5-Hydroxymethylcytosine: a stable or transient DNA modification?

The DNA base 5-hydroxymethylcytosine (5hmC) is produced by enzymatic oxidation of 5-methylcytosine (5mC) by 5mC oxidases (the Tet proteins). Since 5hmC is recognized poorly by DNA methyltransferases, DNA methylation may be lost at 5hmC sites during DNA replication. In addition, 5hmC can be oxidized further by Tet proteins and converted to 5-formylcytosine and 5-carboxylcytosine, two bases that can be removed from DNA by base excision repair. The completed pathway represents a replication-independent DNA demethylation cycle. However, the DNA base 5hmC is also known to be rather stable and occurs at substantial levels, for example in the brain, suggesting that it represents an epigenetic mark by itself that may regulate chromatin structure and transcription. Focusing on a few well-studied tissues and developmental stages, we discuss the opposing views of 5hmC as a transient intermediate in DNA demethylation and as a modified DNA base with an instructive role.

The role of 5-hydroxymethylcytosine in human cancer.

The patterns of DNA methylation in human cancer cells are highly abnormal and often involve the acquisition of DNA hypermethylation at hundreds or thousands of CpG islands that are usually unmethylated in normal tissues. The recent discovery of 5-hydroxymethylcytosine (5hmC) as an enzymatic oxidation product of 5-methylcytosine (5mC) has led to models and experimental data in which the hypermethylation and 5mC oxidation pathways seem to be connected. Key discoveries in this setting include the findings that several genes coding for proteins involved in the 5mC oxidation reaction are mutated in human tumors, and that a broad loss of 5hmC occurs across many types of cancer. In this review, we will summarize current knowledge and discuss models of the potential roles of 5hmC in human cancer biology.

Methods for genome-wide analysis of DNA methylation in intestinal tumors.

Recent studies show that colorectal cancer is strongly associated with aberrant DNA methylation, which has been linked to the origin and progression of the disease. This fact indicates a need for deep analysis of DNA methylation alterations during colorectal carcinogenesis. The knowledge obtained from such studies will elucidate the mechanisms of epigenetic changes and, through the identification and characterization of DNA methylation markers and disease-specific methylation patterns, will help improve the diagnosis and treatment options for patients. The introduction of new methods for genome-wide analysis of DNA methylation has been an important step towards achieving these goals. In this review, we discuss the role of DNA methylation in intestinal carcinogenesis as well as the different methodological approaches that are currently being used for methylation analysis on a genome-wide scale.

Reprogramming of DNA methylation at NEUROD2-bound sequences during cortical neuron differentiation.

The characteristics of DNA methylation changes that occur during neurogenesis in vivo remain unknown. We used whole-genome bisulfite sequencing to quantitate DNA cytosine modifications in differentiating neurons and their progenitors isolated from mouse brain at the peak of embryonic neurogenesis. Localized DNA hypomethylation was much more common than hypermethylation and often occurred at putative enhancers within genes that were upregulated in neurons and encoded proteins crucial for neuronal differentiation. The hypomethylated regions strongly overlapped with mapped binding sites of the key neuronal transcription factor NEUROD2. The 5-methylcytosine oxidase ten-eleven translocation 2 (TET2) interacted with NEUROD2, and its reaction product 5-hydroxymethylcytosine accumulated at the demethylated regions. NEUROD2-targeted differentially methylated regions retained higher methylation levels in Neurod2 knockout mice, and inducible expression of NEUROD2 caused TET2-associated demethylation at its in vivo binding sites. The data suggest that the reorganization of DNA methylation in developing neurons involves NEUROD2 and TET2-mediated DNA demethylation.

Identification of Tissue-Specific DNA Methylation Signatures for Thyroid Nodule Diagnostics.

PURPOSE: Thyroid cancer is frequently difficult to diagnose due to an overlap of cytologic features between malignant and benign nodules. This overlap leads to unnecessary removal of the thyroid in patients without cancer. While providing some improvement over cytopathologic diagnostics, molecular methods frequently fail to provide a correct diagnosis for thyroid nodules. These approaches are based on the difference between cancer and adjacent thyroid tissue and assume that adjacent tissues are the same as benign nodules. However, in contrast to adjacent tissues, benign thyroid nodules can contain genetic alterations that can be found in cancer.Experimental Design: For the development of a new molecular diagnostic test for thyroid cancer, we evaluated DNA methylation in 109 thyroid tissues by using genome-wide single-base resolution DNA methylation analysis. The test was validated in a retrospective cohort containing 65 thyroid nodules. RESULTS: By conducting reduced representation bisulfite sequencing in 109 thyroid specimens, we found significant differences between adjacent tissue, benign nodules, and cancer. These tissue-specific signatures are strongly linked to active enhancers and cancer-associated genes. Based on these signatures, we developed a new epigenetic approach for thyroid diagnostics. According to the validation cohort, our test has an estimated specificity of 97% [95% confidence interval (CI), 81-100], sensitivity of 100% (95% CI, 87-100), positive predictive value of 97% (95% CI, 83-100), and negative predictive value of 100% (95% CI, 86-100). CONCLUSIONS: These data show that epigenetic testing can provide outstanding diagnostic accuracy for thyroid nodules.See related commentary by Mitmaker et al., p. 457.

