Regulation of the neuronal transcription factor NPAS4 by REST and microRNAs

https://doi.org/10.1016/j.bbagrm.2013.11.004Get rights and content

Highlights

  • 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.

Abstract

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.

Introduction

Neuronal activity dependent transcription is critical for the development and maintenance of synapses in the developing central nervous system [1], [2]. This process is reliant upon calcium influx through voltage gated calcium channels, which stimulates the expression of intermediate early genes, in turn modulating downstream synapse effectors [3], [4]. These mechanisms are active during development and throughout adulthood, and are thought to underlie memory and learning [5], [6]. The homeostatic balance between excitatory and inhibitory inputs during these development and maintenance periods is also thought to be an important contributor to neuropsychiatric disease [7], [8]. Indeed it has been recently demonstrated that optogenetic disruption of the excitatory/inhibitory balance in mice can lead to social dysfunction [9], [10].

NPAS4 (Neuronal Per-ARNT-SIM homology domain 4) is a bHLH/PAS (basic Helix-Loop-Helix/Per-Arnt-Sim) transcription factor that is highly restricted to the central nervous system and specifically coupled to calcium influx following neuronal activity [11], [12], [13], [14], [15]. NPAS4 mRNA can be stimulated by a number of stressors, including ischemia and seizure, as well as physiological inducers such as light, odor or paradigms of memory formation in mice [11], [12], [15], [16], [17], [18]. In addition, NPAS4 expression appears to be developmentally controlled, being induced around birth and predominantly restricted to cortical and hippocampal areas of the rodent brain [12], [19]. Increased NPAS4 mRNA following excitatory synapse activation leads to rapid, transient protein expression which in turn activates a number of genes involved in the formation of inhibitory synapses [11], [12]. NPAS4 increases the number of GABA-releasing synapses following neuron activation to maintain activity homeostasis, i.e. the excitatory/inhibitory balance in neurons. Consequently, NPAS4 null mice are hyperactive, prone to seizures and exhibit a number of defects in social interaction and memory formation [11], [12], [16]. Therefore the precise spatial and temporal expression of NPAS4 is a likely determinate in regulating proper synapse formation and maintenance both during development and in response to neuronal activity. Here we identify mechanisms which restrict NPAS4 expression to the CNS. We show that the transcription repressor REST inhibits NPAS4 expression in non-neuronal and undifferentiated cells. Furthermore, we show that NPAS4 expression can be modulated by miR-224, which is expressed from an intron of the GABAA receptor epsilon gene and is enriched in the midbrain/hypothalamus region of the mouse CNS. Conversely expression of NPAS4 is high within the cortex and low within the midbrain/hypothalamus, suggesting miR-224 may play a role in regionalizing NPAS4 expression in vivo.

Section snippets

Generation of DNA constructs

All DNA constructs generated by PCR were amplified using Phusion High-Fidelity DNA polymerase (New England Biosciences) and sequence verified. To create pEFIRESpuro-NPAS4-2Myc, mouse NPAS4 cDNA was PCR amplified using mNPAS4 XhoI F 5′ CCGCTCGAGGTCATGTACCGATCCACC 3′ and mNPAS4 SmaI R 5′ GTCCCCCGGGAAACGTTGGTTCCCCTC 3′ and cloned into XhoI/SmaI digested pEFIRESpuro-SIM2-2Myc [20]. pCI-6xmyc-REST (a gift from David Anderson) was subcloned into pENTR1a (Invitrogen) using BamHI/XbaI and 6xMyc-REST

NPAS4 expression is temporally and spatially restricted

NPAS4 expression has been previously shown to be restricted to the central nervous system in rodents and dramatically increased upon KCl-induced depolarization in cultured neurons [11], [12], [15], [30]. To further investigate developmental and depolarisation induced expression, NPAS4 mRNA was compared in mouse embryonic stem cells, primary cortical neurons and regional areas of brain tissue. NPAS4 was barely detectable in pluripotent embryonic stem cells, but readily expressed in unstimulated

Discussion

NPAS4 is a critical player in neuronal activity homeostasis and not surprisingly its expression is tightly regulated (Fig. 1) [11], [12]. The spatiotemporal expression of NPAS4 is characterized by precise restriction to the CNS and tight transient induction following neuronal activity or various forms of physiological and pathological stress [11], [12], [13], [15], [45]. The mechanisms underlying this restriction to CNS and transient response to stressors have been previously unknown. Here we

Funding

This research was supported by the Australian Research Council.

Acknowledgements

We would like to thank Dr. N. Ooe (Sumitomo Chemical), Dr. A.S. Yoo (Washington University), Prof. G.R. Crabtree (Stanford University) and Prof. D. Anderson (California Institute of Technology) for providing reagents/DNA clones.

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