Transcriptional regulation of rat CYP2A3 by nuclear factor 1: identification of a novel NFI-A isoform, and evidence for tissue-selective interaction of NFI with the CYP2A3 promoter in vivo.
Rat CYP2A3 and its mouse and human orthologs are expressed preferentially in the olfactory mucosa. We found previously that an element in the proximal promoter region of CYP2A3 (the nasal predominant transcriptional activating (NPTA) element), which is similar to a nuclear factor 1 (NFI)-binding site, is critical for transcriptional activation of CYP2A3 in vitro. We proposed that this element might be important for tissue-selective CYP2A3 expression. The goals of the present study were to characterize NPTA-binding proteins and to obtain more definitive evidence for the role of NFI in the transcriptional activation of CYP2A3. The NPTA-binding proteins were isolated by DNA-affinity purification from rat olfactory mucosa. Mass spectral analysis indicated that isoforms corresponding to all four NFI genes were present in the purified NPTA-binding fraction. Further analysis of NPTA-binding proteins led to the identification of a novel NFI-A isoform, NFI-A-short, which was derived from alternative splicing of the NFI-A transcript. Transient transfection assay showed that NFI-A2, an NFI isoform previously identified in the olfactory mucosa, transactivated the CYP2A3 promoter, whereas NFI-A-short, which lacks the transactivation domain, counteracted the activation. Chromatin immunoprecipitation assays indicated that NFI proteins are associated with the CYP2A3 promoter in vivo, in rat olfactory mucosa, but essentially not in the liver where the CYP2A3 promoter is hypermethylated and CYP2A3 is not expressed. These data strongly support a role for NFI transcription factors in the transcriptional activation of CYP2A3.
Ling
Guoyu
G
New York State Department of Health, and School of Public Health, Wadsworth Center, State University of New York, Empire State Plaza, Albany, NY 12201, USA.
Hauer
Charles R
CR
Gronostajski
Richard M
RM
Pentecost
Brian T
BT
Ding
Xinxin
X
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D003062:Codon; D065637:Cytochrome P-450 CYP2A6; D003596:Cytosine / Q000378:metabolism; D019175:DNA Methylation; D019281:Dimerization; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D002843:Chromatin; D003062:Codon; D051839:NFI Transcription Factors; C106508:Nfia protein, rat; D020033:Protein Isoforms; D012333:RNA, Messenger; D012097:Repressor Proteins; D003596:Cytosine; D006899:Mixed Function Oxygenases; D008156:Luciferases; C585519:Cyp2a3 protein, rat; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6