Multiple sites required for expression in 5'-flanking region of the hMLH1 gene.
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.
Arita
Michitsune
M
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
Zhong
Xiaoling
X
Min
Zhenghua
Z
Hemmi
Hiromichi
H
Shimatake
Hiroyuki
H
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
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