Abstract
Neuronal Per-Arnt-Sim homology (PAS) Factor 4 (NPAS4) is a neuronal activity-dependent transcription factor which heterodimerises with ARNT2 to regulate genes involved in inhibitory synapse formation. NPAS4 functions to maintain excitatory/inhibitory balance in neurons, while mouse models have shown it to play roles in memory formation, social interaction and neurodegeneration. NPAS4 has therefore been implicated in a number of neuropsychiatric or neurodegenerative diseases which are underpinned by defects in excitatory/inhibitory balance. Here we have explored a broad set of non-synonymous human variants in NPAS4 and ARNT2 for disruption of NPAS4 function. We found two variants in NPAS4 (F147S and E257K) and two variants in ARNT2 (R46W and R107H) which significantly reduced transcriptional activity of the heterodimer on a luciferase reporter gene. Furthermore, we found that NPAS4.F147S was unable to activate expression of the NPAS4 target gene BDNF due to reduced dimerisation with ARNT2. Homology modelling predicts F147 in NPAS4 to lie at the dimer interface, where it appears to directly contribute to protein/protein interaction. We also found that reduced transcriptional activation by ARNT2 R46W was due to disruption of nuclear localisation. These results provide insight into the mechanisms of NPAS4/ARNT dimerisation and transcriptional activation and have potential implications for cognitive phenotypic variation and diseases such as autism, schizophrenia and dementia.
Highlights
Basic Helix-Loop-Helix Per-Arnt-Sim homology proteins are signal regulated and/or tissue specific dimeric transcription factors involved in a diverse array of physiological and pathological functions [1,2,3]
The overwhelming majority of variants were able to activate the reporter to a similar extent as wild type (WT) Neuronal PAS factor 4 (NPAS4), variant F147S completely ablated the ability of NPAS4 to activate the reporter (Fig. 1B)
NPAS4 expression most strongly overlaps with ARNT2 within the brain, in biochemical experiments NPAS4 appears to show little bias between binding and activating transcription with ARNT1 or ARNT2 [53,54]
Summary
Basic Helix-Loop-Helix Per-Arnt-Sim homology (bHLH-PAS) proteins are signal regulated and/or tissue specific dimeric transcription factors involved in a diverse array of physiological and pathological functions [1,2,3]. They mediate processes such as the cellular response to hypoxia (Hypoxia Inducible Factors (HIF1a/HIF2a)) [2], the maintenance of circadian rhythms (Circadian Locomotor Output Cycles Kaput (CLOCK)) [4], the clearance of environmental pollutants (Aryl hydrocarbon Receptor (AhR)/Dioxin Receptor (DR)) [1], and appetite control (Single minded 1 (Sim1)) [5,6]. Conditional deletion of NPAS4 in the CA3 region of the hippocampus in adult mice has shown it is required for contextual memory formation [11]
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