The CYP2B2 Phenobarbital Response Unit Contains an Accessory Factor Element and a Putative Glucocorticoid Response Element Essential for Conferring Maximal Phenobarbital Responsiveness
1998; Elsevier BV; Volume: 273; Issue: 14 Linguagem: Inglês
10.1074/jbc.273.14.8528
ISSN1083-351X
AutoresCatherine M. Stoltz, Marie-Hélène Vachon, Eric Trottier, Stéphane Dubois, Yanick Paquet, Alan Anderson,
Tópico(s)Estrogen and related hormone effects
ResumoHepatic cytochrome P450s play a critical role in the metabolism of hydrophobic xenobiotics. One of the major unsolved problems in xenobiotic metabolism is the molecular mechanism whereby phenobarbital induces hepatic enzymes, particularly CYP2B1 and CYP2B2 in rat liver. By using primary rat hepatocytes for transfection analyses, we previously identified in the CYP2B2 5′-flank a 163-base pair Sau3AI fragment that confers phenobarbital inducibility on a cat reporter gene and that has the properties of a transcriptional enhancer. Transfection experiments with sub-regions of the Sau3AI fragment now indicate that a central core together with an upstream or downstream accessory element within the fragment can confer phenobarbital responsiveness. One such accessory element, AF1, was identified and localized. DNase I footprinting analysis revealed the presence of a footprint overlapping this AF1 element. It also identified three other major protected regions, two of which are putative recognition sites for known transcription factors. Site-directed mutagenesis indicated that a putative glucocorticoid response element as well as a nuclear factor 1 site and an associated nuclear receptor hexamer half-site are essential for conferring maximal phenobarbital inducibility. Taken together, the results indicate that phenobarbital induction of CYP2B2requires interactions among multiple regulatory proteins and cis-acting elements constituting a phenobarbital response unit. Hepatic cytochrome P450s play a critical role in the metabolism of hydrophobic xenobiotics. One of the major unsolved problems in xenobiotic metabolism is the molecular mechanism whereby phenobarbital induces hepatic enzymes, particularly CYP2B1 and CYP2B2 in rat liver. By using primary rat hepatocytes for transfection analyses, we previously identified in the CYP2B2 5′-flank a 163-base pair Sau3AI fragment that confers phenobarbital inducibility on a cat reporter gene and that has the properties of a transcriptional enhancer. Transfection experiments with sub-regions of the Sau3AI fragment now indicate that a central core together with an upstream or downstream accessory element within the fragment can confer phenobarbital responsiveness. One such accessory element, AF1, was identified and localized. DNase I footprinting analysis revealed the presence of a footprint overlapping this AF1 element. It also identified three other major protected regions, two of which are putative recognition sites for known transcription factors. Site-directed mutagenesis indicated that a putative glucocorticoid response element as well as a nuclear factor 1 site and an associated nuclear receptor hexamer half-site are essential for conferring maximal phenobarbital inducibility. Taken together, the results indicate that phenobarbital induction of CYP2B2requires interactions among multiple regulatory proteins and cis-acting elements constituting a phenobarbital response unit. Many different cytochrome P450s (CYPs) 1The abbreviations used are: CYP, cytochrome P450; PB, phenobarbital; bp, base pair(s); NF1, nuclear factor 1; PBRU, phenobarbital response unit; GRU, glucocorticoid response unit; PEPCK, phosphoenolpyruvate carboxykinase; oligo, oligodeoxyribonucleotide; CAT, chloramphenicol acetyltransferase; GRE, glucocorticoid response element; HNF-4, hepatocyte nuclear factor-4; dATPαS, deoxyadenosine 5′-(α-thio)triphosphate. are involved in the hepatic metabolism of a wide variety of xenobiotic substances including drugs, plant metabolites, and chemical carcinogens (1Guengerich F.P. Mammalian Cytochromes P-450. 1. CRC Press, Inc., Boca Raton, FL1987Google Scholar, 2Guengerich F.P. Shimada T. Chem. Res. Toxicol. 