Mechanism and Function of Chromatin Positional Dynamics in Interphase

描述（由申请人提供）：拟议研究的目标是（1）测量哺乳动物细胞在体内间期的空间和时间分辨的染色质动力学，以及（2）通过将来自物理科学的定量方法与来自分子生物学和生物化学的工具相结合，确定这些动力学行为的起源和功能。 染色质（DNA和相关蛋白）的动态行为传统上通过核蛋白、单基因、亚染色体病灶和染色体区域的活细胞显微镜进行研究[马歇尔等人，1997;贝尔蒙特等人，1998; Levi等人，2005; Kumaran等人，2008; Stixova等人，2011]。这些研究提供了大量信息，但实际上只能同时调查几个地点。提供整体染色质动态图像的方法发展缓慢[Abney et al. 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法（Velocity）[Zidovska et al. 2012 a]，结合粒子图像测速法（PIV）和时空图像相关光谱法（STICS）等已建立的方法，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变（目的1）。在先前使用ESTO的工作中，我们发现时空染色质动力学中的局部相干性，表明染色质动力学在微米和秒级上相关[Zidovska et al. 2012 b]。我们的初步研究还表明，染色质动力学使用ATP，但不依赖于细胞骨架，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的核ATP酶（聚合酶II，DNA聚合酶和拓扑异构酶），并分析测量的染色质动力学模式的时空变化来探测观察到的染色质动力学的起源（目的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JQ 1阻断，该小分子JQ 1特异性结合四种BET家族溴结构域蛋白，拮抗其与组蛋白的结合[Filippakopoulos et al. 2010]。通过研究JQ 1如何阻断染色质运动，我们将获得关于BET蛋白在染色质动力学中的作用的新信息。由于JQ 1已在低分化癌、多发性骨髓瘤和急性白血病的转化模型中证明了疗效[Delmore et al. 2011; Zuber et al. 2011]，因此这项工作也可能有助于阐明一种有前景的抗癌药物的细胞效应。 最后，我们将这种方法扩展到一小组癌症和非癌症细胞系（目的3），以询问癌症中染色质动力学是否受到干扰。如果是这样的话，可重复性分析可能被证明是一种有用的诊断工具。我们在这个项目中的发现将提供一个框架，为哺乳动物细胞中染色质动力学的起源和功能重要性的机制图片。

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.