cis-Acting Elements Regulating Developmental Control of Replication Timing

 描述(申请人提供)：DNA复制是基因组完整性的核心，与大规模3D染色体组织和细胞谱系规范密切相关，但缺乏探索因果关系的工具限制了在理解其规则方面的进展。特别是，迫切需要确定调节复制的顺式元件 计时(RT)作为解决与染色体结构和基因调控的因果联系的第一步。我们的长期目标是了解RT与染色体结构、表观遗传状态和疾病的关系。我们的直接目标是识别调节RT中发育程序性变化的元件，并检查它们在基因组组织和转录中的作用。我们的中心假设是离散的功能元素决定了 RT中独立于转录的发育程序性变化。这一建议的基本原理是，识别调节RT的顺式元件对于确定将RT与更高阶染色体折叠和基因表达联系起来的因果路径至关重要。初步数据确定了设计基因组删除、倒置和异位插入的可行性，并确定了在小鼠胚胎干细胞(MESC)分化过程中进行RT调节所必需和/或足够的DNA片段。我们还提供了转录对于RT改变既不必要也不充分的证据。AIM1将使用CRIPR/Cas9介导的染色体工程来产生相邻区域内和相邻区域之间的进一步缺失和反转，并评估它们对RT、TAD结构和亚核间隔的影响。AIM2将克隆的基因组和/或合成修饰的DNA序列从发育调节的复制结构域引入到构成复制的结构域中，以描绘足以将发育RT控制转移到异位位置的序列，并确定3D染色体结构的哪些方面与RT调节共转移。AIM3将使用这些相同的工具来评估控制转录的顺式元件可以在多大程度上调节RT开关，反之亦然。这一贡献将是重大的，因为识别必要和足够的RT调控DNA序列以前是不可能的，这是从分子上理解RT发育控制及其与染色体结构和最终人类疾病的联系的必要的第一步。建议的工作在结合新的简便的染色体工程方法和特征良好的定向mESC分化系统方面具有创新性，以揭示在RT中引发发育程序性变化的途径。这一认识将对我们理解RT及其与3D染色体组织的关系产生重大影响，它将指导未来探索RT异常在人类疾病中的意义的研究，并将有助于染色体“域工程”的创新，这将影响染色体生物学的许多方面。

 DESCRIPTION (provided by applicant): DNA replication is central to genome integrity and intimately tied to large-scale 3D chromosome organization and cell lineage specification, but a lack of tools with which to probe causality have limited progress in under- standing its regulation. In particular, there is a critical need to identify cis-elements regulating replication timing (RT) as a first step toward addressing causal linkages to chromosome architecture and gene regulation. Our long-term goal is to understand the relationship of RT to chromosome architecture, epigenetic states and disease. Our immediate goal is to identify elements regulating developmentally programmed changes in RT and examine their role in genome organization and transcription. Our central hypothesis is that discrete functional elements dictate developmentally programmed changes in RT independently from transcription. The rationale for this proposal is that identifying cis-elements regulating RT is essential to identify causal pathways linking RT to higher order chromosome folding and gene expression. Preliminary data establish feasibility to engineer genome deletions, inversions and ectopic insertions and identify DNA segments that are necessary and/or sufficient for RT regulation during murine embryonic stem cell (mESC) differentiation. We also provide evidence that transcription is neither necessary nor sufficient for RT changes. Aim1 will use CRIPR/Cas9-mediated chromosome engineering to generate further deletions and inversions within and between adjacent domains and evaluate their consequences to RT, TAD structure and sub-nuclear compartment. Aim2 will introduce cloned genomic and/or synthetically modified DNA sequences from a developmentally regulated replication domain into a constitutively replicated domain to delineate sequences sufficient to transfer developmental RT control to the ectopic site and to determine what aspects of 3D chromosome structure co-transfer with RT regulation. Aim3 will use these same tools to evaluate the extent to which cis-elements controlling transcription can regulate RT switches and vice versa. This contribution will be significant because identifying necessary and sufficient RT regulatory DNA sequences has not previously been possible and it is the essential first step toward a molecular understanding of RT developmental control and its links to chromosome architecture and, ultimately, human disease. The proposed work is innovative in combining novel facile chromosome engineering methods with well-characterized directed mESC differentiation systems to uncover pathways eliciting developmentally programmed changes in RT. This knowledge will have major impact on our understanding of RT and its relationship to 3D chromosome organization, it will guide future studies probing the significance of RT aberrations in human disease, and it will contribute innovations in chromosome "domain engineering" that will impact many facets of chromosome biology.