Mechanisms of Specificity in Neuronal Activity-regulated Gene Transcription

Abstract

The ability to convert sensory stimuli into long-lasting changes in brain function is essential for animals to interact with and learn from their environment. This process is achieved by encoding sensory stimuli into temporal patterns of neuronal activity, which in turn modulate the connectivity and strength of neural circuits in the brain. These long-term plastic changes in the brain are known to depend on the neuronal activity-regulated transcription of new gene products. My dissertation research sought to elucidate how the timing and level of transcriptional responses following neuronal activity can be precisely regulated to form proper neuronal connections. In the first part of this dissertation, I investigated the role of the developmentally regulated GluN3A subunit in NMDAR-induced transcription. I observed that neurons lacking the transcription factor CaRF showed enhanced NMDAR-induced expression of Bdnf and Arc both in cultured neurons and following sensory stimulation in the developing brain in vivo. I identified GluN3A as a regulatory target of CaRF and found that neurons lacking GluN3A showed selective enhancement of NMDAR-induced transcription. GluN3A limited synaptic activity-induced transcription by inhibiting both NMDAR-induced nuclear translocation of the p38 MAP kinase and activation of the transcription factor MEF2C. These data demonstrate that GluN3A negatively regulates NMDAR-dependent activation of gene transcription and reveal a novel mechanism that regulates the level of NMDAR-induced transcriptional response in the developing brain. In the second part of my dissertation, I examined the role of enhancer histone acetylation in neuronal activity-regulated gene transcription. I applied quantitative single-molecule fluorescence in situ hybridization to measure neuronal activity-induced gene transcription at the single neuron level, taking advantage of the intrinsic stochasticity of transcription to quantify the effects of enhancer regulation on the dynamics of promoter state transitions. Locally-induced enhancer histone acetylation by CRISPR-mediated epigenome editing was sufficient to increase Fos mRNA expression both under basal conditions and following membrane depolarization in primary hippocampal neurons, via a mechanism that involves enhancer recruitment of Brd4, increased transcriptional elongation by the release of paused polymerase, and prolonged activation of Fos promoters. These data indicate that enhancer histone acetylation plays a causative role in the induction of neuronal activity-regulated gene transcription and open up the possibility to specifically control the level and timing of the neuronal activity-induced transcriptional response. Taken together my dissertation works elucidate mechanisms that control the specificity, timing, and amplitude of transcriptional responses to neuronal activity, revealing novel information about the dynamic range of this fundamental cellular process.