Hydroxymethylation of DNA: an epigenetic marker

Abstract

DNA methylation, an epigenetic mechanism is claimed to play essential roles in development, aging and disease over the past few decades. Cytosines (C) were known to exist in two functional states: unmethylated or methylated (5mC) in the mammalian genome for a very long time. However, the mechanisms controlling 5mC dynamics remain undefined. Recent studies of genomic DNA on human and mouse brain, neurons and from mouse embryonic stem cells have shown that 2-oxoglutarate and Fe(II)-dependent oxygenases of the ten-eleven translocation (Tet) proteins can catalyze the oxidation of 5mC at cpG dinucleotides to form 5-hydroxymethylcytosine (5-hmC). The exhilarating discovery of these novel 5-hmC has begun to focus on the dynamic nature of 5mC. The prevailing evidence has shown that Tet family proteins and 5-hmC are involved in the normal development as well as in many diseases. This review presents an overview of the role of Tet family proteins and 5-hmC. It also discusses their role as an epigenetic marker and the techniques used for their analysis.

Abbreviations

5caC : 5-carboxycytosine; Dnmt : DNA methyltransferase; ESCs : embryonic stem cells; 5fC : 5-formylcytosine; 5-hmC : 5-hydroxymethylcytosine; 5mC : 5-methylcytosine; PGCs : Primordial germ cells

Introduction

DNA methylation, i.e., methylation of cytosine at CpG sequences, and, to a lesser extent, in CpHpG or CpHpH (where H is A, C or T) sequences, is an epigenetic modification linked to gene expression, regulation and developmental processes in various eukaryotes (Bird, 2002[9]; Feng et al., 2010[22]; He et al., 2011[37]; Law and Jacobsen, 2010[61]; Lister et al., 2009[67]; Ramsahoye et al., 2000[94]; Suzuki and Bird, 2008[109]; Tomizawa et al., 2011[116]; Zemach et al., 2010[140]). It is a highly dynamic process during germ cell and preimplantation development but is relatively static during development of somatic tissues in case of mammals. 5mC has been found to be the sole methylation product in higher eukaryotes, where the methylation occurs not only in sequence-specific but also in a locus-specific manner. In mammalian genomes (especially in somatic cells), methylation of cytosine predominantly occurs at CpG dinucleotides, whereas, a fraction of 5mC is associated with non-CG contexts in embryonic stem cells (Lister et al., 2009[67]). CpG motifs are underrepresented in mammals and tend to cluster in the high density regions, referred to as CpG islands. Studies related to 5mC distribution have shown that the majority (70–80 %) of CpGs are methylated, except for those localized in CpG islands. Traditionally, 5mC when localized at CpG islands are important transcriptional silencers at gene promoters. The cytosine methylation typically leads to strong and heritable gene silencing. The epigenetic role of 5-methylcytosine has been widely studied and was found to be crucial in a variety of cellular processes including development gene regulation, differentiation, genomic imprinting, silencing of transposable elements, X-chromosome inactivation and others (Bird, 2002[9]). Because of its significance and heritability, 5mC is often called the fifth base of DNA. The occurrence of 5mC containing photoproducts in UV irradiated DNA is still controversial (Rastogi et al., 2010[95]). However, on the basis of HPLC and Mass spectrometry analyses a new photoproduct of 5mC has also been characterized (Su et al., 2010[107]). It has been assumed by various group of workers that 5mC display a certain level of plasticity during development (Bernstein et al., 2007[4]; Mohn and Schubeler, 2009[71]), therefore, they needs to be rapidly removed by DNA demethylation processes at certain developmental stages. However, the mechanisms of DNA demethylation have always remained controversial (Ooi and Bestor, 2008[86]; Wu and Zhang, 2010[134]).

DNA hydroxymethylation, is a recently identified type of DNA modification in which the hydrogen atom at the C5-position in cytosine is replaced by a hydroxymethyl group, and whose, importance to biology and its role as an epigenetic marker have only been appreciated during the past few years. The presence of 5-hydroxymethylcytosine (5-hmC) has been observed in the genomes of many different organisms, and in mammals its levels are the highest in the brain and other tissues of the central nervous system (Globisch et al., 2010[27]; Kinney et al., 2011[52]; Kriaucionis and Heintz, 2009[58]; Michaeli et al., 2013[70]). 5-hmC is generated through oxidation of 5mC by the Ten-Eleven Translocation (TET) family of methylcytosine dioxygenases (Guo et al., 2011[30]; Hashimoto et al., 2012[35]; Wu and Zhang, 2011[132]) and is considered to be a possible intermediate step in a replication-independent DNA demethylation pathway (Guo et al., 2011[30]; Tahiliani et al., 2009[113]; Wu and Zhang, 2010[134]).

