Project Grant R01MH135952

THE IMPACT OF DNA SUPERCOILING AND TOPOISOMERASES ON NEURONAL ACTIVITY-DEPENDENT CHROMATIN ORGANIZATION - PROJECT SUMMARY EXPERIENCE-DRIVEN CHANGES TO CHROMATIN ARCHITECTURE HELP ACTIVATE GENE TRANSCRIPTION PROGRAMS THAT ALLOW ANIMALS TO LEARN AND DEVELOP ADAPTIVE BEHAVIORS. YET THE FORCES THAT REMODEL CHROMATIN IN RESPONSE TO NEURONAL ACTIVITY REMAIN OBSCURE. TORSION FROM DNA SUPERCOILING INJECTS FREE ENERGY INTO THE DNA AND HAS THE POTENTIAL TO ORGANIZE CHROMATIN, YET HOW DYNAMIC SUPERCOILING DISTRIBUTES AND ACTS WITHIN THE NEURONAL GENOME ARE POORLY UNDERSTOOD. RNA POLYMERASES (RNAPS) AND DNA TOPOISOMERASES GENERATE AND RESOLVE SUPERCOILS, RESPECTIVELY, AND THEIR CONCERTED ACTIONS AFFECT DYNAMIC SUPERCOILING DISTRIBUTION. HOWEVER, TO WHAT EXTENT SUPERCOILS PROPAGATE FROM THE SITES OF RNAP ACTIVITY, AND HOW CHROMATIN STRUCTURE AFFECTS SUPERCOIL DISTRIBUTION REMAIN UNEXPLORED. FURTHERMORE, WHILE SUPERCOILING IS A UBIQUITOUS FEATURE OF DNA TOPOLOGY, HOW TORSIONAL FREE ENERGY FROM SUPERCOILING AFFECTS CHROMATIN STRUCTURE AND GENE ACTIVITY PATTERNS IS ALSO UNKNOWN. TO ADDRESS THESE ISSUES, PSORALEN CROSSLINKING AND SEQUENCING (TMP-SEQ) WAS RECENTLY PERFORMED TO MAP THE DISTRIBUTION OF UNDERWOUND (NEGATIVELY SUPERCOILED) DNA AT HIGH RESOLUTION IN CULTURED MOUSE CORTICAL NEURONS UNDER BASAL CONDITIONS. ADDITIONALLY, NEW METHODS WERE DEVELOPED TO MAP GENOME-WIDE SITES OF CATALYTICALLY ENGAGED TOPOISOMERASES (TOP1CC-SEQ AND TOP2BCC-SEQ). THESE STUDIES REVEALED THAT SUPERCOILS TRANSMIT WIDELY (> 200 KB) FROM THE SITES OF ACTIVE RNAPS, WHILE TOPOISOMERASES ONLY ACT AT SELECTIVE LOCATIONS TO RESOLVE SUPERCOILS. THESE DATA INDICATE THAT TORSIONAL FREE ENERGY FROM SUPERCOILING COULD BE WIDELY AVAILABLE TO INFLUENCE CHROMATIN STRUCTURE. BASED ON THESE RESULTS, THIS PROJECT WILL PERFORM TMP-SEQ AT VARIOUS TIMES FOLLOWING NEURONAL STIMULATION AND ASSESS HOW ACTIVITY-DRIVEN SUPERCOILING DISTRIBUTES WITHIN THE GENOME. ADDITIONALLY, TOP1CC-SEQ AND TOP2BCC-SEQ WILL BE PERFORMED UNDER THE SAME CONDITIONS TO DETERMINE HOW TOPOISOMERASES ARE UTILIZED TO REGULATE DYNAMIC SUPERCOILING IN STIMULATED NEURONS. WHILE SUPERCOILS GENERALLY DISTRIBUTE FREELY WITHIN CHROMATIN, PRELIMINARY DATA INDICATE THAT THEY ALSO ACCRUE AT SPECIFIC NUCLEOSOME CONFIGURATIONS, PARTICULARLY AT ACTIVE PROMOTERS WITH BROAD H3K4ME3 DISTRIBUTIONS AND IN REGIONS FLANKING H3K27ME3-RICH CHROMATIN. INTRIGUINGLY, A BROADENING OF H3K4ME3 HAS BEEN OBSERVED FOLLOWING NEURONAL STIMULATION IN VIVO, SUGGESTING THAT SPECIFIC STIMULUS-DRIVEN EPIGENETIC CHANGES COULD "CONSTRAIN" DYNAMIC SUPERCOILS. THE PROPOSED RESEARCH WILL TEST THIS IDEA BY ASSESSING HOW KNOCKDOWN OF WDR5, WHICH MEDIATES H3K4 METHYLATION, AND EXPRESSION OF JMJD3, WHICH ERASES H3K27ME3, AFFECT NEURONAL ACTIVITY-DEPENDENT SUPERCOILING PATTERNS. FINALLY, TOPOISOMERASE INHIBITORS AND LOCUS-SPECIFIC MODULATORS OF DNA SUPERCOILING WILL BE USED TO PERTURB SUPERCOILING PATTERNS IN STIMULATED NEURONS, AND CHROMOSOME CONFORMATION CAPTURE (MICRO-C), ATAC-SEQ, AND NASCENT TRANSCRIPTION ANALYSIS (FASTGRO) WILL PERFORMED TO TEST THE IDEA THAT DYNAMIC SUPERCOILING HAS A CAUSAL ROLE IN NEURONAL ACTIVITY-DRIVEN CHROMATIN RECONFIGURATION. TOGETHER, THESE EFFORTS WILL PROVIDE NEW INSIGHTS INTO HOW DNA MECHANICS REGULATES NEURONAL FUNCTIONS DURING DEVELOPMENT AND DISEASE.