DNA Methylation - DNA analogues for Epigenetic Resarch
About DNA methylation
Epigenetic, the study of heritable changes in gene expression and regulation that are not due to changes in the DNA sequence itself, plays an important role in biology and cancer research. While the underlying genetic code defines which proteins and gene products are synthesized, it is the post-synthetic modification such as DNA methylation, the epigenetic control primarily involves methylation of cytidine in CpG islands and the modification of histones, defines when and where they are expressed. This dynamic control of gene expression is responsible for in a number of biological porcesses such as , X chromosome inactivation, embryogenesis, cellular differentiation and tumor suppressor gene silencing in cancerous cells. Aberrant methylation of DNA is associated with a variety of disorders such as Beckwith-Wiedermann, Prader-Willi, and Angelman syndromes, as well as cancers such as neuroblastoma, Wilms tumour, and osteosarcoma, among many others. A few of the chronic diseases associated with dysregulation of DNA methylation patterns include cardiovascular disease, obesity, lupus, and certain types of leukemia.
DNA methylation mechanism
Epigenetic control is generally mediated by methylation of cytidine to 5-methyl-dC in CpG islands, a CG upstream of the promoter greion and post-translational modification of histones. Methylation of CpG sites near promoters is associated with gene silencing, as is deacetylation of histones. While a number of mammalian DNA methyltransferases are known in the DNMT family, the enzymes responsible for demethylation in mammals have yet to be identified conclusively since none has shown clear activity in vitro 1. However, Kriaucionis demonstrated that 40% of the purported 5-methyl-dC in Purkinje neurons was actually 5-hydroxymethyl-dC 2 (5-HOMedC) While most prominent in neuronal tissues such as the brain and spine, 5hmdC has also been identified in a broad range of tissues such as the bladder, heart, liver and pituitary glands7. This hydroxymethyl cytidine prevents the targeting of a number of transcriptional repressors specific for 5-Me-dC, it appears to serve a subtle regulatory role9. Removal of the methyl group from 5-Me-dC (demethylation) also plays an important role in cellular reprogramming, embryogenesis, autoimmune disorders and is critical in the establishment of maternal and paternal methylation patterns.
DNA demethylation occurs through active and passive mechanisms. Passive mechanisms simply involve the synthesis of DNA with the absence of methylation. Active DNA demethylation is thought to occur through a number of different mechanisms such as base excision repair (BER), nucleotide excision repair (NER), or the direct removal of the methyl group from 5-Me-dC6. Direct demethylation from cytidine requires the breaking of a carbon-carbon bond through an unidentified mechanism. More likely, the removal of the methyl group involves the hydroxylation of 5-Me-dC to 5-hmdC or deamination of 5-Me-dC to thymidine and subsequent BER. An alternative demethylation pathway could involve the oxidation of 5-Me-dC to 5-formyl-dC or 5-carboxy-dC with subsequent decarboxylation to cytosine, though these active demethylation intermediates were not detected using a sensitive HPLC-MS assay7,8.
Bio-Synthesis offer several Modified Base Oligonucleotide Synthesis for epigenetic research:
Properties of 5-Carboxy-dC
The incorporation of 5-carboxy-dC into a DNA duplex has a stabilizing effect on DNA as indicated by a modest increase in the Tm (~2 °C per incorporation) 10 despite its extra negative charge of the 5-carboxylic acid. The 5-carboxy moiety of cytidine projects into the major groove of a duplex, minimizing the potential negative steric effect of the modification. In comparison, the incorporation of 5-carboxy-dC into triplex forming oligos (TFO) as the third strand has a destabilizing effect, lowering the melting temperature by approximately 4-5° C. This is most likely because of the repulsion of the extra negative charge with the phosphate backbone and the crowding of the Hoogesten base pairing.
