title: Effects of NAD(P)H and its derivatives on the DNA-binding activity of NPAS2, a mammalian circadian transcription factor.
abstract: NPAS2 is a transcription factor that regulates mammalian circadian rhythms. It has been suggested that NPAS2 DNA-binding activity is regulated by the intracellular redox state of NAD(P)H, although the mechanism remains unclear. To investigate the NAD(P)H interaction site of murine NPAS2, we performed electrophoretic mobility shift assays using several truncation mutants of the NPAS2 bHLH domain. Among the mutants, NPAS2 containing the N-terminal 61 residues formed a heterodimer with BMAL1 to bind DNA, and NAD(P)H enhanced the binding activity, while NAD(P)H inhibited the DNA-binding activity of the BMAL1 homodimer in a dose-dependent manner. NAD(P)H derivatives such as 2',5'-ADP, nicotinamide, nicotinic acid and nicotinic acid adenine dinucleotide (NAAD) did not affect the DNA-binding activity. Interestingly, NAD(P)(+), previously reported as an inhibitor, did not affect NPAS2 binding activity in the presence or absence of NAD(P)H in our system. These results suggest that NPAS2 DNA-binding activity is specifically enhanced by NAD(P)H independently of NAD(P)(+) and that the N-terminal 1-61 amino acids of NPAS2 are sufficient to sense NAD(P)H.
authors.affiliation: Graduate School of Life and Environmental Science, Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Yoshii
authors.forename: Sumio
authors.initials: S
authors.lastname: Ishijima
authors.forename: Ikuko
authors.initials: I
authors.lastname: Sagami
mesh_terms: D056930:ARNTL Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D002940:Circadian Rhythm / Q000235:genetics / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D051379:Mice; D009249:NADP / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D055503:Protein Multimerization / Q000235:genetics; D017384:Sequence Deletion; D015854:Up-Regulation / Q000235:genetics
chemical_list: D056930:ARNTL Transcription Factors; D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D009249:NADP; D009419:Nerve Tissue Proteins; C538960:BMAL1 protein, human; C491259:Npas2 protein, mouse
keywords: Clock genes; DNA-binding activity; E-box; EMS; HAT; NAAD; NAD(P)H cofactor; NPAS2; PAS domain; Transcription; electrophoretic mobility shift; histone acetyltransferase; neuronal PAS domain protein 2; nicotinic acid adenine dinucleotide