Identifying the mechanism of olfactory receptor gene regulation in olfactory neurons with live-cell imaging

Project Summary/Abstract Smell is a fundamental human sense. Even though humans do not rely on smell for survival, olfaction is necessary for physical and behavioral health. Detection and identification of volatile chemicals by the olfactory system is built upon the “one receptor per neuron” rule, whereby each mature olfactory sensory neuron expresses a single olfactory receptor (OR) gene from one allele. Singular expression is critical for olfactory perception, since it defines both the receptive field of the OSN and the circuitry of its axon. OR gene choice and singular expression is contingent on orchestrated changes in nuclear architecture. Using live-cell imaging, I will elucidate how this changing nuclear architecture leads to changes in gene expression. In Aim 1, I will image the endogenous transcription factors (Lhx2/Ebf/Ldb1) and the actively expressed OR DNA to determine how these transcription factors promote the expression of a single OR allele. My preliminary results suggest that these transcription factors come together to form an activating hub, and that this hub represents a greater enrichment of transcription factors than can be explained simply by the stoichiometry of enhancer binding sites. I will image the relationship between the actively expressed OR and heterochromatin markers to determine if association with the activating hub isolates this allele from heterochromatin. In Aim 2, I will investigate the nature of the biomolecular interactions that allow this activating hub to recruit Lhx2/Ebf/Ldb1 beyond the stoichiometry of binding sites through single-particle tracking of transcription factor mutants. I will determine if the amplification in recruitment is due to phase separation of the intrinsically-disordered domains of Lhx2/Ebf/Ldb1 or if it is due to cooperative protein-protein interactions. Finally, in Aim 3, the independent phase of this proposal, I will integrate the imaging of these transcription factors with imaging of the genome to extend my research towards a complete characterization of the nucleoprotein dynamics regulating OR gene expression. Specifically, I will begin by characterizing the interactions of OR enhancers and OR mRNA with Lhx2/Ebf/Ldb1 and the actively expressed OR using live-cell imaging. I am determined to lead an independent research laboratory at an academic institution, working at the interface of OR gene regulation and optical microscopy. I am optimally positioned to achieve this goal, working as I am with Dr. Stavros Lomvardas at Columbia University. Dr. Lomvardas is an expert in olfaction and genome organization, and during the K99, I will receive technical training from him in advanced sequencing technologies. I have also assembled an advisory team consisting of Drs. Elizabeth Hillman, Richard Axel, Carol A. Mason, and Anum Glasgow. This advisory team will guide me in professional training and transitioning to independence. In addition to elucidating general principles of how nuclear organization can dictate transcriptional specificity, the experiments in this proposal will enable a mechanistic interrogation of the molecular interactions that regulate singular OR transcription.