Regulation of the HumanO 6-Methylguanine-DNA Methyltransferase Gene by Transcriptional Coactivators cAMP Response Element-binding Protein-binding Protein and p300

O 6-Methylguanine-DNA methyltransferase (MGMT)1, a ubiquitous DNA repair protein, removes O 6-alkylguanine from DNA, including cytotoxic O 6-chloroethylguanine induced by chemotherapeutic N-alkylN-nitrosourea-type drugs, e.g.1,3-bis(2-chloroethyl)-1-nitrosourea. Treating the pancreatic carcinoma cell line MIA PaCa-2 with trichostatin A (TSA), a specific inhibitor of histone deacetylase, increased MGMT mRNA and protein levels by 2–3-fold. Surprisingly, TSA treatment increased MGMT promoter-dependent luciferase activity by some 40-fold in a transient reporter expression assay. Deletion and point mutation analysis showed that two AP-1 binding sites in the MGMT promoter are involved in activation by TSA. Ectopic expression of the transcriptional coactivators cAMP response element-binding protein-binding protein (CBP) and p300, which have intrinsic histone acetyltransferase activity, enhanced luciferase expression. Overexpression of adenovirus E1A, which binds CBP/p300, strongly inhibited both basal and TSA-inducible MGMT promoter activity, while a mutant E1A, defective in binding CBP/p300, did not. Chromatin immunoprecipitation assays revealed that TSA treatment increased histone acetylation in the endogenous MGMT promoter region, which also showed association with CBP/p300. Taken together, our results indicate that targeted histone acetylation results in the remodeling of chromatin by recruitment of the coactivator CBP/p300, and constitutes an important step in regulating MGMT expression. O 6-Methylguanine-DNA methyltransferase (MGMT)1, a ubiquitous DNA repair protein, removes O 6-alkylguanine from DNA, including cytotoxic O 6-chloroethylguanine induced by chemotherapeutic N-alkylN-nitrosourea-type drugs, e.g.1,3-bis(2-chloroethyl)-1-nitrosourea. Treating the pancreatic carcinoma cell line MIA PaCa-2 with trichostatin A (TSA), a specific inhibitor of histone deacetylase, increased MGMT mRNA and protein levels by 2–3-fold. Surprisingly, TSA treatment increased MGMT promoter-dependent luciferase activity by some 40-fold in a transient reporter expression assay. Deletion and point mutation analysis showed that two AP-1 binding sites in the MGMT promoter are involved in activation by TSA. Ectopic expression of the transcriptional coactivators cAMP response element-binding protein-binding protein (CBP) and p300, which have intrinsic histone acetyltransferase activity, enhanced luciferase expression. Overexpression of adenovirus E1A, which binds CBP/p300, strongly inhibited both basal and TSA-inducible MGMT promoter activity, while a mutant E1A, defective in binding CBP/p300, did not. Chromatin immunoprecipitation assays revealed that TSA treatment increased histone acetylation in the endogenous MGMT promoter region, which also showed association with CBP/p300. Taken together, our results indicate that targeted histone acetylation results in the remodeling of chromatin by recruitment of the coactivator CBP/p300, and constitutes an important step in regulating MGMT expression. 1,3-bis(2-chloroethyl)-1-nitrosourea acetylated histone H4 acid-urea-Triton base pair(s) cAMP response element-binding protein-binding protein chromatin immunoprecipitation histone acetyltransferase histone deacetylase O 6 -methylguanine-DNA methyltransferase MGMT-luciferase p300/CBP-associated factor trichostatin A glucocorticoid-responsive element polyacrylamide gel electrophoresis Antitumor alkylating drugs of the 2-haloethyl-N-nitrosourea class, such as 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU),1 induceO 6-chloroethylguanine in DNA, which in a second reaction forms DNA cross-links, the ultimate cytotoxic lesion (1Ludlum D.B. Mutat. Res. 1990; 233: 