Molecular analysis of NPAS3 functional domains and variants

Abstract

BackgroundNPAS3 encodes a transcription factor which has been associated with multiple human psychiatric and neurodevelopmental disorders. In mice, deletion of Npas3 was found to cause alterations in neurodevelopment, as well as a marked reduction in neurogenesis in the adult mouse hippocampus. This neurogenic deficit, alongside the reduction in cortical interneuron number, likely contributes to the behavioral and cognitive alterations observed in Npas3 knockout mice. Although loss of Npas3 has been found to affect proliferation and apoptosis, the molecular function of NPAS3 is largely uncharacterized outside of predictions based on its high homology to bHLH–PAS transcription factors. Here we set out to characterize NPAS3 as a transcription factor, and to confirm whether NPAS3 acts as predicted for a Class 1 bHLH–PAS family member.ResultsThrough these studies we have experimentally demonstrated that NPAS3 behaves as a true transcription factor, capable of gene regulation through direct association with DNA. NPAS3 and ARNT are confirmed to directly interact in human cells through both bHLH and PAS dimerization domains. The C-terminus of NPAS3 was found to contain a functional transactivation domain. Further, the NPAS3::ARNT heterodimer was shown to directly regulate the expression of VGF and TXNIP through binding of their proximal promoters. Finally, we assessed the effects of three human variants of NPAS3 on gene regulatory function and do not observe significant deficits.ConclusionsNPAS3 is a true transcription factor capable of regulating expression of target genes through their promoters by directly cooperating with ARNT. The tested human variants of NPAS3 require further characterization to identify their effects on NPAS3 expression and function in the individuals that carry them. These data enhance our understanding of the molecular function of NPAS3 and the mechanism by which it contributes to normal and abnormal neurodevelopment and neural function.Electronic supplementary materialThe online version of this article (10.1186/s12867-018-0117-4) contains supplementary material, which is available to authorized users.

Subcellular Localization Signals of bHLH-PAS Proteins: Their Significance, Current State of Knowledge and Future Perspectives

“…Very recently, Luoma et al tested the localization of the canonical human NPAS3 isoform (933aa) in HEK293T cells. The bHLH domain was localized in the cytoplasm, what they discussed as consistent with the result of their predictions with NetNES server [77]. However, no experimental verification of predicted NES was performed.…”

“…The expressed C-terminal part of protein (451-951aa) was detected exclusively in the nucleus. Further analysis revealed that NPAS3 alone is present both in nucleus and cytoplasm, while co-expression with ARNT resulted in mainly nuclear localization [77]. Our predictions (Table 1, Supplementary Materials 1, Supplementary Materials Summary) revealed high probability of the presence of NLSs in bHLH domain (29-78aa), PAS-1 domain (130-161aa), linker between PAS domains (266-297aa) and C-terminal part of protein (727-773aa).…”

The Participation of the Intrinsically Disordered Regions of the bHLH-PAS Transcription Factors in Disease Development

“…In fact, V304I was identified as an NPAS3 missense variant associated with psychiatric disorders. Although the V304I mutation located in the PAS linker did not alter the protein’s molecular function, mutation in the disordered region of NPAS3 led to the aggregation of this protein, which resulted in schizophrenia [ 111 , 112 ]. This has led us to hypothesize that some mutations could impact IDRs, thus promoting their misfolding and aggregation.…”

The Participation of the Intrinsically Disordered Regions of the bHLH-PAS Transcription Factors in Disease Development

“…V304I mutation located in PAS linker did not altered protein' molecular function, however was sequestered in the insoluble fraction of cell lysates, suggesting aggregation of protein. The second mutation A552P located in C-terminus, was documented to be sufficient for activation of reporter gene expression [105].…”