The CYP2B2 Phenobarbital Response Unit Contains an Accessory Factor Element and a Putative Glucocorticoid Response Element Essential for Conferring Maximal Phenobarbital Responsiveness

Abstract

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. 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 were from Life were from The for hepatocyte and of D.J. S. Biochem. J. 1990; 271: PubMed Scopus Google Scholar), as well as for transfection J. Biol. Chem. Full Text PDF PubMed Google Scholar) and PB have been (9Trottier E. Belzil A. Stoltz C. Anderson A. Gene (Amst .). 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar). were using a was by the of Mol. Cell. Biol. 2: PubMed Scopus Google Scholar) by that of and B. Gene (Amst .). PubMed Scopus Google Scholar). or of the 163-bp Sau3AI fragment were by restriction or The for of such was the by the fragment into the site of The or of the were used to other by obtain the and the were and the were for In a of and were used as to obtain the to obtain it and to obtain it were into the 163-bp Sau3AI fragment using the in In the oligos that in the are indicated in by of this in to the a in the NF1 it was used to a Sau3AI fragment that NF1 it a A was also into the core of the of the CYP2B2 promoter using Deletion and were by into the site of the which contains bp of the CYP2B2 5′-flanking region upstream of the cat reporter of (9Trottier E. Belzil A. Stoltz C. Anderson A. Gene (Amst .). 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar). The to obtain by and by an for the with and between the of the of the 163-bp Sau3AI fragment and the site used for The and of the in the reporter constructs were confirmed by DNA extracts were H. M. M. M. H. M. L. Mol. Cell. Biol. PubMed Google Scholar) from of two or three or A. Anderson A. DNA PubMed Scopus Google Scholar) nuclear extracts were by on using The used for DNase I footprinting were from or from a 163-bp Sau3AI fragment, by a fragment with and using the fragment of DNA I or using the fragment was by on a DNase I footprinting was H. M. M. M. H. M. L. Mol. Cell. Biol. PubMed Google Scholar) in 1 of were was Transfection analysis was with of the 163-bp Sau3AI fragment to identify sequences within it that confer PB responsiveness. from the to the site were in conferring PB the response by was by as with that of the fragment constructs with a of and of or from the to an site or to to of 1 to were also the response by constructs and was by as with that of the fragment 1 constructs with a of and of or from the to the site 1 or to not to of results that DNA elements that are essential for PB responsiveness are in the central core between and was is the as that of the and is 1 bp upstream of that of the was also 1 the base pair the of the and constructs not for the of the the central core is by to PB responsiveness. The analysis the to for the PB responsiveness by the 163-bp Sau3AI fragment. A central together with an element or elements upstream of or with an element or elements downstream of can confer PB responsiveness. In of this was was in a PB response 1 This indicated that is a DNA element between and that confers PB responsiveness with the central The element between and is to PB responsiveness by it is present in the 1 it is an accessory site for conferring PB and we have it as AF1 1 and an accessory factor to the AF1 as well as to for of other the 163-bp Sau3AI fragment was to DNase I footprinting analysis using rat liver nuclear the fragment was on the the using revealed a of bp from to of extracts of protected and within this protected region was with the and protected region, was revealed of the is also the footprint in A and to and were identified by the of the region to is also in C. The of the and with the AF1 site by transfection analysis of PB to not the with nuclear extracts of not or with a nuclear by and were by nuclear extracts from and and not The DNA of the 163-bp Sau3AI fragment M. Mager W.H. Scholte B.J. Civil A. Planta R.J. Gene Expr. 1992; 2: 353-363PubMed Google Scholar) also that it contains putative recognition sites for transcription of which are in regulatory were identified by and by of a for DNA sequences for to for transcription factor binding sites in the base H. E. T. Res. 1995; PubMed Scopus Google Scholar). to a NF1 site R.M. S. J. Mol. Cell. Biol. PubMed Scopus 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 hexamer half-site for of the nuclear receptor Biochem. PubMed Scopus Google Scholar, L. Sladek F.M. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). We to the hexamer half-site and NF1 site as the are in an with a two other sequences to In between the site and the on a to a glucocorticoid receptor binding site to on the and to on the The central core between and by the transfection the glucocorticoid receptor binding sites and of the The and from to a region which and the complex of the footprint was by a CTF/NF1 consensus it is in by an NF1 The of and to the two of the and and were by and are by of the nuclear receptor binding to the two of for the for the and are The region of the footprint was using of nuclear a protected region was with the as a of binding of an NF1 the footprint was in results were with extracts from and from a in which NF1 binding was was the on the upstream was Thus, the on the upstream is to binding of a or proteins to a including the nuclear receptor hexamer with this is the that a of the A and and the were by with an