Invited critical review
Advances in DNA methylation: 5-hydroxymethylcytosine revisited

https://doi.org/10.1016/j.cca.2011.02.013Get rights and content

Abstract

Mammalian DNA contains two modified cytosine bases; 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Both of these have been known for decades but have received very different levels of attention in the scientific literature. 5mC has been studied extensively, and its role as an epigenetic modification involved in gene regulation, X-chromosome inactivation, genomic imprinting, long-term silencing of transposons and cancer development is well described. 5hmC, on the other hand, has only recently entered center stage when it was shown that the Ten-Eleven-Translocation (TET) family of oxygenases catalyzes the conversion of 5mC to 5hmC, and that one of these enzymes, TET2, is frequently mutated in myeloid neoplasms. The formation of 5hmC can lead to demethylation of DNA, which may contribute to the dynamics of DNA methylation. 5hmC has been found in many cell types and tissues, with particularly high levels in the brain, and TET1 has been shown to be important for self-renewal and maintenance of embryonic stem cells. Future challenges include better understanding the normal molecular, cellular and physiological roles of 5hmC and TET proteins, understanding the exact roles of TET proteins in cancer development, and developing sequencing methodologies that can accurately distinguish among cytosine, 5mC and 5hmC at single-base-pair resolution.

Introduction

The information contained in eukaryotic DNA is stored and expressed in tightly controlled ways, primarily through post-replicative methylation of DNA and post-translational modification of histones [1]. These modifications are collectively known as epigenetic marks, which regulate chromatin organization and gene expression patterns without altering the primary nucleotide sequence of DNA. The best characterized epigenetic mark is a methyl group at the 5-position of cytosine bases. The pattern of 5-methylcytosine (5mC) in the genome is accurately preserved by mitotic inheritance through the action of DNA methyltransferases (DNMTs), which catalyze the covalent addition of methyl groups to cytosine in newly synthesized DNA (Fig. 1) [2]. In general, dense methylation of gene promoters is associated with gene silencing, and thus the distribution of methyl groups within the genome defines regions of varying transcriptional potential. DNA methylation is established in tissue-specific patterns during embryonic development, which may contribute to important aspects of cellular differentiation [3].

Cytosine methylation plays important roles in key biological processes. Several gene-regulatory mechanisms entirely depend on DNA methylation, including those responsible for X-chromosome inactivation in females, allele-specific silencing of imprinted genes and transcriptional repression of transposons. A number of human syndromes are caused by disturbances in the methylation status of imprinted genes [4] or by mutations in genes encoding components of the DNA-methylation machinery [5], [6]. In addition, alterations in DNA methylation patterns have been implicated in autoimmune diseases [7], neurological and psychiatric conditions [8] and cancer [9], [10].

The genomes of tumor cells are generally characterized by a global loss of methylation (hypomethylation), accompanied by focal increases in methylation (hypermethylation) [9], [10]. Although it has been a long-standing discussion whether perturbations of DNA methylation patterns during tumorigenesis represent “driver” events (i.e., events that are required for the formation and maintenance of a tumor cell) or “passenger” events (i.e., events that are not necessary for the tumor cell) [11], there is strong evidence suggesting that methylation events contribute to the cancer phenotype. Global hypomethylation may lead to genomic instability (a hallmark of cancer) [12], and hypermethylation of gene promoters may lead to transcriptional silencing of tumor suppressor genes [9], [10]. Other methylation events may affect tissue-specific differentiation, which, according to a prevailing model [13], is the main mechanism by which epigenetic changes cause cancer.

