Transcriptional Regulation of Rat CYP2A3 by Nuclear Factor 1
2004; Elsevier BV; Volume: 279; Issue: 27 Linguagem: Inglês
10.1074/jbc.m403705200
ISSN1083-351X
AutoresGuoyu Ling, Christoph Hauer, Richard M. Gronostajski, Brian T. Pentecost, Xinxin Ding,
Tópico(s)Biochemical Analysis and Sensing Techniques
ResumoRat 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. 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. The cytochrome P450 (CYP) 1The abbreviations used are: CYP, cytochrome P450; OM, olfactory mucosa; MS, mass spectral; EMSA, electrophoretic mobility shift assays; RT, reverse transcription; NFI, nuclear factor 1; ChIP, chromatin immunoprecipitation; NPTA, nasal predominant transcriptional activating; PBS, phosphate-buffered saline; PIPES, piperazine-1,4-bis(2-ethanesulfonic acid); PMSF, phenylmethanesulfonyl fluoride; IP, immunoprecipitation; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. 1The abbreviations used are: CYP, cytochrome P450; OM, olfactory mucosa; MS, mass spectral; EMSA, electrophoretic mobility shift assays; RT, reverse transcription; NFI, nuclear factor 1; ChIP, chromatin immunoprecipitation; NPTA, nasal predominant transcriptional activating; PBS, phosphate-buffered saline; PIPES, piperazine-1,4-bis(2-ethanesulfonic acid); PMSF, phenylmethanesulfonyl fluoride; IP, immunoprecipitation; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. superfamily of heme-containing monooxygenases metabolize a wide variety of exogenous as well as endogenous compounds. For example, enzymes of the CYP2A subfamily metabolize xenobiotic compounds such as coumarin, aflatoxin B1, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, nicotine, and cotinine and endogenous compounds such as testosterone, progesterone, and other steroid hormones (1Ding X. Kaminsky L.S. Annu. Rev. Pharmacol. Toxicol. 2003; 43: 149-173Crossref PubMed Scopus (643) Google Scholar, 2Fernandez-Salguero P. Gonzalez F.J. Pharmacogenetics. 1995; 5: S123-S128Crossref PubMed Scopus (92) Google Scholar, 3Honkakoski P. Negishi M. Drug Metab. Rev. 1997; 29: 977-996Crossref PubMed Scopus (73) Google Scholar). Several members of the CYP2A subfamily, including rat CYP2A3, mouse Cyp2a5, rabbit CYP2A10, and human CYP2A13, are expressed preferentially in the respiratory tract with the most abundant expression in the olfactory mucosa (OM) (4Ding X. Dahl A.R. Doty R.L. Handbook of Olfaction and Gustation. Marcel Dekker, Inc., New York2003: 51-73Google Scholar). The tissue-selective expression of these enzymes is believed to play an important role in the tissue-specific toxicity of numerous xenobiotic compounds in the respiratory tract (1Ding X. Kaminsky L.S. Annu. Rev. Pharmacol. Toxicol. 2003; 43: 149-173Crossref PubMed Scopus (643) Google Scholar, 4Ding X. Dahl A.R. Doty R.L. Handbook of Olfaction and Gustation. Marcel Dekker, Inc., New York2003: 51-73Google Scholar), and a genetic polymorphism of CYP2A13 has been linked to decreased risks of smoking-related lung adenocarcinoma in humans (5Wang H. Tan W. Hao B. Miao X. Zhou G. He F. Lin D. Cancer Res. 2003; 63: 8057-8061PubMed Google Scholar). The mechanisms that regulate the basal expression and the tissue selectivity of the CYP2A genes are not clear. Previously, we identified a conserved DNA element (named the nasal predominant transcriptional activating element, or NPTA element) in the rat CYP2A3 promoter that interacted with OM-enriched proteins (6Zhang J. Ding X. J. Biol. Chem. 1998; 273: 23454-23462Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The NPTA element, which is similar to the nuclear factor 1 (NFI)-binding sites (7Gronostajski R.M. Gene (Amst.). 