117-126Crossref PubMed Scopus (225) Google Scholar).O 6-Methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, repairs the mutagenic, carcinogenic and cytotoxic O 6-alkylguanine adducts including the primary alkyl adducts induced by alkylnitrosoureas (2Mitra S. Kaina B. Prog. Nucleic Acids Res. Mol. Biol. 1993; 44: 109-142Crossref PubMed Scopus (188) Google Scholar, 3Pegg A.E. Byers T.L. FASEB J. 1992; 6: 2302-2310Crossref PubMed Scopus (267) Google Scholar). MGMT acts by transferring the O 6-alkyl group to a specific cysteine residue within its own sequence in a single step, stoichiometric reaction (4Foote R.S. Mitra S. Pal B.C. Biochem. Biophys. Res. Commun. 1980; 97: 654-659Crossref PubMed Scopus (135) Google Scholar). This transfer irreversibly inactivates MGMT. Hence, MGMT is a major contributor to cellular protection from the mutagenic, carcinogenic, and cytotoxic effects of DNA-alkylating agents. MGMT expression is highly variable in normal tissues as well as in tumor cells (5Citron M.R. Decker S. Chen S. Schneider S. Graver M. Kleynerman L. Khan L.B. White A. Schoenhaus M. Yarosh D. Cancer Res. 1991; 51: 4131-4134PubMed Google Scholar, 6Washington W.J. Foote R.S. Dunn W.C. Generoso W.M. Mitra S. Mech. Ageing. Dev. 1989; 48: 43-52Crossref PubMed Scopus (45) Google Scholar). A fraction of primary tumor cells, and 20% of human tumor cell lines, lack expression of MGMT (7Day III, R.S. Ziolkowski C.H.J. Seudiero D.A. Meyer S.A. Lubiniecki A.S. Giardi A.J. Galloway S.M. Bynum G.D. Nature. 1980; 288: 724-727Crossref PubMed Scopus (403) Google Scholar, 8Silber J.R. Blank A.M. Bobola S. Mueller D.A. Kolstoe D.D. Ojemann G.A. Berger M.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6941-6946Crossref PubMed Scopus (75) Google Scholar). These MGMT-defective (Mex−) cell lines are highly sensitive to alkylating agents and nitrosourea-type drugs (8Silber J.R. Blank A.M. Bobola S. Mueller D.A. Kolstoe D.D. Ojemann G.A. Berger M.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6941-6946Crossref PubMed Scopus (75) Google Scholar). Conversely, some tumor cells express MGMT at a high level and are highly resistant to chemotherapy with BCNU (9Kokkinakis D.M. Ahmed M.M. Delgado R. Fruitwala M.M. Mohiuddin M. Albores-Saavedra J. Cancer Res. 1997; 57: 5360-5368PubMed Google Scholar). Thus, elucidating the molecular mechanisms controlling MGMT expression is of major clinical significance. The MGMT gene encoding an mRNA of 950 nucleotides consists of five exons, and spans more than 170 kilobase pairs (10Tano K. Shiota S. Collier J. Foote R.S. Mitra S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 686-690Crossref PubMed Scopus (295) Google Scholar, 11Nakatsu Y. Hattori K. Hayakawa H. Shimizu K. Sekiguchi M. Mutant. Res. 1993; 293: 119-132Crossref PubMed Scopus (54) Google Scholar). The 5′-regulatory sequence (including its promoter) has been cloned (12Harris L.C. Potter P.M. Tano K. Shiota S. Mitra S. Brent T.P. Nucleic Acids Res. 1991; 19: 6163-6167Crossref PubMed Scopus (140) Google Scholar). The promoter is extremely GC-rich, and lacks both TATA and CAAT boxes. Several cis elements were identified, including six putative Sp1 sites within the CpG island, two glucocorticoid-responsive elements (GRE), and two each of putative AP-1 and AP-2 elements (12Harris L.C. Potter P.M. Tano K. Shiota S. Mitra S. Brent T.P. Nucleic Acids Res. 1991; 19: 6163-6167Crossref PubMed Scopus (140) Google Scholar). The potential function of each of the GRE and AP-1 sites in activation of MGMT has been investigated previously (13Biswas T. Ramana C.V. Srinivasan G. Boldogh I. Hazra T.K. Chen Z. Tano K. Thompson E.B. Mitra S. Oncogene. 