In contrast to genetic changes, which cause permanent damage to the genome, changes in DNA methylation patterns are potentially reversible and, therefore, obvious targets for therapeutic intervention [14]. This has led to an intensive search for drugs that target components of the epigenetic machinery. Two inhibitors of DNA methylation, 5-azacytidine and 5-aza-2’-deoxycytidine, which have been known for decades [15], have now have been approved by the US Food and Drug Administration (FDA) for treatment of patients with myelodysplastic syndrome (MDS). It was recently demonstrated in a large clinical trial that 5-azacytidine, as the first drug ever, can improve overall survival in high-risk MDS [16], and it is now considered the drug of choice by many institutions for treatment of this cancer. Both 5-azacytidine and 5-aza-2’-deoxycytidine are inhibitors of DNMTs and can reactivate silenced tumor suppressor genes. However, recent evidence suggests that there may be other important mechanisms of these drugs, including the degradation of small regulatory RNAs and the stimulation of immune responses through upregulation of cancer-testis antigens [17], [18], [19].

For more than six decades, 5mC has been recognized as “the fifth base” in mammalian DNA. However, early work suggested [20], and recent work has confirmed [21], [22], the existence of a sixth base, 5-hydroxymethylcytosine (5hmC; Fig. 1). This review discusses the known roles and implications of 5mC and the emerging roles of 5hmC.

Section snippets

The distribution and function of 5mC in mammalian genomes

Cytosine methylation in somatic tissues of mammals occurs almost exclusively in the context of the symmetric cytosine-guanine (CpG) dinucleotide. Embryonic stem (ES) cells exhibit significant levels of non-CpG methylation (~ 20% of total 5mC), which disappears upon cellular differentiation [23], [24]. The distribution of methyl marks throughout the genome is not random. The majority of 5mCs are located in transposons, which are repeated sequences that comprise > 40% of the human genome and

5hmC − “the sixth base” in mammalian DNA

The hydroxylated form of 5mC, 5hmC, was first reported in DNA from T-even bacteriophages [33]. In these organisms, 5hmC is often modified by glycosylation mediated by glucosyltransferases, which serves to render the phage genome resistant to degradation by host restriction enzymes after infection [34]. 5hmC in mammalian DNA was first described in the early 1970'ies by Penn et al. [20], who found that this modified base accounted for ~ 15% of total cytosines in DNA extracted from the brains of

Conversion of 5mC to 5hmC by TET oxygenases

Oxidative damage can lead to the formation of a variety of modified bases in DNA, including the oxidation of guanine to 8-oxoguanine and the oxidation of 5mC to 5hmC, suggesting that the presence of 5hmC in some cells could be the result of oxidative stress. However, Kriaucionis and Heintz [21] showed that the presence of 5hmC was not accompanied by the accumulation of other damage products, and no correlation was found between the age of adult mice and the amount of 5hmC in Purkinje and

TET alterations in hematopoietic malignancies

The first evidence suggesting that TET proteins could be involved in human cancer came with the identification of TET1 as a fusion partner in a rare translocation in leukemia. Specifically, TET1 was found to be fused with the myeloid/lymphoid or mixed-lineage leukemia gene (MLL) in acute myeloid leukemia (AML) patients carrying a t(10;11)(q22;q23) translocation [48], [50], [51]. MLL-TET1 translocations have also later been reported in a few cases of acute lymphocytic leukemia (ALL) [52]. MLL is

5mC or 5hmC – a technical challenge

Over the past decade, there has been a revolution in the techniques and approaches used to investigate DNA methylation. A wealth of simple PCR techniques can provide information on the methylation status of specific loci [61], and more recent developments have made it possible to characterize entire “methylomes” at single-base-pair resolution [24]. However, as most current techniques do not distinguish between 5mC and 5hmC (see below), the recognition that mammalian DNA contains both of these

Conclusion

The “rediscovery” of 5hmC in mammalian tissues [21], [22] has added a new and potentially important dimension to our perception of DNA methylation in human development and disease. However, there are still many unresolved issues and there may be more questions than answers: Is 5hmC confined to CpG dinucleotides, like 5mC? What are the target genes of the TET proteins? Are there functions of the TET proteins other than the 5hmC-forming activity? How do the levels of 5hmC and TET2 mutations in

Acknowledgements

Work that forms the basis of this discussion is supported by grants from the Danish Cancer Society and the Neye Foundation.

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