The discovery of a hydroxylated form of 5mC (5-hmC) (Kriaucionis and Heintz, 2009[58]) and the TET family of enzymes required for its conversion (Tahiliani et al., 2009[113]) has now offered insight into DNA methylation pathway involved in many biological processes including those pertaining to nervous system function (Kass et al., 2013[50]). These modified bases may then function as DNA demethylation intermediates subject to deamination, glycosylase-dependent excision, and repair resulting in a reversion back to unmodified cytosine (Bhutani et al., 2011[8]; Branco et al., 2012[15]). However, recently, the DNA methyltransferases (DNMT) enzymes (Tan and Shi, 2012[114]) has also been ascribed a role in the 5-hmC production, suggesting that this species may not be exclusively considered an intermediate of the 5mC demethylation process, but also an important epigenetic marker enriched within gene bodies, promoters, and transcription factor binding sites, where it may influence gene expression (Hahn et al., 2013[32]; Mellén et al., 2012[69]; Szulwach et al., 2011[110]) and thus, regulates the pluripotency of stem cells, cellular development, aging, and carcinogenesis (Liutkeviciute et al., 2009[68]). Three major types of DNA methyltransferases are active on mammalian genomes (Figure 1A(Fig. 1), modified from Kriukiene et al., 2012[59]). DNMT1, the maintenance methyltransferase, maintains the genome-wide methylation patterns during replication as it preferentially methylates hemi-methylated cytosines in CpG sequences (Bestor, 1992[6]; Li et al., 1992[64]). DNMT3A and DNMT3B can methylate unmethylated CpG sequences and hence function as de novo methyltransferases (Okano et al., 1998[84]). DNMT3L has no catalytic activity but recruits DNMT3A and DNMT3B to their targets by recognizing nucleosomes that carry unmethylated histone H3 lysine 4 (H3K4) (Aapola et al., 2000[1]; Bourc’his and Bestor, 2004[13]; Bourc’his et al., 2001[14]; Hata et al., 2002[36]; Ooi et al., 2007[87]).

Figure 1. Schematic representation of the human DNA methyltransferase (A) and Tet family proteins (B). Dnmts share a conserved catalytic domain (MTase), but differ in their N-terminal regulatory regions. Dnmt1 contains a proliferating cell nuclear antigen (PCNA) binding domain, a pericentric heterochromatin targeting sequence (TS), a CXXC domain, and two bromo adjacent homology domains (BAH). Dnmt3A and 3B comprise a PWWP domain named after a conserved Pro-Trp-Trp-Pro motif and an ATRX-Dnmt3-Dnmt3L (ADD) domain also known as the PHD (plant homeodomain) domain. Tet 1, 2, 3 proteins contain a cysteine-rich (Cys) region and a double-stranded ß-helix (DSBH) fold responsible for catalytic activity (modified from Kriukiene et al., 2012).

Domain structure and distribution of TET-family proteins

TET proteins are mammalian homologs of the trypanosome base J binding proteins, JBP1 and JBP2, that contain catalytic motifs typical of Fe(II)- and 2-oxoglutarate (2OG)-dependent dioxygenases and require α-ketoglutarate as a co-substrate for enzymatic activity (Figure 1B(Fig. 1)) (Iyer et al., 2009[46]; Tahiliani et al., 2009[113]). In the N-terminal part, the Tet proteins possess a CXXC domain, a binuclear Zn-chelating domain found in certain chromatin-associated proteins such as Dnmt1 methyltransferase, and other elements which mediate interactions with multiple components in the cell (Xu et al., 2011[137]; Zhang et al., 2010[141]). Unlike the CXXC domains in other proteins, such as, DNMT1, myeloid/lymphoid or mixed-lineage leukemia (MLL) and CXXC finger protein 1 (CFP1, or CXXC1), which are known to bind unmethylated CpG dinucleotides (Cierpicki et al., 2010[17]; Song et al., 2011[105]; Xu et al., 2011[136]), the function of this domain in Tet1 and Tet3 is largely unknown.Various group of workers have reported that CXXC domain of TET1 recognizes not only unmodified cytosine but also 5mC and 5-hmc, and it prefers to bind to regions in the genome of high CpG content (Xu et al., 2011[137]; Zhang et al., 2010[141]). Depending on this feature, genome-wide mapping of Tet1 binding by ChIP-seq approaches revealed its enrichment around transcription start sites (TSSs) in mouse ES cells (Figure 2(Fig. 2), modified from Tan and Shi, 2012[114]) (Xu et al., 2011[137]; Williams et al., 2011[128]; Wu et al., 2011[131]). In addition, to the functional domains of Tet protein action, a spacer region, common to all TET family members but with varying length bridges the two parts of the disconnected DSBH enzymatic domain. The functional significance of this spacer region is currently unknown. However, recently, Upadhyay et al. (2011[118]) have revealed that the spacer region of Tet1 has significant sequence similarity to the C-terminal domain (CTD) of RNA polymerase II of Saccharomyces cerevisiae. All three members of the TETs (TET1–TET3) have been shown to specifically catalyze the conversion of 5mC to 5-hmC as well as its further oxidation into 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC), in the presence of ATP respectively (Figure 3(Fig. 3)) (Ito et al., 2010[44]; 2011[45]; He et al., 2011[38]).

Figure 2. Structure and function of Tet family proteins in mouse.The Tet protein family contains three members: Tet1, Tet2 and Tet3. All Tet proteins contains a catalytic C-terminal CD domain having cysteine-rich and a double-stranded-ß-helix, DSBH regions) that exhibits 2-oxoglutarate (2-OG)- and iron (II)-dependent dioxygenase activity. A spacer region of varying length is also present in the CD domain. The N-termini of Tet1 and Tet3, but not Tet2, contain a CXXC domain, which mediates their direct DNA-binding ability. There is also an unique PRK12323 (DNA polymerase III subunits gamma and tau; provisional) domain in Tet3, whose function is still not clear. a.a.: amino acids (modified from Tan and Shi, 2012).