1999; 18: 525-532Crossref PubMed Scopus (66) Google Scholar, 14Boldogh I. Ramana C.V. Chen Z. Biswas T. Hazra T.K. Grosch S. Grombacher T. Mitra S. Kaina B. Cancer Res. 1998; 58: 3950-3956PubMed Google Scholar). However, the molecular basis for the lack of expression of MGMT in Mex− cell lines, in which no deletion or gross rearrangement in the gene was observed, is not understood (15von Wronski M.A. Harris L.C. Tano K. Mitra S. Bigner D.D. Brent T.P. Oncol. Res. 1992; 4: 167-174PubMed Google Scholar). Reporter gene expression driven by the MGMT promoter indicates that Mex− cells do not lack necessary trans-acting factors (16Harris L.C. Potter P.M. Remack S.O. Brent T.P. Cancer Res. 1992; 52: 6404-6406PubMed Google Scholar). This suggests that gene silencing results from modification of cis elements, by mechanisms such as CpG methylation (17Qian C.X Brent T.P. Cancer Res. 1997; 57: 3672-3677PubMed Google Scholar) and/or chromatin alteration (18Costello J.F. Futscher B.W. Kroes R.A. Pieper R.O. Mol. Cell. Biol. 1994; 14: 6515-6521Crossref PubMed Google Scholar). 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A is that targeted histone acetylation is by recruitment of acetyltransferase to the for was by the that including p300, and of have intrinsic activity, and are to the promoter in a Cell. Mol. Sci. 1998; PubMed Scopus Google Scholar). The transcriptional coactivators and p300, as adenovirus R. D. M. J. D.M. Dev. 1994; PubMed Scopus Google have been as for gene by factors and the basal and are and to as J.R. Nature. PubMed Scopus Google Scholar). not by with a specific DNA with factors including AP-1 via and T. Y. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar, A.J. T. J. 14: PubMed Scopus Google Scholar, R. T. Nature. 1996; PubMed Scopus Google Scholar). The for the of Y. Cell. 1996; 87: PubMed Scopus Google Scholar) and have been with in of and M. Nature. 1997; PubMed Scopus Google Scholar, Cell. Mol. Sci. 1998; PubMed Scopus Google Scholar). the of chromatin remodeling to transcriptional of the human MGMT in that histone MGMT gene in MIA PaCa-2 cells, and transcriptional is involved in both basal and MGMT promoter also the for the remodeling of chromatin of the MGMT gene that is to its expression. MIA PaCa-2 cells were at in high with and and A was from of cellular with of was a and by electrophoresis J. T. A and with MGMT or as The MGMT was an of (10Tano K. Shiota S. Collier J. Foote R.S. Mitra S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 686-690Crossref PubMed Scopus (295) Google Scholar). and were at with and the was to the The was by or cells were by with and inhibitor for of and the were at and the and at protein was with was of analysis was with MGMT as previously (13Biswas T. Ramana C.V. Srinivasan G. Boldogh I. Hazra T.K. Chen Z. Tano K. Thompson E.B. Mitra S. 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Dev. 1993; PubMed Scopus Google Scholar). treatment of cells in the with TSA for the and cells were in at for to of including to The cells were and in of and inhibitor and for at of the by of chromatin was with the chromatin was for at with of protein as in which was with the chromatin was at with of histone H4 The was by binding to of protein as for at the were by with with and with a the were with and the with two of at for The were to for to the and with for The DNA was with with and the in of of DNA was with and as and the two AP-1 sites in the MGMT promoter that was in the the with of DNA were The were by gel electrophoresis and some cells were with MGMT and the was as the for to of the MGMT promoter and the to a sequence in the of of the the was and the was MIA PaCa-2 cells were in of at and a and to The was with TSA or at TSA treatment for the cells were and with Reporter and the luciferase activity of the was in a the luciferase The luciferase activity was with to the protein of the some of expression was in the that activity to for in The were in the the deletion of MGMT-luciferase and cloned in have been previously (13Biswas T. Ramana C.V. Srinivasan G. Boldogh I. Hazra T.K. Chen Z. Tano K. Thompson E.B. Mitra S. Oncogene. 