1991; 4: 391-407Crossref PubMed Scopus (1054) Google Scholar). The genes encoding these enzymes are either expressed constitutively or are induced by various chemicals (3Okey A.B. Pharmacol. Ther. 1990; 45: 241-298Crossref PubMed Scopus (633) Google Scholar). One of the major unsolved problems in the study of the induction of CYP proteins is the molecular mechanism whereby phenobarbital (PB) induces the closely related CYP2B1 and CYP2B2 forms in rat liver (4Denison M.S. Whitlock Jr., J.P. J. Biol. Chem. 1995; 270: 18175-18178Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). Although it has long been known that PB induces CYP2B1 and CYP2B2 mRNAs by increasing transcription of their genes (5Adesnik M. Bar-Nun S. Maschio F. Zunich M. Lippman A. Bard E. J. Biol. Chem. 1981; 256: 10340-10345Abstract Full Text PDF PubMed Google Scholar, 6Hardwick J.P. Gonzalez F.J. Kasper C.B. J. Biol. Chem. 1983; 258: 8081-8085Abstract Full Text PDF PubMed Google Scholar), details of the transcriptional control of CYP2B1 and CYP2B2 have not been forthcoming. This is largely because, until recently, it was not possible to obtain PB induction of the endogenous CYP2B1 and CYP2B2genes in cultured cells (4Denison M.S. Whitlock Jr., J.P. J. Biol. Chem. 1995; 270: 18175-18178Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 7Waxman D.J. Azaroff L. Biochem. J. 1992; 281: 577-592Crossref PubMed Scopus (543) Google Scholar, 8Sinclair P.R. Bement W.J. Haugen S.A. Sinclair J.F. Guzelian P.S. Cancer Res. 1990; 50: 5219-5224PubMed Google Scholar). We have transfected reporter gene constructs into cultured adult rat hepatocytes and localized, in the CYP2B2 5′-flanking region, a 163-base pair (bp)Sau3AI fragment that confers PB inducibility on acat reporter gene (9Trottier E. Belzil A. Stoltz C. Anderson A. Gene (Amst .). 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar). The Sau3AI fragment, which is situated between 2155 and 2317 bp upstream of the CYP2B2transcription start point, in the vicinity of a liver-specific DNase I-hypersensitive site (10Luc P.-V.T. Adesnik M. Ganguly S. Shaw P.M. Biochem. Pharmacol. 1996; 51: 345-356Crossref PubMed Scopus (51) Google Scholar), has the properties of a transcriptional enhancer (9Trottier E. Belzil A. Stoltz C. Anderson A. Gene (Amst .). 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar). The capacity of the Sau3AI fragment to confer PB responsiveness on a heterologous promoter has been confirmed in a quite different assay system involving in situ DNA injection into rat liver (11Park Y. Li H. Kemper B. J. Biol. Chem. 1996; 271: 23725-23728Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Furthermore, the homologous region of the 5′-flanking region of the PB-inducible mouse Cyp2b10 gene contains a segment 91% identical to the rat CYP2B2 163-bp fragment that also confers PB inducibility on heterologous promoters and possesses the properties of a transcriptional enhancer (12Honkakoski P. Negishi M. J. Biol. Chem. 1997; 272: 14943-14949Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The CYP2B2 163-bp Sau3AI fragment contains a functional nuclear factor 1 (NF1) site (9Trottier E. Belzil A. Stoltz C. Anderson A. Gene (Amst .). 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar, 13Hoffmann M. Mager W.H. Scholte B.J. Civil A. Planta R.J. Gene Expr. 1992; 2: 353-363PubMed Google Scholar) and recognition sites for other sequence-specific DNA binding factors present in rat liver nuclear extracts (9Trottier E. Belzil A. Stoltz C. Anderson A. Gene (Amst .). 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar). In the present study, we sought to define the elements within the 163-bp Sau3AI fragment which confer PB responsiveness. Deletion constructs of the Sau3AI fragment, as well as constructs in which putative recognition sites for DNA binding factors had been mutated, were transfected into adult rat hepatocytes, and their effect in conferring PB responsiveness was analyzed. DNase I footprinting experiments were used to identify potential regulatory elements by defining interactions between rat liver nuclear proteins and the Sau3AI fragment. A putative glucocorticoid response element as well as an NF1 site and an associated nuclear receptor hexamer half-site were found to be essential for conferring maximal phenobarbital inducibility. Taken together, the results indicate that the Sau3AI fragment is a multicomponent enhancer and that multiple regulatory proteins and their cognate recognition sequences within it are critical for obtaining maximal PB responsiveness. Thus, the Sau3AI fragment constitutes a PB response unit (PBRU), analogous to the complex glucocorticoid response unit (GRU) required for glucocorticoid induction of transcription of the rat gene for phosphoenolpyruvate carboxykinase (PEPCK) (14Imai E. Stromstedt P.