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

描述(申请人提供)：研究方案基于最近对人类癌症的全基因组测序研究，越来越多的人认识到哺乳动物的SWI/SNF复合体，一种依赖于ATP的染色质重构体，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复合体在大约20%的人类癌症中发生了突变。人们非常感兴趣的是Brg(SMARCA4)所起的作用，它是mSWI/SNF复合体的ATPase。Brg的ATP结合口袋含有几个经典的ATPase基序，如Walker A、Walker B和SF2解旋酶家族的保守Loop Ia。在人类癌症中，突变倾向聚集在一系列恶性肿瘤中这些保守的基序周围，这表明Brg ATP结合口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。基于公开的人类肿瘤测序数据，我们已经确定了Brg的ATP结合口袋中的28个突变，位于保守的Walker A/B和Loop Ia基序及其周围。我们建议通过在活细胞中成像Brg的癌症突变来检查mSWI/SNF复合体的天然染色质环境中的这些与癌症相关的突变。我们将产生一个与荧光蛋白融合的突变Brg构建库，并在内源性Brg被移除的活细胞中遵循这些突变的动态。我们将使用FRAP检测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，我们将测试两个假说来解释在癌症中观察到的遗传显性。此外，我们将利用现代成像技术的进步，利用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过程中微观核组织中的缺陷。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，与由于未能完成ATP水解循环而改变的对染色质的亲和力一致。因此，我们的初步工作证实，ATPase缺陷改变了活细胞的动态参数。通过对这些突变在ATP水解循环中的动态影响和特定缺陷进行分类，我们将生成综合的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、直接地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影响，并提供对依赖于ATP的染色质重构体突变如何促进人类恶性肿瘤的机械性洞察。培训、设施、发展和职业我选择导师和合作导师的目的是让我在癌症/染色质生物学和尖端成像技术方面拥有专业知识。在指导K99阶段，我将花费大部分时间开发和筛选构建物，并在我的主要导师克拉布特里博士的实验室进行初步的成像研究。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。为了最后一个目的，我将参观史洛夫博士在NIBIB的实验室，学习第三个目的的技术和仪器。对于所有这三个目标，设施和设备已经到位，并可用于拟议的研究。由于我在研究生毕业后换了专业，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。通过将这两个领域的专业知识结合起来，我将为追求自己的独立职业生涯做好独特而有力的准备。我计划在2015年秋季申请研究型教师的职位，因此我认为，该奖项的指导和独立阶段的时机非常适合我早期的职业时间表。

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.