HBx induces hypomethylation of distal intragenic CpG islands required for active expression of developmental regulators Sun-Min Leea, Young-gun Leeb, Jae-Bum Baea,1, Jung Kyoon Choia,2, Chiharu Tayamac, Kenichiro Hatac, Yungdae Yund, Je-Kyung Seonge, and Young-Joon Kima,b,3 aDepartment of Biochemistry, College of Life Science and Technology, Yonsei University, Seoul 120-749, Korea; bDepartment of Integrated Omics for Biomedical Science, World Class University Program, Yonsei University, Seoul 120-749, Korea; cDepartment of Maternal-Fetal Biology, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan; dDepartment of Life Science and the Center for Cellular Homeostasis, Ewha Woman’s University of Life Science, Seoul 120-750, Korea; and eLaboratory of Developmental Biology and Genomics, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea Edited* by Roger D. Kornberg, Stanford University School of Medicine, Stanford, CA, and approved May 19, 2014 (received for review January 13, 2014) Epigenetic alterations caused by viral oncoproteins are strong Some unmethylated intragenic and intergenic CGIs contain distinct initiation factors for cancer development, but their mechanisms epigenetic markers and work as enhancers or alternative promoters, are largely unknown. To identify the epigenetic effects of viral demonstrating the regulatory potential of CGIs. Although most hepatitis B virus X (HBx) that lead to hepatocellular carcinoma (HCC), promoter associate CGIs are unmethylated, about 20–30% of CGIs we profiled the DNA methylomes of normal and HBx transgenic found in the gene body are highly methylated (13) and suggested as mouse liver. Intriguingly, severe hypomethylation of intragenic CpG marking actively transcribed regions (14) or as suppressing pro- islands (CGIs) was observed in HBx liver before the full development moters for antisense transcripts (15). of HCC. Normally, these CGIs were highly methylated (mCGIs) by the The HBV x (HBx) protein, which plays a critical role in the DNMT3L complex and marked with epigenetic signatures associated development of HCC, was shown to interact with several epi- with active expression, such as H3K36me3. Hypomethylation of genetic factors, such as DNMT3A and HDAC1 (16). However, mCGI was caused by the downregulation of Dnmt3L and Dnmt3a the physiological significance of these interactions and the reg- due to HBx bound to their promoters, along with HDAC1. These ulatory mechanism leading to gene silencing are not clear. HBx events lead to the downregulation of many developmental regu- was shown to recruit DNMT3A to certain promoters to repress lators that could facilitate tumorigenesis. Here we provide an their activities through de novo DNA methylation; however, intriguing epigenetic regulation mediated by mCGI that is required HBx also causes hypomethylation in certain promoters, possibly via the redistribution of DNMT3A to other promoters (16). In for cell differentiation and describe a previously unidentified epige- netic role for HBx in promoting HCC development. Significance DNA methylation | methylated CpG island | viral protein Epigenetic dysregulation by oncoviral protein plays a key role epatocellular carcinoma (HCC) is one of the most danger- in tumor development. DNA methylome analysis of hepatitis B Hous cancers that threaten many people, especially those with virus X (HBx)-induced hepatocellular carcinoma (HCC) revealed hepatitis B or C virus (HBV or HCV) (1–3). However, the exact drastic changes in host epigenome, but in an unconventional mechanisms underlying HCC are not clear because there are way: intragenic CpG islands (CGIs) were dramatically deme- multiple factors, including chronic inflammation (4), genetic al- thylated. We showed methylated intragenic CGIs as previously teration caused by viral integration into the host genome (5), and unidentified regulatory elements associated with active ex- pression. The methylated CGIs are marked with distinct epi- the oncogenic actions of viral proteins (6). Although many genetic signatures and require DNA methyltransferase (DNMT) studies have previously demonstrated that these factors, alone or 3L complex for their high methylation levels. By directly sup- in combination with other factors, are able to initiate tumori- pressing Dnmt3L and Dnmt3a promoters, HBx induces hypo- genesis (1), the oncogenic potential of viral protein in cancer methylation of the intragenic CGIs and downregulation of the development is one of the most interesting exogenic factors that associated developmental regulators. We provide previously facilitates tumorigenesis, in particular, through the epigenetic unreported functional identification of intragenic CGIs that regulation of infected cells (7). may enhance our understanding of epigenetic regulation and Epigenetic alterations in cancer cells are now regarded as one a new epigenetic role for HBx in promoting HCC development. of the most important factors driving cancer initiation. Abnormal DNA methylation is the most frequently found change in many Author contributions: S.