-E. Quinn P.G. Carlstedt-Duke J. Gustafsson J.-Å. Granner D.K. Mol. Cell. Biol. 1990; 10: 4712-4719Crossref PubMed Scopus (267) Google Scholar, 15Hall R.K. Sladek F.M. Granner D.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 412-416Crossref PubMed Scopus (204) Google Scholar, 16Wang J.-C. Strömstedt P.-E. O'Brien R.M. Granner D.K. Mol. Endocrinol. 1996; 10: 794-800PubMed Google Scholar, 17Scott D.K. Mitchell J.A. Granner D.K. J. Biol. Chem. 1996; 271: 31909-31914Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Chee's medium for hepatocyte culture as well as restriction and DNA-modifying enzymes were from Life Technologies, Inc. The double-stranded CTF/NF1 consensus oligodeoxyribonucleotide (oligo) (5′-CCTTTGGCATGCTGCCAATATG-3′) was from Promega or was purchased as complementary single-stranded oligos from Life Technologies. Other oligos were also from Life Technologies, except as noted. [γ-32P]ATP (6000 Ci/mmol), [α-32P]dATP (3000 Ci/mmol), [α-35S]dATPαS (1250 Ci/mmol), [14C]dichloroacetylchloramphenicol (60 Ci/mmol), and [3H]dichloroacetylchloramphenicol (30 Ci/mmol) were from NEN Life Science Products. Male Sprague-Dawley rats (150–180 g) were from Charles River Canada. The methods for hepatocyte isolation and culture, essentially those of Waxman et al. (18Waxman D.J. Morrissey J.J. Naik S. Jauregui H.O. Biochem. J. 1990; 271: 113-119Crossref PubMed Scopus (170) Google Scholar), as well as those for liposome-mediated transfection (19Jacoby D.B. Zilz N.D. Towle H.C. J. Biol. Chem. 1989; 264: 17623-17626Abstract Full Text PDF PubMed Google Scholar) and PB treatment have been described (9Trottier E. Belzil A. Stoltz C. Anderson A. Gene (Amst .). 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar). Plasmids were purified using a plasmid purification kit (Qiagen). CAT activity was assayed by the method of Gorman et al. (20Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (6475) Google Scholar) and, occasionally, by that of Seed and Sheen (21Seed B. Sheen J.-Y. Gene (Amst .). 1988; 67: 271-277Crossref PubMed Scopus (923) Google Scholar). Sequential 5′ or 3′ deletions of the 163-bp Sau3AI fragment (−2317/−2155) were generated by restriction enzyme or Bal31 digestion (Fig. 1). The starting point for construction of such deletion mutants was the pSa-Sa163 plasmid, obtained by subcloning the 163-bpSau3AI fragment into the SmaI site of pBluescript KS (Stratagene). The pSa-Sa163 plasmid or similar pBluescript KS derivatives containing specified portions of the CYP2B25′-flank were used to generate other deletions by polymerase chain reaction-mediated amplification. To obtain −2257/−2208 (Fig. 1) the starting pBluescript derivative contained −2257/−2012, and the primers were KS (Stratagene) and oligo HX (GATCCACTGTGCCAAGGTCAGGA −2226, lower stand; the underlined nucleotides were added for convenience). In a series of amplifications (Fig. 1), primers 2B2Nco (−2257 CATGGTGATTTCAGGCA) and SK (Stratagene) were used as follows: to obtain −2257/−2207 the starting pBluescript derivative contained −2317/−2207; to obtain −2257/−2188 it contained −2317/−2188, and to obtain −2257/−2172 it contained −2317/−2172. Point mutations were introduced into the 163-bp Sau3AI fragment using the Altered Sites II in vitro Mutagenesis System (Promega). In the mutant oligos that follow, substitutions in the wild-type sequence are indicated in boldface: GREm (−2253 GTGATTTCAGGCGTGGACTCTGTACTT), NF1m1, synthesized by Michel Lambert of this Research Center (−2218 TTGGCACAGTGCTTCCATCAACTTGA), NF1dm2 (−2224 CTGACCTTGGTACAGTGCTTC), HXm (−2232 GTACTTTCCTGAGATTGGCACAGT), HXm-NF1dm2 (−2232 GTACTTTCCTGAGATTGGTACAGT). Oligo HXm-NF1dm2 contains, in addition to the HXm mutation, a single mutation in the NF1 sequence; since it was used to mutate a Sau3AI fragment that already carried NF1 m1, it generated a triple mutant sequence. A 3-bp tandem point mutation was also introduced into the AAAG core of the Barbie box of the CYP2B2 promoter using oligo BBm (−94 AGTGAATAGCCAGCTCAGGAGGCGTGA). Deletion and point mutants were subcloned by blunt-ended ligation into the EcoRV site of the non-PB responsive Ev construct which contains 1681 bp of the CYP2B2 5′-flanking region cloned upstream of the cat reporter of pBSCAT (9Trottier E. Belzil A. Stoltz C. Anderson A. Gene (Amst .). 