Multiple sites required for expression in 5'-flanking region of the hMLH1 gene

Gene. 2003 Mar 13;306:57-65. doi: 10.1016/s0378-1119(03)00385-8.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5' Flanking Region / genetics*
  • Adaptor Proteins, Signal Transducing
  • Base Sequence
  • Binding Sites / genetics
  • CCAAT-Binding Factor / metabolism
  • Carrier Proteins
  • DNA / genetics
  • DNA / metabolism
  • Electrophoretic Mobility Shift Assay
  • Gene Expression
  • Humans
  • Luciferases / genetics
  • Luciferases / metabolism
  • Molecular Sequence Data
  • MutL Protein Homolog 1
  • Mutation
  • Neoplasm Proteins / genetics*
  • Nuclear Proteins
  • Promoter Regions, Genetic / genetics
  • Protein Binding
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Deletion
  • Transcription, Genetic
  • Tumor Cells, Cultured

Substances

  • Adaptor Proteins, Signal Transducing
  • CCAAT-Binding Factor
  • Carrier Proteins
  • MLH1 protein, human
  • Neoplasm Proteins
  • Nuclear Proteins
  • Recombinant Fusion Proteins
  • DNA
  • Luciferases
  • MutL Protein Homolog 1