Transcriptional Regulation of Mouse μ-Opioid Receptor Gene

Abstract

Previously, the existence of dual promoters was reported in mouse μ-opioid receptor (mor) gene, with mor transcription in the mouse brain predominantly initiated by the proximal promoter. In this study, we further analyzed the proximal promoter region, base pairs −450 to −249, to identifycis-DNA regulatory elements and trans-acting protein factors that are important for mor promoter activity. The results revealed that a mor inverted GA (iGA) motif and a canonical Sp1 binding site are required for the promoter activity. Using electrophoretic mobility shift analysis, we identified nuclear proteins that specifically bind to the mor iGA motif and that are immunologically related to Sp1 and Sp3. Mutation of the mor iGA motif, resulting in a loss of Sp binding, led to a 50% decrease in activity. Mutation of the canonical Sp1 binding site yielded a lesser (approximately 25%) loss of activity. Mutation of both motifs together resulted in an approximately 70% decrease in activity. In cotransfection assays using Drosophila SL2 cells, Sp1trans-activated the promoter in a manner dependent on the presence of mor iGA and canonical Sp1 binding motifs. Sp3 can alsotrans-activate the promoter, and furthermore, Sp1 and Sp3 can trans-activate the mor promoter additively. Our results suggest that combined or cooperative interaction of Sp transcription factors within the proximal promoter is necessary for activation of mor gene transcription. Previously, the existence of dual promoters was reported in mouse μ-opioid receptor (mor) gene, with mor transcription in the mouse brain predominantly initiated by the proximal promoter. In this study, we further analyzed the proximal promoter region, base pairs −450 to −249, to identifycis-DNA regulatory elements and trans-acting protein factors that are important for mor promoter activity. The results revealed that a mor inverted GA (iGA) motif and a canonical Sp1 binding site are required for the promoter activity. Using electrophoretic mobility shift analysis, we identified nuclear proteins that specifically bind to the mor iGA motif and that are immunologically related to Sp1 and Sp3. Mutation of the mor iGA motif, resulting in a loss of Sp binding, led to a 50% decrease in activity. Mutation of the canonical Sp1 binding site yielded a lesser (approximately 25%) loss of activity. Mutation of both motifs together resulted in an approximately 70% decrease in activity. In cotransfection assays using Drosophila SL2 cells, Sp1trans-activated the promoter in a manner dependent on the presence of mor iGA and canonical Sp1 binding motifs. Sp3 can alsotrans-activate the promoter, and furthermore, Sp1 and Sp3 can trans-activate the mor promoter additively. Our results suggest that combined or cooperative interaction of Sp transcription factors within the proximal promoter is necessary for activation of mor gene transcription. μ-opioid receptor inverted GA mutated GA motif electrophoresis mobility shift assay polymerase chain reaction base pair(s) fragment b oligonucleotides containing the canonical Sp1 binding site oligonucleotides containing 3-bp mutation at the canonical Sp1 site. Opioids can relieve pain without affecting sensory modalities such as vision and hearing, making these drugs the preferred clinical analgesics for severe pain. Three major types of opioid receptors, μ, δ, and κ, have recently been cloned and shown to belong to the G-protein-coupled receptor superfamily (1Kieffer B.L. Cell. Mol. Neurobiol. 1995; 15: 615-635Crossref PubMed Scopus (355) Google Scholar). Based largely on pharmacological and clinical observations, μ-opioid receptor (mor)1 has traditionally been considered the main site of interaction of the major clinically used analgesics, particularly morphine (2Wood P.L. Iyengar S. Pasternak G.W. The Opiate Receptors. 1988: 307-356Crossref Google Scholar). The critical role of mor in analgesia, as well as in the development of tolerance and dependence, has been further confirmed by pharmacological analysis of knockout mice in which this receptor has been eliminated (3Matthes H.W. Maldonado R. Simonin F. Valverde O. Slowe S. Kitchen I. Befort K. Dierich A. Le Meur M. Dolle P. Tzavara E. Hanoune J. Roques B.P. Kieffer B.L. Nature. 1996; 383: 819-823Crossref PubMed Scopus (1424) Google Scholar, 4Sora I. Takahashi N. Funada M. Ujike H. Revay R.S. Donovan D.M. Miner L.L. Uhl G.