Single base resolution analysis of 5-methylcytosine and 5-hydroxymethylcytosine by RRBS and TAB-RRBS.

Sodium bisulfite-assisted deamination of cytosine forms the basis for conducting single base resolution analysis of 5-methylcytosine in DNA. The TET family of proteins represents a group of enzymes that can oxidize 5-methylcytosine to 5-hydroxymethylcytosine. A modification of the bisulfite-based DNA methylation mapping technique employs TET1-mediated oxidation of 5-methylcytosine (TET-assisted bisulfite sequencing) for single base analysis of 5-hydroxymethylcytosine. Whole genome analysis of cytosine modifications with bisulfite sequencing techniques still is challenging and expensive. Reduced representation bisulfite sequencing (RRBS) has been used to limit the complexity of the analysis to mostly CpG-rich genomic fragments flanked by restriction enzyme cleavage sites, for example MspI (5'CCGG). In this chapter, we describe detailed methods used in our laboratory for analysis of 5-methylcytosine and 5-hydroxymethylcytosine combined (RRBS) and for specific analysis of 5-hydroxymethylcytosine (TAB-RRBS).

Loss of the polycomb mark from bivalent promoters leads to activation of cancer-promoting genes in colorectal tumors.

In colon tumors, the transcription of many genes becomes deregulated by poorly defined epigenetic mechanisms that have been studied mainly in established cell lines. In this study, we used frozen human colon tissues to analyze patterns of histone modification and DNA cytosine methylation in cancer and matched normal mucosa specimens. DNA methylation is strongly targeted to bivalent H3K4me3- and H3K27me3-associated promoters, which lose both histone marks and acquire DNA methylation. However, we found that loss of the Polycomb mark H3K27me3 from bivalent promoters was accompanied often by activation of genes associated with cancer progression, including numerous stem cell regulators, oncogenes, and proliferation-associated genes. Indeed, we found many of these same genes were also activated in patients with ulcerative colitis where chronic inflammation predisposes them to colon cancer. Based on our findings, we propose that a loss of Polycomb repression at bivalent genes combined with an ensuing selection for tumor-driving events plays a major role in cancer progression.

Dynamics of 5-hydroxymethylcytosine and chromatin marks in Mammalian neurogenesis.

DNA methylation in mammals is highly dynamic during germ cell and preimplantation development but is relatively static during the development of somatic tissues. 5-hydroxymethylcytosine (5hmC), created by oxidation of 5-methylcytosine (5mC) by Tet proteins and most abundant in the brain, is thought to be an intermediary toward 5mC demethylation. We investigated patterns of 5mC and 5hmC during neurogenesis in the embryonic mouse brain. 5hmC levels increase during neuronal differentiation. In neuronal cells, 5hmC is not enriched at enhancers but associates preferentially with gene bodies of activated neuronal function-related genes. Within these genes, gain of 5hmC is often accompanied by loss of H3K27me3. Enrichment of 5hmC is not associated with substantial DNA demethylation, suggesting that 5hmC is a stable epigenetic mark. Functional perturbation of the H3K27 methyltransferase Ezh2 or of Tet2 and Tet3 leads to defects in neuronal differentiation, suggesting that formation of 5hmC and loss of H3K27me3 cooperate to promote brain development.

Expression of lactoperoxidase in differentiated mouse colon epithelial cells.

Lactoperoxidase (LPO) is known to be present in secreted fluids, such as milk and saliva. Functionally, LPO teams up with dual oxidases (DUOXs) to generate bactericidal hypothiocyanite in the presence of thiocyanate. DUOX2 is expressed in intestinal epithelium, but there is little information on LPO expression in this tissue. To fill the gap of knowledge, we have analyzed Lpo gene expression and its regulation in mouse intestine. In wild-type (WT) C57BL/6 (B6) mouse intestine, an appreciable level of mouse Lpo gene expression was detected in the colon, but not the ileum. However, in B6 mice deficient in glutathione peroxidase (GPx)-1 and -2, GPx1/2-double-knockout (DKO), which had intestinal pathology, the colon Lpo mRNA levels increased 5- to 12-fold depending on mouse age. The Lpo mRNA levels in WT and DKO 129S1/SvlmJ (129) colon were even higher, 9- and 5-fold, than in B6 DKO colon. Higher levels of Lpo protein and enzymatic activity were also detected in the 129 mouse colon compared to B6 colon. Lpo protein was expressed in the differentiated colon epithelial cells, away from the crypt base, as shown by immunohistochemistry. Similar to human LPO mRNA, mouse Lpo mRNA had multiple spliced forms, although only the full-length variant 1 was translated. Higher methylation was found in the 129 than in the B6 strain, in DKO than in control colon, and in older than in juvenile mice. However, methylation of the Lpo intragenic CpG island was not directly induced by inflammation, because dextran sulfate sodium-induced colitis did not increase DNA methylation in B6 DKO colon. Also, Lpo DNA methylation is not correlated with gene expression.