Figure 3. TET proteim mediated oxidation reaction.TET family proteins oxidize 5mC into 5hmC in the presence of 2-OG and iron. 5mC and 5hmC can also be further oxidized mainly to 5caC by Tet proteins in the presence of ATP.

The distribution of TET1/2/3 proteins vary in different tissue (Cimmino et al., 2011[18]; Szwagierczak et al., 2010[112]; Tahiliani et al., 2009[113]) (Table 1(Tab. 1), modified from Saitou et al., 2012[99]). 5-hmC is abundant in the brain (~40 % and ~13 % as abundant as 5mC in Purkinje and granule cells, respectively), but is present at lower levels in other mouse tissues (Kriaucionis and Heintz, 2009[58]). 5-hmc is detected in mouse ESCs (~7-10 % as abundant as 5mC) but is undetectable in human T cells and in mouse dendritic cells (Tahiliani et al., 2009[113]).

Table 1. Proteins involved in cytosine modification (modified from Saitou et al., 2012).

Table 1 (references)

DNMT 1: Bestor et al., 1988[5]; Lei et al., 1996[62]

DNMT 2: Goll et al., 2006[28]; Okano et al., 1998[85]

DNMT 3A: Kaneda et al., 2004[51]; Okano et al., 1998[84], 1999[83]

DNMT 3B: Kaneda et al., 2004[51]; Okano et al., 1998[84],1999[83]; Ueda et al., 2006[117]

DNMT 3L: Bourc’his and Bestor, 2004[13]; Hata et al., 2002[36]; Webster et al., 2005[126]

NP95 (UHRF1): Bostick et al., 2007[12]; Fujimori et al., 1998;[26] Sharif et al., 2007[101]

TET 1: Dawlaty et al., 2011[20]; Ito et al., 2010[44]; Koh et al., 2011[56]; Szwagierczak et al., 2010[112]; Tahiliani et al., 2009[113]; Xu et al., 2011[137]

TET 2: Szwagierczak et al., 2010; Figueroa et al., 2010[24]; Ito et al., 2010[44]; Ko et al., 2010[55]; Koh et al., 2011[56]; Langemeijer et al., 2009[60]; Moran-Crusio et al., 2011[72]; Quivoron et al., 2011[93]

TET 3: Gu et al., 2011[29]; Iqbal et al., 2011[43]; Wossidlo et al., 2011[130]; Ito et al., 2010[44]; Szwagierczak et al., 2010[112]

AID: Muramatsu et al., 2000[78]; Morgan et al., 2004[73]; Bhutani et al., 2010[7]; Popp et al., 2010[92]; Guo et al., 2011[30]

APOBEC1: Hirano et al., 1996[39]; Morrison et al., 1996[74]; Navaratnam et al., 1993[80]; Teng et al.,1993[115]; Morgan et al., 2004[73]; Guo et al., 2011[30]

TET1 was first reported to have a role in maintaining the pluripotent state of embryonic stem cells (ESCs) (Ito et al., 2010[44]), and together with TET2, it also regulates the cell lineage commitment of ESCs (Koh et al., 2011[56]). TET2 modulates the balance between self-renewal and differentiation in hematopoietic stem cells, making them critical for normal myelopoiesis; TET2 mutations are seen in multiple types of leukemia and also in patients with diverse myeloid malignancies (Ko et al., 2010[55], 2011[54]; Li et al., 2011[65]; Moran-Crusio et al., 2011[72]; Quivoron et al., 2011[93]; Shide et al., 2012[102]). Shide et al. (2012[102]) reported the critical role of TET2 in survival and HSC homeostasis after in vivo analysis of its function in Ayu17-449 (TET2 trap) mice. TET3 contributes to the global DNA methylation erasure during the zygote stage of embryonic development (Gu et al., 2011[29]; Inoue and Zhang, 2011[42]). These studies together, demonstrates the critical role of TET-mediated 5-hmC modification in developmental processes and the possibility that dysregulation of 5-hmC may be associated with disease.

In mouse ESCs, Tet1 and Tet2 are highly expressed but are rapidly downregulated upon differentiation (Ito et al., 2010[44]; Koh et al., 2011[56]; Saitou et al., 2012[99]; Tahiliani et al., 2009[113]), whereas Tet3 is expressed at very low levels in ES cells and upregulated during ES cell differentiation. Dawlaty et al. (2011[20]) demonstrated that Tet1 knockout ESCs showed a ~35 % reduction in 5-hmc levels, and exhibit only subtle changes in gene expression, are pluripotent and support full-term mouse development in the Tetraploid complementation assay. Koh et al. (2011[56]) reported that both Tet1 and Tet2 are downstream targets of key pluripotency factors.