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1544-1549Crossref PubMed Scopus (473) Google Scholar). Mor is mainly expressed in the central nervous system, with receptors varying in densities in different regions and perhaps playing different roles (5Mansour A. Fox C.A. Akil H. Watson S.J. Trends Neurosci. 1995; 18: 22-29Abstract Full Text PDF PubMed Scopus (1086) Google Scholar, 6Minami M. Satoh M. Neurosci. Res. 1995; 23: 121-145Crossref PubMed Scopus (282) Google Scholar, 7Delfs J.M. Kong H. Mestek A. Chen Y., Yu, L. Reisine T. Chesselet M.F. J. Comp. Neurology. 1994; 345: 46-68Crossref PubMed Scopus (203) Google Scholar). For example, mor located in the periaqueductal gray has been suggested to mediate analgesia (2Wood P.L. Iyengar S. Pasternak G.W. The Opiate Receptors. 1988: 307-356Crossref Google Scholar, 8Basbaum A.I. Kuhar M.J. Pasternak G.W. Analgesics: Neurochemical, Behavioral, and Clinical Perspectives. Raven Press, New York1984: 97-117Google Scholar), whereas mor located in the locus coeruleus and ventral tegmental areas may be involved in the development of tolerance and physical dependence (9Maldonado R. Saiardi A. Valverde O. Samad T.A. Roques B.P. Borrelli E. Nature. 1997; 388: 586-589Crossref PubMed Scopus (378) Google Scholar, 10Nestler E.J. Alreja M. Aghajanian G.K. Brain Res. Bull. 1994; 35: 521-528Crossref PubMed Scopus (123) Google Scholar, 11Hyman S.E. Science. 1996; 273: 611-612Crossref PubMed Scopus (32) Google Scholar). The development of different roles for mor in different central nervous system areas must ultimately depend on how its expression is regulated in these areas. It is likely that this expression is modified by fluctuating levels of various agents in certain brain regions (12Azaryan A.V. Coughlin L.J. Buzas B. Clock B.J. Cox B.M. J. Neurochem. 1996; 66: 443-448Crossref PubMed Scopus (78) Google Scholar, 13Delfs J.M., Yu, L. Ellison G.D. Reisine T. Chesselet M.F. J. Neurochem. 1994; 63: 777-780Crossref PubMed Scopus (41) Google Scholar, 14Azaryan A.V. Clock B.J. Cox B.M. Neurochem. Res. 1996; 21: 1411-1415Crossref PubMed Scopus (35) Google Scholar, 15Ronnekleiv O.K. Bosch M.A. Cunningham M.J. Wagner E.J. Grandy D.K. Kelly M.J. Neurosci. Lett. 1996; 216: 129-132Crossref PubMed Scopus (37) Google Scholar), as well as in certain disease state (16Maekawa K. Minami M. Masuda T. Satoh M. Pain. 1996; 64: 365-372Abstract Full Text PDF PubMed Scopus (54) Google Scholar). This raises the possibility of increasing the clinical efficacy of morphine and other opioids, and perhaps also of treating certain diseases, by manipulation of mor levels. We have previously isolated and partially sequenced the mouse mor gene, which is over 53 kilobases long and its coding sequence divided into four exons (17Min B.H. Augustin L.B. Felsheim B.F. Fuchs J.A. Loh H.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9081-9085Crossref PubMed Scopus (162) Google Scholar). Using deletional and transient transfection assays, a distal and a proximal promoter of mouse mor gene were identified in the cell lines endogenously expressing mor (18Ko J.L. Minnerath S.R. Loh H.H. Biochem. Biophys. Res. Commun. 1997; 234: 351-357Crossref PubMed Scopus (56) Google Scholar, 19Liang Y. Mestek A., Yu, L. Carr L.G. Brain Res. 1995; 679: 82-88Crossref PubMed Scopus (59) Google Scholar). The distal promoter initiated the mor transcription from a single transcription initiation site, which was located 794 bp upstream of the translation initiation site (19Liang Y. Mestek A., Yu, L. Carr L.G. Brain Res. 1995; 679: 82-88Crossref PubMed Scopus (59) Google Scholar). The proximal promoter initiated mor transcription from four major transcription initiation sites, located in a region 291–268 bp upstream of the translation initiation site (17Min B.H. Augustin L.B. Felsheim B.F. Fuchs J.A. Loh H.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9081-9085Crossref PubMed Scopus (162) Google Scholar). Furthermore, utilizing the reverse transcription-polymerase chain reaction (RT-PCR) assay, we found that mor mRNA in the brain was predominantly initiated by the proximal promoter, in a ratio of approximately 20:1 (proximal:distal) (18Ko J.L. Minnerath S.R. Loh H.H. Biochem. Biophys. Res. Commun. 1997; 234: 351-357Crossref PubMed Scopus (56) Google Scholar). Thus, the proximal promoter appears to be the principal promoter of regulation of the mor gene in the brain. DNA sequence analysis indicates that the proximal promoter (17Min B.H. Augustin L.B. Felsheim B.F. Fuchs J.A. Loh H.