Regulation of the human O6-methylguanine-DNA methyltransferase gene by transcriptional coactivators cAMP response element-binding protein-binding protein and p300

Kishor K. Bhakat, Sankar Mitra

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Abstract

O6-Methylguanine-DNA methyltransferase (MGMT)1, a ubiquitous DNA repair protein, removes O6-alkylguanine from DNA, including cytotoxic O6-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.

Original languageEnglish (US)
Pages (from-to)34197-34204
Number of pages8
JournalJournal of Biological Chemistry
Volume275
Issue number44
DOIs
StatePublished - Nov 3 2000

Fingerprint

Cyclic AMP Response Element-Binding Protein
Methyltransferases
trichostatin A
Protein Binding
Carrier Proteins
Genes
DNA
Acetylation
Luciferases
Histones
p300-CBP Transcription Factors
Chromatin
Assays
Protein Methyltransferases
Histone Acetyltransferases
Carmustine
Histone Deacetylase Inhibitors
Chromatin Assembly and Disassembly
O-(6)-methylguanine
Sequence Deletion

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Regulation of the human O6-methylguanine-DNA methyltransferase gene by transcriptional coactivators cAMP response element-binding protein-binding protein and p300",
abstract = "O6-Methylguanine-DNA methyltransferase (MGMT)1, a ubiquitous DNA repair protein, removes O6-alkylguanine from DNA, including cytotoxic O6-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.",
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AU - Bhakat, Kishor K.

AU - Mitra, Sankar

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N2 - O6-Methylguanine-DNA methyltransferase (MGMT)1, a ubiquitous DNA repair protein, removes O6-alkylguanine from DNA, including cytotoxic O6-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.

AB - O6-Methylguanine-DNA methyltransferase (MGMT)1, a ubiquitous DNA repair protein, removes O6-alkylguanine from DNA, including cytotoxic O6-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.

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