CAREER: Establishment of Replication Timing and Patterning in Mammalian Cells

智力优势：真核细胞细胞核中发现的染色质（基因组DNA和蛋白质的复合体）的结构控制着包括DNA复制、转录和转录后基因调控在内的细胞过程。但是，如何做到这一点是一个重要的问题，仍有待明确阐明。在哺乳动物细胞中，染色体结构域占据特定的核位置，并在细胞分裂周期的s期的特定时间复制。给定细胞类型内DNA复制的时空模式是可遗传的，并在细胞周期的G1期建立。然而，对控制DNA复制时空模式的事件和分子的理解却很少。起源识别复合体（ORC）在DNA复制起始、染色质组织和细胞周期进程中起着至关重要的作用。人类ORC的最大亚基Orc1与染色质的结合是G1期预rc组装的必要步骤。同样，Orc2-5以细胞周期依赖的方式显示与染色质的动态关联。ORC和控制复制时间和模式的染色质结构之间的相互作用仍有待阐明。本项目的目的是确定ORC在G1期间如何贡献和/或读取表观遗传标记。该项目将解决ORC在G1中建立空间模式和复制时间的作用，并确定染色质结构在调节ORC与染色质结合中的作用。这项研究在分子生物学和生物化学与最先进的活细胞成像相结合方面具有创新性。这个项目非常重要，因为它旨在促进我们对活细胞中染色质组织和复制如何协调的理解。更广泛的影响：这项研究将具有强大而积极的推广潜力，因为它利用了广泛的高分辨率显微镜和延时活细胞成像，这不仅对理解复制的关键生物学问题至关重要，而且还为本科生提供了一个欣赏生物学的平台。这种方法将在几个方面得到应用，包括通过教授本科生，通过为伊利诺伊大学和社区学院的学生提供研究机会，通过指导未来的研究人员，以及通过组织研讨会让学生欣赏基础研究的兴奋。这些活动都将涉及妇女和少数民族学生。该项目整合了生物化学，细胞生物学和分子生物学的应用，以吸引来自不同背景的学生，包括生物学，物理学，化学和生物工程的兴趣。只要投入一点时间，让年轻聪明的学生在生命的早期进行研究将是非常有益的，并向他们灌输科学意识。

Intellectual merit:The structure of chromatin, the complex of genomic DNA and proteins found in the nucleus of eukaryotic cells, governs cellular processes including DNA replication, transcription and post-transcriptional gene regulation. But, how that is accomplished is an important question that remains to be clearly elucidated. In mammalian cells, chromosomal domains occupy a specific nuclear position and replicate at defined times during S-phase of the cell division cycle. The spatiotemporal pattern of DNA replication within a given cell type is heritable and is established during the G1 phase of the cell cycle. There is, however, minimal understanding of the events and molecules that govern the spatiotemporal patterning of DNA replication. The Origin Recognition Complex (ORC) plays critical roles in the initiation of DNA replication, chromatin organization and cell cycle progression. The binding of the largest subunit of human ORC, Orc1 to chromatin is an obligatory step for pre-RC assembly during G1. Similarly, Orc2-5 display dynamic association with chromatin in a cell cycle-dependent manner. The interplay between ORC and chromatin structure that govern replication timing and patterning remain to be elucidated. The objective of the present project is to determine how ORC contributes to and/or reads epigenetic marks during G1. The project will address the role of ORC in establishing spatial patterning and timing of replication in G1 and determine the role of chromatin architecture in modulating ORC binding to chromatin. This research is innovative in its combination of molecular biology and biochemistry with state of the art live cell imaging. This project is highly significant as it aims to advance our understanding of how chromatin organization and replication are coordinated in living cells.Broader impacts:This research will have a strong and active outreach potential, since it utilizes extensive high-resolution microscopy as well as time-lapse live cell imaging that is crucial not only to understand key biological questions on replication, but also to serve as a platform for undergraduate students to appreciate biology. This approach will be applied in several ways including by teaching undergraduate students, by providing research opportunities to University of Illinois and community college students, by mentoring future researchers, and by organizing a symposium for students to appreciate the excitement of basic research. These activities will all involve women and minority students. This project integrates the use of Biochemistry, Cell Biology and Molecular Biology to capture the interest of students from varied backgrounds, including Biology, Physics, Chemistry and Bioengineering. With a small investment of time, exposing young and bright students to research early on in life will be highly beneficial and instill in them a sense of scientific awareness.