Wang et al. (2012[123]) have reported that 5-hmC-mediated epigenetic pathways play evolutionarily conserved roles in the mammalian brain. In neuronal cells, 5-hmC is not enriched at enhancers but associates preferentially with gene bodies of activated neuronal function-related genes. Within these genes, gain of 5-hmC is often accompanied by loss of H3K27me3. Enrichment of 5-hmC is not associated with substantial DNA demethylation suggesting the potential of 5-hmc as 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 5-hmC and loss of H3K27me3 cooperate to promote brain development (Hahn et al., 2013[32]).

The presence of 5-hydroxymethylcytosine in DNA was first observed in T-even bacteriophages (Wyatt and Cohen, 1953[135]). In T-even phages, 5-hydroxymethylated cytosine is incorporated into the genome during DNA synthesis, and is subsequently modified by phage α- and ß-glucosyltransferases, creating a highly glucosylated DNA containing 5-glucosyloxymethylcytosine (glc-hmC) residues. The precursor nucleotide, 2’-deoxy-5-hydroxymethylcytidine- 5’-monophosphate (dhmCMP), is produced by enzymatic addition of a methylene group at the 5-position of dCMP. This reaction is mechanistically similar to DNA C5-methylation as it involves the formation of a covalent intermediate between a cysteine residue from an enzyme and the C6-position of cytosine (Warren, 1980[125]). A similar modified base J (5-(ß-D-glucosyl)-oxymethyluracil) is present in DNA of flagellated protozoa of the order of Kinetoplastida and unicellular alga Euglena gracilis (Borst and Sabatini, 2008[11]). However, discovery of physiologically relevant DNA modification in mammals, such as in mouse neurons and ESCs (Kriaucionis and Heintz, 2009[58]; Tahiliani et al., 2009[113]), is a more recent novel finding.

The chemical oxidation of 5mC and thymine is considered to be the major source of these hydroxymethylated nucleobases in higher eukaryotes. 5-hmC can be formed at 5mC sites in response to oxidative stress in vitro (Wagner and Cadet, 2010[121]). Oxidation of thymine residues in DNA to hmU was considered to be an important source of endogenous oxidative DNA damage. 5mC is slightly more reactive towards hydroxyl radical attack than is thymine and it was predicted that in human cells, ~20 5mC residues would be oxidized to hmC per cell per day (Planechaud et al., 1997[91]).

Valinluck et al. (2004[120]) have reported that 5-hmC may also be a biological end-product of demethylation, as methyl-CpG binding proteins have a significantly lower affinity for 5-hmC. The ability of DNMT1 to recognize 5-hmC under in vitro condition is very poor (Valinluck and Sowers, 2007[119]); thus, 5-hmc might passively convert into cytosine during replication. However, recently, it has been worked out by Frauer et al. (2011[25]) that Np95, which recruits DNMT1 to replication foci, recognizes 5-hmC as efficiently as it does 5mC, raising the possibility that 5-hmC may have the same capacity as 5-mC for 5-mC propagation during replication.

Szulwach et al. (2011[111]) reported that 5-hmC-mediated epigenetic modification is critical in neurodevelopment and diseases both in mice and human. The kinetics study of hydroxymethylation during the first cell stage have shown that hydroxymethylation remains very low in the maternal pronucleus while it increases in the paternal one, in parallel to demethylation (Inoue and Zhang, 2011[42]; Iqbal et al., 2011[43]; Wossidlo et al., 2011[130]; Ruzov et al., 2011[98]; Gu et al., 2011[29]). Based on their work on mouse zygote Salviang et al. (2012[100]) hypothesized that 5-hydroxymethylcytosine is not a simple intermediate in an active demethylation process and could play a role of its own during early development.

The presence of 5-hmC within eukaryotic genomes was unknown mainly because standard methods for detection of 5-methylcytosine (5-mC) cannot distinguish between this base and 5-hmC (Huang et al., 2010[40]; Münzel et al., 2010[76]; Jin et al., 2010[48]). A newly developed method for single-nucleotide resolution 5-hmC sequencing, based on oxidative conversion of 5-hmC to 5-formyl-cytosine and its further bisulfite conversion to uracil was given by Booth et al. (2012[10]). Overall, 5-hmC content might also be determined via selective chemical labeling with ß-glucosyltransferase of 5-hmC by a glucose moiety containing an azide group modified, for example, by biotin (Pastor et al., 2011[89]; Song et al., 2011[104]). Wanunu et al. (2010[124]) reported another approach using electric signal for detection on a DNA solution flown through nanopores. However, to understand the principles of recognition and binding of 5-hmC in biological context requires its atomic structure. The interaction of 5-hmC-modified DNA with the SET and RING-associated domain of the Uhrf1 protein that belongs to the big family of 5-mC binding proteins was studied by crystal structure and molecular dynamics analyses (Frauer et al., 2011[25]) and it was found that 5-hmC base is flipped out of the DNA double helix and that the 5-hmC hydroxyl group participates in hydrogen bonding, which helps to stabilize the interaction. Expansion of studies on 5-hmC was enabled by the development of suitable phosphoramidites (Münzel et al., 2011[77]). These authors found that both 5-hmC and 5-mC were not causing any insertion of wrong nucleotides even by low-fidelity polymerases, and therefore, concluded that position 5 of cytosine is the ideal place to store epigenetic information. By using X-ray diffraction Renciuk et al. (2013[96]) reported that the cytosine modification (5mC and 5-hmC) neither influences the helical structure of B-DNA nor the thermodynamics.