2000; 249: 31-45Crossref PubMed Scopus (420) Google Scholar), was crucial for the activity of the CYP2A3 promoter in vitro. The NPTA-binding proteins have not been fully characterized, but in vitro studies showed that at least some of the NPTA-binding complexes are recognized by an antibody to NFI (8Zhang J. Zhang Q.-Y. Guo J. Zhou Y. Ding X. J. Biol. Chem. 2000; 275: 8895-8902Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). There are four NFI genes in mammals, NFI-A, -B, -C, and -X, from which numerous NFI isoforms are generated through alternative splicing or alternative promoter usage. NFI proteins are highly conserved in the amino-terminal DNA binding/dimerization domains, whereas the carboxyl termini contain diverse transactivation/repression domains (7Gronostajski R.M. Gene (Amst.). 2000; 249: 31-45Crossref PubMed Scopus (420) Google Scholar). NFI isoforms are thought to be involved in the regulation of developmental and tissue-specific gene expression (7Gronostajski R.M. Gene (Amst.). 2000; 249: 31-45Crossref PubMed Scopus (420) Google Scholar, 9Chaudhry A.Z. Lyons G.E. Gronostajski R.M. Dev. Dyn. 1997; 208: 313-325Crossref PubMed Scopus (174) Google Scholar, 10Kannius-Janson M. Johansson E.M. Bjursell G. Nilsson J. J. Biol. Chem. 2002; 277: 17589-17596Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Recent studies using knockout mouse models have demonstrated that the NFI genes play important roles in the development of tissues such as brain, lung, and tooth (11das Neves L. Duchala C.S. Tolentino-Silva F. Haxhiu M.A. Colmenares C. Macklin W.B. Campbell C.E. Butz K.G. Gronostajski R.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11946-11951Crossref PubMed Scopus (179) Google Scholar, 12Grunder A. Ebel T.T. Mallo M. Schwarzkopf G. Shimizu T. Sippel A.E. Schrewe H. Mech. Dev. 2002; 112: 69-77Crossref PubMed Scopus (83) Google Scholar, 13Steele-Perkins G. Butz K.G. Lyons G.E. Zeichner-David M. Kim H.J. Cho M.I. Gronostajski R.M. Mol. Cell. Biol. 2003; 23: 1075-1084Crossref PubMed Scopus (155) Google Scholar). The expression of the NFI genes in the OM has been studied using molecular cloning and in situ hybridization; all four NFI genes are expressed in the OM (14Baumeister H. Gronostajski R.M. Lyons G.E. Margolis F.L. Brain Res. Mol. Brain Res. 1999; 72: 65-79Crossref PubMed Scopus (29) Google Scholar, 15Behrens M. Venkatraman G. Gronostajski R.M. Reed R.R. Margolis F.L. Eur. J. Neurosci. 2000; 12: 1372-1384Crossref PubMed Scopus (36) Google Scholar). However, the identities of the specific isoforms have not been determined, with the exception of NFI-A2, which is a recently identified NFI isoform that is detected in the OM (14Baumeister H. Gronostajski R.M. Lyons G.E. Margolis F.L. Brain Res. Mol. Brain Res. 1999; 72: 65-79Crossref PubMed Scopus (29) Google Scholar, 16Xie Y. Madelian V. Zhang J. Ling G. Ding X. Biochem. Biophys. Res. Commun. 2001; 289: 1225-1228Crossref PubMed Scopus (8) Google Scholar). Most interesting, we found that NFI-A2 was capable of activating a reporter construct containing the NPTA element in yeast one-hybrid assays (16Xie Y. Madelian V. Zhang J. Ling G. Ding X. Biochem. Biophys. Res. Commun. 2001; 289: 1225-1228Crossref PubMed Scopus (8) Google Scholar). In the present study, 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 isoform, named NFI-A-short, that has a truncated transactivation/repression domain. The tissue distribution of NFI-A-short and the levels of NFI-A2, NFI-A-short, and total NFI-A mRNAs were determined by reverse transcription (RT)-PCR, and the functions of the two NFI-A isoforms at the CYP2A3 promoter were analyzed by using transient transfection assays in cultured mammalian cells. Furthermore, chromatin immunoprecipitation (ChIP) assays using rat liver and OM tissues demonstrated a tissue-selective association of NFI with the CYP2A3 promoter in vivo. In addition, the methylation