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9081-9085Crossref PubMed Scopus (162) Google Scholar) of the mouse mor gene lacks a consensus TATA box (20Gill G. Tjian R. Curr. Opin. Genet. Dev. 1992; 2: 236-242Crossref PubMed Scopus (104) Google Scholar), a consensus initiator (21Smale S.T. Baltimore D. Cell. 1989; 57: 103-113Abstract Full Text PDF PubMed Scopus (1151) Google Scholar, 22Javahery R. Khachk A. Lo K. Zenzie-Gregory B. Smale S.T. Mol. Cell. Biol. 1994; 14: 116-127Crossref PubMed Scopus (597) Google Scholar), or GC-rich sequences (23Briggs M.R. Kadonaga J.T. Bell S.P. Tjian R. Science. 1986; 234: 47-52Crossref PubMed Scopus (1059) Google Scholar). However, analysis indicated that this promoter does possess a canonical Sp1 binding site, (G/T)(G/A)GGC(G/T)(G/A)(G/A)(G/T) (24Faisst S. Meyer S. Nucleic Acids Res. 1992; 20: 3-26Crossref PubMed Scopus (938) Google Scholar). It has been suggested that the Sp1 transcription factor could tether preinitiation complexes to the promoter by interacting with TFIID (20Gill G. Tjian R. Curr. Opin. Genet. Dev. 1992; 2: 236-242Crossref PubMed Scopus (104) Google Scholar, G. E. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1994; Scopus (473) Google Scholar, N. D. M.F. Chen J.L. A. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar), which is for the transcription at both TATA and promoters B.F. Tjian R. Dev. PubMed Scopus Google Scholar). a of and were which or A. Mol. Cell. Biol. 1992; PubMed Scopus Google Scholar, G. S. M. G. Nucleic Acids Res. 1992; 20: PubMed Scopus Google Scholar). In the study, was to DNA elements and which were required for the expression of mouse mor gene, with on the proximal promoter. We that a mor iGA region, the of transcription sites, is for mor gene This site Sp1 and Sp3 or related In a canonical Sp1 binding site, located bp upstream of the mor iGA motif, also to mor promoter activity. of Sp transcription factors to both has an on activation of transcription. Sp proteins are the transcription factors A. Mol. Cell. Biol. 1992; PubMed Scopus Google Scholar, G. S. M. G. Nucleic Acids Res. 1992; 20: PubMed Scopus Google Scholar, S.P. Mol. Cell. Biol. PubMed Scopus Google Scholar), are as of the expression of the mor Furthermore, of regulatory proteins S. Mol. Cell. Biol. PubMed Scopus Google Scholar, K. R. S. B. J. Biol. 1994; Full Text PDF PubMed Google Scholar, M. A. F. R. Y. 1989; Full Text PDF PubMed Scopus Google Scholar) or proteins D. S. E. M. G. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus (32) Google Scholar, A. N. J. J. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus (54) Google Scholar), which may with Sp binding motifs or with Sp transcription raises the possibility that these Sp binding a role in the of mor were containing a upstream sequence bp −450 to to the translation site, or upstream regulatory sequences of the mouse mor The and and and were by with of the upstream the site and the site. of the were cloned into The sequences of were confirmed by and into of a The mutation was into the to and The containing the site with a at site, was for the sequence by with the were further confirmed by and into The mutation was by into to and for containing base was used in the The for also base for base The mutation was by into were sequenced the −450 to bp region to of base at the a promoter containing the mor iGA motif upstream of the promoter, oligonucleotides containing the mor iGA motif were and and The resulting was into the site of the containing the promoter. were in with in an of and at Drosophila from were at in containing were using the as previously (18Ko J.L. Minnerath S.R. Loh H.H. Biochem. Biophys. Res. Commun. 1997; 234: 351-357Crossref PubMed Scopus (56) Google Scholar). with approximately were with an of The of DNA used was within the of the the and the of to were and with for in transfection from to a ratio of containing the gene by the promoter was in transfection and used for SL2 were with to the for of were with and at for to SL2 were and in SL2 transient transfection the by M.R. J. J.A. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). transfection were or with utilizing that were at The and of were as by the and were from or mouse brain using the by S. 1995; Google Scholar). were to and with of the were at The were in and The was at for to the which were with without The were in and by of to the with for on a of and was and the was at of the were at was with oligonucleotides that were with nuclear in and For analysis, a of oligonucleotides were also to the at for the was analyzed on For assays, of or was to the The reaction was at for complexes and DNA were on in and at and were by of from were with the and and for The were The was in The was in in at with or was the The were using a We previously that deletional containing the proximal promoter −450 to to the of containing both distal and proximal promoters in (18Ko J.L. Minnerath S.R. Loh H.H. Biochem. Biophys. Res. Commun. 