Project Grant R35GM153268

EPIGENOMIC CONSEQUENCES OF BREAKAGE-FUSION-BRIDGE CYCLING - PROJECT SUMMARY CHROMOSOME BREAKAGE CAN GENERATE EXTRAORDINARY GENETIC NOVELTY THAT IS SELECTED UPON DURING TUMOR PROGRESSION. ONE COMMONLY OBSERVED FEATURE OF CANCER IS THE BREAKAGE-FUSION-BRIDGE (BFB) CYCLE, WHERE DOUBLE-STRAND BREAKS SET OFF A CHAIN REACTION THAT LEADS TO COPY NUMBER VARIATIONS AND COMPLEX CHROMOSOME SHATTERING EVENTS. THE BFB CYCLE WAS FIRST DESCRIBED IN MAIZE BY BARBARA MCCLINTOCK IN THE 1940S, AND LED TO THE DISCOVERY OF TRANSPOSABLE ELEMENTS (TES). TWO TYPES OF TES, ACTIVATOR/DISSOCIATION (AC/DS) AND SUPPRESSOR-MUTATOR (EN/SPM), BURST FROM HER BFB SAMPLES, LEADING HER TO HYPOTHESIZE THAT CHROMOSOME BREAKAGE CAN LEAD TO A GENOME-WIDE RESPONSE SHE CALLED "GENOME SHOCK". HER OBSERVATION THAT BFB ACTIVATES PREVIOUSLY DORMANT TRANSPOSABLE ELEMENTS SUGGESTS THAT MAJOR EPIGENETIC CHANGES ACCOMPANY BFB, LIKELY COMPROMISING DNA METHYLATION AND HETEROCHROMATIN. DNA DEMETHYLATION AND TE ACTIVATION ARE ALSO OBSERVED IN MANY CANCERS, BUT CAUSAL RELATIONSHIPS BETWEEN BFB AND DEMETHYLATION HAVE NOT BEEN CLEARLY ESTABLISHED. UNLIKE IN ANIMALS, MAIZE TOLERATES THE TRANSMISSION OF MAJOR CHROMOSOMAL ABNORMALITIES THROUGH GAMETOGENESIS, GREATLY FACILITATING DOWNSTREAM STUDIES. WE HAVE DEVELOPED A SYSTEM FOR INDUCING THE BFB CYCLE IN MAIZE AND DEMONSTRATED ITS ABILITY TO INDUCE GENETIC INSTABILITY AND LARGE CHROMOSOMAL ABNORMALITIES. IN OUR APPROACH, AN ARRAY OF LEXO REPEATS INSERTED ON A CHROMOSOME ARM IS COMBINED WITH A LEXA- CENH3 TRANSGENE AND INITIATES A SECOND CENTROMERE. WITH LONG READ SEQUENCING AND CHIP-SEQ ASSAYS, WE WILL ANALYZE THE GENOMIC AND EPIGENOMIC CHANGES THAT FOLLOW BFB OVER MULTIPLE GENERATIONS. WE WILL ALSO DEPLOY HIGH-SENSITIVITY SCREENS FOR TE ACTIVATION. THESE APPROACHES WILL ALLOW US TO TEST AT THE MOLECULAR LEVEL THE EXTENT TO WHICH GENOME REORDERING BY BFB LEADS TO GENETIC AND EPIGENETIC CHANGES IN OTHERWISE WILD TYPE GENETIC BACKGROUNDS. THE PROPOSED RESEARCH WILL ESTABLISH A NEW MODEL FOR INVESTIGATING BFB IN COMPLEX EUKARYOTES AND PROVIDE GREATER INSIGHT INTO HOW THE BFB CYCLE IMPACTS EPIGENETIC PROCESSES THAT HAVE THE POTENTIAL TO FURTHER DESTABILIZE THE GENOME. IN ADDITION, THE WORK WILL PROVIDE A DIRECT TEST OF THE GENOME SHOCK HYPOTHESIS, WHICH HAS PROVIDED AN IMPORTANT GUIDEPOST FOR INTERPRETING THE CHROMOSOME REARRANGEMENTS IN CANCER AS WELL AS HUNDREDS OF OTHER GENOME RESTRUCTURING EVENTS IN ANIMALS, PLANTS, AND FUNGI.