Roles of 5-hmc

(a) DNA methylation and gene expression

Multiple pathways have been suggested by various workers for 5-hmc mediated regulation of the DNA methylation dynamics and gene transcription. 5-hmC was found to be more sensitive to deamination than 5mC by activation-induced deaminase (AID), and the deamination product, 5-hydroxymethyluracil (5hmU), activates base-excision repair (BER) pathway-mediated demethylation (Guo et al., 2011[30]), suggesting one possible way to acheive DNA demethylation. Recently, it has been shown that Tet proteins can oxidize 5mC not only to 5-hmc but also to 5fC and 5caC, which can subsequently be recognized and excised by thymine DNA glycosylase (TDG) in vitro and in vivo (He et al., 2011[38]; Ito et al., 2011[45]; Pfaffeneder et al., 2011[90]; Zhang et al., 2012[142]). The gradual loss of 5-hmC, 5fC and 5caC, associated with the paternal genome in the zygote during preimplantation development suggested a dominant mechanism of passive DNA demethylation at this developmental stage (Inoue and Zhang, 2011[42]; Inoue et al., 2011[41]; Oda et al. 2009[82]). 5-hmC is important for proper gene transcription as it directs the dynamic remodeling and organization of the chromatin structures (Tan and Shi, 2012[114]). Jin et al. (2010[48]) reported that many methyl-CpG-binding domain (MBD) proteins, such as MBD1, MBD2, methyl CpG-binding protein 2 (MECP2) and MBD4, do not bind 5-hmC in vitro. However, recently it has been proposed that Tet1 is associated with the Mbd3/NuRD complex in mouse ES cells and that Mbd3 can bind 5-hmC and unmodified cytosine, but not 5mC, in vitro (Yildirim et al., 2011[138]). Hashimoto et al. (2012[35]) based on his studies on DNA methyltranferases reported the high intrinsic activity of DNMT1 towards the hemi-methylated CpG substrate but a greatly reduced activity for the hemihydroxymethylated CpG substrate in methyl transfer assays in vitro, whereas the DNMT3A and DNMT3B exhibited equal activities on unmodified, hemimethylated and hemi-hydroxymethylated CpG substrates. Based on these studies it can be considered that 5-hmC acts as a stable epigenetic marker that directly influence genome structure and function, instead of being simply an intermediate of DNA demethylation.

(b) Mammalian development

(i) Mouse primordial germ cells and gametes

Gene knockout strategies have been used to investigate the role of DNA methylation and demethylation involving TET family proteins and 5-hmc in mammalian development. The mouse oocyte and sperm develop from primordial germ cells (PGCs) which are specified from epiblast cells and migrate into the gonad region at embryonic day (E) 7.25. The genome-wide DNA demethylation, erasure of genomic imprints, and large-scale chromatin remodeling takes place at E11.5 (Hajkova et al., 2008[33]; Kota and Feil, 2011). Both Tet1 and Tet2 are expressed in E11.5 and E12.5 PGCs but less so in oocytes and zygotes, whereas Tet3 is highly expressed in oocytes but not in PGCs (Hajkova et al., 2010[34]; Surani and Hajkova, 2010[108]; Gu et al., 2011[29]). The development of PGCs, and subsequently of spermatocytes or oocytes, is normal in Tet1 or Tet2 knockout mice (Dawlaty et al., 2011[20]; Ko et al., 2011[54]; Li et al., 2011[65]; Moran-Crusio et al., 2011[72]; Quivoron et al., 2011[93]), whereas some workers established PGC-conditional Tet3 knockout mice and found that oogenesis and spermatogenesis are normal when Tet3 is absent in PGCs (Gu et al., 2011[29]).

(ii) Mouse zygote

When DNA methylation ends up in parental genome rapidly after fertilization, then DNA demethylation takes place (Oswald et al., 2000[88]). Recently, it has been reported that the oxidation of 5mc to 5-hmc takes place only in paternal but not in the maternal pronucleus (Iqbal et al., 2011[43]; Wossidlo et al., 2011[130]). In contrast to Tet1 and Tet2, TET 3 are highly expressed in oocytes and fertilized zygotes but disappears rapidly during cleavage (Gu et al., 2011[29]; Iqbal et al., 2011[43]; Wossidlo et al., 2011[130]). Gu et al. (2011[29]) showed maternal Tet3 to be the key enzyme catalyzing 5mC into 5-hmC in the paternal pronuclei of developing zygotes. Nakamura et al. (2007[79]) reported the presence of DNA demethylation and 5-hmc in the maternal pronuclei of Stella (Dppa3) null zygotes. Gu et al. (2011[29]) reported the requirement of oxidation of 5mc to 5-hmc in oocytes during somatic cell nuclear transfer.

