Genetic dissection of cis-acting elements controlling DNA replication timing and genome architecture

项目摘要/摘要 DNA复制对于基因组的结构和功能的完整性以及对基因组的时空控制至关重要 DNA复制(复制定时，RT)与3D基因组结构和组织密切相关， 然而，我们对它是如何监管的知之甚少。实验室最近发现了顺式作用元件，这些元件 对控制小鼠胚胎干细胞(MESCs)的RT、染色体结构和转录至关重要， 称为早期复制控制元件(ERCE)。ERCEs含有许多活性染色质标记，例如 乙酰化组蛋白残基，由多能因子Oct4、Sox2和Nanog(OSN)共同占据。ERCEs 还含有转录起始位点(TSS)，这可能是它们在转录调控中的作用。ERCEs 形成独立于建筑蛋白、CTCF和粘附素的3D相互作用。尚不清楚的是 ERCEs是一个协调调节RT、架构和转录的功能单位，或者如果它们是 由独立地调节不同核功能的离散的、可分离的元素组成。这个 假设ERCEs由多个元件组成，如OSN结合位点和TSS，它们可以是 解偶联，以确定它们在调节RT、体系结构和转录中的单独角色。其基本原理是 RT、基因组结构和转录在疾病状态下通常都会被破坏，因此 了解它们是如何调控的将有助于更好地理解细胞和分子基础 疾病。AIM 1将使用CRISPR对ERCEs进行基因解剖，以确定含有活性的序列 ERCE活性所必需的，如RT和转录。AIM 2将转移ERCE或ERCEs的组件 检测异位位点的充分性以促进早期复制和转录并改变染色质 异位基因组位置的建筑。这一贡献将是重大的，因为它将解决 DNA结构和功能的调节，以及这种调节在疾病中是如何被干扰的。拟议的研究 是创新的，因为ERCEs的发现提供了新的生物学问题和研究方法 染色体结构和功能的调节。

PROJECT SUMMARY/ABSTRACT DNA replication is essential for the structural and functional integrity of genomes, and spatiotemporal control of DNA replication (replication timing, RT) is intimately related to 3D genome architecture and organization, however, we know little about how it is regulated. The lab has recently identified cis-acting elements that are crucial for controlling RT, chromosome architecture, and transcription in mouse embryonic stem cells (mESCs), termed early replication control elements (ERCEs). ERCEs contain many active chromatin marks, such as acetylated histone residues and are co-occupied by pluripotency factors Oct4, Sox2, and Nanog (OSN). ERCEs also contain transcription start sites (TSSs), which may account for their role in regulating transcription. ERCEs form 3D interactions independent of architectural proteins CTCF and cohesin. What is unknown is whether ERCEs are one functional unit that coordinately regulates RT, architecture, and transcription, or if they are composed of discrete, separable elements that independently regulate different nuclear functions. The hypothesis is that ERCEs are composed of multiple elements, such as OSN binding sites and TSSs, that can be uncoupled, to determine their individual roles in regulating RT, architecture, and transcription. The rationale is that RT, genome architecture, and transcription are all commonly disrupted in disease states, and so understanding how they are regulated will lead to a better understanding of the molecular and cellular basis of disease. AIM 1 will use CRISPR to genetically dissect ERCEs in order to identify the sequences harboring activity necessary for ERCE activity, such as RT and transcription. AIM 2 will transfer ERCEs or components of ERCEs to ectopic sites and assay their sufficiency to promote early replication and transcription and to alter chromatin architecture at the ectopic genomic location. This contribution will be significant because it will address the regulation of DNA structure and function and how that regulation is perturbed in disease. The proposed research is innovative because the discovery of ERCEs provide novel biological questions and approaches to investigate the regulation of chromosome structure and function.