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar). The strategy employed to obtain deletions by Bal31 digestion and by amplification generated an additional 8-nucleotide sequence (5′-GGGGGATC) or, for the deletions obtained with oligo HX, and additional 12-nucleotide sequence (5′-GATCGGGGGATC), between the 3′ end of the deleted portion of the 163-bp Sau3AI fragment and the EcoRV site used for subcloning. The normal orientation and integrity of the subcloned fragments in the reporter constructs were confirmed by DNA sequence analysis. Nuclear extracts were prepared (22Bernier D. Thomassin H. Allard D. Guertin M. Hamel D. Blaquière M. Beauchemin M. LaRue H. Estable-Puig M. Bélanger L. Mol. Cell. Biol. 1993; 13: 1619-1633Crossref PubMed Google Scholar) from pooled livers of two or three untreated or PB-treated (23Labbé D. Jean A. Anderson A. DNA (N. Y.). 1988; 7: 253-260Crossref PubMed Scopus (36) Google Scholar) rats. Where noted, nuclear protein extracts were fractionated by chromatography on heparin-Sepharose using Hitrap Heparin columns (Amersham Pharmacia Biotech). The fragments used for DNase I footprinting analyses were prepared from pSa-Sa163, or from similar plasmids containing a mutated 163-bp Sau3AI fragment, by releasing a 230-bp fragment with EcoRI and EagI digestions. After 3′ end-labeling using the Klenow fragment of Escherichia coli DNA polymerase I or 5′ end-labeling using T4 polynucleotide kinase, the fragment was purified by electrophoresis on a 5% polyacrylamide gel. DNase I footprinting was performed (22Bernier D. Thomassin H. Allard D. Guertin M. Hamel D. Blaquière M. Beauchemin M. LaRue H. Estable-Puig M. Bélanger L. Mol. Cell. Biol. 1993; 13: 1619-1633Crossref PubMed Google Scholar) in 12.5 mm Hepes, pH 7.6, 70 mm KCl, 3.5 mm MgCl2, 5 μm ZnSO4, 1 mm EDTA, 5% glycerol, 0.5 mm NaMoO4, 0.075 mmNonidet P-40, 0.5 mm dithiothreitol, 0.25 mmphenylmethylsulfonyl fluoride containing 5 μg of poly(dI-dC). Reaction volumes were typically 70 μl. Na2β-glycerophosphate, when present, was at 10–20 mm. Transfection analysis was performed with subfragments of the 163-bp Sau3AI fragment to identify sequences within it that might confer PB responsiveness. Sequential deletions from the 5′ end up to the internal NcoI site (coordinate −2257) were all active in conferring PB responsiveness, although the response conferred by construct −2257/−2155 was reduced by about 2-fold as compared with that of the full-length fragment (Fig.1 A, constructs with a common 3′ end point of −2155 and 5′ end points of −2290, −2283, −2273, −2264, or −2257). Further deletion from the 5′ end, to an internalRsaI site (coordinate −2230) or to −2180, led to complete loss of activity (Fig. 1 A). Similarly, sequential 3′ deletions up to −2207 were also active, although the response conferred by constructs −2317/−2188 and −2317/−2207 was again reduced by about 2-fold as compared with that of the full-length fragment (Fig. 1 A, constructs with a common 5′ end point of −2317 and 3′ end points of −2172, −2188, or −2207). Further deletion from the 3′ end, to the RsaI site (coordinate −2231; Fig. 1 A) or to −2253 (construct −2600/−2253; data not shown), led to complete loss of activity. These results suggested that DNA sequence elements that are essential for PB responsiveness are localized in the 51-bp central core between −2257 and −2207. However, construct −2257/−2208 was completely inactive (Fig. 1), although its 5′ extremity is the same as that of the active −2257/−2155 construct and its 3′ extremity is 1 bp upstream of that of the active −2317/−2207 construct. Since construct −2257/−2207 was also inactive (Fig. 1 B), the single base pair difference at the 3′ ends of the −2257/−2208 and −2317/−2207 constructs does not account for the inactivity of the former. Hence, the central core is insufficient by itself to elicit PB responsiveness. The