status of the CYP2A3 promoter region in these two tissues was also compared, so as to derive a better understanding of the mechanistic basis of the tissue-specific interaction of the promoter with NFI. These studies provide compelling evidence for the in vivo role of NFI transcription factors in the expression of CYP2A3. Nuclear Extract Preparation and Electrophoretic Mobility Shift Assays (EMSA)—Heads of adult male Wistar rats (Harlen Bioproducts) were frozen in liquid nitrogen immediately after decapitation. Frozen rat heads were partially thawed at 4 °C before dissection of OM. Nuclear extract was prepared according to the protocol of Kudrycki et al. (17Kudrycki K. Stein-Izsak C. Behn C. Grillo M. Akeson R. Margolis F.L. Mol. Cell. Biol. 1993; 13: 3002-3014Crossref PubMed Scopus (101) Google Scholar). EMSA was performed as described previously, with a double-stranded NPTA element (6Zhang J. Ding X. J. Biol. Chem. 1998; 273: 23454-23462Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Reporter Plasmid Construction, Cell Culture, Transfection, and Luciferase Reporter Assays—For the preparation of the CYP2A3-luciferase reporter gene construct, a 254-bp CYP2A3 proximal promoter region (–254 to +1), with either a wild-type (pGL2A3) or a mutated (pGL2A3M) NPTA element (6Zhang J. Ding X. J. Biol. Chem. 1998; 273: 23454-23462Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), was inserted into the pGL3-Basic vector (Promega). Expression vectors for NFI-A2 (pNFI-A2) and NFI-A-short (pNFI-A-short) were modified from the pCHNFI-A1.1 vector that has a cytomegalovirus promoter and the mouse NFI-A1 coding sequence (9Chaudhry A.Z. Lyons G.E. Gronostajski R.M. Dev. Dyn. 1997; 208: 313-325Crossref PubMed Scopus (174) Google Scholar). pNFI-A2 was prepared by replacement of the NFI-A1 sequence with NFI-A2, as a 1.6-kb NotI-KpnI fragment (16Xie Y. Madelian V. Zhang J. Ling G. Ding X. Biochem. Biophys. Res. Commun. 2001; 289: 1225-1228Crossref PubMed Scopus (8) Google Scholar). pNFI-A-short was generated by replacement of the EcoRV-KpnI fragment of NFI-A1 with an ∼600-bp EcoRV-KpnI fragment of NFI-A-short; the mouse NFI-A1 sequence (NotI-EcoRV) that remained in the vector encodes the same amino acids as in rat NF1-A1. A vector with no NFI insert (pCMV), used as a control, was prepared by modification of pCHNFI-A1.1. All constructs were sequenced for confirmation of structural integrity. Human choriocarcinoma JEG-3 cells (HTB-36, ATCC) were cultured at 37 °C in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin (Sigma). Firefly luciferase reporter gene constructs, NFI expression constructs, and a Renilla luciferase construct pRL-SV40 (Promega) were co-transfected into JEG-3 cells with use of LipofectAMINE 2000 (Invitrogen), according to the manufacturer's instructions. Transfections were done in duplicate using at least two different DNA preparations. Cells were harvested 48 h after transfection. The Dual Luciferase Reporter Assay System (Promega) was used for determination of the relative luciferase activities. Luminescence was measured using a luminometer (LB9501, Berthold). For each sample, the activity of the firefly luciferase was normalized by that of the Renilla luciferase. Chromatin Immunoprecipitation Assay—ChIP was performed as described (18Wells J. Farnham P.J. Methods. 