5-methylcytosine (5mc) is the methylated form of DNA at the 5-position of the DNA base cytosine found in mammals. Its significance is in epigenetic modification, which demonstrates an important role in development and genome regulation. Furthermore the oxidation of 5-methylcytosine in Tet catalyzed reactions has been suggested to play an vital role in the regulation of transcription and gene expression, and DNA de-methylation (Wu and Zhang, 2011). There has been a considerable amount of research into 5-methylcytosine oxidation; clearly indicating that 5-methylcytosine oxidation in the genome has an effect on mammalian development due to its contribution to normal mammalian development as well as being associated with disease. This is a review of recent research in the key roles of 5-methylcytosine oxidation products in the development of mammals.
As described by Liu et al., (2013) the addition of a methyl group during DNA methylation in mammals occurs at the position of 5th carbon of cytosine residues primarily at CpG dinucleotide regions. Methylation of DNA plays a role in repressing gene expression including repressing transposable elements (TEs) (Ito et el., 2011). This process of methylation is first established during embryonic development in embryogenesis and then retained during cell division due to the presence of various de novo DNA methyltransferases (DNMT). Research shows the significance of cytosine methylation in mammalian development as it was observed that mice that lack DNA methyltransferases will die at the age of 4 weeks (Liu et al., 2013).
5-methylcytosine is a crucial epigenetic marker, as methylation of cytosine in DNA has a main role in gene expression due to methylated genes in the DNA being able to express differently even though the DNA sequence remains the same. It has also been recognised that CpGs can be methylated in various areas of the genome due to differences in cell type and in points of development (Xu et al., 2013).
Recent studies within the past have suggested that aberrance in DNA methylation pattern can cause the process to become deficient through either passive or active mechanisms. Passive cytosine DNA demethylation refers to removal of DNMT1 activity during cell division. Active cytosine DNA demethylation refers to the 5-methylcytosine being converted to cytosine due to the removal of a methyl group, which is independent of DNA replication. These mechanisms of DNA demethylation are associated with defects in development (Liu et al., 2013).
A series of enzymatic oxidation reactions in the genome using ten-eleven translocation 1-3 proteins, also known as TET dioxygenases, allow 5-methylcytosine to produce 5-hydroxymethylcytosine (5HmC), 5-formylcytosine (5FoC), and 5-carboxylcytosine (5CaC). The Tet-catalysed process relies on iron and alpha-ketoglutarate dependent oxidation. This sequence of oxidation reactions is said to be linked to active mammalian cytosine demethylation (Ito et al., 2011).
DNA demethylation can be categorised as either global referring to genome wide, or locus specific referring to just certain sequences being methylated. In mammals, genome wide DNA de-methylation is said to occur in mouse primordial germ cells (PGCs) in embryos as early as E8.5-E11.5 days (Schomacher 2011).
During early embryogenesis it has been suggested that removal followed by re-establishment of cytosine methylation occurs in a process of major reprogramming. Due to the ten-eleven translocation proteins having the ability to convert 5-methylcytosine to 5-hydroxymethylcytosine, there is a possibility that 5-hydroxymethylcytosine may work in an epigenetic manner and may contribute to dynamic alterations in the regulation of transcription and in DNA methylation during embryogenesis. Research shows that embryonic stem cells express high levels of the Tet dioxygenases Tet1, and reasonably high levels of 5-hydroxymethylcytosine compared to many differential cells. The large distribution of Tet1 and 5-hydroxymethylcytosine throughout the embryonic stem cells of the mouse genome, demonstrate the role of Tet proteins and 5-hydroxymethylcytosine in regulating gene expression associated with cellular differentiation and pluripotency (Wu and Zhang, 2011)2.
The occurrence of oxidation of 5-methylcytosine and 5-hydroxymethylcytosine in DNA to 5-carboxylcytosine, and subsequent thymine DNA glycosylase (TDG) excision of 5-carboxycytosine is said to establish a route for active DNA demethylation. Moreover study into TDG reduction in mouse embryonic stem cells has been found to cause an evident build-up of 5-carboxylcytosine. Research showed that 5-carboxylcytosine was absent in the embryonic stem cells and neurons of mice who presented high levels of Tet dioxygenases. However 5-carboxylcytosine was seen to be chemically stable and did not freely decarboxylate to cytosine, implying that in genomic DNA, 5-carboxylcytosine may be actively removed directly after being generated in cells (He et al., 2011).
