Overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, associated with DNA hypomethylation on pericentromeric satellite regions during human hepatocarcinogenesis

Abstract

DNA hypomethylation on pericentromeric satellite regions is an early and frequent event associated with heterochromatin instability during human hepatocarcinogenesis. A DNA methyltransferase, DNMT3b, is required for methylation on pericentromeric satellite regions during mouse development. To clarify the molecular mechanism underlying DNA hypomethylation on pericentromeric satellite regions during human hepatocarcinogenesis, we examined mutations of the DNMT3b gene and mRNA expression levels of splice variants of DNMT3b in noncancerous liver tissues showing chronic hepatitis and cirrhosis, which are considered to be precancerous conditions, and in hepatocellular carcinomas (HCCs). Mutation of the DNMT3b gene was not found in HCCs. Overexpression of DNMT3b4, a splice variant of DNMT3b lacking conserved methyltransferase motifs IX and X, significantly correlated with DNA hypomethylation on pericentromeric satellite regions in precancerous conditions and HCCs (P ‫؍‬ 0.0001). In particular, the ratio of expression of DNMT3b4 to that of DNMT3b3, which is the major splice variant in normal liver tissues and retains conserved methyltransferase motifs I, IV, VI, IX, and X, showed significant correlation with DNA hypomethylation (P ‫؍‬ 0.009). Transfection of human epithelial 293 cells with DNMT3b4 cDNA induced DNA demethylation on satellite 2 in pericentromeric heterochromatin DNA. These results suggest that overexpression of DNMT3b4, which may lack DNA methyltransferase activity and compete with DNMT3b3 for targeting to pericentromeric satellite regions, results in DNA hypomethylation on these regions, even in precancerous stages, and plays a critical role in human hepatocarcinogenesis by inducing chromosomal instability. D NA methylation plays important roles in gene silencing, chromatin remodeling, and genome stability (1-4). Aberrant DNA methylation is one of the most consistent epigenetic changes in human cancers (1-4). Generally, the overall level of DNA methylation is lower in cancer cells than in normal cells (5, 6), although a number of tumor suppressor genes are silenced by DNA methylation on CpG islands around their promoter regions in cancer cells (1-4, 7, 8).We have carefully examined alterations of DNA methylation status on pericentromeric satellite regions and CpG islands of specific genes, and expression of DNA methyltransferases and methyl-CpG-binding proteins, in noncancerous liver tissues showing chronic hepatitis and cirrhosis, which are considered to be precancerous conditions (9, 10), and in hepatocellular carcinomas (HCCs) (11-17). Among these alterations, DNA hypomethylation on pericentromeric satellite regions was detected even in precancerous conditions and appears to be one of the earliest epigenetic changes during human hepatocarcinogenesis (17). Satellite regions are located in pericentromeric heterochromatin DNA, and DNA hypomethylation on these regions is known to result in centromeric decondensation, enhancing chromosome recombinations (18,19). In fact, frequent chromosome 1q copy gain wi...

Cancer cells express aberrant DNMT3B transcripts encoding truncated proteins

“…DNMT3B transcripts involving aberrant splicing events at the 5 0 end of the gene could be detected in all of the samples tested, except in HepG2 and Alexander cells, derived from hepatocellular carcinomas. Alexander cells are known to express DNMT3B4, which encodes a catalytically inactive DNMT3B isoform (Saito et al, 2002). All of the primary leukemia samples expressed a transcript containing aberrant splicing between exons 9 and 13 as originally observed, confirming that the expression of these novel mRNAs was not due to an artifact of in vitro culture.…”

“…The only tumor type in which we did not observe novel DNMT3B transcripts was hepatocellular carcinoma. Interestingly, Alexander cells, derived from hepatocellular carcinomas, are known to express DNMT3B4, which encodes a catalytically inactive DNMT3B isoform (Saito et al, 2002). Furthermore, recently described transcripts from non-small-cell lung cancers, DDNMT3B5-7, are also predicted to encode truncated DNMT3B proteins lacking the catalytic domain (Wang et al, 2006a, b).…”

“…Mutations of the DNMT genes are rare in cancer cells (Kanai et al, 2003). Several groups have examined the expression of DNMT transcripts and have not found a correlation with DNA methylation levels in cancer cells (Robertson et al, 1999;Saito et al, 2002;Ehrlich et al, 2006). However, expression of transcripts that originate from a promoter within intron 4, DDNMT3B1-7, correlates with the DNA methylation states of the p16 and RASSF1A promoters in non-small-cell lung cancers (Wang et al, 2006a).…”

MTSS1, a novel target of DNA methyltransferase 3B, functions as a tumor suppressor in hepatocellular carcinoma

“…This suggested that the epigenetic mechanism by which DNMT3B inactivates TSGs may be an important way in which TSGs are silenced in HCC. In HCC, the correlation between DNMTs' expression and the frequency of gene promoter methylation remains to be examined (Saito et al, 2002). In this study, we found that DNMT3B suppressed metastasis suppressor 1 (MTSS1) expression by directly binding to the promoter region of MTSS1, rather than DNA methylation.…”

“…Mouse studies have shown that both Dnmt3a and Dnmt3b are required for the establishment and maintenance of genomic methylation patterns and proper murine development (Bachman et al, 2001;Gowher and Jeltsch, 2002;Kaneda et al, 2004). DNMT3A and DNMT3B are upregulated in certain types of hematological malignancies and solid tumors, including leukocythemia (Mizuno et al, 2001;Claus and Lubbert, 2003), colorectal cancer Takeshima et al, 2006), lung cancer, breast cancer (Girault et al, 2003;Shafiei et al, 2008) and HCC (Saito et al, 2002;Choi et al, 2003). Specifically, DNMT3B contributes to oncogenic phenotypes (Linhart et al, 2007) and tumorigenesis in vivo by gene-specific de novo methylation and transcriptional silencing (Wang et al, 2007).…”