deletion analysis presented above suggests the following model to account for the PB responsiveness conferred by the 163-bp Sau3AI fragment. A central core, together with an element or elements extending upstream of coordinate −2257 or with an element or elements extending downstream of coordinate −2207, can confer PB responsiveness. In support of this model, although construct −2257/−2188 was inactive, construct −2257/−2172 was active in eliciting a PB response (Fig. 1 B). This result indicated that there is a DNA sequence element between coordinates −2188 and −2172 that confers PB responsiveness when combined with the central core. The element between −2188 and −2172 is insufficient to elicit PB responsiveness by itself, because it is present in the inactive −2230/−2155 construct (Fig. 1 A). Hence, it is an accessory site for conferring PB responsiveness, and we have designated it as AF1 (Figs. 1 B and 2). To ascertain whether an accessory factor binds to the AF1 site, as well as to look for evidence of other protein-DNA interactions, the 163-bp Sau3AI fragment was subjected to DNase I footprinting analysis using rat liver nuclear extracts. First, the fragment was labeled on the lower strand at the 3′ end (Fig. 3 A). Analysis using crude extract revealed a series of almost continuous footprints over some 85 bp extending from about −2285 to about −2200 (Fig. 3 A). Use of heparin-Sepharose-fractionated extracts facilitated resolution of protected regions F1, F2, F3, and F4 within this segment: protected region F2 was virtually undetectable with the fractionated extracts, whereas F1, F3 and F4 remained visible (Fig. 3 A). An additional protected region, F0, was revealed after 5′ end-labeling of the lower strand (Fig. 3 B). F0 is also visible above the F1 footprint in Fig. 3 A (crude extract lane). Protected regions F1′, F2′, and F3′, corresponding to F1, F2, and F3 plus F4, respectively, were identified by labeling the 5′ end of the upper strand (Fig. 3 C). Protected region F0′ (corresponding to F0) is also evident above F1′ in Fig. 3 C. The positions of the F0 and F0′ footprints overlap with the AF1 site defined by transfection analysis (Fig. 2). Addition of PB to incubation mixtures did not appreciably change the footprints obtained with crude nuclear extracts of untreated rats (data not shown) or with a nuclear extract partially purified by heparin-Sepharose fractionation (Fig.3 A, lanes PB), and similar footprints were generated by crude nuclear extracts from untreated and PB-treated rats (Fig. 3 B and data not shown). The DNA sequence of the 163-bp Sau3AI fragment (13Hoffmann M. Mager W.H. Scholte B.J. Civil A. Planta R.J. Gene Expr. 1992; 2: 353-363PubMed Google Scholar) (see also Fig. 2) reveals that it contains putative recognition sites for several transcription factors, only some of which are shown in Fig. 2. Potential regulatory motifs were identified by inspection and by application of MatInspector (matrix similarity threshold, 0.85), a search tool for scanning DNA sequences for matches to nucleotide distribution matrices for transcription factor binding sites accessible in the TRANSFAC data base (24Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2432) Google Scholar). Immediately adjacent to a perfectly symmetrical NF1 site (−2217 TGGN7CCA) (25Gronostajski R.M. Adhya S. Nagata K. Guggenheimer R.A. Hurwitz J. Mol. Cell. Biol. 1985; 5: 964-971Crossref PubMed Scopus (107) Google Scholar), previously identified (9Trottier E. Belzil A. Stoltz C. Anderson A. Gene (Amst .). 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar, 13Hoffmann M. Mager W.H. Scholte B.J. Civil A. Planta R.J. Gene Expr. 1992; 2: 353-363PubMed Google Scholar), is a perfect hexamer half-site (AGGTCA −2223, lower strand) for orphan members of the nuclear receptor superfamily (26Tsai M.