2002; 26: 48-56Crossref PubMed Scopus (207) Google Scholar), with modifications. Approximately 1 g of freshly dissected rat liver or OM from four rats was used for the preparation of chromatin. Tissues were minced on ice and transferred to 20 ml of Dulbecco's modified Eagle's medium in a 50-ml tube, to which formaldehyde was added to a final concentration of 1% (v/v), for cross-linking of proteins to DNA. The mixture was incubated at room temperature for 15 min with constant rocking. Reactions were stopped by the addition of 0.125 m glycine. The tissues were collected by a brief spin, washed with ice-cold phosphate-buffered saline, and homogenized in 4 ml of phosphate-buffered saline containing a protease inhibitor mixture (1836153, Roche Applied Science) using a Dounce homogenizer (type A pestle, 10 strokes). The dissociated cells were filtered through four layers of cheesecloth, transferred to microcentrifuge tubes, and collected by spinning at 2000 × g for 10 min. Cells were washed with RSB buffer (10 mm Tris-HCl, pH 7.5, containing 10 mm NaCl, 3 mm MgCl2, supplemented with the Roche Applied Science protease inhibitor mixture) and were incubated with cell lysis buffer (5 mm PIPES buffer, pH 8.0, containing 85 mm KCl, 0.5% Nonidet P-40, supplemented with the protease inhibitor mixture from Roche Applied Science) for 10 min at 4 °C. After incubation, the nuclei were collected by centrifugation and were resuspended in 500 μl of immunoprecipitation (IP) buffer (25 mm Tris-HCl, pH 8.0, containing 2 mm EDTA, 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 2.5 mm PMSF, and the protease inhibitor mixture (Roche Applied Science)) in 1.5-ml microcentrifuge tubes. About 0.1 g of glass beads (G-1277, Sigma) were added to each tube, and the nuclei were sonicated on ice 20 times for 15 s each time, at 30% of maximum power, with a VibraCell ultrasonic processor (600 watts) equipped with a microtip (Sonics & Materials). Fragmented chromatin was separated from the glass beads and intact nuclei by centrifugation at 12,000 × g for 15 min at 4 °C. Aliquots of chromatin suspension were stored at –80 °C until use. Sonicated chromatin, equivalent to ∼60 mg of tissue for each tube, was diluted to 1.5 ml with the IP buffer and was mixed at 4 °C for 2 h with 0.1 ml of protein A-Sepharose (Amersham Biosciences) that had been pretreated with 0.4 mg/ml sonicated salmon sperm DNA and 1 mg/ml bovine serum albumin. After a brief centrifugation, two 0.7-ml aliquots of the supernatant were transferred to two new tubes, which were incubated overnight on a rotating platform with 3 μg of either normal rabbit IgG or the anti-NFI antibody H300 (both from Santa Cruz Biotechnology). The H300 antibody is against the conserved amino-terminal half of an NFI protein. The mixtures were then incubated with 30 μl of protein A-Sepharose at 4 °C for 2 h, followed by a brief centrifugation. An aliquot of the supernatant from the samples incubated with normal IgG was saved as the “input” chromatin. The pellets were washed twice with 1.4 ml of wash buffer I (50 mm Tris-HCl, pH 8.0, containing 2 mm EDTA, 0.2% Sarkosyl, and 1 mm PMSF) and four times with 1.4 ml of wash buffer II (100 mm Tris-HCl, pH 9.0, containing 500 mm LiCl, 1% Nonidet P-40, 1% deoxycholic acid, and 1 mm PMSF). The immobilized immune complexes were dissociated by incubating twice in 250 μl of elution buffer (0.1 m NaHCO3 and 1% SDS) at room temperature for 15 min each. The cross-link was subsequently reversed by addition of NaCl to the eluted fractions to a final concentration of 300 mm and then heating at 68 °C for 6 h in the presence of 20 μg/ml RNase (Roche Applied Science, catalog number 1579681). DNA was then precipitated by ethanol, resuspended in 100 μl of PK buffer (10 mm Tris-HCl, pH 7.5, containing 5 mm EDTA, 0.25% SDS, and 20 μg of proteinase K), and after a 1-h incubation at 45 °C, purified using QIAquick columns (Qiagen). RT-PCR—Real time quantitative PCR was performed using a Light-Cycler and the LightCycler FastStart DNA Master SYBR Green I kit (Roche Applied Science). The reaction mixtures contained 2 μl of DNA templates, 1.5 mm MgCl2, 0.5 μm each of the primers, and 1 μl of 1× DNA Master SYBR Green I in a total volume of 10 μl. The following primers were used to quantitate the promoter fragments in immunoprecipitated samples: for CYP2A3, 5′-tccctgttaatctgtcatggag-3′ and 5′-tgggatgacagacacagtga-3′, amplifying –169 to +16 of the CYP2A3 promoter; for CYP1A2, 5′-tggaactgagggatcatggctt-3′ and 5′-aagctaaggtgggtccctgttt-3′, amplifying –142 to +140 of the CYP1A2 promoter; and for immunoglobulin-β (IgB), 5′-ttcaagtcctagcagaccag-3′ and 5′-agggtctggggcaaaacatg-3′, amplifying –173 to +5 of the IgB promoter (19Osano K. Ono M. Eur. J. Biochem. 