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，并且不同类型的染色质在S期期间的不同时间组装。此外，许多研究已经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相关联，但没有一项研究能够解决伴随这些变化的中间状态。机制研究将需要一个系统，其中这些变化可以引起足够的同步性和同质性，以允许生物化学和分子分析。我们在本提案中描述了这样一个系统。我们检测到在一个细胞周期内复制timin的动态变化，并与小鼠ES细胞分化为神经前体细胞过程中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相一致，而晚期到早期的变化与神经谱系的定型和神经特异性基因的上调相一致。由于复制时间是在大的染色体结构域的水平上进行调节的，我们的研究有可能在基因调控方面开辟新的篇章。我们的工作假设是，在分化过程中的复制时间的变化，加强了遗传性的染色质结构的变化，在整个大的染色体结构域，反过来又调节基因的反应，在干细胞的承诺。在目标1中，我们将在分化过程中的关键阶段进行全基因组的复制时间，转录和染色质状态分析，以确定生物学意义的关系。我们证明了缺乏G9 a组蛋白甲基转移酶的ES细胞在S期早期复制神经诱导基因的一个子集，这表明组蛋白甲基化和复制之间存在联系。这些基因之一，多效营养蛋白（Ptn）基因位于500 kb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切换到早期复制。有趣的是，在定形外胚层样阶段，在复制转换之前的1-2个细胞周期，非编码转录波开始通过该染色质结构域。我们提出了一个模型，在该模型中，非编码转录eldance组蛋白修饰的变化，积累，直到它们触发复制定时的开关，反过来将染色质状态传输到整个域，提交域的响应染色质状态。目的2解决了转录在重塑全域染色质结构中的作用，而目的3解决了G9 a组蛋白甲基转移酶在调节大染色质结构域水平上的复制时机和染色质结构中的作用。外行相关性：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征方式将其与蛋白质包装成“染色质”。在每次细胞分裂过程中，染色质被拆除并重新组装，我们已经发现，DNA片段包装成染色质的顺序随着干细胞变成不同的细胞类型而发生变化。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.