NF1 consensus of in the NF1 site on the and the fragment or the fragment the NF1 was with nuclear of from of were on the the presence of a of the NF1 consensus The of the footprint is by and a the fragment or the fragment the was were on the DNase I in the of nuclear and control and PB DNase I with nuclear extracts of from and the presence of a of the NF1 consensus The of the footprint is by a and that of the footprint NF1 binding was by or is by the role of the complex in conferring PB the NF1 site was in two as in the and in the and The NF1 a DNase I-hypersensitive site and the footprint not the of the footprint to NF1 sequences was by with an NF1 consensus the the footprint was to that with the in the presence of an NF1 consensus the NF1 it a footprint on the upstream to the binding of a or proteins to a including the nuclear receptor hexamer half-site and as identical results were using nuclear extracts from of and The NF1 and as well as two of the in the hexamer and in the hexamer half-site and in not PB responsiveness by transfection analysis of these the of not results that elements of the complex be to a maximal response to The consensus for the glucocorticoid response element is M. Cell. Full Text PDF PubMed Scopus Google Scholar). The two glucocorticoid receptor binding sites found by within the central core by transfection analysis putative of on the the consensus of This was to the to of This to of the PB response This that a binding in the region of the putative is required to confer PB responsiveness. It has been that the J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) is involved in conferring PB responsiveness on the rat CYP2B1 and CYP2B2 genes J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). the core of the CYP2B2 was mutated, PB responsiveness was has that an upstream enhancer element, between and 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 (10Luc P.-V.T. Adesnik M. Ganguly S. Shaw P.M. Biochem. Pharmacol. 1996; 51: 345-356Crossref PubMed Scopus (51) Google Scholar). sites are a of regulatory F. Cell. 51: Full Text PDF PubMed Scopus Google Scholar). Furthermore, in a rat CYP2B2 including the bp of the 5′-flank was not a of 5′-flank the was J. Biol. Chem. Full Text PDF PubMed Google Scholar). The experiments in (9Trottier E. Belzil A. Stoltz C. Anderson A. Gene (Amst .). 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar) and in from two other (11Park Y. Li H. Kemper B. J. Biol. Chem. 1996; 271: 23725-23728Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, P. Negishi M. J. Biol. Chem. 1997; 272: 14943-14949Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), as well as a of that upstream control elements confer PB responsiveness on the rat CYP2B2 and The mouse Cyp2b10 sequences are situated between and and 91% to of CYP2B2 (12Honkakoski P. Negishi M. J. Biol. Chem. 1997; 272: 14943-14949Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). in this is the that results have been in three different using different and PB-inducible genes of two different The results that three and elements within the 163-bp Sau3AI fragment are required to confer maximal PB the of this multicomponent enhancer. to the now for the receptor to or response elements upstream of the gene and as a transcriptional enhancer Biochem. PubMed Scopus Google Scholar). of a variety of transcription factors with response elements have been known for M. C. PubMed Scopus Google Scholar), and in a of genes cis-acting elements in to transcription Li M. Mol. Endocrinol. 1997; Google Scholar). The such is that of the Full glucocorticoid induction of transcription is by a complex of two glucocorticoid response as well as three accessory factor binding sites (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, 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). We to the CYP2B2 Sau3AI fragment as a element (9Trottier E. Belzil A. Stoltz C. Anderson A. Gene (Amst .). 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar), in the of we now to it as a A. E. Stoltz C. Y. S. J. 1997; Scholar, and A. on and Scholar). The homologous mouse Cyp2b10 has been a (12Honkakoski P. Negishi M. J. Biol. Chem. 1997; 272: 14943-14949Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, P. Negishi M. J. Biochem. Mol. Toxicol. PubMed Scopus Google Scholar). that can as accessory factors in the glucocorticoid response hepatocyte nuclear J.-C. Strömstedt P.-E. O'Brien R.M. Granner D.K. Mol. Endocrinol. 1996; 10: 794-800PubMed Google Scholar) and the upstream promoter transcription factor R.K. Sladek F.M. Granner D.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 412-416Crossref PubMed Scopus (204) 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) and hepatocyte nuclear R.K. Sladek F.M. Granner D.