Characterization of functional heterodimer partners in brain for a bHLH-PAS factor NXF

NXF, a brain-specific bHLH-PAS transcription factor, can regulate the transcription of target genes forming heterodimer complexes, along with several other bHLH-PAS family members (Arnt1, Arnt2, BMAL1) in vitro. To characterize its dimerization partner protein(s) in vivo, we performed a co-immunoprecipitation analysis of whole brain extracts using anti-NXF IgG. In the protein fraction co-precipitating with the NXF protein, in addition to the major precipitate of Arnt2 protein, a faint protein band of Arnt1 protein was consistently observed. The following in vitro co-precipitation analysis with recombinant proteins and yeast-two-hybrid analysis confirmed the specific physical associations. Reporter gene analyses further revealed comparable levels of transcriptional activity with Arnt1:NXF and Arnt2:NXF combinations. mRNA expression for Arnt1 was found in several NXF-containing regions in brain, even an example with no Arnt2 expression. The data thus suggest that Arnt1, as well as Arnt2, could have the separate significance for NXF signaling, with Arnt1:NXF heterodimer complexes in vivo.