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，但没有一个研究能够解决伴随这些变化的中间状态。机械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引起，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，并与小鼠胚胎干细胞向神经前体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es特异性基因的多能性丧失和不可逆的下调相一致，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水平上调控的，我们的研究有可能在基因调控方面打开新的篇章。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区域染色质结构变化的遗传性，从而调节了干细胞承诺过程中基因的反应性。在目标1中，我们将在分化的关键阶段对复制时间、转录和染色质状态进行全基因组分析，以确定生物学上重要的关系。我们证明，缺乏G9a组蛋白甲基转移酶的胚胎干细胞在s期更早地复制一组神经诱导基因，这表明组蛋白甲基化与复制之间存在联系。其中一个基因，多营养蛋白（Ptn）基因位于一个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，在最终的外胚层样阶段，非编码转录波在复制开关之前的1-2个细胞周期开始通过该染色质结构域。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，这些变化积累直到它们触发复制时间的开关，从而将染色质状态传递到整个结构域，使结构域进入响应性染色质状态。Aim 2研究转录在染色质结构域重塑中的作用，Aim 3研究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间和染色质结构的作用。Lay Relevance：所有细胞都含有相同的遗传信息（DNA），但将其与蛋白质一起打包成“染色质”，以特有的方式定义每种细胞类型。在每次细胞分裂过程中，染色质会被分解和重新组装，我们已经发现，当干细胞变成不同的细胞类型时，DNA片段被包装成染色质的序列会发生变化。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.