Dynamic effects of cancer mutations on the mammalian SWI/SNF ATPase Brg

癌症突变对哺乳动物 SWI/SNF ATPase Brg 的动态影响

基本信息

批准号：
8902078
负责人：
Hamilton Courtney Hodges
金额：
$ 17.05万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8902078
关键词：
ATP Hydrolysis ATP phosphohydrolase Affinity Alanine Alleles Area Award Binding Biological Assay Biology Cell Cycle Cell Nucleus Cells Chemicals Chimeric Proteins Chromatin Chromatin Structure Cloning Complex DNA Data Defect Development Environment Equipment Faculty Failure Family Fluorescence Microscopy Fluorescence Recovery After Photobleaching Gene Expression Genetic Genetic Recombination Health Human Image Imaging Techniques In Vitro Interphase Kinetics Laboratories Lead Learning Libraries Life Life Cycle Stages Light Lighting Malignant Neoplasms Mammalian Cell Measurement Mediating Mentors Meta-Analysis Microscopic Microscopy Mitosis Modeling Mutate Mutation National Institute of Biomedical Imaging and Bioengineering Nuclear Optics Pathway interactions Phase Play Positioning Attribute Preparation Proteins Radiolabeled Research Research Proposals Resolution Resources Role SMARCA4 gene Scanning Schools Structure Techniques Testing Time TimeLine Training Visit Walkers Work base cancer imaging cancer type career career development cellular imaging chromatin remodeling design embryonic stem cell falls genome-wide helicase in vivo insight instrumentation interest mathematical model melanoma mutant particle radiotracer screening treatment strategy tumor tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Research Proposal Based on recent genome-wide sequencing studies of human cancers, there is growing recognition that the mammalian SWI/SNF complex, an ATP-dependent chromatin remodeler, plays a widespread role in human malignancy. In particular, we recently found through a meta-analysis of 44 studies that the mSWI/SNF complex is mutated in ~20% of all human cancers. Of great interest is the role played by Brg (SMARCA4), the ATPase of the mSWI/SNF complex. The ATP binding pocket of Brg contains several classic ATPase motifs such as Walker A, Walker B, and the conserved Loop Ia of the SF2 helicase family. In human cancers, mutation propensities cluster around these conserved motifs in a range of malignancies, suggesting mutation of the Brg ATP binding pocket is a common pathway to many diverse cancers. However, the specific effects of these mutations remain uncertain. Based on publicly available human tumor sequencing data, we have identified 28 mutations in the ATP-binding pocket of Brg, in and around the conserved Walker A/B and Loop Ia motifs. We propose to examine these cancer-associated mutations in the native chromatin environment of the mSWI/SNF complex by imaging cancer mutants of Brg in live cells. We will generate a library of mutant Brg constructs fused to a fluorescent protein t follow the dynamics of these mutants in live cells in which endogenous Brg has been removed. We will examine changes in their dynamics on chromatin using FRAP, and explore in vitro the enzymological defects associated with each mutation. During these studies, we will test two hypotheses to explain the genetic dominance observed in cancer. Additionally, we will take advantage of modern imaging advances to characterize defects in the microscopic nuclear organization during interphase and mitosis by using super-resolution imaging techniques and light-sheet fluorescence microscopy. Preliminary studies with K785R Brg, observed in melanoma, show profound changes in the dynamics of Brg, consistent with altered affinity to chromatin as a result of its failure to complete the ATP hydrolytic cycle. Thus, our preliminary efforts confirm that ATPase defects alter dynamic parameters in live cells. By classifying these mutations with regard to their dynamic effects and specific defects in the ATP hydrolytic cycle, we will generate integrative mathematical models to explain their specific dynamic defects in living cells. Through careful, direct observation of each cancer-associated Brg mutant, we will reveal the specific effect each mutation has, and provide mechanistic insight into how mutation of an ATP-dependent chromatin remodeler promotes human malignancy. Training, Facilities, Development, and Career My choice of mentor and co-mentor is designed to give me expertise in both cancer/chromatin biology and cutting-edge imaging techniques. In the mentored K99 phase, I will spend the majority of my time developing and screening constructs, and performing preliminary imaging studies in the laboratory of my primary mentor, Dr. Crabtree. The first two of my proposed aims will be performed at Stanford, where there is also a specific training plan in place. For the last aim, I will visit the laboratory of Dr. Shroff at the NIBIB, to learn the techniques and instrumentation for the third aim. For all three aims, the facilities and equipment are already in place and operational for the proposed research. Because I changed fields after graduate school, the additional mentored time in the K99 phase will allow me to learn the techniques and the instrumentation necessary to bring these techniques to my own lab during the R00 phase. By blending the expertise in both areas, I will be provided a unique and powerful preparation to pursue my own independent career. I plan to apply for research faculty positions during Fall 2015, so I believe the timing of the mentored and independent phase of the award is ideally suited for my early career timeline.

