cis-Acting Elements Regulating Developmental Control of Replication Timing

调节复制时间发育控制的顺式作用元件

• 批准号: 9296144
• 负责人: David M Gilbert
• 金额: $ 32.6万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9296144
• 关键词: Address; Affect; Architecture; Biology; CRISPR/Cas technology; Cell Differentiation process; Cell Lineage; Cell Nucleus; Cells; Characteristics; Chromatin; Chromosome Structures; Chromosomes; Complex; DNA; DNA Sequence; DNA biosynthesis; Data; Development; Disease; Dissection; Elements; Engineering; Epigenetic Process; Etiology; Future; Gene Expression; Gene Expression Regulation; Genetic; Genetic Transcription; Genome; Genome engineering; Genomics; Goals; Knowledge; Link; Location; Malignant Neoplasms; Mammalian Chromosomes; Mediating; Methods; Mission; Molecular; Molecular Conformation; Mus; Nuclear; Nuclear Lamina; Pathogenesis; Pathway interactions; Process; Public Health; Regulation; Replicon; Role; S Phase; Site; Structure; Structure-Activity Relationship; System; Time; Transplantation; United States National Institutes of Health; Work; cell type; cis acting element; embryonic stem cell; experimental study; genome integrity; human disease; innovation; insight; novel; programs; public health relevance; relating to nervous system; tool

 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.

 描述（由申请人提供）：DNA 复制对于基因组完整性至关重要，并且与大规模 3D 染色体组织和细胞谱系规范密切相关，但缺乏探测因果关系的工具限制了理解其调控的进展。特别是，迫切需要识别调节复制的顺式元件 计时（RT）作为解决染色体结构和基因调控因果关系的第一步。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。我们的近期目标是确定调控 RT 发育程序变化的元件，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 RT 的发育程序变化独立于转录。该提议的基本原理是，识别调节 RT 的顺式元件对于识别将 RT 与更高阶染色体折叠和基因表达联系起来的因果途径至关重要。初步数据证实了基因组删除、倒位和异位插入工程的可行性，并鉴定了在小鼠胚胎干细胞 (mESC) 分化过程中 RT 调节所必需和/或充分的 DNA 片段。我们还提供证据表明转录对于 RT 变化既不是必要的也不是充分的。 Aim1 将使用 CRIPR/Cas9 介导的染色体工程在相邻结构域内和相邻结构域之间产生进一步的缺失和倒位，并评估其对 RT、TAD 结构和亚核区室的影响。 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组成型复制结构域，以描绘足以将发育 RT 控制转移到异位位点的序列，并确定 3D 染色体结构与 RT 调节共转移的哪些方面。 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调节 RT 开关的程度，反之亦然。这一贡献将具有重要意义，因为以前不可能确定必要且足够的 RT 调控 DNA 序列，这是从分子角度理解 RT 发育控制及其与染色体结构以及最终与人类疾病的联系的重要的第一步。所提出的工作具有创新性，将新颖的简便染色体工程方法与特征明确的定向 mESC 分化系统相结合，以揭示引起 RT 发育程序变化的途径。这些知识将对我们对 RT 及其与 3D 染色体组织的关系的理解产生重大影响，它将指导未来探索 RT 畸变在人类疾病中的重要性的研究，并将贡献染色体“域工程”的创新，从而影响染色体生物学的许多方面。