AN INDUCIBLE MOLECULAR MEMORY SYSTEM TO RECORD TRANSIENT STATES OF CNS CELLS

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 DESCRIPTION (provided by applicant): The brain is a remarkably complex organ comprised of hundreds of unique cell types that are organized to form sophisticated neural circuits. Although we have made progress toward understanding brain function and development, it is clear there is still much to be learned. Currently, all genome-wide methods that could be brought to bear on functional studies of the brain are destructive, meaning that as a genomic analysis is performed on a population of cells, the cells are destroyed. This fact limits our ability to connec early molecular events in the cells of the brain with later behavioral or cellular changes. For example, it is currently impossible to connect transcriptional changes in a neuron with knowledge of whether or not the cell was successfully incorporated into a memory trace. Similarly, it is not feasible to connect the early molecular events that occur in a neuronal progenitor cell with the final cell fate decision made by the cell. We have set out to develop a transformative technology that can record molecular events at the time that they occur and can then be read out later after any defined period of time. We have a novel technology called transposon `Calling Cards' that, in culture, provides cells with a molecular memory of protein-DNA interactions that occur at a particular moment in time. Here, we propose to adapt this technology for use in vivo enabling a retrospective genomic analysis of molecular events. We will demonstrate the utility of this technology by completing four test-case experiments that cannot be done with existing methods. Specifically, we will test the method by: 1) retrospectively identifying candidate transcription factors that control the specification of cell types in the CNS 2) identifying features that distinguish neurons resistant to neurodegeneration in vivo, 3) identifying the neurons that become active during mouse vocalization behavior while simultaneously mapping the genome-wide binding of activity-dependent transcription factors in these neurons, and 4) identifying the molecular features that distinguish neurons that were incorporated into a fear memory trace from those that were not incorporated.