描述（由申请人提供）：研究计划基于最近对人类癌症的全基因组测序研究，人们越来越认识到哺乳动物 SWI/SNF 复合物（一种 ATP 依赖性染色质重塑剂）在人类恶性肿瘤中发挥着广泛的作用。特别是，我们最近通过对 44 项研究的荟萃分析发现，mSWI/SNF 复合物在约 20% 的人类癌症中发生突变。人们非常感兴趣的是 Brg (SMARCA4)（mSWI/SNF 复合物的 ATP 酶）所发挥的作用。 Brg 的 ATP 结合口袋包含几个经典的 ATP 酶基序，例如 Walker A、Walker B 和 SF2 解旋酶家族的保守环 Ia。在人类癌症中，一系列恶性肿瘤中的突变倾向集中在这些保守基序周围，表明 Brg ATP 结合袋的突变是许多不同癌症的常见途径。然而，这些突变的具体影响仍不确定。根据公开的人类肿瘤测序数据，我们在 Brg 的 ATP 结合口袋、保守的 Walker A/B 和 Loop Ia 基序内部和周围确定了 28 个突变。我们建议通过对活细胞中 Brg 的癌症突变体进行成像来检查 mSWI/SNF 复合物天然染色质环境中的这些癌症相关突变。我们将生成一个与荧光蛋白融合的突变 Brg 构建体文库，以跟踪这些突变体在活细胞中的动态，其中内源 Brg 已被去除。我们将使用 FRAP 检查它们在染色质上的动态变化，并在体外探索与每个突变相关的酶学缺陷。在这些研究中，我们将测试两个假设来解释在癌症中观察到的遗传优势。此外，我们将利用现代成像技术的进步，通过超分辨率成像技术和光片荧光显微镜来表征间期和有丝分裂期间微观核组织的缺陷。在黑色素瘤中观察到的 K785R Brg 的初步研究表明 Brg 的动力学发生了深刻的变化，这与由于其未能完成 ATP 水解循环而导致的染色质亲和力的改变一致。因此，我们的初步努力证实 ATP 酶缺陷会改变活细胞中的动态参数。通过根据这些突变的动态效应和 ATP 水解循环中的特定缺陷对这些突变进行分类，我们将生成综合数学模型来解释它们在活细胞中的特定动态缺陷。通过仔细、直接观察每个与癌症相关的 Brg 突变体，我们将揭示每个突变的具体影响，并提供有关 ATP 依赖性染色质重塑剂的突变如何促进人类恶性肿瘤的机制见解。培训、设施、发展和职业我选择的导师和共同导师旨在为我提供癌症/染色质生物学和尖端成像技术方面的专业知识。在 K99 指导阶段，我将花费大部分时间开发和筛选构建体，并在我的主要导师 Crabtree 博士的实验室中进行初步成像研究。我提出的前两个目标将在斯坦福大学实现，那里也有一个具体的培训计划。对于最后一个目标，我将参观NIBIB的Shroff博士的实验室，学习第三个目标的技术和仪器。对于所有这三个目标，设施和设备均已就位并可用于拟议的研究。因为我在研究生毕业后改变了领域，所以 K99 阶段的额外指导时间将使我能够学习必要的技术和仪器，以便在 R00 阶段将这些技术带到我自己的实验室。通过融合这两个领域的专业知识，我将为追求自己的独立职业生涯提供独特而有力的准备。我计划在 2015 年秋季申请研究教员职位，因此我相信该奖项的指导和独立阶段的时机非常适合我的早期职业生涯时间表。