HBx induces hypomethylation of distal intragenic CpG islands required for active expression of developmental regulators

Abstract

Epigenetic alterations caused by viral oncoproteins are strong initiation factors for cancer development, but their mechanisms are largely unknown. To identify the epigenetic effects of viral hepatitis B virus X (HBx) that lead to hepatocellular carcinoma (HCC), we profiled the DNA methylomes of normal and HBx transgenic mouse liver. Intriguingly, severe hypomethylation of intragenic CpG islands (CGIs) was observed in HBx liver before the full development of HCC. Normally, these CGIs were highly methylated (mCGIs) by the DNMT3L complex and marked with epigenetic signatures associated with active expression, such as H3K36me3. Hypomethylation of mCGI was caused by the downregulation of Dnmt3L and Dnmt3a due to HBx bound to their promoters, along with HDAC1. These events lead to the downregulation of many developmental regulators that could facilitate tumorigenesis. Here we provide an intriguing epigenetic regulation mediated by mCGI that is required for cell differentiation and describe a previously unidentified epigenetic role for HBx in promoting HCC development.DNA methylation | methylated CpG island | viral protein H epatocellular carcinoma (HCC) is one of the most dangerous cancers that threaten many people, especially those with hepatitis B or C virus (HBV or HCV) (1-3). However, the exact mechanisms underlying HCC are not clear because there are multiple factors, including chronic inflammation (4), genetic alteration caused by viral integration into the host genome (5), and the oncogenic actions of viral proteins (6). Although many studies have previously demonstrated that these factors, alone or in combination with other factors, are able to initiate tumorigenesis (1), the oncogenic potential of viral protein in cancer development is one of the most interesting exogenic factors that facilitates tumorigenesis, in particular, through the epigenetic regulation of infected cells (7).Epigenetic alterations in cancer cells are now regarded as one of the most important factors driving cancer initiation. Abnormal DNA methylation is the most frequently found change in many cancers and is believed to control associated gene expression without altering the DNA sequence itself (8). The transcriptional silencing of tumor suppressor genes associated with the hypermethylation of promoter regions is a typical epigenetic change in many cancers (9). The DNA hypomethylation in repeat sequences and transposable elements is known to induce chromosomal instability and mutation events (10) that lead to cancer development and progression (11). In addition, various types of cancer cells exhibit abnormal expression levels of DNA methyltransferase (DNMT) families, which probably causes global changes in DNA methylation (12).In mammalian cells, large clusters of CpG dinucleotides known as CpG islands (CGIs) appear to act as a key epigenetic element regulating gene expression. Most CGIs are found at the 5′ end of transcripts and behave as functional promoters (13).Some unmethylated intragenic and intergenic CGIs contain distinct epige...

The Regulatory Mechanisms of Intragenic DNA Methylation

“…However, contrary to expectations, intragenic methylation negatively correlated with promoter methylation of the same genes, and often induced transcription [13]. Finally, intragenic CGI DNA methylation was negatively and positively related to H3K27me3 and H3K36me3 levels, respectively [14]. These results highlight the function of intragenic methylation in transcriptional activation.…”

“…We observed that HBx, an oncoprotein of the hepatitis B virus, represses Dnmt3L expression, resulting in hypomethylation of intragenic CGIs [14]. The genes associated with hypomethylated intragenic CGIs in the HBx-induced liver cancer were developmental regulators related to the morphogenesis and cell adhesion, and their expression was decreased in HBx-mediated hepatocellular carcinoma.…”

DDB1 Stimulates Viral Transcription of Hepatitis B Virus via HBx-Independent Mechanisms

“…After centrifugation, pellets were resuspended in 1ϫ TE buffer containing an EDTA-free protease inhibitor cocktail (Roche) and sonicated in a UP200S sonication device with 5 cycles at an amplitude of 50%, a 0.5-s pulse during 30 s, and 30 s on ice. Lysates were centrifuged and diluted with 2ϫ radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-Cl [pH 7.6], 1 mM EDTA [pH 8.0], 0.1% Na-deoxycholate, 1% Triton X-100, and an EDTA-free protease inhibitor cocktail [Roche]) (28). Chromatin was subjected to immunoprecipitation overnight at 4°C with 3 g of nonspecific immunoglobulins (Sigma) as a negative control, an anti-H4 antibody (Active Motif) as a positive control, or an anti-DDB1 antibody (Santa Cruz).…”

ChARM: Discovery of combinatorial chromatin modification patterns in hepatitis B virus X-transformed mouse liver cancer using association rule mining

“…Genome-wide DNA methylation [49], histone methylations (H3K4Me3, H3K27Me3, and H3K36Me3), and serine 2 and 5 phosphorylation of RNA polymerase II were profiled from the livers of 3-month old wild-type and HBx transgenic (TG) mice. The gene expression data were downloaded from the Gene Expression Omnibus (GEO accession number: GSE48052 for RNA-Seq [49]).…”