2003; 270: 2532-2539Crossref PubMed Scopus (9) Google Scholar). The CYP2A3 and CYP1A2 PCR products include NFI-like elements, whereas the IgB PCR product (used as a negative control) does not. The annealing temperature for these primer pairs was 65 °C. Cycle numbers were used for calculation of the amounts of specific DNA sequences in the immunoprecipitated samples, relative to the amounts present in total input, as described previously (20Frank S.R. Schroeder M. Fernandez P. Taubert S. Amati B. Genes Dev. 2001; 15: 2069-2082Crossref PubMed Scopus (418) Google Scholar). Standard curves for quantitation were generated from serial dilutions of the input chromatin. Fold enrichment of a promoter fragment is the fold difference between its amount in the NFI antibody-precipitated DNA and its amount in the control IgG-precipitated DNA. Restriction Analysis of DNA Methylation—Genomic DNA from rat OM and liver (50 μg each) was digested overnight with SacI and PvuII, 250 units each, in a total volume of 100 μl. After confirmation of complete digestion, the DNA was precipitated by ethanol and was resuspended in water. The resuspended DNA was then divided into two aliquots, which were incubated overnight with 200 units of either HpaII (methylation-sensitive) or MspI (methylation-insensitive). After digestion, the enzymes were inactivated by heating at 65 °C, for 20 min, and the DNA fragments were detected on Southern blots, with a 2070-bp SacI-PvuII CYP2A3 promoter fragment as the probe. Other Methods—For SDS-PAGE, proteins were separated in 10% BisTris NuPage gels, according to the manufacturer's instruction (Invitrogen), and detected using a Colloidal Blue kit or a SilverQuest silver staining kit from Invitrogen. Western blot analysis was carried out using an ECL reagent (Amersham Biosciences), with use of the anti-NFI antibody (H300). RNA was prepared from adult Wistar rats (Charles River Breeding Laboratories), with use of TRIzol reagent (Invitrogen). A 3′-rapid amplification of cDNA end kit (Invitrogen) was used to clone the putative, short NFI isoforms, with an NFI-A genespecific primer (5′-ctgatggtgaacgccttgtg-3′) and the universal amplification primer. PCR products were gel-purified and then cloned into the TOPO XL vector (Invitrogen). Experimental procedures for the purification and MS analysis of NPTA-binding proteins and methods for RT-PCR quantification of NFI-A mRNAs are described in the Supplemental Material. 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 OM. A DNA fragment containing 12 copies of the NPTA element was prepared and was used to generate NPTA-Sepharose and NPTA-Dynabead. Nuclear extract (∼100 mg of total protein, from ∼200 rat heads) was fractionated on a 1-ml NPTA-Sepharose column; bound proteins were eluted by increasing the KCl concentration stepwise, in 100 mm increments. Column fractions were analyzed for NPTA binding activity by EMSA. The highest activity was found in the 0.6 m KCl fraction (Fig. 1A). Additional clean-up steps before the NPTA-Sepharose column, either anion-exchange or cation-exchange column chromatography, did not improve yield (data not shown). Proteins in the 0.5–0.7 m KCl fractions were pooled and dialyzed to remove KCl and were further purified using NPTA-Dynabeads. Strong NPTA