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 412-416Crossref PubMed Scopus (204) Google Scholar). The of the to PB of CYP2B2 not that recognition site is between the two response that PB induction of CYP2B2 interactions among multiple regulatory proteins and cis-acting elements constituting a The essential element of the is that element within the 163-bp Sau3AI fragment is required to confer PB responsiveness. results we that a central core of the fragment, between and is can confer PB responsiveness either with upstream or downstream accessory elements within the fragment. In of this such accessory AF1, has been between and AF1 an accessory factor binding The for the footprint also be for AF1 these with the AF1 The of PB responsiveness of is the as that of 1 and are the This that sequences between and are not essential for maximal PB responsiveness that a accessory element be present between and PB responsiveness is in constructs to the putative accessory element between and it is also possible that the responsiveness of the of the is a of of essential sequences the of the central are to these In the of the accessory sites not PB responsiveness. of the accessory sites not glucocorticoid responsiveness 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). results indicate that the complex is required for maximal PB responsiveness NF1 proteins are transcription factors S. S. Res. 1992; PubMed Scopus Google Scholar), different of which are of a U. U. Res. 1990; PubMed Scopus (163) Google Scholar). The NF1 consensus binding sites are of two and by a or and the a region this from DNase I R.M. S. J. Mol. Cell. Biol. PubMed Scopus Google Scholar). NF1 is for of the footprint and the role of NF1 is which NF1 or NF1 binding not PB responsiveness This is to that of and Negishi (12Honkakoski P. Negishi M. J. Biol. Chem. 1997; 272: 14943-14949Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) with a different NF1 that not PB responsiveness by a of the of the CYP2B2 fragment. In of the nuclear receptor half-site element, as in or of that element and NF1, as in also PB that is have to of of NF1 In the mouse Cyp2b10 and Negishi (12Honkakoski P. Negishi M. J. Biol. Chem. 1997; 272: 14943-14949Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) a effect of such in PB responsiveness. the complex is essential for conferring maximal PB responsiveness. The binding to the hexamer half-site the of the footprint is are of the the complex a recognition site for the nuclear transcription factor H. M. M. M. H. M. L. Mol. Cell. Biol. PubMed Google Scholar, L. L. J. S. L. Mol. Cell. Biol. 1996; PubMed Google Scholar), and a to recognition sites for three other including upstream promoter transcription factor and experiments have revealed binding of Cytochromes CYP2B2 Scholar) and transcription factor L. L. and A. to the double-stranded of these or other proteins with the complex to maximal PB responsiveness to be The of the proteins for the and is are of the central core identified as essential for PB responsiveness the accessory AF1 The for the is a of the nuclear receptor of the with site and the footprint is by the the hexamer The region is not required for PB it is not an essential it be an accessory were by and Negishi (12Honkakoski P. Negishi M. J. Biol. Chem. 1997; 272: 14943-14949Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) with to the that of a putative to a major in PB responsiveness is particularly as it the that a glucocorticoid be involved in conferring PB responsiveness. The putative the consensus of the of the (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), it is not a has been on particularly the J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar) and their possible role in conferring PB responsiveness on the rat CYP2B1 and CYP2B2 of the in the of the CYP2B2 promoter and 5′-flank not PB responsiveness either in rat hepatocytes transfected by injection in situ (11Park Y. Li H. Kemper B. J. Biol. Chem. 1996; 271: 23725-23728Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) or in transfected primary rat hepatocytes sequences are required for transcriptional (9Trottier E. Belzil A. Stoltz C. Anderson A. Gene (Amst .). 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar, P. Negishi M. J. Biochem. Mol. Toxicol. PubMed Scopus Google Scholar), is present for their in conferring PB responsiveness. The sequences of the of CYP2B1 and CYP2B2 are identical Y. Y. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Y. Y. M. Y. Proc. Natl. Acad. Sci. U. S. A. 1983; PubMed Scopus Google Scholar), and this 1 of their A. E. Anderson A. DNA PubMed Scopus Google Scholar) and of their 5′-flanking M. Mager W.H. Scholte B.J. Civil A. Planta R.J. Gene Expr. 1992; 2: 353-363PubMed Google Scholar, P.M. M. J. Adesnik M. 1996; PubMed Scopus Google Scholar). the that the of liver CYP2B1 and CYP2B2 to PB are the of CYP2B2 is that of CYP2B1 M. Biochem. 258: PubMed Scopus Google Scholar, M. Biochem. 256: PubMed Scopus Google CYP2B1 and as were a gene L. P. S. P.G. S.A. A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar, J. Kemper B. J. Biol. Chem. 1997; 272: PubMed Scopus Google Scholar). are in the of CYP2B1 and in CYP2B2 is not is expressed constitutively is not PB-inducible M. Biochem. 256: PubMed Scopus Google Scholar). the 163-bp Sau3AI fragment is except for a identical in in CYP2B1 and CYP2B2 M. Mager W.H. Scholte B.J. Civil A. Planta R.J. Gene Expr. 1992; 2: 353-363PubMed Google Scholar, P.M. M. J. Adesnik M. 1996; PubMed Scopus Google Scholar) also we have to to the between CYP2B1 and CYP2B2 in the It that the CYP2B2 and are in the and that is on of the presence of other transcription the of which on of the between the CYP2B1 and CYP2B2 5′-flanking The molecular details as to the presence of PB to of transcription the to be a of results by in footprinting that PB the of transcription factors with the in J. Kemper B. J. Biol. Chem. 1997; 272: PubMed Scopus Google Scholar). Although this for the role of the in transcriptional of the proteins that to it be to the We and of for and to for rat for the for on DNase I Belzil for and for and and of the for