Mechanism and Function of Chromatin Positional Dynamics in Interphase

间期染色质位置动力学的机制和功能

基本信息

批准号：
8915221
负责人：
Alexandra Zidovska
金额：
$ 24.9万
依托单位：
NEW YORK UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8915221
关键词：
ATP phosphohydrolase Acute leukemia Address Aftercare Antineoplastic Agents Award BRD2 gene Behavior Binding Biochemical Biochemistry Biological Biological Process Biology Bromodomain Cancer cell line Carcinoma Cell Cycle Regulation Cell Line Cell Nucleus Cell physiology Cells Cellular biology Centromere Chromatin Chromosome Territory Chromosomes Complement Complex Cultured Cells Cytoskeleton DNA DNA Packaging DNA Polymerase II DNA Repair DNA Topoisomerases Data Diagnostic Doctor of Philosophy Embryo Evolution Family Fellowship Foundations Future Gene Expression Genes Genetic Transcription Goals Hela Cells Histone H2B Histones Image In Situ Interphase Knowledge Label Lamins Laws Learning Life Malignant Neoplasms Mammalian Cell Maps Measurement Measures Mentors Methodology Methods Microscopy Modeling Molecular Molecular Biology Motor Multiple Myeloma Nature Nuclear Nuclear Fusion Nuclear Proteins Paper Pattern Phase Physics Poison Polymers Positioning Attribute Postdoctoral Fellow Property Protein Binding Proteins Proteomics Publications RNA Interference Research Resolution Role Site Spectrum Analysis System Testing Time Training Velocimetries Work abstracting analytical tool cancer cell cell motility cell type chromatin remodeling condensed matter physics fly in vivo inhibitor/antagonist knock-down particle particle spectroscopy physical model physical science post-doctoral training screening segregation single molecule skills small molecule telomere tool

项目摘要

Project Summary/Abstract The goal of the proposed research is (1) to measure spatially and temporally resolved chromatin dynamics in mammalian cells in interphase in vivo and (2) determine the origin and function of these dynamical behaviors by combining quantitative approaches derived from physical sciences with tools from molecular biology and biochemistry. The dynamic behavior of chromatin (DNA and associated proteins) has traditionally been studied by live cell microscopy of nuclear proteins, single genes, subchromosomal foci and chromosomal territories [Marshall et al. 1997; Belmont et al. 1998; Levi et al. 2005; Kumaran et al. 2008; Stixova et al. 2011]. Such studies are highly informative, but in practice one can only investigate a few sites simultaneously. Approaches that provide a picture of an overall chromatin dynamics have been slow to develop [Abney et al. 1997]. In the proposed research we will use our newly developed method of velocity correlation spectroscopy (VCS) [Zidovska et al. 2012a], combined with established methods like particle image velocimetry (PIV) and spatio-temporal image correlation spectroscopy (STICS), to measure high-resolution chromatin velocity maps and spatio-temporal evolution of chromatin dynamics over an entire nucleus for the first time (aim 1). In previous work using VCS we found local coherence in spatio-temporal chromatin dynamics, showing that chromatin dynamics is correlated over micron and second scale [Zidovska et al. 2012b]. Our preliminary studies also showed that chromatin dynamics uses ATP, but is independent of the cytoskeleton, hinting at nuclear ATPases being responsible for the observed dynamics. We will probe the origin of the observed chromatin dynamics by specifically inhibiting major nuclear ATPases (polymerase II, DNA polymerase and topoisomerase) using molecular poisons or RNA interference and analyzing the spatio-temporal changes in the pattern of measured chromatin dynamics (aim 2). An exciting preliminary finding showed that chromatin dynamics are largely blocked by a recently-described small molecule JQ1, which binds specifically to four BET family bromodomain proteins, antagonizing their binding to histones [Filippakopoulos et al. 2010]. By investigating how JQ1 blocks chromatin motility we will gain new information on the role of BET proteins in chromatin dynamics. Since JQ1 has demonstrated efficacy in translational models of poorly differentiated carcinoma, multiple myeloma and acute leukemia [Delmore et al. 2011; Zuber et al. 2011], this work may also help elucidate the cellular effects of a promising anti-cancer agent. Finally, we extend this approach to a small panel of cancer and non-cancer cell lines (aim 3) to ask whether chromatin dynamics are perturbed in cancer. If so, VCS analysis might prove useful as a diagnostic tool. Our discoveries in this project will provide a framework for a mechanistic picture of the origins and functional importance of chromatin dynamics in mammalian cells.

项目摘要/摘要拟议的研究的目的是（1）在空间和时间分辨的染色质动力学中测量体内相间的哺乳动物细胞，（2）确定这些动力学行为的起源和功能通过将源自物理科学衍生的定量方法与分子生物学的工具相结合，生物化学。传统上已经研究了染色质（DNA和相关蛋白）的动态行为核蛋白，单基因，亚染色体灶和染色体区域的活细胞显微镜检查 [Marshall等。 1997; Belmont等。 1998; Levi等。 2005; Kumaran等。 2008; Stixova等。 2011]。这样的研究非常有用，但实际上，只能同时研究一些站点。方法提供总体染色质动力学的图片的发展缓慢[Abney等。 1997]。在拟议的研究中，我们将使用我们新开发的速度相关光谱方法（VC）[Zidovska等。 [2012a]，结合了诸如粒子图像速度法（PIV）和时空图像相关光谱（Stics），以测量高分辨率染色质速度图和染色质动力学在整个核上首次在整个核上的时空演化（AIM 1）。在以前使用VC的工作我们在时空染色质动力学中发现了局部连贯性，表明这一点表明染色质动力学在微米和第二刻度上相关[Zidovska等。 2012b]。我们的初步研究还表明，染色质动力学使用ATP，但与细胞骨架无关，暗示核ATPases负责观察到的动力学。我们将探测观察到的起源染色质动力学通过特异性抑制主要的核ATPases（聚合酶II，DNA聚合酶和拓扑异构酶）使用分子毒物或RNA干扰并分析时空变化测得的染色质动力学模式（AIM 2）。令人兴奋的初步发现表明染色质动力学在很大程度上被最近描述的小分子JQ1阻止，该分子与四个下注专门结合家族溴生型蛋白，拮抗其与组蛋白的结合[Filippakopoulos等。 2010]。经过研究JQ1如何阻止染色质运动，我们将获得有关BET蛋白在染色质动力学。由于JQ1在分化差的翻译模型中表现出功效癌，多发性骨髓瘤和急性白血病[Delmore等。 2011; Zuber等。 2011年]，这项工作也可能有助于阐明有前途的抗癌剂的细胞作用。最后，我们将这种方法扩展到一小部分癌症和非癌细胞系（目标3）以询问染色质动力学是否在癌症中干扰。如果是这样，VCS分析可能被证明可作为诊断工具。我们在这个项目中的发现将为起源的机理图片提供一个框架染色质动力学在哺乳动物细胞中的功能重要性。