Enhancer-Mediated BDNF Regulation

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ABSTRACT: Brain Derived Neurotrophic Factor (BDNF) plays essential roles in neuronal survival, differentiation, and plasticity, and it contributes to learning and memory. The expression of BDNF is highly induced by neuronal activity, allowing it to function in temporal coordination with the reception of sensory stimuli. Proper control of Bdnf expression is essential to its function, because expressing too little or too much BDNF at the wrong times or in the wrong places has been shown to impair brain development, plasticity, and function. Neuronal activity- dependent induction of BDNF expression is mediated primarily at the transcriptional level, via the regulation of stimulus-modulated transcription factors. Although prior studies sought evidence for mechanisms of Bdnf regulation at this gene’s promoters, recent evidence shows increased long-range chromatin interactions with Bdnf upon neuronal activation, which suggests the potential importance of transcriptional regulation at distal activity-regulated enhancers. Using previously published ChIP-seq datasets and the chromatin conformation data from these recent studies, I have identified six putative distal enhancer regions. Given the function of enhancers in conferring specificity upon the regulation of gene transcription, I hypothesize that these enhancers contribute to the stimulus-specific control of Bdnf transcription. In Aim 1, I will test the function and neuronal activity-dependent regulation of these distal enhancers in mouse cortical neurons both using reporter assays and in their endogenous chromatin context by activating or repressing the enhancers with dCas9-fusion proteins. In Aim 2, I will focus on an intragenic enhancer of BDNF to test the hypothesis that the binding of the transcription factor NPAS4 to this enhancer mediates a cell-type specific mechanism of regulation in the hippocampus in the context of fear in vivo. This aim will take advantage of transgenic dCas9/CRISPR mice that I will use for enhancer regulation in the context of neural circuits in vivo. Completion of these two aims will elucidate the specificity provided by enhancers in Bdnf regulation. This proposal will also highlight the significance of transcriptional regulatory mechanisms in coupling upstream stimuli to its appropriate downstream response.