5hmC-The "Sixth Base" in mammalian DNA
5-hydroxymethylcytosine (5hmC) in mammalian DNA was first described in the early 1970s by Penn et al. (Penn et al., 1972). However, as this finding could not be reproduced (Kothari and Shankar, 1976), the topic received scant attention over the next three decades until 2009 when Kriaucionis and Tahiliani demonstrated that 5hmC is present in mouse Purkinje and granule neurons and in embryonic stem cells (Kriaucionis and Heintz, 2009; Tahiliani et al., 2009).
5hmC has since been detected at substantial levels in other mammalian tissues (Globisch et al., 2010). High levels are observed in the central nervous system and the spinal cord (0.3-0.7% of dG), moderate levels in kidney, nasal epithelium, bladder, heart, skeletal muscle and lung (0.15 -0.17%), and low levels in liver, spleen and the endocrine glands (0.03-0.06%).
Furthermore, using a more sensitive method based on chemical tagging of 5hmC, Song et al. detected 5hmC in HeLa and human embryonic kidney (HEK293) cells at levels of ~0.001% of total nucleotides (Song et al., 2011).
Conversion of 5mC to 5hmC
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 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 granule cells (Kriaucionis and Heintz, 2009), as would be expected if oxidative damage was the driving force. An important step towards understanding the occurrence of 5hmC was provided when Tet proteins were recently identified as 5mC hydrolases that catalyze the conversion of 5mC to 5hmC. The mammalian TET family contains three members, Tet1, Tet2 and Tet3, which share significant sequence homology at their c-terminal catalytic domains (Ito et al., 2010; Tahiliani et al., 2009) and display the typical features of 2-oxyglutarate (2OG)- and Fe(II)-dependent dioxygenases (2OGFeDO) (Aravind and Koonin, 2001; Loenarz and Schofield, 2009). 5hmC is formed through the action of TET proteins, which utilize molecular oxygen to transfer a hydroxyl group to 5mC.
Legend: Cytosine species in mammalian DNA. Cytosine exists as a free nucleotide that is incorporated into DNA during replication. 5-mthylcytosine (5mC) is formed by the post-replicative addition of a methyl group to cytosine through the action of DNA methyltransferases (DNMT), which use S-adenosyl methionine (SAM) as the methyl donor. 5-hydroxymethylcytosine (5hmC) is formed through the action of TET proteins, which utilize molecular oxygen to transfer a hydroxyl group to 5mC.
Biological roles of 5hmC
5hmC can influence both long and short-term regulation of gene expression, which will likely have biological significance in vivo.
Conversion of 5mC to 5hmC may facilitate passive DNA demethylation by excluding maintenance of DNA methylation during cell division by DNA methyltransferases 1 (DNMT1), which recognizes 5hmC poorly. Even a minor reduction in the fidelity of maintenance methylation would be expected to result in an exponential decrease in CpG methylation over the course of many cell cycles and thus may lead to passive demethylation.
Possible biological roles of 5hmC-A: 5hmC is not recognized by DNA methyltransferases (DNMT), which will prevent maintenance methylation during DNA replication, resulting in passive DNA demethylation.
Conversely, 5hmC has been shown to yield cytosine through loss of formaldehyde in photooxidation experiments (Privat and Sowers, 1996) and at high pH (Alegria, 1967; Flaks and Cohen, 1959), leaving open the possibility that 5hmC could convert to cytosine under certain conditions in cells. A related possibility is that 5hmC could also be a key intermediate in a possible active DNA demethylation pathway during DNA repair. (Tahiliani et al., 2009). In support of this hypothesis, a glycosylase activity specific for 5hmC was reported in bovine thymus extracts (Cannon et al., 1988). Moreover, several DNA glycosylases, including TDG and MBD4, have been implicated in DNA demethylation, although none of them has shown convincing activity on 5mC in in vitro enzymatic assays (Kangaspeska et al., 2008; Metivier et al., 2008; Zhu et al., 2000). Cytosine deamination of hmC yields hmU, and high levels of hmU:G glycosylase activity have been reported in fibroblast extracts (Rusmintratip and Sowers, 2000).
Possible biological roles of 5hmC-B: 5hmC may be recognized by DNA repair proteins, eg. A 5hmC-specific DNA glycosylase (5hmC-DG), which will convert 5hmC to cytosine, leading to active DNA demethylation.
Finally, as a potentially stable base, 5hmC may influence chromatin structure and local transcriptional activity by recruiting selective 5hmC-binding proteins or excluding methyl-CpG-binding proteins (MBPs) that normally recognize 5mC, thus displacing chromatin-modifying complexes recruited by MBPs. Indeed, it has already been demonstrated that the methyl-binding protein MeCP2 does not recognize 5hmC (Valinluck et al., 2004).