-J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2727) Google Scholar, 27Jiang G.Q. Nepomuceno L. Hopkins K. Sladek F.M. Mol. Cell. Biol. 1995; 15: 5131-5143Crossref PubMed Scopus (175) Google Scholar). We refer to the combined hexamer half-site and NF1 site (Fig. 2) as the HX·NF1 complex. There are also, in an unusual everted repeat arrangement with a 7-bp spacing (ER-7), two other AGGTCA sequences (coordinates −2282 to −2264) (Fig. 2). In addition, between the internal NcoI site and the HX·NF1 complex, MatInspector finds on each strand a match to a glucocorticoid receptor binding site (TRANSFAC matrix GR_Q6; coordinates −2244 to −2226 on the upper strand and −2246 to −2228 on the lower strand) (Fig. 2). The 51-bp central core between −2257 and −2207 defined by the transfection analyses described above excludes ER-7 but includes the candidate glucocorticoid receptor binding sites and most of the HX·NF1 complex. The F1 and F1′ footprints extend from −2230 to −2192, a region which includes and extends beyond the HX·NF1 complex (Fig. 2). Most of the F1 footprint was eliminated by a CTF/NF1 consensus oligo competitor (Fig. 3 A, lane NF1). Therefore, it is caused in part at least by an NF1 protein. The positions of F3/F4 and F3′ correspond to the two halves of the ER-7 sites, and both F3 and F4 were competed by oligo HX (Fig. 3 A, lane HX). Hence F3/F4 and F3′ are presumably caused by protein(s) of the nuclear receptor superfamily binding to the two halves of ER-7. No good candidates for the protein(s) responsible for the F2 and F2′ footprints are yet available. The region of the F1′ footprint was analyzed using varying amounts of nuclear protein. At lower protein levels, a reduced protected region was observed with the wild-type sequence, presumably as a result of binding of an NF1 protein, whereas at higher protein levels, the footprint was extended in both directions (Fig.4 A). Similar results were obtained with protein extracts from untreated and from PB-treated rats (Fig. 4 A). When a mutant sequence in which NF1 binding was abolished was analyzed, the extended protection on the upstream side was still evident at higher protein levels (Fig. 4 B). Thus, the extended protection on the upstream side is due to binding of a protein or proteins to a sequence including the nuclear receptor hexamer half-site. Consistent with this conclusion is the observation that only a part of the F1 (Figs. 3 A and 5 A) and the F1′ (Fig. 5 B) footprints were eliminated by competition with an NF1 consensus oligo.Figure 5Effect of mutations in the NF1 site on the F1 and F1′ footprints. A, the wild-type fragment or the fragment carrying the NF1 m1 mutation was treated with unfractionated nuclear extract (100 μg of protein) from livers of untreated rats. Fragments were 3′ end-labeled on the lower strand. NF1denotes the presence of a 500-fold molar excess of the NF1 consensus oligo competitor. The position of the F1 footprint is shown by abox, and HS at coordinate −2207 denotes a hypersensitive site. B, the wild-type fragment or the fragment carrying the NF1dm2 mutation was used. Fragments were 5′ end-labeled on the upper strand. BSA denotes DNase I treatment in the absence of nuclear proteins, and control and PB denote DNase I treatment with nuclear extracts (100 μg of protein) from untreated and PB-treated rats, respectively.NF1 denotes the presence of a 500-fold molar excess of the NF1 consensus oligo competitor. The position of the F1′ footprint is shown by a box and that of the footprint remaining after NF1 binding was eliminated by competition or mutation is denoted by abracket labeled HX.View Large Image Figure ViewerDownload (PPT) To investigate the role of the HX·NF1 complex in conferring PB responsiveness, the NF1 site was mutated in two steps as follows: first in the distal portion (NF1m1; Fig. 6) and then in both the distal and proximal portions (NF1dm2; Fig. 6). The NF1 m1 mutation created a new DNase I-hypersensitive site at −2207 and modified the F1 footprint but did not abolish it; furthermore, the portion of the modified F1 footprint corresponding to NF1 sequences was eliminated by competition with an NF1 consensus oligo (Fig.5 A). With the NF1dm2 mutant sequence, the F1′ footprint was reduced to that seen with the wild-type sequence in the presence of an NF1 consensus oligo competitor (Fig. 5 B). Hence, the NF1dm2 mutation completely eliminated detectable NF1 binding, but it left a footprint on the upstream side, corresponding to the anticipated binding of a protein or proteins to a sequence including the nuclear receptor hexamer half-site (Figs. 4 B and 5 B). Here, as elsewhere, identical results were obtained using nuclear extracts prepared from livers of untreated