Transcription factor NF2d9 (LBP-1a) interacts with the positive regulatory element for the xenobiotic responsive element

Biochimica et Biophysica Acta 1727 (2005) 141 – 144 http://www.elsevier.com/locate/bba

Promoter paper

Transcription factor NF2d9 (LBP-1a) interacts with the positive regulatory element for the xenobiotic responsive element Kouichi Kurose, Masahiro Tohkin*, Ryuichi Hasegawa Division of Medicinal Safety Science, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Received 6 July 2004; received in revised form 12 December 2004; accepted 16 December 2004 Available online 30 December 2004

Abstract PREX is a positive regulatory element for xenobiotic responsive element (XRE)-mediated gene expression that is located upstream of the XRE in the CYP2A8 gene. Using gel mobility shift assays, we demonstrated that NF2d9 (LBP-1a), a transcription factor related to CP2 (LBP-1c/LSF), bound directly to PREX and also interacts indirectly with XRE. Luciferase-reporter gene assays showed that the overexpression of NF2d9 enhanced PREX and XRE-driven CYP2A8 gene transcriptional induction. These findings suggest that the interaction of NF2d9 with PREX and XRE enhances XRE-driven CYP2A8 gene transcriptional induction. D 2004 Elsevier B.V. All rights reserved. Keywords: Arylhydrocarbon receptor; CYP2A8; NF2d9; PREX; Xenobiotic responsive element

Several members of the CYP family, including CYP1A1, 1A2, 1B1, and 2A8, are known to be markedly induced by aromatic hydrocarbons such as 3-methylcholanthrene (3MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin [1,2]. The inducer binds to the arylhydrocarbon receptor (AhR), which heterodimerizes with the AhR nuclear translocator (Arnt). The AhR!Arnt complex then binds to the enhancer element, known as the xenobiotic responsive element (XRE), located in the 5V-flanking region of the gene and induces the expression of target genes. We have studied the induction mode of CYP2A8, a major form of cytochrome P450 that is induced by 3-MC in Syrian hamster liver [1,3,4], and have identified a functional XRE in the regulatory region of the CYP2A8 gene. We also found a novel element, which we designated as PREX (positive regulatory element for XREmediated gene expression), located upstream of XRE in the CYP2A8 gene and demonstrated the presence of unidenti-

Abbreviations: 3-MC, 3-methylcholanthrene; AhR, arylhydrocarbon receptor; Arnt, AhR nuclear translocator; PREX, positive regulatory element for XRE-mediated gene expression; XRE, xenobiotic responsive element * Corresponding author. Tel.: +81 3 3700 1141; fax: +81 3 3700 9788. E-mail address: [email protected] (M. Tohkin). 0167-4781/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbaexp.2004.12.003

fied PREX-binding proteins that are involved in PREX/ XRE-mediated CYP2A8 gene activation but these proteins have not been identified [4]. The PREX sequence includes a hyphenated DNA sequence element composed of a fourbase pair direct repeat motif that is separated by a linker of 5 or 6 nucleotides {CNRG-N5–6-CNR(G/C)}; this DNA sequence is similar to a consensus DNA-binding sequence of the LBP-1 family [5–12] involving NF2d9, a nuclear transcription factor that regulates mouse cyp2d9 gene expression [13]. Therefore, the LBP-1 family might be involved in the activity of the PREX-binding proteins. In this study, the mechanism by which PREX enhances XREmediated gene activation was further clarified by characterizing the nuclear proteins that interact with PREX. To analyze the PREX-binding protein, we performed gel mobility shift assays using hamster liver nuclear extracts and a labeled PREX oligonucleotide as a probe (Fig. 1). Since PREX includes a DNA-binding sequence of the LBP1 family involving NF2d9, anti-NF2d9 antibody was used in the gel-shift assays. The 32P-labeled PREX oligonucleotide probe (5V-TGCCAAGGTTCACAGCTGTGTTGCAATTTACC-3V, 1 pmol, 20,000–30,000 cpm) was mixed with 2 Ag of poly(dI-dC)poly(dI-dC), and 1 Ag of hamster liver nuclear extract in a final volume of 20 Al of solution