binding activity was recovered in the 0.6 m NaCl fraction (Fig. 1B). This fraction contained numerous proteins, most of which had molecular masses between 20 and 50 kDa (Fig. 1C). The sequence of the NPTA element is similar to sequences of known NFI-binding sites. Therefore, we suspected that the affinity-purified NPTA-binding proteins contain multiple NFI isoforms. Indeed, NFI proteins were highly enriched in the 0.6 m NaCl fraction, as indicated by Western blot analysis using an anti-NFI antibody that recognizes all NFI isoforms (Fig. 1D). Proteins encoded by each of the four NFI genes were detected in the affinity-purified fractions, as indicated by the presence of peptides unique to each gene (Table I), although the peptides detected did not permit the distinction among specific NFI proteins of each type (such as NFI-A1 and NFI-A2). However, data from MS analysis did not indicate the presence of any other known transcription factors, which suggested that the NFI proteins are the major NPTA-binding proteins. Furthermore, the relative abundance of the unique NFI-A peptides was 2–8-fold greater than the abundance of peptides of the other NFI proteins in three independent experiments (data not shown), suggesting that, in rat OM, NFI-A is the most abundant among the four types of NFI proteins. Also of note, various peptides derived from NFI-A were found with either unmodified or phosphorylated serines at residues 191 and 193. Moreover, all of the NFI-related peptides identified in the affinity-purified fractions were from the 30-kDa NH2-terminal half of full-length NFI proteins, as shown in Fig. 1E for peptides that correspond to an NFI-A isoform.Table INFI gene-specific peptides detected in affinity-purified NPTA-binding proteins of rat OMGeneGenBank™ accession numberNFI gene-specific peptides detected by MS analysisNFI-ABAA11203AVKDELLSEKPEVK, DELLSEKPEVK, DIRPEYREDFVLTVTGKNFI-BAAF23586AVKDELLSEKPEIKNFI-CAAF23587EDFVLAVTGK, APGCVLSNPDQK, FVLAVTGKNFI-XBAA25295AVKDELLGEKPEIK, DIRPEFR, EDFVLTITGK Open table in a new tab Identification of A Novel NFI-A Isoform That Contains Truncated Activation/Repression Domain—The absence of any peptides corresponding to the carboxyl-terminal portion of the identified NFI proteins suggested that proteolysis was occurring during purification, although efforts had been made to minimize protein degradation. However, this finding also suggested the possible occurrence of unidentified, shorter NFI isoform(s) corresponding to the ∼30-kDa proteins detected by SDS-PAGE and Western blot analysis of both the purified fraction and OM nuclear extract. NFI isoforms with truncated carboxyl-terminal activation/repression domains have been reported in other species (7Gronostajski R.M. Gene (Amst.). 2000; 249: 31-45Crossref PubMed Scopus (420) Google Scholar, 21Grunder A. Qian F. Ebel T.T. Mincheva A. Lichter P. Kruse U. Sippel A.E. Gene (Amst.). 2003; 304: 171-181Crossref PubMed Scopus (21) Google Scholar). To detect mRNAs encoding putative, short NFI isoforms, we performed 3′-rapid amplification of cDNA end experiments with rat OM RNA and primers specific for NFI-A. Several cDNA clones with sequences corresponding to a novel, short NFI-A isoform (designated NFI-A-short; GenBank™ accession number AY572794) were isolated. A full-length NFI-A-short cDNA (∼0.9 kb) was subsequently obtained by RT-PCR, using OM RNA as template, with a primer corresponding to the 5′-end of NFI-A1 (GenBank™ accession number D78017) and an NFI-A-short-specific primer, derived from the putative 3′-untranslated region of the short mRNA. A comparison of the cDNA sequence of NFI-A-short with that of NFI-A1 revealed that the two sequences are identical, except that in NFI-A-short exon 5 was spliced to an alternative exon 6 (Fig. 2A), which