Project Grant DP2GM149752

HARNESSING THE CHROMATIN CONFORMATIONAL CODE FOR EPIGENETIC REGULATION - PROJECT SUMMARY EUKARYOTIC DNA IS WRAPPED AROUND NUCLEOSOMES, WHICH FORM CHAINS OF CHROMATIN THAT ARE FURTHER FOLDED INTO THREE-DIMENSIONAL ASSEMBLIES. THE ARCHITECTURE OF THESE ASSEMBLIES REGULATES MANY NUCLEAR FUNCTIONS, INCLUDING GENOME 3D FOLDING AND TRANSCRIPTION, AND ULTIMATELY DICTATES CELLULAR IDENTITY. NUCLEOSOMES ARE A WELL-KNOWN HUB OF CHROMATIN REGULATION, MOST OF WHICH IS THOUGHT TO OCCUR VIA A VARIETY OF POST-TRANSLATIONAL MODIFICATIONS ON THE PROTRUDING FLEXIBLE TAILS OF HISTONES. BASED ON THE ASSUMPTION THAT MOST REGULATION OF CHROMATIN'S STRUCTURE AND INTERACTIONS WITH OTHER FACTORS OCCURS AT THESE HISTONE TAILS, THE GLOBULAR CORE OF NUCLEOSOMES HAS BEEN CONSIDERED RIGID AND MINIMALLY REGULATORY. EXCITINGLY, MY RECENT WORK HAS REVEALED A NEW INSIGHT: THAT THE NUCLEOSOME CORE IS MALLEABLE AND THAT THIS PLASTICITY REGULATES CHROMATIN FOLDING AND GENE REPRESSION. I THEREFORE PROPOSE THAT THE GLOBULAR, MALLEABLE CORE OF NUCLEOSOMES IS A HUB FOR GENETIC AND EPIGENETIC REGULATION AS WELL AS A POTENTIAL NOVEL THERAPEUTIC TARGET. TO TEST THIS PROVOCATIVE HYPOTHESIS THAT CHALLENGES THE TEXTBOOK PARADIGM OF CHROMATIN REGULATION, NOVEL TOOLS CAPABLE OF PROBING BOTH IN VITRO AND IN VIVO ATOMIC-SCALE DYNAMICS OF LARGE MACROMOLECULAR ASSEMBLIES SUCH AS CHROMATIN MUST BE DEVELOPED. MY LAB WILL CLOSE THIS GAP BY DEVELOPING CONFORMATION-SPECIFIC NANOBODIES (NANONUCS) THAT ACT AS SENSORS OF DISTINCT NUCLEOSOME CONFORMATIONS. NANONUCS WILL BE DISCOVERED FROM A SYNTHETIC LIBRARY CONTAINING >2 X 109 DISTINCT NANOBODIES. WE WILL EMPLOY NANONUCS TO GAIN STRUCTURAL AND BIOPHYSICAL INSIGHTS INTO NUCLEOSOME CONFORMATIONAL DYNAMICS AND TO PROBE AND PERTURB THE NUCLEOSOME CONFORMATIONAL CODE IN CELLS. SPECIFICALLY, WE WILL: (I) OBTAIN ATOMIC UNDERSTANDING OF NUCLEOSOME ALTERNATIVE STATES BY COMBINING NMR, HDX-MS, AND CRYO-EM; (II) IDENTIFY CHROMATIN FACTORS THAT SENSE AND LEVERAGE NUCLEOSOME PLASTICITY; (III) SEARCH FOR NUCLEOSOME CONFORMATIONS THAT ARE BIOLOGICAL OR PATHOLOGICAL BIOMARKERS; AND (IV) DEVELOP A NOVEL STRATEGY TO MANIPULATE NUCLEOSOME SHAPES AND CHROMATIN STATES IN CELLS. BY CARRYING OUT THIS HIGHLY AMBITIOUS, INTEGRATED, AND MULTIDISCIPLINARY RESEARCH PROGRAM, MY LAB WILL UNVEIL THE MOLECULAR MECHANISMS AND THERAPEUTICAL POTENTIAL OF THE NUCLEOSOME CONFORMATIONAL CODE. I ANTICIPATE THAT THESE HIGH-RISK, HIGH-REWARD INVESTIGATIONS WILL REVEAL NEW FUNDAMENTAL PRINCIPLES OF GENOME REGULATION THAT SHIFT THE LONG-STANDING PARADIGM OF RIGID HISTONE UNITS AND THAT WILL BROADLY IMPACT BIOMEDICAL SCIENCE OVER THE SHORT AND LONG TERMS. EXPLORING THE STRUCTURAL FLEXIBILITY OF NUCLEOSOMES REPRESENTS AN OPPORTUNITY TO IDENTIFY NOVEL THERAPEUTIC BIOMARKERS AND DRUGS FOR DISEASES LINKED TO EPIGENETICS DEFECTS, SUCH AS CANCER. ULTIMATELY, WITH CRITICAL SUPPORT FROM THE NIH DIRECTOR'S NEW INNOVATOR PROGRAM, OUR STUDIES WILL ENRICH OUR KNOWLEDGE OF THE FUNCTION AND PHYSIOLOGY OF CHROMATIN WITH ATOMIC-SCALE BIOPHYSICAL INSIGHTS INTO THE CHROMATIN ARCHITECTURE ITSELF.