Furthermore, it is suggested that oxidation products 5-formylcytosine and 5-carboxylcytosine can partake in the base excision repair (BER) mechanism. This allows 5-formylcytosine and 5-carboxylcytosine to be excised followed by being repaired leading to regeneration as unmodified cytosines by thymine DNA glycosylase. Research, using genome wide distribution maps, into TDG deficient embryonic stem cells, found that reduction of TDG in mouse embryonic stem cells caused noticeable build-up of 5-formylcytosine and 5-carboxylcytosine in genes. Therefore, these results imply that active DNA demethylation is TDG dependent and occurs widely in the mammalian genome (Shen et el., 2013).
Additionally, in order to determine if oxidation of 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine occurs in the zygote in vivo, research was conducted in which antibodies were produced specific for 5-formylcytosine and 5-carboxylcytosine. An immuno-staining technique determined that the depletion of 5-methylcytosine in the mouse paternal pronucleus is concomitant with the presence of 5-formylcytosine and 5-carboxylcytosine. It was notably significant that rather than being instantly removed, both oxidation products displayed dilution which was replication-dependent during preimplantation development in mice. (Inoue et al., 2011)
It is well recognised that 5-hydroxymethylcytosine is associated with mammalian development, as studies show the importance of 5-hydroxymethylcytosine activity in both passive and active DNA demethylation, during phases of reprogramming in development. It has also been found that brain tissue has copious amount of 5-hydroxymethylcytosine suggesting that the mammalian brain relies on 5-hydroxymethylcytosine for development. Recently, 5-hydroxymethylcytosine has also been associated with a potential role in cancer as current research has suggested that the levels of 5-hydroxymethylcytosine are considerably reduced in tumour cells. In addition it has been proposed that mutations in the Tet2 protein can cause lack of function which may also be implicated in tumour suppression (Pfeifer et al., 2013).
In conclusion, modified versions of cytosine due to oxidation by Tet proteins, are important in the roles of DNA demethylation and reprogramming of stem cells. Hence, future additional research into the function of Tet proteins and further advanced stem cell research could benefit by acquiring more knowledge into alterations in DNA methylation. This will greatly develop understanding of epigenetic regulation in normal mammalian development and disease.
Wu, H. and Zhang, Y. (2011) ‘Mechanisms and functions of Tet protein-mediated 5-methylcytosine oxidation’ Genes Dev, 25 (23), pp. 2436-2452
Liu, S., Wang, J., Su, Y., Guerrero, C., Zeng, Y., Mitra, D., Brooks, P. J., Fisher, D. E., Song, H. and Wang, Y. (2013) ‘Quantitative assessment of Tet-induced oxidation products of 5-methylcytosine in cellular and tissue DNA’ Nucleic acids research, 41 (13), pp. 6421-6429
Ito, S., Shen, L., Dai, Q., Wu, S. C., Collins, L. B., Swenberg, J. A., He, C. and Zhang, Y. (2011) ‘Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine’ Science, 333 (6047), pp. 1300-1303
Xu, Y., Wu, F., Tan, L., Kong, L., Xiong, L., Deng, J., Barbera, A. J., Zheng, L., Zhang, H., Huang, S. and Others. (2011) ‘Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells’ Molecular cell, 42 (4), pp. 451-464
Schomacher, L. (2013) ‘Mammalian DNA demethylation’ Epigenetics, 8 (7), pp. 679-684
Wu, H. and Zhang, Y. (2011) ‘Tet1 and 5-hydroxymethylation’ Cell Cycle, 10 (15), pp. 2428-2436
He, Y., Li, B., Li, Z., Liu, P., Wang, Y., Tang, Q., Ding, J., Jia, Y., Chen, Z., Li, L. and Others. (2011) ‘Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA’ Science, 333 (6047), pp. 1303-1307
Shen, L., Wu, H., Diep, D., Yamaguchi, S., D’Alessio, A. C., Fung, H., Zhang, K. and Zhang, Y. (2013) ‘Genome-wide analysis reveals TET-and TDG-dependent 5-methylcytosine oxidation dynamics’ Cell, 153 (3), pp. 692-706
Inoue, A., Shen, L., Dai, Q., He, C. and Zhang, Y. (2011) ‘Generation and replication-dependent dilution of 5fC and 5caC during mouse preimplantation development’ Cell research, 21 (12), pp. 1670-1676
Pfeifer, G. P., Kadam, S. and Jin, S. (2013) ‘5-hydroxymethylcytosine and its potential roles in development and cancer’ Epigenetics Chromatin, 6 (10), pp. 1-9.