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

描述（由申请人提供）：研究建议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，mSWI/SNF复合物在约20%的人类癌症中发生突变。非常感兴趣的是Brg（SMARCA 4），mSWI/SNF复合物的ATP酶所起的作用。Brg的ATP结合口袋包含几个经典的ATP酶基序，如步行者A、步行者B和SF 2解旋酶家族的保守环Ia。在人类癌症中，突变倾向聚集在一系列恶性肿瘤中的这些保守基序周围，表明Brg ATP结合口袋的突变是许多不同癌症的共同途径。然而，这些突变的具体影响仍然不确定。基于公开的人类肿瘤测序数据，我们在Brg的ATP结合口袋中，在保守的步行者A/B和Loop Ia基序中及其周围鉴定了28个突变。我们建议通过成像活细胞中Brg的癌症突变体来检查mSWI/SNF复合物的天然染色质环境中的这些癌症相关突变。我们将产生与荧光蛋白融合的突变体Brg构建体的文库，以跟踪这些突变体在已去除内源性Brg的活细胞中的动力学。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，通过使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期间微观核组织中的缺陷。在黑色素瘤中观察到的K785 R Brg的初步研究显示，Brg的动力学发生了深刻的变化，这与其未能完成ATP水解循环而改变的染色质亲和力一致。因此，我们的初步努力证实，ATP酶缺陷改变活细胞中的动力学参数。通过对这些突变的动态效应和ATP水解循环中的特定缺陷进行分类，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ATP依赖性染色质重塑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。在指导K99阶段，我将花费大部分时间开发和筛选结构，并在我的主要导师Crabtree博士的实验室进行初步成像研究。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，我将访问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，设施和设备已经到位，并可用于拟议的研究。因为我在研究生院毕业后改变了领域，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R 00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强大的准备。我计划在2015年秋季申请研究教师职位，所以我相信该奖项的指导和独立阶段的时间非常适合我早期的职业生涯时间轴。

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.