Learning requires the conversion of transient experiences into long-lasting changes in neural circuitry. Animal behavior triggers changes in gene expression in small populations of neurons and behaviorally induced genes regulate synapses and neuronal morphology. Yet, it is unclear if changes in gene expression are the cause of behavioral plasticity, or the consequence. This project will develop a new genre of fluorescent reporters that enable the visualization and manipulation of endogenous transcription factors in individual neurons, in real time, and within the brain of behaving animals. During the award period, candidate reporters will be made that recognize six different transcription factors. These reporters will have widespread utility for investigating the molecular mechanisms that support learning in vivo and analysis of populations of neurons that are active during a learning paradigm. The development of these reporters includes ongoing training of undergraduate, graduate, and postgraduate scientists. Student training is optimized with guidance from the CREATE STEM Success Initiative on the UCSD campus.
Inducible transcription factors (ITFs) translate signals that last milliseconds or seconds into changes in cellular function that may persist for hours, days, or longer. This project will develop genetically encoded transcription factor reporters (GETFaRs) that are designed to visualize or manipulate an ITF. GETFaRs are based on molecular scaffolds, engineered through a process of synthetic affinity maturation of camelid nanobodies (Nbs) which bind the endogenous ITF. The Nb protein will be fused to a fluorophore or DNA modifying enzyme, allowing users to visualize or manipulate endogenous transcription factors. A degradation signal (degron) will be incorporated into the Nb near the ITF binding site. Consequently, GETFaRs will be constitutively expressed and rapidly degraded in the cytoplasm. When the ITF is expressed, the GETFaR-ITF interaction will mask the degron, stabilizing the complex. The ITF's nuclear localization signal will translocate the complex into the nucleus, resulting in stabilized GETFaRs that accumulate in the nucleus and stoichiometrically reflect ITF expression. Candidate GETFaRs will be validated in vitro using standard biochemical and imaging techniques and in vivo using two photon imaging of neurons in head fixed mice. Optimal GETFaRs will enable research that 1) monitors or manipulates transcriptional states during learning, 2) studies the emergence of ensembles of co-active neurons within a circuit, 3) probes the dynamics of chromatin and nuclear organization, and 4) analyzes the genome of defined populations of neurons responding to complex, natural stimuli.
