Regulation of the human O(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-alkyl N-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.
Bhakat
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Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555, USA.
Mitra
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D000107:Acetylation; D017500:Adenovirus E1A Proteins / Q000502:physiology; D001483:Base Sequence; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry / Q000235:genetics; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006657:Histones / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000502:physiology*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D015534:Trans-Activators / Q000502:physiology*
D017500:Adenovirus E1A Proteins; D002843:Chromatin; D017362:Cyclic AMP Response Element-Binding Protein; D017931:DNA Primers; D006657:Histones; D009687:Nuclear Proteins; D015534:Trans-Activators; D019853:O(6)-Methylguanine-DNA Methyltransferase