Aberrant epigenetic modifications in hepatocarcinogenesis induced by hepatitis B virus X protein.
OBJECTIVE
The involvement of the hepatitis B virus X (HBx) protein in epigenetic modifications during hepatocarcinogenesis has not been previously characterized. The aim of the present study was to identify the involvement of HBx in regional hypermethylation and global hypomethylation during the formation of hepatocellular carcinoma (HCC).


METHODS
Liver cell lines were transiently or stably transfected with an HBx-expressing vector. DNA methyltransferase (DNMT) promoter activity changes were examined by luciferase assay and chromatin immunoprecipitation. The methylation status of insulin-like growth factor binding protein-3 was examined by methyl-specific polymerase chain reaction and bisulfite sequencing. Global DNA methylation levels were examined using 5-methylcytosine dot blot and methylation-sensitive Southern blot analysis. HBx-mediated DNA methylation abnormalities were confirmed in patient HCC samples using methyl-specific polymerase chain reaction and 5-methylcytosine dot blot analysis.


RESULTS
HBx expression increased total DNMT activities by up-regulation of DNMT1, DNMT3A1, and DNMT3A2 and selectively promoted regional hypermethylation of specific tumor suppressor genes. HBx specifically repressed insulin-like growth factor-3 expression through de novo methylation via DNMT3A1 and DNMT3A2 and by inhibiting SP1 binding via recruiting methyl CpG binding protein 2 to the newly methylated SP1 binding element. HBx also induced global hypomethylation of satellite 2 repeat sequences by down-regulating DNMT3B. The prevalence of these specific methylation abnormalities by HBx was significantly correlated with HBx expression in HBV-infected HCC patients.


CONCLUSIONS
Targeted deregulation of DNMTs by HBx promotes both specific regional hypermethylation and global hypomethylation. These epigenetic modulations by HBx may suggest a mechanism for epigenetic tumorigenesis during HBV-mediated hepatocarcinogenesis.
Park
In Young
IY
Liver Cell Signal Transduction Laboratory, Molecular Cancer Research Center, KRIBB, Daejeon, Korea.
Sohn
Bo Hwa
BH
Yu
Eunsil
E
Suh
Dong Jin
DJ
Chung
Young-Hwa
YH
Lee
Je-Ho
JH
Surzycki
Stefan J
SJ
Lee
Young Ik
YI
D015139:Blotting, Southern; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D004273:DNA, Neoplasm / Q000235:genetics*; D018450:Disease Progression; D004591:Electrophoresis, Polyacrylamide Gel; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins
D004273:DNA, Neoplasm; C000717967:DNMT3A protein, human; D018972:Insulin-Like Growth Factor Binding Protein 3; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; C000619380:DNMT1 protein, human