(iii) Mouse pre- and post-implantation embryos and in ES cells

High levels of Tet1 and 5-hmC are found in mouse ES cells, inner cell mass of the mouse blastocyst, and in cells of the early mouse epiblast (Ito et al., 2010[44]; Koh et al., 2011[56]; Ruzov et al., 2011[98]). Recently, it has been found that the shRNA-mediated knockdown of Tet1, but not Tet2 or Tet3, led to the spontaneous differentiation of mouse ES cells even in the presence of LIF (Ito et al., 2010). However, others believe that Tet1 is not required for maintaining the self-renewal of mouse ES cells (Koh et al., 2011[56]; Williams et al., 2011[128]; Xu et al., 2011[137]). The role of knockdown and knockout of Tet1 in forcing the transdifferentiation of mouse ES cells into trophoblasts has not been detected in vivo (Dawlaty et al., 2011; Ito et al., 2010), and Tet1–/– and Tet2–/– mice appeared to develop normally, appear healthy through adulthood and were found to be fertile (Dawlaty et al., 2011[20]; Li et al., 2011[66]; Moran-Crusio et al., 2011[72]; Quivoron et al., 2011[93]). Gu et al. (2011[29]) showed that homozygous mutation of Tet3 led to neonatal lethality, highlighting its importance in the development of multiple organs during embryogenesis. Thus, Tet1/Tet2 and 5-hmC plays an important but redundant role in the regulation of postimplantation embryonic development.

(iv) Mouse postnatal development

The role of 5-hmC dynamics in postnatal neurodevelopment and brain function and their abnormality leading to neural disorders has been worked out by various workers. 5-hmc is found to be present in almost all organs and tissues of mice but with varying levels (Ito et al., 2010[44]; Li et al., 2011[66]). The genome-wide analysis of brain revealed that 5-hmC levels in the cerebellum and hippocampus of adult mice are higher than those observed in postnatal day (P) 7 mice (Szulwach et al., 2011[111]). MECP2, a methyl-CpG-binding protein plays an important role in neural development and brain function (Lewis et al., 1992[63]). Amir et al. (1999[3]) reported that humans suffer from Rett syndrome, a neurodevelopmental disorder due to the mutations in MECP2. Szulwach et al. (2011[111]) using Mecp2 knockout and knockin mice found that the overall abundance of 5-hmC in the cerebellum was negatively correlated with Mecp2 dosage. Recent evidence suggests that Tet2 and 5-hmC are also associated with postnatal development of the hematopoietic lineage, and alteration of Tet2 expression or the levels of 5-hmC may disrupt the homeostasis of cells and ultimately lead to abnormalities in hematopoietic cell proliferation and maturation (Albano et al., 2011[2]; Chou et al., 2011[16]; Weissmann et al., 2012[127]). In TET knockout phenotype (Tet2–/–) of mice an abnormality in myeloid lineage differentiation and spontaneous leukemia but with a overall normal growth appeared (Ko et al., 2011[54]; Li et al., 2011[66]; Moran-Crusio et al., 2011[72]; Quivoron et al., 2011[93]). Gu et al. (2011[29]) showed that although the Tet3–/– phenotype was neonatal lethal, the exact functions of Tet3 protein in postnatal development are largely unknown and need further investigation.

(v) Human cerebellum development

5-hmC is abundant in human ESCs (hESCs) and brain regions, and the genomic features of 5-hmC in hESCs has been well characterized by Szulwach et al. (2011[110]) and Stroud et al. (2011[106]). Similar to hESCs, in cerebellum, 5-hmC displayed a bimodal distribution around transcription start sites (TSS) and was enriched in gene body regions. In comparison with hESCs, less 5-hmC was found at CpG islands and CpG shores, suggesting that CpG islands and shores may have a specific protective mechanism against 5-hmC modification in cerebellum. Wang et al. (2012[123]) have reported a positive correlation between 5-hmC levels and cerebellum development in humans. Genome-wide profiling reveals that 5-hmC is highly enriched on specific gene regions including exons and particularly the untranslated regions (5’UTRs), but it is depleted on introns and intergenic regions in both the fetal and adult cerebellum samples. Furthermore, they have also identified fetus-specific and adult-specific differentially hydroxymethylated regions (DhMRs), most of which overlap with genes and CpG island shores. Surprisingly, during development, DhMRs are highly enriched in genes encoding mRNAs that can be regulated by fragile X mental retardation protein (FMRP), some of which are disrupted in autism, as well as in many known autism genes.

(c) Human diseases

The crucial role of 5-hmc for normal neuronal function being evident with the mutation of MeCP2 protein, causing Rett syndrome, an autism disorder characterized by severe deterioration of neuronal function after birth (Szulwach et al., 2011[111]). The role of Tet proteins and 5-hmC has also been studied in the context of haematopoiesis and cancer. Aberrant DNA methylation is a hallmark of cancer, and cancer cells often display global hypomethylation and promoter hypermethylation (Esteller, 2008[21]). The Tet1 gene was originally identified through its translocation in acute myeloid leukemia (AML). Later, somatic Tet2 mutations were tified in patients with a variety of myeloid malignancies, including myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), acute myeloid leukemias and many others (Ko et al., 2010[55]). Figueroa et al. (2010[24]) demonstrated that Tet2 mutations in AML patients are predominantly associated with a DNA hypermethylation phenotype.

