Transcriptionally active heterodimer formation of an Arnt-like PAS protein, Arnt3, with HIF-1a, HLF, and clock.
We isolated a cDNA clone encoding a polypeptide of 626 amino acids containing basic helix-loop-helix (bHLH) and PAS domains from a mouse cDNA library of P19 cells. This protein, termed Arnt3, showed the highest similarity to Arnt and Arnt2 in the bHLH and PAS regions. Arnt3 mRNA was expressed in brain, skeletal muscle, 13.5-day embryos, and P19 cells treated with retinoic acid. The partner PAS proteins of Arnt3 were searched for by the two-hybrid system in yeast, and HIF-1 alpha, HLF, and Clock among various bHLH/PAS proteins were found. Gel mobility shift analysis using nuclear extracts from 293T cells cotransfected with Arnt3 and HIF-1 alpha (or HLF) expression plasmids revealed that these complexes specifically bound the hypoxia-response element (HRE). Coexpression of Arnt3 and HIF-1 alpha (or HLF) in Arnt-deficient c4 cells enhanced transcription of a reporter gene driven by the HRE sequences. We also showed that Arnt3 contained an activation domain at the C-terminal region and a repression domain between the PAS-A and PAS-B regions.
Department of Chemistry, Graduate School of Science, Tohoku University, Sendai, Japan.
S
S
Takahata
K
K
Sogawa
A
A
Kobayashi
M
M
Ema
J
J
Mimura
N
N
Ozaki
Y
Y
Fujii-Kuriyama
D056930:ARNTL Transcription Factors; D000595:Amino Acid Sequence; D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D050976:Basic-Leucine Zipper Transcription Factors; D056926:CLOCK Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D005656:Fungal Proteins / Q000096:biosynthesis; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016350:Leucine Zippers; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic
D056930:ARNTL Transcription Factors; C538960:BMAL1 protein, human; C538961:Bmal1 protein, mouse; D050976:Basic-Leucine Zipper Transcription Factors; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; C497442:HIF1A protein, human; C490671:HLF protein, human; C497443:Hif1a protein, mouse; C490670:Hlf protein, mouse; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D056926:CLOCK Proteins; C538943:CLOCK protein, human; C538944:Clock protein, mouse