Visualizing the dynamics of chromatin and chromatin remodeling proteins

描述（由申请人提供）：基因组的复制、转录、修复和维持是维持细胞活力的关键过程。在真核生物中，这些功能在组织成高度浓缩的染色质的DNA上进行。在其他必需因子进入DNA之前，必须使用专门的蛋白质来修饰染色质的结构。随着越来越多的疾病相关基因的鉴定，染色质在人类疾病中的重要性已经变得非常清楚，并且几种疾病已经与染色质重塑缺陷有关。例如，与染色质重塑蛋白的Snf 2家族相关的突变可引起使人衰弱的遗传性疾病，并且与癌症相关。了解参与这些过程的蛋白质的基本特性对于揭示产生疾病表型的潜在缺陷至关重要。然而，染色质重塑的机制细节仍然难以捉摸。这是因为大多数用于研究重塑的方法只能探测核小体的初始和最终状态，并且通常不能揭示反应的动态方面。我们的假设是Snf 2蛋白通过与DNA易位偶联的机制使核小体和其他核蛋白复合物不稳定，从而允许其他细胞组分不受阻碍地进入DNA。为了验证这一假设，我们将使用单分子光学显微镜测定直接可视化Snf 2重塑蛋白Rdh 54和RSC，因为它们与裸DNA和染色质底物相互作用。这些实验将依赖于格林实验室开发的一项新技术，该技术使我们能够直接可视化数百个真实的蛋白质-DNA相互作用。这种方法的好处是，我们可以探测染色质重塑的动力学，使我们能够从数百个单独的生物分子中快速收集统计相关信息，并且我们可以以前所未有的细节水平研究这些反应。公共卫生相关性：染色质重塑酶的缺陷可导致极其严重的人类疾病，这与它们作为基因组结构的全局调节剂的作用一致。作为开发可用于有效预防或治愈这些衰弱性疾病的靶向疗法的第一步，了解染色质重塑蛋白本身的基本生化特性至关重要。为了帮助扩展我们对染色质重塑的理解，我们开发了基于荧光的方法来直接观察蛋白质和单个DNA分子之间的相互作用，我们的重点是了解与人类生物学和疾病相关的生化反应。

DESCRIPTION (provided by applicant): Replication, transcription, repair and maintenance of the genome are processes critical for maintaining cellular viability. In eukaryotes, these functions are carried out on DNA that is organized into highly condensed chromatin. Specialized proteins must be employed to modify the structure of chromatin before other essential factors can access the DNA. With the identification of an increasing number of disease-associated genes, the importance of chromatin in human disorders has become abundantly clear, and several diseases have been linked to defects in chromatin remodeling. For example, mutations associated with the Snf2 family of chromatin remodeling proteins can cause debilitating genetic disorders and are associated with cancers. Understanding the basic properties of the proteins involved in these processes will be essential for revealing the underlying defects that produce the disease phenotypes. However, the mechanistic details of chromatin remodeling have remained elusive. This is because most methods used for studying remodeling can only probe the initial and final states of nucleosomes, and often don't reveal dynamic aspects of the reactions. Our hypothesis is that Snf2 proteins destabilize nucleosomes and other nucleoprotein complexes via a mechanism coupled to DNA translocation, thereby allowing other cellular components to gain unimpeded access to the DNA. To test this hypothesis we will use a single-molecule optical microscopy assay to directly visualize the Snf2 remodeling proteins Rdh54 and RSC as they interact with both naked DNA and also with chromatin substrates. These experiments will rely on a new technology developed in the Greene laboratory, which allows us to directly visualize hundreds of individual protein-DNA interactions in real time. The benefits of this approach are that we can probe the dynamics of chromatin remodeling in a format that allows us to rapidly gather statistically relevant information from hundreds of individual biomolecules and we can study these reactions with an unprecedented level of detail. PUBLIC HEALTH RELEVANCE: Defects in chromatin remodeling enzymes can result in extremely severe human diseases, which is consistent with their roles as global regulators of genome structure. As a first step towards developing targeted therapies that can be used to effectively prevent or cure these debilitating disorders it is essential to understand the basic biochemical properties of the chromatin remodeling proteins themselves. To help extend our understanding of chromatin remodeling we have developed fluorescence-based approaches for directly observing the interactions between proteins and individual DNA molecules, and our emphasis is placed on understanding biochemical reactions relevant to human biology and disease.