1997; 234: 351-357Crossref PubMed Scopus (56) Google Scholar). the the region of initiation these results that elements important for the proximal are in the −450 to bp In to the and trans-acting proteins required for the of proximal promoter, and were in this shown in a to a to resulted in a decrease of to whereas the to a major decrease The in that of the to resulted in a in over that of the whereas a to the promoter results suggested that the of to bp the important elements for the activity. we analyzed the of with in this shown in the to whereas the to In reverse of the to bp fragment resulted in activity. In the sequence and an important role in proximal promoter of the mor gene, and the promoter in an the of mor gene expression in the of the to bp region to the promoter were with the fragment of to bp as the and cell nuclear shown in the major by and complexes by and were The indicated in by and indicated by were a of to fragment of the sequence from to bp revealed a region, which the mor iGA motif and the sequence to this motif has such as Sp1 E. N. Mol. Cell. Biol. PubMed Google Scholar), have been shown to bind to a We the possibility that Sp1 or a protein was the factor to this shown in a consensus GC-rich Sp1 binding site the shift by and The suggested that these complexes Sp1 or assay was to the of Sp factors (23Briggs M.R. Kadonaga J.T. Bell S.P. Tjian R. Science. 1986; 234: 47-52Crossref PubMed Scopus (1059) Google Scholar, A. Mol. Cell. Biol. 1992; PubMed Scopus Google Scholar, G. S. M. G. Nucleic Acids Res. 1992; 20: PubMed Scopus Google Scholar). shown in and a was using by the in and the at of and also by in and the of of the reaction with both Sp1 and Sp3 led to an of the major indicated by the other with the or in the of these factors are in the this analysis was using the nuclear from the of approximately and were the was with or The of Sp factor is with the (23Briggs M.R. Kadonaga J.T. Bell S.P. Tjian R. Science. 1986; 234: 47-52Crossref PubMed Scopus (1059) Google Scholar, A. Mol. Cell. Biol. 1992; PubMed Scopus Google Scholar, G. S. M. G. Nucleic Acids Res. 1992; 20: PubMed Scopus Google Scholar). Thus, four Sp transcription factors are in the cells, Sp1 and Sp3 are the trans-acting factors to this sequence of proximal promoter of the mor the binding motif within the to bp region, oligonucleotides the region were and as and and were for to with the to bp for the of major complexes in the The of complexes indicated by was by the of a of of fragment a or fragment suggested that the binding of Sp proteins in the to the Sp1 and Sp3 binding motif the region was further into of mor iGA the mor iGA and mor iGA assay was The of major complexes indicated by was partially by the of a of by or that the mor iGA motif is important for the binding of Sp However, the containing this mor iGA motif was as as the sequences the mor iGA motif to be important as further for this were using the as a and nuclear from cells, as well as from mouse brain. shown in the electrophoretic was to that using the fragment of to bp as the The GC-rich Sp1 binding site the shift with the major indicated by the was by using and Sp3 or Using mouse brain nuclear the binding of Sp1 and Sp3 was also the other using the as a the was with the that binding of Sp1 and Sp3 required the mor iGA the of mor iGA motif to the of proximal promoter, the region of was mutated using a site. shown in the mutated at the region of the in the mutated at the region of in suggested that the mor iGA motif is important for the proximal promoter activity. the of mor iGA motif to the promoter and to the possibility of a transcription factor binding site, were and shown in the in which in the mor iGA motif was to the The in which was to in the region in as with that of results with the was used to the mutation of mor iGA motif as in resulted in of Sp transcription factor shown in using the oligonucleotides as a the of the major by was by of or by the in which the mor iGA motif was the that mutation of the mor iGA motif, which binding of resulted in an approximately 50% decrease in promoter activity. the mor iGA motif could of a promoter, we a in which the mor iGA region, was of the promoter The resulting that of promoter Thus, the mor iGA motif can mediate within the of the mor gene promoter also within a promoter. The analysis suggested that elements located in the to bp region approximately of the proximal promoter mutation of the mor iGA motif resulted in a 50% decrease in This suggest the of other DNA sequences analysis indicated a canonical Sp1 binding site, located bp of the mor iGA motif, also in the to bp region (17Min B.H. Augustin L.B. Felsheim B.F. Fuchs J.A. Loh H.