Possible biological roles of 5hmC-C: 5hmC is not recognized by methyl-CpG-binding proteins (MBDs), including MBD1, MBD2, MBD4 and MeCP2, which will prevent the recruitment of histone deacetylases (HDAC), leading to the formation of transcriptionally competent chromatin. -CH3, methyl group; -CH2OH, hydroxymethyl group.
The Genomic location of 5hmC and its relationship to gene expression levels
5hmC in gene bodies and its relationship to gene expression levels
5hmC was enriched specifically in gene bodies (Jin et al., 2011; Pastor et al., 2011; Song et al., 2011; Xu et al., 2011). Further analysis also reveals that intragenic enrichment of 5hmC is associated with expressed genes, consistent with a potential role for 5hmC in activating and/or maintaining gene expression (Jin et al., 2011; Song et al., 2011). It is possible that conversion of 5mC to 5hmC is a pathway to offset the gene repression effect of 5mC during this process without going through demethylation (Wu and Zhang, 2010).
However, the 5hmC level within gene bodies is not a simple reflection of associated gene expression level. Xu et al. found that the 5hmC level is very low within the bodies of a set of genes with constantly high expression levels, such as housekeeping genes (Xu et al., 2011).
5hmC in gene promoters and its relationship to gene expression levels
Song et al. observed enrichment of 5hmC in proximal upstream and downstream regions relative to TSS, TTS and distal regions (Song et al., 2011). Further analysis reveals that proximal enrichment of 5hmC is associated with more highly expressed genes, consistent with a role for 5hmC in maintaining and/or promoting gene expression. Ficz et al. also showed that the presence of 5hmC in promoter regions was associated with high levels of transcription. This effect is also partially dependent on promoter CpG density. Promoters enriched for both 5hmC and 5mC were also associated with higher levels of transcription than promoters specifically enriched for 5mC, suggesting that the presence of 5hmC partially overcomes the silencing effect of 5mC. Consistent with these observations, promoters that are high in 5hmC are enriched in the activating histone mark H3K4me3, whereas those enriched in 5mC are depleted of H3K4me3 (Ficz et al., 2011).
Interestingly, it was observed that promoters of pluripotency-related genes including Esrrb, Prdm14, Dppas, Klfz, TCL1 and ZFP43 that were down regulated during ES cell differentiation had a marked decrease of 5hmC enrichment levels, which was accompanied by a significant increase in 5mC levels (Ficz et al., 2011).
Enrichment of 5hmC at bivalent domains and its correlation with gene expression levels
5hmC is especially enriched at the start sites of genes whose promoters bear dual histone H3 lysine 27 trimethylation (H3K27me3) marks and histone H3 lysine 4 trimethylation (H3K4me3) (Pastor et al., 2011). Williams et al. demonstrated that Tet1 binds a significant proportion of polycomb group target genes (Williams et al., 2011). Therefore, 5hmC may contribute to the "poised" chromatin signature found at developmentally-regulated genes in ES cells and have a probable role in transcriptional regulation.
Enrichment of 5hmC at enhancers and a potential role of 5hmC in gene regulation
5hmC is relatively more enriched in enhancers (defined H3K4me1 in the absence of H3K4me3) than 5mC, strongly indicating a connection between 5hmC and regulatory elements (Pastor et al., 2011). Stroud et al. also observed a positive correlation between 5hmC and H3K4me1 and H3K27ac and found that 5hmC marks active or poised enhancers (Stroud et al., 2011).
Distribution of 5hmC at promoters of testis-species genes
Promoters of testis-specific genes showed strong 5mC peaks in brain DNA. Testis-specific genes and other germ line-specifically expressed genes are often silenced by promoter DNA methylation in somatic tissues (De Smet et al., 1999). These genes are remarkably devoid of 5hmC peaks at their promoters. Testis-specific genes are characterized by the absence of 5hmC at their promoters.
Dynamic regulation of 5hmC in ES cells and during differentiation
5hmC is uniquely associated with a "poised" chromatin conformation and with lineage-specific genes that are upregulated upon differentiation, and may thus be involved in priming loci for rapid activation in response to appropriate signals. Activation of lineage-specific genetic loci upon differentiation could occur via a postulated 5mC "demethylation" pathway (5mC to 5hmC to cytosine) or through recruitment of transcriptional regulators that specifically recognize 5hmC and are activated in response to differential signals (Pastor et al., 2011). Alternatively, upon differentiation into EBs, 5hmC enrichment from promoters of pluripotency related gene in ES cells decreases in these regions, concomitant with a gain of 5mC (Ficz et al., 2011).
