Activation of Galectin-1 gene in human hepatocellular carcinoma involves methylation-sensitive complex formations at the transcriptional upstream and downstream elements.
The expression of Galectin-1 (Gal-1) mRNA was activated in primary hepatocellular carcinomas (HCCs) compared to matched non-tumorous liver tissues. To elucidate the mechanism of Gal-1 activation in HCC cells, DNA methylation encompassing the transcriptional start site (-165/+151) was examined. Among 12 CpG dinucleotides on both DNA strands, those at positions -116, -109, -52, -41, -36, +35 and +43 were preferentially methylated in non-tumorous liver tissue, while hypomethylated in the matched HCC tissue. Transient transfection of a series of deleted GAL-1 promoters revealed that both an upstream (-57/-31) and a downstream (+10/+57) elements accounted for efficient promoter activity. Electrophoretic mobility shift assay of the upstream element (-63/-30) using nuclear extracts from three HCC cell lines (HLF, HuH7 and HepG2) and normal liver cells revealed at least two complexes (alpha and (beta) interacted with the upstream element in all of the nuclear extracts. Competition experiments revealed that the complex beta preferentially attached to the upstream element harboring unmethylated CpGs. On the other hand, at least three complexes (I, II and III) interacted with the downstream element in all of the nuclear extracts. Competition experiments revealed that complex I specifically attached to the downstream element harboring unmethylated CpG at +35. Furthermore, a DNase I protection assay revealed that a methylation-associated conformational alteration occurred near the CpG site at +35 in HLF cells. Thus, the specific interaction of methylation-sensitive factors to the upstream and downstream elements may be essential for the activation of the Gal-1 gene in HCC cells.
Kondoh
Nobuo
N
Department of Biochemistry II, National Defense Medical College, Saitama 359-8513, Japan.
Hada
Akiyuki
A
Ryo
Akihide
A
Shuda
Masahiro
M
Arai
Masaaki
M
Matsubara
Osamu
O
Kimura
Fumihiro
F
Wakatsuki
Toru
T
Yamamoto
Mikio
M
D001483:Base Sequence; D001667:Binding, Competitive; D015152:Blotting, Northern; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D018899:CpG Islands; D019175:DNA Methylation; D003850:Deoxyribonuclease I / Q000378:metabolism; D037483:Galectin 1 / Q000378:metabolism*; D017353:Gene Deletion; D006801:Humans; D008099:Liver / Q000473:pathology; D008113:Liver Neoplasms / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014162:Transfection
D037483:Galectin 1; D012313:RNA; D008156:Luciferases; D003850:Deoxyribonuclease I