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9081-9085Crossref PubMed Scopus (162) Google Scholar). We the role of this canonical Sp1 binding site in the proximal promoter activity. Sp1 or a factor to the canonical Sp1 binding site, an was The within the to bp region, the canonical Sp1 site, indicated by the of the with nuclear resulted in a major by in The of the major was by the of a of GC-rich Sp1 as well as by that factor to this fragment be Sp1 or This was using the The major was using The or in The that in to the mor iGA motif, Sp1 transcription factor can also bind to a canonical Sp1 binding site located in the to bp In the Sp3 transcription factor bind to this canonical Sp1 site to the mor iGA the the canonical Sp1 binding and mor iGA we the role of the canonical Sp1 binding site to the proximal promoter activity. The Sp1 binding site in the was mutated by of which resulted in the Mutation of these binding of Sp1 to its binding site The Sp1 binding site also a decrease in promoter (approximately to the the interaction these Sp1 binding sites, the mor iGA and a canonical Sp1 site, a mutation was shown in the mutation a decrease (approximately in that of the (approximately or the (approximately that the Sp binding can in an We have shown the of both mor iGA motif and canonical Sp1 binding site to the proximal promoter and the binding of transcription factor Sp1 Sp3 to these results suggested that Sp factors were the promoter binding to these motifs. this we assays in Drosophila SL2 cells, which Sp1 and shown in Sp1 can specifically trans-activate the promoter of the in a Mutation of the mor iGA motif, canonical Sp1 binding site, or both by The of of is by was by which may the binding of Sp1 to within the promoter. In to Sp1 Sp3 was also found to bind to the mor iGA Sp3 is a transcription factor that can or transcription G. S. M. G. J. 1994; PubMed Scopus Google Scholar, B. P. L. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), we the role of Sp3 on the proximal promoter using assays in SL2 shown in Sp3 can trans-activate the promoter of the Sp3 at the has a Mutation of the mor iGA motif the by Sp3. of a of or and different of together with Sp1 and Sp3 were to trans-activate the promoter in an manner these results that mor iGA and canonical Sp1 motifs can mediate by interaction with Sp1 and Sp3 can bind to the mor iGA motif and an the promoter activity. In this we have identified DNA sequences that bind Sp1 Sp3 factors and are necessary for the of the proximal promoter of mouse mor gene of a iGA is to the transfection analysis of deletional and mutation that the mor iGA motif an important role in the proximal promoter. Mutation of the mor iGA motif in Drosophila SL2 cells, that the mor iGA motif is of by whereas a of the mor iGA motif upstream of the promoter was also to this promoter. Using with as we the presence of a major that was specifically in by Sp1 and that was with Sp1 and Sp3. suggest that the major both Sp1 and Sp3. Sp1 is to be modified Tjian R. Cell. Full Text PDF PubMed Scopus Google Scholar), the other complexes may modified of proteins in major complexes or other factors T. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), or may of In to is a transcription factor G. S. M. G. J. 1994; PubMed Scopus Google Scholar, B. P. L. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), Drosophila SL2 cell indicated that the expression of Sp3 the proximal promoter of mor gene binding to the mor iGA However, Sp3 a of the mor gene Sp1 does at the Furthermore, Sp1 and Sp3 the mor gene promoter in an Our results that the DNA canonical Sp1 binding located bp upstream of the mor iGA motif, the mor iGA motif, which is found in and as well as the canonical Sp1 binding site may be to the mouse mor gene, the consensus Sp1 sequence is in J. G. A. T. P. Biochem. Biophys. Res. Commun. 1995; PubMed Scopus Google Scholar) or mor promoter. However, the Sp1 factor can bind to and mor promoter to be Our that both transcription binding are required for proximal promoter the mor iGA motif a by the canonical site. we the possibility that other elements by of Sp1 is expressed in or cell brain S.P. Mol. Cell. Biol. PubMed Scopus Google Scholar), and to be a for a factor expression of the mor gene proximal promoter. 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