Furthermore, it is proposed that hydroxymethylation (5hmC) could have a role in erasing methylation marks from promoters of pluripotency-related genes during the generation of induced pluripotent stem cells (IPSs) (Meissner et al., 2008) and have a role in the large-scale erasure of methylation in primordial germ cells (Iqbal et al., 2011; Ito et al., 2010).
Oxygen-sensing and regulation
Song et al. observed an enrichment of 5hmC in genes linked to hypoxia and angiogenesis. The oxidation of 5mc to 5hmC by Tet proteins requires dioxygen (Ito et al., 2010; Tahiliani et al., 2009). A well-known oxygen sensor in mammalian systems that are involved in hypoxia and angiogenesis is the HIF protein, which belongs to the same mononuclear iron-containing dioxygenase superfamily as the active domain of the Tet proteins. It is tempting to speculate that oxidation of 5mC to 5hmC by Tet proteins may constitute another oxygen-sensing and regulation pathway in mammalian cells (Song et al., 2011).
Epigenetic reprogramming in fertilized oocytes
The epigenomes of early mammalian embryos are extensively reprogrammed to acquire a totipotent developmental potential. 5-hydroxymethylcytosine (5hmC) in the mammalian zygote is linked with epigenetic reprogramming.
Genome-wide erasure of DNA cytosine-5 methylation has been reported to occur along the paternal pronucleus in fertilized oocytes in an apparently replication-dependent manner. However, Wosido et al. show that in advanced pronuclear-stage zygotes, 5hmC accumulates in the paternal pronucleus along with a reduction of 5mC. Itwas further demonstrated that 5hmC is derived from the enzymatic oxidation of 5mC by Tet3 oxidase in the paternal pronucleus.
The role of 5hmC in the paternal pronucleus is currently unknown. One immediate effect of this oxidation step should be the neutralization of the functional role of 5mC in gene suppression. Embryonic genome activation in the mouse takes place at the two-cell stage and it is expected that many genes that are methylation-suppressed during spermatogenesis (e.g. Oct4 and Nanog) will need to be activated to allow development to process. After oxidation of 5mC, the 5hmC-containing sequences will no longer be capable of interacting with repressor proteins that are known to bind to 5mC (Jin et al., 2010; Valinluck et al., 2004). Alternatively, DNA sequences containing 5hmC in place of 5mC are not substrates for the maintenance methylatransferase activity of DNMT1 (Valinluck and Sowers, 2007). This finding means that the formation of 5hmC may serve to dilute DNA CpG methylation during replication in early embryos, even in the presence of any nuclear DNMT activity.
Neuronal development and maturation
Song et al. observed the developmental stage-dependent increase of 5hmC in mouse cerebellum. Compared to postnatal day 7 at a time of massive cell proliferation in the mouse cerebellum, adult cerebellum has a significantly increased level of 5hmC, suggesting that 5hmC might be involved in neuronal development and maturation.
Role of 5hmC in diseases
Role of 5hmC in age-related neurodegeneration
Gene ontology pathway analysis of the 5425 genes acquiring 5hmC during aging identified significant enrichment of pathways associated with age-related neurodegenerative disorders, angiogenesis and hypoxia response. Furthermore, an assessment of the gene list revealed that 15/23 genes previously identified as the cause of axia and Purkinje cell degeneration in mouse and human acquired intragenic 5hmC in adult mice (Lim et al., 2006). The association of 5hmC with genes that have been implicated in neurodegenerative disorders suggests that this base modification (5hmC) could potentially contribute to the pathogenesis of human neurological disorders.
Role of 5hmC in cancer
5hmC is significantly reduced in cancerous colorectal tissue and even decreased to an undetectable level in colon cancer cell lines. The levels detected with an increased number of samples (38 colon cancer tissues and 8 normal colon tissues) further confirmed that the 5hmC content in colon cancer is four-fold lower than that in normal colon tissues. These results suggested that 5hmC may negatively regulate cancer formation and development at least in colorectal tissues. It is possible that the 5hmC reduction in cancerous colorectal tissues damages the reactivation of methylation-mediated silencing of tumor suppression and apoptosis genes through 5hmC-mediated methylation turnover, which would help the cancer cells to escape form tumor suppression and apoptosis caused by products of these genes (Li and Liu, 2011).
5hmC may participate directly in hematopoietic malignancies. TET2, resides on chromosome 4q24, a region that is commonly deleted or involved in chromosomal rearrangement in patients with myelodysplastic disorders (Viguie et al., 2005). Tet2 missense mutations were found primarily in the c-terminal region containing the 2OGFeDO domain and interfere with the ability of TET2 to convert 5mC to 5hmC (Ko et al., 2010). Bone marrow samples from AML patients with TET2 mutations had significantly lower levels of 5hmC in genomic DNA compared with healthy controls (Ko et al., 2010). All these results strongly indicate that 5hmC may have a role in AML.
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