title: Activity-Dependent Dynamics of the Transcription Factor of cAMP-Response Element Binding Protein in Cortical Neurons Revealed by Single-Molecule Imaging.
abstract: Transcriptional regulation is crucial for neuronal activity-dependent processes that govern neuronal circuit formation and synaptic plasticity. An intriguing question is how neuronal activity influences the spatiotemporal interactions between transcription factors and their target sites. Here, using a single-molecule imaging technique, we investigated the activity dependence of DNA binding and dissociation events of cAMP-response element binding protein (CREB), a principal factor in activity-dependent transcription, in mouse cortical neurons. To visualize CREB at the single-molecule level, fluorescent-tagged CREB in living dissociated cortical neurons was observed by highly inclined and laminated optical sheet microscopy. We found that a significant fraction of CREB spots resided in the restricted locations in the nucleus for several seconds (dissociation rate constant: 0.42 s-1). In contrast, two mutant CREBs, which cannot bind to the cAMP-response element, scarcely exhibited long-term residence. To test the possibility that CREB dynamics depends on neuronal activity, pharmacological treatments and an optogenetic method involving channelrhodopsin-2 were applied to cultured cortical neurons. Increased neuronal activity did not appear to influence the residence time of CREB spots, but markedly increased the number of restricted locations (hot spots) where CREB spots frequently resided with long residence times (>1 s). These results suggest that neuronal activity promotes CREB-dependent transcription by increasing the frequency of CREB binding to highly localized genome locations.
  
  
  
  The transcription factor, cAMP response element-binding protein (CREB) is known to regulate gene expression in neuronal activity-dependent processes. However, its spatiotemporal interactions with the genome remain unknown. Single-molecule imaging in cortical neurons revealed that fluorescent-tagged CREB spots frequently reside at fixed nuclear locations in the time range of several seconds. Neuronal activity had little effect on the CREB residence time, but increased the rapid and frequent reappearance of long-residence CREB spots at the same nuclear locations. Thus, activity-dependent transcription is attributable to frequent binding of CREB to specific genome loci.
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Osaka 585-0871, Japan, and.
authors.forename: Hironobu
authors.initials: H
authors.lastname: Kitagawa
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Osaka 585-0871, Japan, and sugo@fbs.osaka-u.ac.jp nobuhiko@fbs.osaka-u.ac.jp.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Sugo
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Osaka 585-0871, Japan, and.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Morimatsu
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Osaka 585-0871, Japan, and.
authors.forename: Yoshiyuki
authors.identifier: 0000-0003-2862-0829
authors.initials: Y
authors.lastname: Arai
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Osaka 585-0871, Japan, and.|Riken Quantitative Biological Center (QBiC), Osaka 565-0874, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Yanagida
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Osaka 585-0871, Japan, and sugo@fbs.osaka-u.ac.jp nobuhiko@fbs.osaka-u.ac.jp.
authors.forename: Nobuhiko
authors.identifier: 0000-0002-1916-932X
authors.initials: N
authors.lastname: Yamamoto
mesh_terms: D000818:Animals; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D000075402:Channelrhodopsins; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D057054:Molecular Imaging; D009154:Mutation / Q000235:genetics; D009474:Neurons / Q000378:metabolism*; D062308:Optogenetics; D061251:Primary Cell Culture; D014157:Transcription Factors
chemical_list: D000075402:Channelrhodopsins; C492004:Creb1 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; D014157:Transcription Factors; D004247:DNA
keywords: CREB; cortex; gene expression; imaging; neuronal activity; single molecule