1999; 18: 525-532Crossref PubMed Scopus (66) Google Scholar). mutation of two AP-1 sites in reporter was by I. Ramana C.V. Chen Z. Biswas T. Hazra T.K. Grosch S. Grombacher T. Mitra S. Kaina B. Cancer Res. 1998; 58: 3950-3956PubMed Google Scholar). and were as encoding adenovirus protein and mutant were of K. of encoding the was by R. H. was from S. Cancer encoding the human and were of Y. of chromatin remodeling via histone acetylation a in MGMT the of a specific histone deacetylase MGMT expression. analysis showed that the level of MGMT with to the level of was at treatment with TSA a in the MGMT protein level by analysis studies the MGMT protein level showed that the increased level of the MGMT protein of TSA with the protein level the by These results the that of histone was for the MGMT TSA was to cell in some cell lines in the or Y. M. S. T. Cell. Res. 1994; PubMed Scopus Google cell to the effects of of TSA cell of MIA PaCa-2 TSA at a cell in cell line TSA for MGMT activation in activation of MGMT mRNA and protein in MIA PaCa-2 level of MGMT mRNA by analysis at treatment with of TSA. The level of was in the to for gel analysis of MGMT in of cells with TSA as of MGMT activation TSA treatment are analysis of MIA PaCa-2 cells with PaCa-2 cells were with TSA or for and for cell by cell in a in a MIA PaCa-2 cells were with TSA or for and for cell by cell in a studies that the primary of histone is to of a of S. 1996; Google activation of MGMT at the promoter MGMT reporter the MGMT promoter sequence from to base pairs (13Biswas T. Ramana C.V. Srinivasan G. Boldogh I. Hazra T.K. Chen Z. Tano K. Thompson E.B. Mitra S. Oncogene. 1999; 18: 525-532Crossref PubMed Scopus (66) Google was for transient expression of luciferase reporter in MIA PaCa-2 cells as promoter-dependent luciferase expression was as the TSA MGMT luciferase expression by some promoter activation with TSA of as as also a in luciferase activity driven by the with an H. H. M. S. Cell. Res. 1998; PubMed Scopus Google Scholar). A reporter expression with TSA showed that activation of luciferase of TSA and the activity the the factors that are for activation of the MGMT promoter deletion and MIA PaCa-2 cells were with a of promoter deletion with and the luciferase activity with A activation of the promoter activity by TSA was with the promoter reporter the was with the promoter reporter a was TSA treatment with the reporter This suggests the of a element in the to of the MGMT The of a element was also in activation of promoter Y. T. S. K. T. H. T. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar). Deletion of the promoter sequence from to base pairs by deletion of the sequence to by from that the cis elements for promoter activation by TSA are within the and and of the and analysis of MGMT promoter in reporter for TSA reporter were The in the to the of the and nucleotides of the MGMT promoter to the of luciferase activity treatment with PaCa-2 cells were with of and luciferase reporter activity was of luciferase activity the of MGMT promoter and of single and AP-1 is The were (13Biswas T. Ramana C.V. Srinivasan G. Boldogh I. Hazra T.K. Chen Z. Tano K. Thompson E.B. Mitra S. Oncogene. 1999; 18: 525-532Crossref PubMed Scopus (66) Google Scholar, 14Boldogh I. Ramana C.V. Chen Z. Biswas T. Hazra T.K. Grosch S. Grombacher T. Mitra S. Kaina B. Cancer Res. 1998; 58: 3950-3956PubMed Google Scholar). The in the to the of the and nucleotides of the MGMT promoter to the in a MIA PaCa-2 cells were with of and luciferase reporter activity was of luciferase activity the of MGMT promoter and of single and AP-1 is of the sequence within the revealed the of in studies showed DNA protein at six Sp1 including in an cell line (18Costello J.F. Futscher B.W. Kroes R.A. Pieper R.O. Mol. Cell. Biol. 1994; 14: 6515-6521Crossref PubMed Google Scholar). of the sequence and revealed sites (12Harris L.C. Potter P.M. Tano K. Shiota S. Mitra S. Brent T.P. Nucleic Acids Res. 1991; 19: 6163-6167Crossref PubMed Scopus (140) Google including two AP-1 sites and two The AP-1 sites and were previously to involved in activation of MGMT with and respectively (13Biswas T. Ramana C.V. Srinivasan G. Boldogh I. Hazra T.K. Chen Z. Tano K. Thompson E.B. Mitra S. Oncogene. 