Gamma irradiation does not induce detectable changes in DNA methylation directly following exposure of human cells.

Environmental chemicals and radiation have often been implicated in producing alterations of the epigenome thus potentially contributing to cancer and other diseases. Ionizing radiation, released during accidents at nuclear power plants or after atomic bomb explosions, is a potentially serious health threat for the exposed human population. This type of high-energy radiation causes DNA damage including single- and double-strand breaks and induces chromosomal rearrangements and mutations, but it is not known if ionizing radiation directly induces changes in the epigenome of irradiated cells. We treated normal human fibroblasts and normal human bronchial epithelial cells with different doses of γ-radiation emitted from a cesium 137 ((137)Cs) radiation source. After a seven-day recovery period, we analyzed global DNA methylation patterns in the irradiated and control cells using the methylated-CpG island recovery assay (MIRA) in combination with high-resolution microarrays. Bioinformatics analysis revealed only a small number of potential methylation changes with low fold-difference ratios in the irradiated cells. These minor methylation differences seen on the microarrays could not be verified by COBRA (combined bisulfite restriction analysis) or bisulfite sequencing of selected target loci. Our study shows that acute γ-radiation treatment of two types of human cells had no appreciable direct effect on DNA cytosine methylation patterns in exposed cells.

Relationship between gene body DNA methylation and intragenic H3K9me3 and H3K36me3 chromatin marks.

To elucidate the relationship between intragenic DNA methylation and chromatin marks, we performed epigenetic profiling of chromosome 19 in human bronchial epithelial cells (HBEC) and in the colorectal cancer cell line HCT116 as well as its counterpart with double knockout of DNMT1 and DNMT3B (HCT116-DKO). Analysis of H3K36me3 profiles indicated that this intragenic mark of active genes is associated with two categories of genes: (i) genes with low CpG density and H3K9me3 in the gene body or (ii) genes with high CpG density and DNA methylation in the gene body. We observed that a combination of low CpG density in gene bodies together with H3K9me3 and H3K36me3 occupancy is a specific epigenetic feature of zinc finger (ZNF) genes, which comprise 90% of all genes carrying both histone marks on chromosome 19. For genes with high intragenic CpG density, transcription and H3K36me3 occupancy were not changed in conditions of partial or intensive loss of DNA methylation in gene bodies. siRNA knockdown of SETD2, the major histone methyltransferase responsible for production of H3K36me3, did not reduce DNA methylation in gene bodies. Our study suggests that the H3K36me3 and DNA methylation marks in gene bodies are established largely independently of each other and points to similar functional roles of intragenic DNA methylation and intragenic H3K9me3 for CpG-rich and CpG-poor genes, respectively.

Methylation of polycomb target genes in intestinal cancer is mediated by inflammation.

Epigenetic changes are strongly associated with cancer development. DNA hypermethylation is associated with gene silencing and is often observed in CpG islands. Recently, it was suggested that aberrant CpG island methylation in tumors is directed by Polycomb (PcG) proteins. However, specific mechanisms responsible for methylation of PcG target genes in cancer are not known. Chronic infection and inflammation contribute to up to 25% of all cancers worldwide. Using glutathione peroxidase, Gpx1 and Gpx2, double knockout (Gpx1/2-KO) mice as a model of inflammatory bowel disease predisposing to intestinal cancer, we analyzed genome-wide DNA methylation in the mouse ileum during chronic inflammation, aging, and cancer. We found that inflammation leads to aberrant DNA methylation in PcG target genes, with 70% of the approximately 250 genes methylated in the inflamed tissue being PcG targets in embryonic stem cells and 59% of the methylated genes being marked by H3K27 trimethylation in the ileum of adult wild-type mice. Acquisition of DNA methylation at CpG islands in the ileum of Gpx1/2-KO mice frequently correlates with loss of H3K27 trimethylation at the same loci. Inflammation-associated DNA methylation occurs preferentially in tissue-specific silent genes and, importantly, is much more frequently represented in tumors than is age-dependent DNA methylation. Sixty percent of aberrant methylation found in tumors is also present in the inflamed tissue. In summary, inflammation creates a signature of aberrant DNA methylation, which is observed later in the malignant tissue and is directed by the PcG complex.