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阶段的不同时间组装。此外，许多研究已经将复制时间的变化与不同细胞系和癌症中基因表达的变化联系起来，但没有一项研究能够解决伴随这些变化的中间状态。机械论研究将需要一个系统，在这个系统中，这些变化能够以足够的同步性和同质性被引发，从而允许进行生化和分子分析。我们在这份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蛋白的动态变化，并与小鼠ES细胞向神经前体细胞分化过程中关键细胞命运的变化相一致。早期到晚期的复制变化与多能性丧失和ES特异性基因的不可逆转下调相一致，而晚期到早期的变化与神经谱系的承诺和神经特异性基因的上调相一致。由于复制时间是在大染色体结构域的水平上调节的，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加强了大染色体结构域中染色质结构变化的遗传性，而这些变化反过来又调节了干细胞承诺期间基因的反应。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，以确定生物学上有意义的关系。我们证明，缺乏G9a组蛋白甲基转移酶的ES细胞在S期较早地复制了神经诱导基因的子集，暗示组蛋白甲基化和复制之间存在联系。其中，Pleiotroin(PTN)基因位于500kb的染色质结构域中，在诱导转录的同一细胞周期中，该结构域作为一个单位从晚期复制到早期复制。有趣的是，在明确的外胚层阶段，在复制开关之前，一波非编码转录开始通过染色质区域1-2个细胞周期。我们提出了一个模型，在这个模型中，非编码转录引起组蛋白修饰的变化，这些变化累积直到它们触发复制时间的切换，进而将染色质状态传递到整个结构域，使结构域处于响应的染色质状态。目的2阐述转录在重塑全区染色质结构中的作用，而目的3阐述G9a组蛋白甲基转移酶在大染色质结构域水平上调节复制时机和染色质结构的作用。层面相关性：所有细胞都包含相同的遗传信息(DNA)，但它们以定义每种细胞类型的独特方式将其与蛋白质打包成“染色质”。在每个细胞分裂过程中，染色质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片段包装成染色质的序列会发生变化。了解如何操纵这个包装过程可能有助于我们设计不同类型的细胞，这是干细胞治疗的中心目标。

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.