To achieve a global non-biased enrichment of the methylated fragments of genomic DNA, methyl-DNA immunoprecipitation (MeDIP) combined with the novel zebrafish CpG island tiling array was used. This method enabled us to perform the first gene-specific large scale analysis and establishment of a comprehensive methylation map at CGIs of the regions 1.5 kb upstream to 1 kb downstream of the TSS in both healthy zebrafish liver and in hepatocellular carcinoma.
Regulation of transcription is a complex procedure. It is partly accomplished by formation of nucleosomes  and by modulating the binding of regulatory factors and the transcription complex to transcriptional response elements, both directly and indirectly . Epigenetic mechanisms such as chromatin modifications, RNA interference (RNAi) and DNA methylation are key modulators of transcription through different mechanisms, such as prohibiting transcription factor access to their binding sites and affecting the formation of nucleosomes [12, 35, 36].
The methylation profiles of cancer cells are extensively distorted . Hypomethylation in tumors is associated with transcriptional activation of previously suppressed genes and is a hallmark of tumorigenesis [1, 11], where active genes are un-methylated with hyperacetylated histones . As shown in Table 1A, GO terms associated with DNA binding and transcription regulation were significantly over-represented amongst genes hypomethylated in HCC samples. Genes including histone deacetylase 4, DNA (cytosine-5-)-methyltransferase 6 and lysine-specific demethylase 4A showed significant decreases in their methylation levels in HCC samples in comparison to healthy liver.
Comparison of the normal hepatic gene methylation pattern between human liver, presented by Archer et al , and zebrafish liver in this study showed a low correlation (r2 = 0.187) implying lack of conservation of methylation patterns between human and zebrafish. However, our comparisons showed that, in zebrafish and human HCC tumors, similar gene families and genes involved in shared pathways are altered in terms of methylation. For example, zebrafish HCC samples showed methylation changes in genes involved in proliferation, cell cycle, metastasis, apoptosis, energy production, adhesion, stress, DNA binding and regulation of transcription (Table 1A, B and Additional file 6, Tables S2 and S3), similar to the biological processes that contain genes with altered methylation in human HCC . Several other genes from families such as ABCA, CHST, DHX, KCTD, MEGF, MYO, NPY, RNF and TBCID were found to be hypermethylated in both zebrafish and human HCC . Ingenuity Pathway Analysis indicated that the genes with altered methylation in zebrafish hepatocellular carcinoma were associated with biological functions such as cell death, cell morphology, inflammatory response, DNA repair and replication and induced molecules involved in cancer formation such as c-jun, shc and pka. These functions and molecules are commonly altered in human cancers. We have associated changes in methylation of these particular genes with changes in gene expression during tumorigenesis. The changes in methylation levels of these particular genes and pathways could be directly or indirectly linked to their altered expression levels during tumorigenesis.
In our study, GO terms associated with cell proliferation were significantly over-represented in the list of hypomethylated genes in tumors which is particularly relevant since an imbalance between regulation of cellular proliferation pathways and cell death by apoptosis can promote the development of tumors . Anti-apoptotic genes, such as BCL-2, and their regulators, are often over expressed in human tumors [40, 41]. Our results showed a significant decrease in the methylation of a positive regulator of the Bcl-2 protein, bcl-2 associated athanogene 5 (baga5) gene . Changes in anti-apoptotic pathways in zebrafish HCC were concurrent with changes in pathways of proliferation. Insulin like growth factors (IGF) and insulin like growth factor binding proteins (IGFBPs) play important roles in organizing cell proliferation, apoptosis and differentiation and are commonly deregulated in human tumors . In zebrafish HCC, genes for several insulin growth factor binding proteins (IGFBPs) such as igfbp2b were significantly hypomethylated. The promoter region of the human IGFBP-2 gene is rich in CpGs and lacks a TATA box . It is therefore plausible that methylation plays an important role in regulating the expression of this gene. Multiple complex IGF-dependent and independent biological functions influenced by the tissue type and pathological status have been identified for IGFBPs [42, 43]. An increased level of IGFBP-2 protein has been reported in liver tissues and serum during human malignancy [42, 43] with a positive correlation to the malignancy status of the tumor . In contrast to its normal role as a negative regulator of growth, increased levels of IGFBPs in tumors have been linked to enhanced proliferation, partly as a response to androgens and hypoxia-inducible factor-1 (HIF-1) protein. Thus, anaerobic conditions as well as IGF, can result in increased amount of HIF-1 . A lack of vascular supply at the early stages of tumorigenesis in highly proliferating tumor cells results in hypoxia . Under hypoxic circumstances glycolysis becomes the dominant pathway for energy production in tumors and glycolytic enzymes are induced. Associated with this, the HIF-1 protein, expressed in an anaerobic environment, initiates the transcription of several genes involved in stress and glycolysis as well as IGFBP2 [43, 44]. This is in accord with our finding that GO terms related to glycolysis and hypoxia pathways were more prevalent in the list of hypomethylated genes in HCC samples. Glycolytic enzymes such as enolase 2 (ENO2) and hexokinase 2 showed significant decreases in methylation of their genes in HCC samples, indicating a potential increase in expression. Increased expression of enolases such as ENO1 and 2, as a response to hypoxia and HIF-1, has been reported in human HCC . As well as its function in energy production, ENO1 has been associated with enhanced proliferation in HCC . Therefore, based on the functions of genes whose methylation was significantly altered in HCC samples it appears that there is a link between induction of IGF, IGFBPs, HIF, anti-apoptotic and glycolytic pathways [43, 44, 46]. This is similar to findings previously reported on gene expression in human HCC implying that differential methylation is at least partially causative of differential gene expression in HCC.