Effects of mutations in the heme domain on the transcriptional activity and DNA-binding activity of NPAS2.
The heme domain of neuronal PAS domain protein 2 (NPAS2), a transcription factor that regulates the mammalian circadian rhythm, has been suggested to act as a sensor for carbon monoxide. To characterize the role of the heme domain in this function, we investigated the effects of PASA domain mutants, in the context of full-length NPAS2, on the transcriptional activity of the mouse Period 1 gene in NIH3T3 cells. Mutation of the endogenous ligand for ferrous heme (H119A or H171A) resulted in remarkably reduced transcriptional activity. In gel-shift assays, H119A or H171A mutants of the isolated basic helix-loop-helix (bHLH)-PASA domain impaired heterodimer formation with BMAL1, resulting in loss of DNA binding to the canonical E-box (CACGTG). These results indicate that the transcriptional activities of the mutants correlated well with their DNA-binding activities, suggesting that local conformational changes near the axial ligands of the PASA domain are responsible for its regulation of transcription.
Ishida
Mashiho
M
Graduate School of Agriculture, Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.
Ueha
Takeshi
T
Sagami
Ikuko
I
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D015533:Transcriptional Activation / Q000502:physiology*
D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D006418:Heme; D009419:Nerve Tissue Proteins; C491259:Npas2 protein, mouse