Among all the members of TET family, TET2 is the most studied genes in MDS and other types of leukemia and whose mutations or dysregulation might lead to lethality. In case of myeloid malignancies, it has been reported as one of the most frequently mutated genes (Albano et al., 2011[2]; Chou et al., 2011[16]; Weissmann et al., 2012)[127]. Some of the mutation sites in TET2 affects the residues within the catalytic domain and could thus damage catalytic activity, whereas, many mutations appear unrelated to enzymatic activity (Ko et al., 2010[55]). Recent studies on isocitrate dehydrogenase (IDH) gene which catalyzes the conversion of isocitrate to 2-oxoglutarate (2OG) also suggests the involvement of 5-hmC in cancer. The mutations in the isocitrate dehydrogenase gene IDH1/2 in patients suffering from AML were found to stimulate the accumulation of 2-hydroxyglutarate (2-HG) in cells (Klose et al., 2006[53]). This metabolite impairs catalytic activity of many 2-oxoglutarate-dependent enzymes, including Tet proteins, by competing with its co-substrate 2OG. Thus, these results suggest that the deficiencies in both IDH and Tet genes contribute to cancer development via a common disease mechanism that leads to altered 5mC and 5-hmC patterns. However, the role of TET1 in hematopoietic development and leukemogenesis still remains uncertain. The levels of 5-hmC have been found to be decreased in a broad range of solid tumors, including glioma, colon cancer, breast cancer and melanoma (Li and Liu, 2011[65]; Jin et al., 2011[47]; Haffner et al., 2011[31]).

(d) Transcriptional regulation

The abundance of Tet1 and 5-hmc at the promoter sites suggests a role for the Tet-mediated hydroxymethylation in transcriptional regulation (Wu and Zhang, 2011[132]). Tet1 and 5-hmc are enriched at 5' gene regulatory regions of various pluripotency factors, such as Nanog, Esrrb and Tcl1 thereby promoting the transcription of the gene. Therefore, the depletion of Tet1/2 has been reported to decrease the expression of a group of genes, including these pluripotency-related factors (Ficz et al., 2011[23]; Ito et al., 2010[44]; Wu et al., 2011[131]). In undifferentiated mouse ES cells, Tet1, probably in conjunction with Tet2, are required for promoting transcription of a cohort of pluripotency factors by maintaining a hypomethylated state at their promoters. Williams et al. (2011[128]) reported that the colocalization of TET1 the SIN3A co-repressor brings about the gene repression.

Methods for in vitro production and analysis of 5-hmc in DNA

The chemical synthesis of hmC containing oligonucleotides was first worked out by Planechaud et al. (1997[91]), and is currently being used by the majority of commercial supplies. DNA polymerase-dependent processes (including PCR) utilising commercial 2'-deoxynucleoside-5'-triphosphate (dhmCTP) are being used for enzymatic incorporation of 5-hmC into DNA. Recently, chemo-enzymatic reaction of DNA cytosine-5 methyltransferases has been developed for in vitro sequence specific incorporation of 5-hmC into DNA duplexes (Liutkeviciute et al., 2009[68]).

Following the discovery of 5-hmc, all the existing DNA methylation investigation methods needed to be re-evaluated as they were designed to distinguish only the two epigenetic states of cytosine: methylated versus unmodified. Therefore, there is a need to develop methods having the ability to discriminate between 5-hmC and 5mC in DNA. Previously, bisulfite sequencing (Huang et al., 2010[40]) were widely used for DNA methylation studies, in which the unmodified cytosine readily forms a 5,6-dihydro-6-sulfonyl adduct in the presence of bisulfite, which undergoes hydrolytic deamination to uracil, and thus appears as T in DNA sequencing. 5mC is very stable to bisulfite-promoted deamination and is subsequently read as normal C. Bisulfite reacts predominantly with the 5-hydroxymethyl group yielding a hydrolytically stable 5-sulfonylmethylcytosine (smC, also called cytosine 5-methylenesulfonate), and thus hmC appears as C (Huang et al., 2010[40]). In contrast, the products of hmC oxidation, caC and fC, are interpreted as unmodified C (Booth et al., 2012[10]) owing to their transient bisulfite-induced conversion to C. Thus, it is evident that bisulfite conversion cannot discriminate between 5mC and hmC (Huang et al., 2010[40]; Jin et al., 2010[48]). Recently, two methods have been proposed that permit detection of hmC in bisulfite sequencing after chemical or enzymatic modifications of hmC (Booth et al., 2012[10]; Yu et al., 2012[139]).

Methyl-sensitive CG-specific restriction endonucleases that differentially cleave unmethylated and methylated target sites are also an important tool for analysis of DNA methylation. Most of the restriction enzymes do not clearly discriminate between 5mC and 5-hmC (Nestor et al., 2010[81]) although some hmC-specific restriction endonucleases have recently been described (Wang et al., 2011[122]). Radiolabeling strategies have also been used for analysis of hmC in DNA. In one such approach named ‘‘nearest neighbour analysis’’, single stranded DNA breaks are randomly introduced by DNaseI and then DNA polymerase and [α-³³P]- dGTP is used to incorporate the radioactively labeled G nucleotide at the DNA breaks in the case when C is in the opposite strand. Then DNA is enzymatically hydrolysed to yield 3'-nucleotides such that the radiolabeled phosphate group appears on a 5'-neighbouring nucleotide (Kriaucionis and Heintz, 2009[58]). Alternatively, another method with higher sensitivity is also being used in which the modified cytosine in CG dinucleotides can be enriched by using R.MspI or R.TaqI restriction endonucleases, that cleaves CCGG or TCGA sites, respectively, between the two pyrimidines, regardless of whether C, 5mC or hmC is present in the second position. The produced DNA fragments with 5'-terminal CG sites are ³³P-labeled and DNA is degraded to 5'-NMPs for thin layer chromatography (TLC) (Tahiliani et al., 2009[113]).