and PB-treated rats (Fig.5 B). The NF1 m1 and NF1dm2 mutations, as well as two additional mutations of the HX·NF1 complex, HXm (mutated in the hexamer half-site) and HXm-NF1dm2 (mutated in the hexamer half-site and in NF1), all reduced but did not abolish PB responsiveness when tested by transfection analysis (Fig. 6). None of these mutations increased the basal level of CAT activity (data not shown). These results demonstrated that both elements of the HX·NF1 complex must be intact to elicit a maximal response to PB. The consensus sequence for the glucocorticoid response element (GRE) is GGTACAnnnTGTTCT (28Beato M. Cell. 1989; 56: 335-344Abstract Full Text PDF PubMed Scopus (3151) Google Scholar). The two candidate glucocorticoid receptor binding sites found by MatInspector within the central core defined by transfection analysis contain putative GREs, one of which, −2244 GGCACAgacTCTGTA on the upper strand, matches the consensus at 7 of 12 positions. This sequence was mutated to GGCGTGgacTCTGTA, thereby reducing the match to 4 of 12. This led to virtual abolition of the PB response (Fig. 6). This suggests that a protein binding in the region of the putative GRE is required to confer PB responsiveness. It has been suggested that the Barbie box (29Liang Q.W. He J.-S. Fulco A.J. J. Biol. Chem. 1995; 270: 4438-4450Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) is involved in conferring PB responsiveness on the rat CYP2B1 and CYP2B2 genes (30He J.S. Fulco A.J. J. Biol. Chem. 1991; 266: 7864-7869Abstract Full Text PDF PubMed Google Scholar). However, when the core sequence (5′-AAAG-3′) of the CYP2B2 Barbie box was mutated, PB responsiveness was retained (Fig. 6). Our previous work has shown that an upstream enhancer element, located between −2317 and −2155 in 5′-flanking region, confers PB responsiveness on the rat CYP2B2 gene (9Trottier E. Belzil A. Stoltz C. Anderson A. Gene (Amst .). 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar). This element is situated in the vicinity of a liver-specific DNase I-hypersensitive site in chromatin (10Luc P.-V.T. Adesnik M. Ganguly S. Shaw P.M. Biochem. Pharmacol. 1996; 51: 345-356Crossref PubMed Scopus (51) Google Scholar). Such sites are a hallmark of regulatory regions (31Grosveld F. van Assendelft G.B. Greaves D.R. Kollias G. Cell. 1987; 51: 975-985Abstract Full Text PDF PubMed Scopus (1608) Google Scholar). Furthermore, in transgenic mice a rat CYP2B2 transgene including only the first 800 bp of the 5′-flank was not PB-inducible, whereas a transgene carrying 19 kb of 5′-flank (and hence the −2317/−2155 segment) was normally inducible (32Ramsden R. Sommer K.M. Omiecinski C.J. J. Biol. Chem. 1993; 268: 21722-21726Abstract Full Text PDF PubMed Google Scholar). The experiments described in our previous report (9Trottier E. Belzil A. Stoltz C. Anderson A. Gene (Amst .). 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar) and in subsequent reports from two other laboratories (11Park Y. Li H. Kemper B. J. Biol. Chem. 1996; 271: 23725-23728Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 12Honkakoski P. Negishi M. J. Biol. Chem. 1997; 272: 14943-14949Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), as well as those described here, provide a strong body of evidence indicating that upstream control elements confer PB responsiveness on the rat CYP2B2 and mouseCyp2b10 genes. The mouse Cyp2b10 active sequences are situated between −2426 and −2250 and display 91% sequence identity to those of CYP2B2 (12Honkakoski P. Negishi M. J. Biol. Chem. 1997; 272: 14943-14949Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Particularly important in this context is the observation that generally similar results have been obtained in three different laboratories using different experimental approaches and PB-inducible CYP2B genes of two different rodent species. The results presented here, showing that at least three and probably more sequence elements within the 163-bp Sau3AI fragment are required to confer maximal PB responsiveness, reveal the surprising complexity of this multicomponent enhancer. According to the now classical model for steroid hormone action, the liganded receptor binds to one or more response elem
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