Regulation of the neuronal transcription factor NPAS4 by REST and microRNAs

• NPAS4 expression is highly restricted to the brain and tightly coupled to neuronal activity.
• NPAS4 is repressed in non-neuronal cells by the REST bound regulatory elements.
• REST binds promoter and intron I sites in NPAS4, correlating with CTCF occupancy.
• miR-224 and miR-203 down regulate NPAS4 expression through its 3′UTR.
• miR-224 is enriched in hypothalamic/midbrain regions and expressed from an intron of GABRE.

NPAS4 is a brain restricted, activity-induced transcription factor which regulates the expression of inhibitory synapse genes to control homeostatic excitatory/inhibitory balance in neurons. NPAS4 is required for normal social interaction and contextual memory formation in mice. Protein and mRNA expression of NPAS4 is tightly coupled to neuronal depolarization and most prevalent in the cortical and hippocampal regions in the brain, however the precise mechanisms by which the NPAS4 gene is controlled remain unexplored. Here we show that expression of NPAS4 mRNA is actively repressed by RE-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) in embryonic stem cells and non-neuronal cells by binding multiple sites within the promoter and Intron I of NPAS4. Repression by REST also appears to correlate with the binding of the zinc finger DNA binding protein CTCF within Intron I of NPAS4. In addition, we show that the 3′ untranslated region (3′UTR) of NPAS4 can be targeted by two microRNAs, miR-203 and miR-224 to further regulate its expression. miR-224 is a midbrain/hypothalamus enriched microRNA which is expressed from an intron within the GABAA receptor epsilon (GABRE) gene and may further regionalize NPAS4 expression. Our results reveal REST and microRNA dependent mechanisms that restrict NPAS4 expression to the brain.

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