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

• 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
• 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

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（申请人提供）：基于人类癌症的最新基因组测序研究的研究建议，人们越来越认识到，哺乳动物SWI/SNF复合体是ATP依赖性的染色质重塑剂，在人类恶性肿瘤中起着广泛的作用。特别是，我们最近通过对44项研究的荟萃分析发现，在所有人类癌症中，MSWI/SNF复合物被突变。令人兴趣的是MSWI/SNF综合体ATPase BRG（Smarca4）所扮演的角色。 BRG的ATP绑定袋中包含几个经典的ATPase图案，例如Walker A，Walker B和SF2解旋酶家族的保守环IA。在人类癌症中，突变倾向聚集在这些保守的基序中，这表明BRG ATP结合口袋的突变是通往许多多种癌症的常见途径。但是，这些突变的具体作用仍然不确定。基于公开可用的人类肿瘤测序数据，我们在BRG的ATP结合袋中鉴定了28个突变，在保守的Walker A/B和环环IA基序中。我们建议通过对活细胞中BRG的癌症突变体进行成像，以检查MSWI/SNF复合物的天然染色质环境中的这些相关突变。我们将生成一个融合到荧光蛋白t的突变体BRG构建体库，遵循这些突变体在活细胞中的动力学，其中已去除内源性BRG。我们将使用FRAP检查其在染色质上的动力学变化，并在体外探索与每个突变相关的酶学缺陷。在这些研究中，我们将检验两个假设，以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的进步来表征在相间和有丝分裂过程中使用超分辨率成像技术和浅层荧光显微镜的缺陷。在黑色素瘤中观察到的K785R BRG的初步研究显示了BRG动力学的深刻变化，这与对染色质的亲和力的改变，因为它未能完成ATP水解循环。因此，我们的初步工作证实了ATPase缺陷会改变活细胞中的动态参数。通过对ATP水解循环中的动态效应和特定缺陷进行分类，我们将生成整合性数学模型，以解释其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具有的特定效果，并提供有关ATP依赖性染色质重塑剂如何促进人类恶性肿瘤的机械洞察力。培训，设施，发展和职业我对导师和同事的选择旨在为我提供癌症/染色质生物学和尖端成像技术的专业知识。在指导的K99阶段，我将花费大部分时间来开发和筛选结构，并在我的主要导师Crabtree博士的实验室中进行初步成像研究。我建议的目标的前两个将在斯坦福大学进行，那里还制定了特定的培训计划。为了最后一个目标，我将访问尼比布（Nibib）的史肖夫（Shroff）博士，以学习第三个目标的技术和仪器。对于所有三个目标，设施和设备已经到位，并为拟议的研究而运营。因为我在研究生院后改变了领域，所以在K99阶段的额外指导时间将使我能够学习在R00阶段将这些技术带到我自己的实验室所需的技术和仪器。通过将两个领域的专业知识融合在一起，我将为我提供独特而有力的准备，以追求自己的独立职业。我计划在2015年秋季申请研究教师职位，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时间表。