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2016-11-18

Neuronal activity mediates the formation of neuronal circuits in the cerebral cortex. These processes are regulated by the transcription factor CREB, which regulates gene expression in neuronal activity-dependent processes. Neuronal activity enhances CREB-mediated transcription but the mechanisms remain unclear. CREB binds to a cAMP response element (CRE) in the promoter region of its target genes. Assembly and disassembly of CREB-CRE interactions control spatiotemporal gene expression in the nucleus. However, how CREB interacts with CRE in activity-dependent mechanisms is not known.

In a new study from Osaka University, led by Nobuhiko Yamamoto, Hironobu Kitagawa, Noriyuki Sugo and colleagues investigated how neuronal activity influences CREB dynamics. Fluorescent-tagged CREB constructs were expressed in primary cortical neurons and visualized by staining fixed cells. Single CREB molecules were predominantly detected in the nucleus and some were observed at sites where gene transcription was taking place.

The movement of single CREB molecules in living cortical neurons was monitored by real-time imaging. Interestingly, some CREB molecules disappeared almost immediately while a fraction remained in the same location for a longer duration. Using mutant forms of CREB that cannot bind to CRE, the researchers showed that these CREB molsecules were interacting with CRE.

To investigate how neuronal activity affects CREB-CRE interactions and subsequent gene transcription, the authors treated cortical neurons with pharmacological agents that alter neuronal activity. Neuronal activity did not affect the time that CREB resided in the nucleus. Next, the research team investigated whether neuronal activity regulates the spatial distribution of CREB in the nucleus. They stimulated cortical neurons and monitored CREB localization by real-time imaging. Interestingly, the number of locations where CREB resided for long periods increased considerably after neuronal activation. These findings indicated that neuronal activity increases CREB-CRE interactions in the nucleus.

The findings provide novel insights into the regulation of gene expression by neuronal activity. Based on the results of this study, neuronal activity may contribute to CREB-dependent gene expression by increasing the binding of CREB to specific genomic sites.

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 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 using a single-molecule imaging technique. 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 (HILO) 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 (CRE), 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.

Figure 1. A model of the frequent interaction of CREB with CRE to promote transcription initiation in response to neuronal activity.

Figure 2

To learn more about this research, please view the full research report entitled “Activity-Dependent Dynamics of the Transcription Factor CREB in Cortical Neurons Revealed by Single-Molecule Imaging” at this page of the Journal of Neuroscience website.


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