encodes three amino acids followed by a stop codon (Fig. 2B). The resulting NFI-A-short protein (about 31 kDa) has an intact DNA binding and dimerization domain (encoded by exon 2) but lacks most of the transactivation/repression domain. Tissue distribution of NFI-A-short mRNA was determined using RT-PCR, with a forward primer derived from exon 5, and a reverse primer that is unique for NFI-A-short mRNA. The PCR product representing NFI-A-short mRNA (∼250 bp) was detected in RNA preparations from all rat tissues examined, including OM, lung, liver, brain, and kidney (Fig. 2C). In experiments not shown, the same primer pair amplified a 1.1-kb fragment from rat genomic DNA. This 1.1-kb PCR product contained an 865-bp intron sequence flanked by exons 5 and 6 of NFI-A-short. A comparison of the sequence of this PCR product with that of the rat NFI-A genomic sequence (GenBank™ accession number NW_043856) indicated that the new exon 6 in NFI-A-short is located at ∼5 kb upstream of the original exon 6 of the NFI-A gene (Fig. 2A). We did not detect a similar NFI-A-short in mice (data not shown), which indicated that this alternative splicing may be specific for rats. The relative mRNA levels of total NFI-A, NFI-A2, and NFI-A-short in rat OM, liver, lung, brain, and kidney were determined by using real time quantitative RT-PCR, with gene-specific or isoform-specific primers. As shown in Table II, of the five tissues, liver had the highest and lung had the lowest levels of total NFI-A mRNA. The expression level of NFI-A-short was low in all tissues examined (∼0.5–2% of total NFI-A mRNA). NFI-A2, which had been shown previously (16Xie Y. Madelian V. Zhang J. Ling G. Ding X. Biochem. Biophys. Res. Commun. 2001; 289: 1225-1228Crossref PubMed Scopus (8) Google Scholar) to be enriched in the OM, accounted for 2.7% of total NFI-A mRNA and was about twice as abundant as NFI-A-short in OM. The presumed low abundance of NFI-A-short and NFI-A2 proteins in the OM is consistent with our failure to detect the unique carboxyl-terminal tryptic peptide of NFI-A-short and the unique amino-terminal tryptic peptide of NFI-A2 by MS analysis of total peptides derived from the affinity-purified OM NPTA-binding proteins.Table IIQuantitative analysis of NFI-A mRNA expression in various rat tissuesTissueTotal NFI-ANFI-A-shortNFI-A2copy number per 103 copies of β-actinOM27 ± 20.35 ± 0.050.72 ± 0.04aSignificantly higher than NFI-A-short; p < 0.01.Liver60 ± 170.59 ± 0.15NDbND, not determined and not detected in a previous study (16).Brain30 ± 70.15 ± 0.02NDLung7.7 ± 0.40.07 ± 0.01NDKidney14 ± 40.29 ± 0.10NDa Significantly higher than NFI-A-short; p < 0.01.b ND, not determined and not detected in a previous study (16Xie Y. Madelian V. Zhang J. Ling G. Ding X. Biochem. Biophys. Res. Commun. 2001; 289: 1225-1228Crossref PubMed Scopus (8) Google Scholar). Open table in a new tab NFI-A2 and NFI-A-short Form Homo- and Heterodimers When Bound to the CYP2A3 Promoter—We transfected human choriocarcinoma JEG-3 cells with plasmids containing NFI-A2 or NFI-A-short cDNA to characterize further the abilities of NFI-A2 and NFI-A-short to bind to the NPTA element and the potential interactions between the two NFI proteins. The expression of NFI protein and the presence of NPTA binding activity in transfected cells were determined by immunoblot analysis (using an antibody that reacts with all NFI proteins) and by EMSA (using 32P-labeled NPTA element), respectively. Extracts of mock-transfected JEG-3 cells had low endogenous NFI proteins (Fig. 3A), and they had no detectable NPTA binding activity (Fig. 3B). NFI-A2 and NFI-A-short proteins were easily detected following transient transfection, and they had the following expecte
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