title: Multiple sites required for expression in 5'-flanking region of the hMLH1 gene.
abstract: Expression of the hMLH1 gene, one of the DNA mismatch repair genes, is frequently repressed in various cancers such as colorectal, ovarian, gastric, and endometrial origins with a microsatellite instable phenotype. In this study, we investigated details of the relationship between the transcriptional activity and the protein-binding sites in the 5'-flanking region of the hMLH1 gene. Luciferase reporter gene analysis with a series of deletion mutants revealed that a region containing -301 to -76 relative to a translation start site is essential for maximal expression. Eight protein-binding sites in this region were identified by in vivo methylation footprinting analysis and homology search. A presence of binding proteins to CCAAT-box at -145 to -139 was confirmed by the electrophoresis mobility shift assay. Partial involvement of NF-Y was seen by the super gel shift assay. Three reporter plasmids having a single site-directed mutation at -163 to -158, -145 to -139, or -96 to -93 showed 14-30% less activities to that of having the wild-type. Dual or triple mutations were no greater effect than the single mutation on the activity. These results indicate that three cis-elements are essential for full expression of the hMLH1 gene and may work co-operatively.
authors.affiliation: Department of Molecular Biology, Toho University School of Medicine, 5-21-16 Ohmori-Nishi, Ohta-ku, Tokyo 143-8540, Japan. cwwarita@med.toho-u.ac.jp
authors.forename: Michitsune
authors.initials: M
authors.lastname: Arita
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Zhong
authors.forename: Zhenghua
authors.initials: Z
authors.lastname: Min
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Hemmi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Shimatake
mesh_terms: D024506:5' Flanking Region / Q000235:genetics*; D048868:Adaptor Proteins, Signal Transducing; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D023081:CCAAT-Binding Factor / Q000378:metabolism; D002352:Carrier Proteins; D004247:DNA / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D000070957:MutL Protein Homolog 1; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics*; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D023081:CCAAT-Binding Factor; D002352:Carrier Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D008156:Luciferases; D000070957:MutL Protein Homolog 1