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.
In cells, a protein called Brg uses the chemical energy of ATP to control access to DNA. In humans, when Brg is mutated and cannot efficiently use ATP, many diverse malignancies can arise, suggesting mutation of Brg is a common pathway to several cancer types. We will directly observe and compare the normal and mutant versions of Brg in live cells to understand how these mutations promote cancer; our observations will provide mechanistic insight into how this widespread pathway to cancer promotes human malignancy, and may ultimately lead to development of new treatment strategies.