HPLC can also be used for the analysis of 5-hmc. hmC 2'-deoxynucleoside elutes closely following, and is partially disguised by, a major peak of 2'-deoxycytidine. Similar elution times on reverse-phase columns and similar UV spectra of C and hmC may complicate detection of hmC in routine analyses of mammalian DNA using UV detection (Ito et al., 2011[45]). But, this problem can be overcome by the use of synthetic stable-isotope labeled internal standards in mass spectrometry detectors for reliable quantification of nucleosides (Münzel et al., 2010[75]).

Genome-wide localization of hmC can also be detected by affinity enrichment based methods (Figure 4(Fig. 4); modified from Kriukiene et al., 2012[59]). Such methods rely on selective binding of short hmC containing DNA fragments (200-800 bp) to hmC-specific antibodies or other hmC-binding proteins permitting their physical extraction from the rest of DNA for analysis using quantitative PCR, DNA microarrays or sequencing. Polyclonal and monoclonal antibodies have been raised against hmC itself or the product of bisulfite treatment of hmC, 5-sulfonylmethylcytosine (smC) (Ficz et al., 2011[23]; Jin et al., 2011[49]). During the last few years, methylated-DNA immunoprecipitation (MeDIP), hMeDIP (hydroxymethylated-DNA immunoprecipitation) are being widely used for 5-hmC profiling studies.

Figure 4. Analytical strategies for labelling and enrichment of hydroxymethylated DNA (modified from Kriukiene et al., 2012).

The enzymatic glucosylation of 5-hmC, using T4 beta-glucosyltransferase (BGT) and the uridine-5’-diphospho-D-glucose (UDP-glc) cofactor, to a much bulkier and distinctive residue, 5-glucosyloxymethylcytosine is highly specific and selective for the 5-hydroxymethyl group of hmC (or hmU). Such treatment can be used to attach tritium labeled glucose moieties from UDP-[3H] glucose to DNA, permitting direct quantification of hmC by scintillation counting (Szwagierczak et al., 2010[112]). JBP1 protein has been reported to selectively bind DNA fragments containing glucosylated hmC which can be used for selective isolation and analysis of hmC containing DNA (Robertson et al., 2011[97]).

Single-nucleotide resolution mapping of hmC residues in the genome can also be done by combining glucosylation of hmC and MspI restriction endonuclease digestion. R.MspI cleaves the CCGG target site if the second cytosine is unmethylated, methylated or hydroxymethylated, but glucosylation of hmC residues renders the sites resistant to MspI cleavage. Thus, glc-hmC DNA can be enriched and analysed using qPCR (Davis and Vaisvila, 2011[19]), microarrays or next-generation sequencing.

The enrichment of 5-hmc in DNA is associated with chemical capture of glucosylated hmC residues (Pastor et al., 2011[89]). Oxidation of the glucose moiety with NaIO₄ creates two reactive aldehyde groups which can be subsequently modified by commercially available aldehyde reactive probes containing biotin (Kriaučionis and Heintz, 2009[58]). Nanopore sequencing has also been shown to directly discriminate 5-hmC from 5mC without any further chemical modifications of the base (Wanunu et al., 2010[124]). Zhao and Xue (2012[143]) developed a highly sensitive fluorescence polarization method for genome-wide quantification of DNA methylation. In addition, to estimation of methylation level of submicrograms of lambda or human DNA, and of a 255-bp DNA segment containing a single HpaII /MspI restriction site, fluorescence polarization (FP) based measurement of DNA methylation (FPDM) was also applied to measure dose-dependent DNA hypomethylation in human embryonic kidney 293T cells treated with the DNA-methyltransferase inhibitor 5-aza-dC. Song et al. (2012[103]) used single-molecule real-time (SMRT) DNA sequencing for detection of 5-hydroxymethylcytosine (5-hmC) in genomic DNA.

Conclusion

5-Hydroxymethylcytosine (5-hmC) a newly discovered modified form of cytosine has been assumed to be an important epigenetic modification having important role in development ageing and diseases. 5-hmC is generated through oxidation of 5mC by the Ten-Eleven Translocation (TET) family of methylcytosine dioxygenases and is considered to be a possible intermediate step in a replication-independent DNA demethylation pathway. The role of Tet proteins and 5hmC in development and disease has been widely studied during the past few years. Moreover, further investigations are needed to distinguish the catalytic activity-dependent and independent functions of Tet proteins. Most studies of Tet proteins and 5hmC have been done on mice, rats and human models, therefore, investigations are needed to be expanded to other model systems (such as Xenopus, zebrafish and even non-vertebrates) in order to elucidate the evolutionally conserved functions of these proteins.

Acknowledgements

Richa and Rajneesh are thankful to the DST (Project No. SR/WOS-A/LS-140/2011) and DBT, Govt. of India, respectively, for the financial assistance in the form of fellowship.