体域水平上调节的，因此我们的研究有可能开启基因调控的新篇章。我们的工作假设是，分化过程中复制时间的变化增强了大染色体区域染色质结构变化的遗传性，进而调节干细胞定型期间基因的反应性。在目标 1 中，我们将对分化过程中关键阶段的复制时间、转录和染色质状态进行全基因组分析，以确定具有生物学意义的关系。我们证明，缺乏 G9a 组蛋白甲基转移酶的 ES 细胞在 S 期早期复制了神经诱导基因的子集，这表明组蛋白甲基化和复制之间存在联系。其中一个基因，多效蛋白 (Ptn) 基因位于 500 kb 的染色质结构域内，该结构域作为一个单位在诱导转录的同一细胞周期内从晚期复制到早期复制。有趣的是，在复制转换之前，在最终的外胚层样阶段，一波非编码转录开始穿过该染色质结构域 1-2 个细胞周期。我们提出了一个模型，其中非编码转录引起组蛋白修饰的变化，这些变化会累积，直到触发复制时间的转换，进而将染色质状态传递到整个域，使该域进入响应性染色质状态。目标 2 解决转录在重塑全域染色质结构中的作用，而目标 3 解决 G9a 组蛋白甲基转移酶在大染色质域水平上调节复制计时和染色质结构中的作用。相关性：所有细胞都含有相同的遗传信息 (DNA)，但以定义每种细胞类型的特有方式将其与蛋白质一起包装到“染色质”中。染色质在每次细胞分裂过程中都会被分解和重新组装，我们发现，随着干细胞转变为不同的细胞类型，DNA片段包装到染色质中的顺序也会发生变化。了解如何操纵这种包装过程可能有助于我们设计不同的细胞类型，这是干细胞治疗的核心目标。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the role of DNA replication in cellular epigenetic states. Chromatin is assembled at the replication fork and different types of chromatin are assembled at different times during S-phase. Moreover, many studies have correlated changes in replication timing to changes in gene expression in different cell lineages and in cancer but none have been able to address the intermediate states that accompany these changes. Mechanistic studies will require a system in which these changes can be elicited with sufficient synchrony and homogeneity as to permit biochemical and molecular analyses. We describe such a system in this proposal. We detect dynamic changes in replication timin within a single cell cycle and coincident with key cell fate changes during the differentiation of mouse ES cells to neural precursors. Early to late replication changes coincide with loss of pluripotence and irreversible down-regulation of ES-specific genes, while late to early changes coincide with commitment to neural lineages and up-regulation of neural specific genes. Since replication timing is regulated at the level of large chromosomal domains, our studies have the potential to open a novel chapter in gene regulation. Our working hypothesis is that changes in replication timing during differentiation reinforce the heritability of changes in chromatin structure across large chromosome domains that in turn modulate the responsiveness of genes during stem cell commitment. In Aim 1 we will perform genome-wide analyses of replication timing, transcription and chromatin states at key stages during differentiation to identify biologically significant relationships. We demonstrate that ES cells lacking the G9a histone methyltransferase replicate a subset of neural-induced genes earlier during S-phase, suggesting a link between histone methylation and replication. One of these genes, the Pleiotrophin (Ptn) gene resides within a 500 kb chromatin domain that switches as a unit from late to early replicating within the same cell cycle in which transcription is induced. Intriguingly, a wave of non-coding transcription begins throughput this chromatin domain 1-2 cell cycles prior to the replication switch, during a definitive ectoderm-like stage. We propose a model in which non-coding transcription elicits changes in histone modifications that accumulate until they trigger a switch in replication timing that in turn transmits the chromatin state to the entire domain, committing the domain to a responsive chromatin state. Aim 2 addresses the role of transcription in remodeling domain-wide chromatin structure while Aim 3 addresses the role of the G9a histone methyltransferase in regulating replication timing and chromatin structure at the level of large chromatin domains. Lay Relevance: All cells contain the same genetic information (DNA) but package it with proteins into "chromatin" in characteristic ways that define each cell type. Chromatin is dismantled and re-assembled during each cell division, and we have discovered that the sequence in which segments of DNA are packaged into chromatin changes as stem cells turn into different cell types. Understanding how to manipulate this packaging process may help us engineer different cell types, a central goal in stem cell therapy.