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Mapping the 3D Genome Landscape

项目来源

美国卫生和人类服务部基金(HHS)

项目主持人

BLONDEL, OLIVIER

项目受资助机构

UNIVERSITY OF SOUTHERN CALIFORNIA

项目编号

5U54DK107981-05

立项年度

2019

立项时间

未公开

项目级别

国家级

研究期限

未知 / 未知

受资助金额

716482.00美元

学科

未公开

学科代码

未公开

基金类别

RESEARCH CENTERS

关键词

未公开

参与者

ALBER, FRANK ; CHEN, LIN

参与机构

NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES

项目标书摘要：? DESCRIPTION: The 3D structural organization of the genome plays a key role in nuclear functions such as gene expression and DNA replication. One of the next grand challenges of biology is to determine the detailed 3D genome architecture and elucidate its functional implications. In this proposal, we aim to develop a suite of novel technologies for comprehensive structural and dynamic analyses of genomes, in two aspects: (1) the spatial interactions and higher-order organization of genomic DNA and (2) the functional organization of protein complexes driving the 3D folding of genomes. The new computational and experimental methods proposed herein integrate multiple experimental inputs and generate physical higher-order models of the 3D nuclear genome organization. Analysis of these models will yield new insights into the principles and structure/functions relationships of the genome's 3D organization in space and time. We have the following aims: (1) Develop technologies for mapping the relative spatial positions of genomic DNA in the nucleus: our focus will be on the three major technical barriers faced by all current mapping technologies, namely inefficient and potentially biased data acquisition, lack of temporal resolution, and missing higher-order contact information. (2) Develop technologies for deciphering the Protein Code of 3D genome organizations: we will employ a proven pipeline for the isolation of native protein complexes, but extensively optimized for the purpose of reading out the chromatin interactome surrounding each particular chromatin interacting region. (3) Develop technologies for modeling and analysis of 3D genome structures: we will develop an integrated platform for population-based modeling of 3D genome structures, and develop a series of computational tools to perform structure-function mapping on the 3D genomes. (4) Develop sold validation techniques for guiding the above technology innovations: we will develop a set of simple-to-implement and easy-to-interpret techniques to validate our novel technologies, and this techniques can be generally adopted by the community for similar validation purposes.

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  • 1.Quantitative Methods to Investigate the4D Dynamics of Heterochromatic Repair Sites in Drosophila Cells

    • 关键词：
    • DOUBLE-STRAND BREAKS; DNA-DAMAGE RESPONSE; HOMOLOGY-DIRECTED REPAIR; CHROMATIN MOBILITY; NUCLEAR PERIPHERY; RECOMBINATIONAL REPAIR; HISTONE H2AX; LIVING CELLS; YEAST; CHECKPOINT
    • Caridi, Christopher P.;Delabaere, Laetitia;Tjong, Harianto;Hopp, Hannah;Das, Devika;Alber, Frank;Chiolo, Irene
    • 《MECHANISMS OF DNA RECOMBINATION AND GENOME REARRANGEMENTS: INTERSECTION BETWEEN HOMOLOGOUS RECOMBINATION, DNA REPLICATION AND DNA REPAIR》
    • 2018年
    • 会议

    Heterochromatin is mostly composed of long stretches of repeated DNA sequences prone to ectopic recombination during double-strand break (DSB) repair. In Drosophila, "safe" homologous recombination (HR) repair of heterochromatic DSBs relies on a striking relocalization of repair sites to the nuclear periphery. Central to understanding heterochromatin repair is the ability to investigate the 4D dynamics (movement in space and time) of repair sites. A specific challenge of these studies is preventing phototoxicity and photobleaching effects while imaging the sample over long periods of time, and with sufficient time points and Z-stacks to track repair foci over time. Here we describe an optimized approach for high-resolution live imaging of heterochromatic DSBs in Drosophila cells, with a specific emphasis on the fluorescent markers and imaging setup used to capture the motion of repair foci over long-time periods. We detail approaches that minimize photobleaching and phototoxicity with a DeltaVision widefield deconvolution microscope, and image processing techniques for signal recovery post-imaging using SoftWorX and Imaris software. We present a method to derive mean square displacement curves revealing some of the biophysical properties of the motion. Finally, we describe a method in R to identify tracts of directed motions (DMs) in mixed trajectories. These approaches enable a deeper understanding of the mechanisms of heterochromatin dynamics and genome stability in the three-dimensional context of the nucleus and have broad applicability in the field of nuclear dynamics.

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