Gelsolin gene silencing involving unusual hypersensitivities to dimethylsulfate and KMnO4 in vivo footprinting on its promoter region.
We previously reported that gelsolin gene expression is reduced in various tumors. In an effort to gain further insights into the mechanism of gelsolin downregulation in tumors, we examined the in vivo properties of the gelsolin promoter in urinary bladder cancer cell lines. Neither mutation nor hypermethylation was responsible for gene silencing at the promoter. After exposure to trichostatin A (TSA), a histone deacetylase inhibitor, gelsolin promoter activity was markedly enhanced in the cancer cells, not in cells derived from normal tissue. Chromatin immunoprecipitation assays revealed that both histones H3 and H4 were hypoacetylated in the promoter region of the cancer cells, and the accumulation of acetylated histones was detected by TSA treatment. In vivo footprinting analysis revealed the presence of dimethylsulfate (DMS) hypersensitive site in the untranslated region around nucleotide--35 only in the cancer cells but not in cells derived from normal tissue, and analysis of KMnO4 reactive nucleotides showed that the stem loop structure could be formed in vivo of the cancer cells. This novel stem loop structure may play a part in regulating the transcription of the gelsolin gene in the cancer cells. These results suggest that nucleosome accessibility through histone deacetylation and structural changes (DMS hypersensitivity and stem loop structure) in the promoter region form the basis of the mechanism leading to the silencing of gelsolin gene in human bladder cancer.
Division of Cancer Gene Regulation, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan.
Kazunori
K
Haga
Hisakazu
H
Fujita
Minoru
M
Nomoto
Ataru
A
Sazawa
Koji
K
Nakagawa
Toru
T
Harabayashi
Nobuo
N
Shinohara
Masato
M
Takimoto
Katsuya
K
Nonomura
Noboru
N
Kuzumaki
D001483:Base Sequence; D002843:Chromatin; D018983:DNA Footprinting; D019175:DNA Methylation; D015536:Down-Regulation; D018260:Gelsolin / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D008969:Molecular Sequence Data; D009153:Mutagens / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011196:Potassium Permanganate / Q000494:pharmacology*; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic / Q000235:genetics; D013463:Sulfuric Acid Esters / Q000494:pharmacology*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000473:pathology*
D002843:Chromatin; D018260:Gelsolin; D009153:Mutagens; D013463:Sulfuric Acid Esters; D011196:Potassium Permanganate; C007482:dimethyl sulfate