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Fig. 1. Gel mobility shift assay of the PREX-binding proteins. Labeled PREX was incubated with Syrian hamster liver nuclear extract in the presence of pre-immune IgG (lane 2) or anti-NF2d9 antibody (lane 3). An excess amount of unlabeled competitor oligonucleotide (PREX; lane 4), unlabeled mutated PREX oligonucleotide (m1; lane 5), or NF2d9 binding element (NF2d9-BE; lane 6) was used, as shown at the top of each lane. The reaction mixtures were then subjected to the gel mobility shift assay.

containing 25 mM HEPES–KOH (pH 7.8), 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10% glycerol (v/v), 50 mM KCl, and 0.5 mM EDTA. AntiNF2d9 antibody (1 Al) or pre-immune IgG (1 Al) were added to the reaction mixture when necessary [13]. Mutated PREX (m1 oligonucleotide, 5V-TGTACGAGTTCACAGCTGTGTTGCAATTTACC-3V, 100 pmol) or the NF2d9 binding element (NF2d9-BE oligonucleotide, 5V-GGCTCCTCCCTATTCCGGGCC-3V, 100 pmol) were added to the reaction mixtures as competitors. The solutions were incubated at 258C for 30 min and then separated by electrophoresis on 4% nondenaturing polyacrylamide gels using 50 mM Tris–HCl (pH 8.5), 0.38 M glycine, and 2 mM EDTA as the running buffer. Gels were then dried and exposed to an imaging plate (Fuji FLA 3000 system) for autoradiography. Two specific bands of PREX–nuclear protein complexes (Fig. 1, lane 1, arrowhead) were outcompeted by unlabeled PREX and the NF2d9 binding element (NF2d9-BE), but not by mutated PREX (m1) (Fig. 1, lanes 4, 5, 6, respectively). Moreover, these two bands of PREX–nuclear protein complexes were super-shifted by the anti-NF2d9 antibody (Fig. 1, lane 3, brackets), but not by pre-immune IgG (Fig. 1, lane 2). These results indicated that NF2d9, or a closely related protein, is involved in the PREX–binding nuclear complexes. Sueyoshi et al. suggested that NF2d9 binds to its binding site as a heteromultimer [13]. Therefore, the two bands observed in our experiment are likely derived from different complexes containing NF2d9.

PREX was identified as the element that enhances XREmediated transcriptional activation by 3-MC and could hardly enhance transcription without XRE [4]. Our previous work using luciferase-reporter constructs pGL2A8-2468, pGL2A8-2409, and pGL2A8-2363, which contain PREX and XRE, XRE alone, and neither, respectively, indicated that pGL2A8-2468, pGL2A8-2409, and pGL2A8-2363 showed full, half, and no 3-MC-inducible transcriptional activities in primary cultured hepatocytes, respectively [4]. Thus, we examined whether the PREX-binding protein, NF2d9, enhances XRE-mediated gene transcriptional activation using these three reporter genes and an NF2d9 expression plasmid. In this NF2d9 expression experiment, we used Drosophila SL2 cells, since we have shown that SL2 cells lack PREX binding factors [4]. Expression plasmids of AhR, Arnt, and Sp1, which are necessary for XRE-mediated gene transcription, were also transfected into SL2 cells because SL2 cells are devoid of these transcription factors [14]. SL2 cells were maintained in Drosophila Schneider’s medium supplemented with 10% fetal bovine serum at 258C. Luciferase reporter gene plasmids (pGL2A82468, pGL2A8-2409, and pGL2A8-2363) and expression plasmids for AhR, Arnt, and Sp1 were prepared as described previously [4]. The NF2d9 expression plasmid, NF2d9/ pCR3, was kindly supplied by Dr. Negishi (National Institute of Environmental Health Sciences, National Institutes of Health, NC USA). Plasmids (3 Ag of each DNA) were introduced into SL2 cells (5106 cells/2 ml of culture medium/35-mm plastic culture dish) using the calciumphosphate method described by Di Nocera and Dawid [15]. After incubation for 72 h, the cells were treated with 1 AM 3MC in 0.1% DMSO or 0.1% DMSO alone for 24 h. Then, a luciferase assay was performed using the Dual-Glo Luciferase assay system (Promega), as described by the manufacturer. As shown in Fig. 2, induction by 3-MC was not observed, regardless of NF2d9 expression, when the reporter construct pGL2A8-2363, which lacked XRE and PREX, was introduced to the SL2 cells. Although the induction of luciferase expression in the presence of pGL2A8-2409, which contained XRE, by 3-MC was less than half of that in the presence of pGL2A8-2468, which contained XRE and PREX, in the primary cultured hepatocytes [4], the induction of the two reporter constructs by 3-MC was almost equivalent in SL2 cells when NF2d9 was not expressed (Fig. 2). Since we have shown that SL2 cells were devoid of PREX-binding factors [4], this result indicated that PREX could not enhance the XRE-mediated CYP2A8 gene in the absence of NF2d9. The overexpression of NF2d9 enhanced the luciferase expression of pGL2A82409 (XRE alone) by two-fold, compared with the absence of NF2d9, while the overexpression of NF2d9 exhibited about a 4-fold enhancement in the luciferase expression of pGL2A8-2468 (both PREX and XRE), compared with the absence of NF2d9 (Fig. 2). These results suggest that NF2d9 may interact not only with PREX, but also with XRE. Therefore, a gel-shift assay was performed using 32P-