The presence of the carboxy moiety does not significantly alter the pKa of the N3 on cytidine with a pKa of 4.4 for cytidine versus a pKa of 4.0 for 5-carboxy-dC (Table 1), as determined by spectral analysis.10 The Watson-Crick hybridization of G-CCOO- is essentially unaffected by the modification.
| Nucleoside |
pKa |
| dC |
4.4 |
| 5-methyl-dC |
4.5 |
| 5-carboxy-dC |
4 |
| 5-formyl-dC |
2.4 |
| Table 1 |
Properties of 5-Formyl-dC
The duplex stability of oligos containing 5-formyl-dC is comparable to oligos containing 5-Me-dC, with the Tm for 5-Me-dC being approximately 1.3° C higher per incorporation.11 Interestingly, the misincorporation of thymidine opposite 5-formyl-dC was 3-4 times higher compared to control oligos containing dC and 5-Me-dC.11 The higher misincorporation rate was speculated to be a result of the changes in shape and the presence of the electron-withdrawing formyl group of 5-formyl-dC in comparison to 5-Me-dC. The overall effect is that 5-formyl-dC is considered highly mutagenic and increases the rate of transition mutations when evaluated by polymerase extension.11
In contrast to 5-carboxy-dC, the presence of the electron-withdrawing formyl group lowers the pKa to 2.4 compared to a pKa of 4.4 for dC and 4.5 for 5-Me-dC (Table 1).The hybridization of G-dCFo is unaffected by the modification as indicated by thermal denaturation.
5-Formyl-dC and 5-carboxy-dC may find uses in research into DNA damage and repair processes.
Properties of 5-Hydroxymethyl-dC (5-HOMedC)
Hydroxymethyl cytidine analog (5-HOMedC) has low affinity for the Methyl-CpG-binding Protein MeCP2, which is a known transcriptional repressor, as well as DNMT1, which is the maintenance DNA methyltransferase3,4. This opens up the possibility that demethylation would be acquired passively over multiple cell cycles if a means could be found to convert 5-methyl-dC to 5-hydroxymethyl-dC. Less than a month after Kriaucionis’ publication2, an enzyme, TET1, was found5 which catalyzes the conversion of 5-methyl-dC to 5-hydroxymethyl-dC in vitro and in vivo, ushering in a new chapter in the field of epigenetics with 5-hydroxymethyl-dC taking center stage.
5-hydroxymethyl-dC also plays an important role in DNA damage. After all, 5-hydroxymethyl-dU is a product of oxidative damage of thymidine by hydroxyl radicals or ionizing radiation. Similarly, 5-hydroxymethyl-dC may be a damaged form of 5-methyl-dC. However, it is unlikely that the 5-hydroxymethyl-dC detected in neuronal cells is formed by oxidative damage since no other damaged nucleosides, e.g., 8-oxo-dG, were detected in these cells.5 Nevertheless, 5-hydroxymethyl-dC has been detected in bacteria9 and it is certainly feasible that it could still be a product of oxidative damage in mammalian cells.
References:
- S.K. Ooi, and T.H. Bestor, Cell, 2008, 133, 1145-8.
- S. Kriaucionis, and N. Heintz, Science, 2009, 324, 929-30.
- V. Valinluck, et al., Nucleic Acids Res., 2004, 32, 4100-8.
- V. Valinluck, and L.C. Sowers, Cancer Res, 2007, 67, 946-50.
- M. Tahiliani, et al., Science, 2009, 324, 930-5.
- K.M. Schmitz, et al., Mol Cell, 2009, 33, 344-53.
- D. Globisch, et al., PLoS One, 2010, 5, e15367.
- S.C. Wu, and Y. Zhang, Nat Rev Mol Cell Biol, 11, 607-20.
- S.G. Jin, X. Wu, A. X. Li, and G.P. Pfeifer, Nucleic Acids Res., 2011.
- M. Sumino, A. Ohkubo, H. Taguchi, K. Seio, and M. Sekine, Bioorganic & Medicinal Chemistry Letters 2008, 18, 274-227
- N. Karino, Y. Ueno, and A. Matsuda Nucleic acids Res., 2001, 29, 2456-2463