Cytoplasmic sequestration of an O6-methylguanine-DNA methyltransferase enhancer binding protein in DNA repair-deficient human cells.
O6-Methylguanine-DNA methyltransferase (MGMT), an enzyme that repairs adducts at O6 of guanine in DNA, is a major determinant of susceptibility to simple methylating carcinogens or of tumor response to anticancer chloroethylating drugs. To investigate the mechanisms underlying cellular expression of this DNA repair enzyme, we focused on the role of a 59-bp enhancer of the human MGMT gene in the regulation of its expression. By using chloramphenicol acetyltransferase reporter assays, we found that the enhancer activity, which was present in both MGMT-expressing (Mer+) and -deficient (Mer-) cells, correlated with the endogenous MGMT activity in Mer+ cell lines. Band-shift assays and deletion analysis of the 59-bp sequence defined a minimal 9-mer cis element (5'-CTGGGTCGC-3') for specific trans factor binding. The MGMT enhancer binding protein (MEBP), 45 kDa by Southwestern blot analysis, was present in the nuclei of all Mer+ cells tested but was apparently restricted to the cytoplasm of Mer- cells. We conclude that the MEBP-enhancer interaction plays an important role in regulating constitutive MGMT expression in Mer+ cells and that MEBP exclusion from the nucleus may account for the down-regulation of MGMT in Mer- cells.
Chen
F Y
FY
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA.
Harris
L C
LC
Remack
J S
JS
Brent
T P
TP
D001483:Base Sequence; D002451:Cell Compartmentation; D003593:Cytoplasm / Q000737:chemistry; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015536:Down-Regulation; D004742:Enhancer Elements, Genetic; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D008780:Methyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011485:Protein Binding; D015534:Trans-Activators / Q000378:metabolism*; D015854:Up-Regulation
D004268:DNA-Binding Proteins; D015534:Trans-Activators; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase