Abstract
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.
Highlights
Rat CYP2A3 and its mouse and human orthologs are expressed preferentially in the olfactory mucosa
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
Purification and Identification of NPTA-binding Proteins—We developed a two-step DNA-affinity chromatography scheme to identify proteins that bind to the NPTA element of CYP2A3 in rat olfactory mucosa (OM)
Summary
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 tissueselective CYP2A3 expression. 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. We used DNA-affinity chromatography to isolate the CYP2A3 NPTA-binding proteins from rat OM for identification by immunoblot and mass spectral (MS) analyses. These efforts have confirmed NFI as the major NPTA-binding proteins and have led to the identification of a novel NFI-A
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