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Fig. 2. Effects of NF2d9 on the transcriptional activities of the reporter plasmids containing XRE alone or both XRE and PREX. Schematic representation of the reporter plasmids with different length 5V-deletions is shown on the left side. These reporter plasmids were cotransfected into SL2 cells alone (shaded bar) or together with an expression plasmid for NF2d9 (closed bar) for 72 h. The SL2 cells were then incubated in the absence or presence of 3-MC for 24 h, and the luciferase activities were measured. The luciferase activity of each reporter construct is presented as a relative value to the pGL2A8-2363 plasmid in the absence of 3-MC and NF2d9. Values are shown as the meanFS.D. from more than three independent experiments.

labeled XRE oligonucleotide (5V-AGAGTGTGTGCCATGCAGTGTCACGCAAACACGCAAAGCT-3V) as a probe. As shown in Fig. 3, several protein–XRE complexes were observed at a slower migrating position than the AhR!Arnt– XRE complex (lane 2, brackets), one of which disappeared with the addition of the anti-NF2d9 antibody (lane 3, arrowhead), while pre-immune IgG showed no effect on the protein–XRE complexes (lane 4). Moreover, the addition of the unlabeled NF2d9-BE disrupted the formation of the slower migration complexes although NF2d9-BE did not affect the binding of AhR!Arnt to the XRE (lane 5,

Fig. 3. Gel mobility shift assay of the XRE-binding protein. Labeled XRE was incubated with Syrian hamster liver nuclear extract in the presence (lane 1) or absence of an excess amount of unlabeled XRE (lane 2). AntiNF2d9 antibody (lane 3) or pre-immune IgG (lane 4) was added to the reaction mixture in the absence of an excess amount of unlabeled XRE. An excess amount of unlabeled NF2d9 binding element (NF2d9-BE) was added to the reaction mixture (lane 5). The reaction mixtures were then subjected to the gel mobility shift assay.

arrowhead), suggesting that NF2d9 or a closely related protein interacted with the XRE–binding nuclear protein complexes. These results suggest that NF2d9 or a closely related protein is involved not only in the PREX-binding protein, but also in the XRE–binding nuclear protein complex. Since NF2d9 appears to co-activate AhR!Arnt– XRE-mediated transcription (Fig. 2) and XRE, unlike PREX, has no recognition sequence for the LBP-1 family, NF2d9 or a closely related protein could bind to the AhR!Arnt nuclear protein complex, although no direct evidence has been obtained to support this binding. In consideration of these results and those of our previous report [4], we propose an induction mechanism for CYP2A8 gene expression in which the interactions of NF2d9 with PREX and with the AhR!Arnt–XRE complex are necessary for the full activation of 3-MC dependent CYP2A8 transcription. XRE is found in the regulatory region of various genes, such as CYP1A1, glutathione S-transferase, UDP-glucuronosyltransferase, and AhR repressor, and activates their transcription [16–19]. While the recognition sequence for the LBP-1 family has not been reported in these genes, considering our study in which XRE and PREX were shown to share DNA-binding proteins, including NF2d9, it is conceivable that the LBP-1 family possibly participates in the regulation of these genes. In conclusion, we demonstrated that NF2d9 or its closely related nuclear protein interacts with PREX and XRE to enhance CYP2A8 gene transcription.

Acknowledgments We thank Dr. Negishi (National Institute of Environmental Health Sciences, National Institutes of Health, NC USA) for providing the anti-NF2d9 antibody and NF2d9

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expression plasmid. This study was supported in part by the Japan Health Sciences Foundation (to MT).

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