1999; 18: 525-532Crossref PubMed Scopus (66) Google Scholar, 14Boldogh I. Ramana C.V. Chen Z. Biswas T. Hazra T.K. Grosch S. Grombacher T. Mitra S. 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PubMed Scopus Google Scholar). of cells with a of MGMT reporter and of an expression showed of luciferase expression in an and a of was with of binds to including the gene K. M. R.A. Nature. PubMed Scopus Google the that inhibited MGMT promoter activity by binding to factors for MGMT in to CBP/p300. cells with an MGMT and an expression for E1A, which was to defective in binding of binding to of the protein M. J. J. 1990; PubMed Google Scholar). in mutant did not MGMT an in promoter activity was Taken together, results that transcriptional coactivator is involved in MGMT promoter expression. Recent indicate that the histone acetyltransferase activity of both and its factor in and in D. A. Chen H. Y. Cell. 1999; PubMed Scopus Google Scholar). the activity with or the was for activation of the MGMT cells with of the MGMT promoter and of the expression the cells were with TSA or for in inhibited of the MGMT promoter in a These the for activation of a promoter in a reporter which inhibited by for of in MGMT promoter the of of and/or reporter Ectopic expression of human increased MGMT luciferase activity by of enhanced MGMT luciferase activity by expression of or a with TSA and of and its factor which also activity, did not a with TSA not more that the of MGMT promoter activity was to of CBP/p300, of the promoter activity in the of Ectopic expression of the of MGMT promoter activity with E1A, that was to of a of endogenous These for of in MGMT promoter of MGMT luciferase expression the that is involved in the function of the MGMT promoter as by cells with a of promoter reporter and of or mutant expression that strongly inhibited MGMT promoter activity, while mutant did not. the mutant protein a the promoter activity as was also with the promoter expression of enhanced the promoter-dependent luciferase activity The promoter is highly GC-rich, and sites in were previously to in (18Costello J.F. Futscher B.W. Kroes R.A. Pieper R.O. Mol. Cell. Biol. 1994; 14: 6515-6521Crossref PubMed Google Scholar). Thus, that the transcriptional is also for the basal promoter activity of the MGMT which acts by Sp1 or by with the basal TSA was to of acetylated histone in cell lines, which by gel electrophoresis M. M. M. T. J. Biol. 1990; PubMed Google Scholar). investigated the of TSA histone acetylation in MIA PaCa-2 cells by from or A that levels of and forms of H4 and were in the cells to the histone H4 a in the binding of factors to DNA in J. 1996; PubMed Scopus Google Scholar). to the of histone H4 TSA analysis was histone H4 which and of histone in 6 TSA treatment a in the level of histone H4 in The that TSA expression of of cellular that histone acetylation is targeted to specific or S. 1996; Google and that such is by recruitment of to the to that TSA increased acetylation of to the MGMT promoter in chromatin immunoprecipitation as in A. MIA PaCa-2 cells were with TSA for and the cells the were with and the chromatin was and with histone H4 of an MGMT promoter sequence was with DNA from the that the MGMT promoter sequence was in the H4 histone from cells with that from the cells in for Thus, or no MGMT promoter