-M.L. and Y.-J.K. designed research; S.-M.L. and J.-B.B. performed cancers and is believed to control associated gene expression research; Y.-g.L., J.K.C., C.T., K.H., Y.Y., and J.-K.S. contributed new reagents/analytic without altering the DNA sequence itself (8). The transcriptional tools; S.-M.L. analyzed data; and S.-M.L. and Y.-J.K. wrote the paper. silencing of tumor suppressor genes associated with the hyper- The authors declare no conflict of interest. methylation of promoter regions is a typical epigenetic change *This Direct Submission article had a prearranged editor. in many cancers (9). The DNA hypomethylation in repeat se- Data deposition: The sequences reported in this paper have been deposited in the Gene quences and transposable elements is known to induce chromo- Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. somal instability and mutation events (10) that lead to cancer GSE48052). development and progression (11). In addition, various types 1Present address: Division of Structural and Functional Genomics, Center for Genome of cancer cells exhibit abnormal expression levels of DNA methyl- Science, Korea National Institute of Health, Osongsaemyeong 2-ro, Osong-eup, transferase (DNMT) families, which probably causes global Cheongwon-gun, Chungcheongbuk-do 363-700, Korea. changes in DNA methylation (12). 2Present address: Department of Bio and Brain Engineering, Korea Advanced Institute of In mammalian cells, large clusters of CpG dinucleotides Science and Technology, Daejeon 305-701, Korea. 3 known as CpG islands (CGIs) appear to act as a key epigenetic To whom correspondence should be addressed. E-mail: [email protected]. GENETICS element regulating gene expression. Most CGIs are found at the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 5′ end of transcripts and behave as functional promoters (13). 1073/pnas.1400604111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1400604111 PNAS | July 1, 2014 | vol. 111 | no. 26 | 9555–9560 Downloaded by guest on October 1, 2021 addition, as a transcriptional activator, HBx modifies diverse HBx13 vs. HBx13). Significant methylation changes were already signal transduction pathways that mediate cellular transformation obvious in the early stage of transformation (Norm3 vs. HBx3) (17, 18). Most HBx transgenic (17) mice spontaneously develop and were maintained during cancer progression (Norm13 vs. HCC at about 1 y of age, providing genetic validation of the HBx13). The methylation levels did not change a lot between oncogenic potential of HBx even in the absence of viral in- different ages compared with the methylation changes caused by tegration and chronic inflammation (6). tumor development. The differentially methylated regions (DMRs) To address the role of HBx viral oncoprotein in the de- were highly enriched in exons, with the most striking changes at velopment of HCC, specifically the aspect of epigenetic regula- CGIs in both age groups (Fig. 1B). To understand the role of DNA tion of the host genome, we examined the differences in DNA methylation changes in the early stage of tumor development, we methylation patterns between normal and HBx-induced liver carried out additional MIRA-seq and RNA-seq analyses of the cancer cells. Intriguingly, DNA methylome analysis revealed 3-mo-old age group to expand the methylation sequence reads dramatic demethylation mainly in the intragenic CGIs with con- up to 170 million for each sample, which covers more than 85% current downregulation of the affected genes. Reanalysis of the of the mouse genome with >70× coverage of the CpG site (SI epigenome data from human cell lines revealed specific epige- Appendix,TableS1B and C). We used these high-density methyl- netic signatures associated with a distinct group of the intragenic omes of 3-mo-old normal and HBx liver to understand the detained CGIs, indicating that they are potential epigenetic regulatory nature of DNA methylation changes caused by HBx. elements for active gene expression. The suppression of Dnmt3L We found 10,553 tumor-associated DMRs (6,668 hyper- and Dnmt3a promoters by HBx appears to cause deficient DNA methylated and 3,885 hypomethylated regions) with differences methyltransferase activity in HBx transgenic (TG) mouse liver, of at least 7 RPKM in the 3-mo-old age group. For comparison, the expected frequency of variations was normalized