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：
8425595
负责人：
Alexandra Zidovska
金额：
$ 9万
依托单位：
HARVARD MEDICAL SCHOOL
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 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8425595
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 analytical tool cancer cell cell motility cell type chromatin remodeling fly in vivo inhibitor/antagonist knock-down particle physical model physical science post-doctoral training public health relevance screening segregation single molecule skills small molecule telomere tool

项目摘要

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：拟议研究的目标是（1）测量体内哺乳动物细胞间期的空间和时间分辨染色质动态，以及（2）通过结合衍生的定量方法确定这些动态行为的起源和功能来自物理科学和分子生物学和生物化学的工具。传统上，染色质（DNA 和相关蛋白）的动态行为是通过核蛋白、单基因、染色体亚灶和染色体区域的活细胞显微镜来研究的[Marshall 等，2017]。 1997；贝尔蒙特等人。 1998；利维等人。 2005年；库马兰等人。 2008年；斯蒂克索娃等人。 2011]。此类研究信息丰富，但实际上只能同时调查几个地点。提供整体染色质动态图景的方法发展缓慢 [Abney 等人。 1997]。在拟议的研究中，我们将使用我们新开发的速度相关谱（VCS）方法[Zidovska 等人。 2012a]，结合粒子图像测速（PIV）和时空图像相关光谱（STICS）等现有方法，首次测量整个细胞核的高分辨率染色质速度图和染色质动力学的时空演化（目标1）。在之前使用 VCS 的工作中，我们发现了时空染色质动力学的局部一致性，表明染色质动力学在微米和秒尺度上是相关的 [Zidovska 等人，2017]。 2012b]。我们的初步研究还表明，染色质动力学使用 ATP，但独立于细胞骨架，暗示核 ATP 酶负责观察到的动力学。我们将通过使用分子毒物或 RNA 干扰特异性抑制主要核 ATP 酶（聚合酶 II、DNA 聚合酶和拓扑异构酶）并分析所测量的染色质动力学模式的时空变化，来探究所观察到的染色质动力学的起源（目标 2）。一项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 阻断，该小分子与四种 BET 家族溴结构域蛋白特异性结合，拮抗它们与组蛋白的结合 [Filippakopoulos 等人，2017]。 2010]。通过研究 JQ1 如何阻断染色质运动，我们将获得有关 BET 蛋白在染色质动力学中的作用的新信息。由于 JQ1 已在低分化癌、多发性骨髓瘤和急性白血病的转化模型中表现出功效[Delmore 等人，2017]。 2011；祖伯等人。 2011]，这项工作也可能有助于阐明一种有前途的抗癌药物的细胞效应。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），以探究癌症中染色质动态是否受到干扰。如果是这样，VCS 分析可能会被证明作为一种有用的诊断工具。我们在这个项目中的发现将为哺乳动物细胞染色质动力学的起源和功能重要性的机制图提供一个框架。