sequence was by the in the of histone H4 of the MGMT promoter sequence was not a was as no was the step was to that TSA treatment enhanced the level of to the MGMT promoter than to the MGMT gene as a the to the of the MGMT promoter sequence and sequence in the histone H4 was in the of histone H4 in the MGMT and TSA treatment and to the acetylation of at the MGMT promoter sequence of the Chromatin immunoprecipitation was MIA PaCa-2 cells with MGMT The MGMT promoter was to with acetylated histone H4 and TSA treatment a in the of acetylated histone H4 with promoter in the level of acetylated histone H4 in the of MGMT was as a of TSA treatment and These results indicate that the MGMT promoter was with histone H4 in the of TSA. that activation of MGMT promoter involved the AP-1 with MIA PaCa-2 cells with MGMT AP-1 in TSA treatment did not the level of acetylated histone H4 the two AP-1 elements were that AP-1 binding elements are for association of acetylated with the MGMT The results of the are with the that TSA increased the level of to the MGMT promoter in both endogenous and and that such acetylation was by the activity of CBP/p300. was important to that is with the MGMT promoter MIA PaCa-2 cells with the reporter by chromatin by of and DNA from the a of the MGMT promoter was by that the MGMT promoter sequence was by treatment with and not with These results that is with the MGMT at in the This the that histone acetylation a in MGMT both endogenous MGMT gene expression and MGMT reporter expression were enhanced by a histone deacetylase However, was that TSA the MGMT promoter in the to a than the promoter of the MGMT was previously that with to that histone deacetylase activity from such Y. T. S. K. T. H. T. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar, S. Mol. Cell. Biol. 1998; 18: PubMed Google Scholar). A for activation of the MGMT promoter by inhibitor is that the of not normal S. A. Nucleic Acids Res. 1994; PubMed Scopus Google histone association is for from acetylation of in a more chromatin in the gene than in the The of activation to the of histone acetylation also a gene to the of with two in the is also that with in a not for the are studies showed that two AP-1 sites in the MGMT promoter are involved in its activation by I. Ramana C.V. Chen Z. Biswas T. Hazra T.K. Grosch S. Grombacher T. Mitra S. Kaina B. Cancer Res. 1998; 58: 3950-3956PubMed Google Scholar). The that AP-1 sites are also involved in activation of the MGMT promoter suggests that the at sites This in targeted acetylation of to of the in the promoter in binding of AP-1 and Sp1 This is by the that expression of the transcriptional coactivator enhanced MGMT promoter activity A and was to and factors T. Y. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar, R. T. Nature. 1996; PubMed Scopus Google Scholar). a in which AP-1 activity histone acetylation in the promoter to a of and binding of the normal the function by an TSA treatment and the levels of for the of activity of was by the that of the adenovirus protein promoter activation This is with the of D. A. Chen H. Y. Cell. 1999; PubMed Scopus Google Scholar) that is a inhibitor of the activity of CBP/p300, as well as that of and to CBP/p300. binds to the of at which and its activity D. A. Chen H. Y. Cell. 1999; PubMed Scopus Google Scholar). of the of in MGMT gene was by our that the MGMT basal promoter is by expression of in a the of the protein to the basal promoter activity suggests that is its binding to CBP/p300. Surprisingly, mutant also increased basal of the MGMT results were previously for and L. G. J. Biol. 1998; PubMed Scopus Google Scholar). have no for from its activity, a specific factors and the via binding to and protein T.K. T. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). 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