Methods for detection of cytosine and thymine modifications in DNA

Abstract

Methylation of cytosine at the 5-position is a common epigenetic marker in mammalian DNA, and plays an important role in regulating gene expression. Oxidised derivatives of 5-methylcytosine have recently been discovered. As well as being intermediates in an active demethylation pathway, some of these oxidised derivatives may function as epigenetic markers in their own right. Oxidised derivatives of thymine are also known as products of DNA damage. There is evidence however that one such derivative, 5-hydroxymethyluracil, may play an epigenetic role. There is a pressing need to learn more about these modifications, due to the role epigenetic markers play in development, and diseases such as cancer. This emerging area of research requires methods for detecting cytosine and thymine modifications in DNA with a high degree of accuracy and sequence specificity. This review will introduce the biochemistry of cytosine and thymine modifications, and discuss new and established detection methods which have been developed to overcome the high degree of difficulty associated with studying these modifications in DNA. Introduction: Cytosine and Thymine Modifications DNA codes for all the proteins necessary for life, but the DNA sequence is not the sole determinant of all the phenotypic traits of cells and organisms. Transcription of DNA is tightly regulated by epigenetic modifications, which play an important role in controlling when and where specific genes are expressed. 1,2 Epigenetic modifications regulate important processes such as cellular differentiation, and are also implicated in various diseases including cancer, autism spectrum disorder, and other developmental diseases such as Rett syndrome. 3-5 Epigenetic control of gene expression is often mediated through covalent modification of DNA itself, or of the histone proteins which pack DNA in the nucleus. These modifications do not result in changes to the DNA sequence, but can affect the binding of certain proteins to the DNA, including transcription factors and proteins which regulate chromatin structure. 6-8 In mammals and many other eukaryotes, the most common epigenetic covalent modification of DNA is methylation of cytosine (C) at the 5-position. Such modifications can be inherited, but can also be enzymatically introduced and removed in response to stimuli. 9 5-Methylcytosine (mC) is introduced by the methylation of cytosine residues by DNA-methyltransferases (DNMTs) with an S-adenosylmethionine cofactor. 5-Methylcytosine is most often found in the context of 5'-cytosinephosphate-guanine-3' (CpG) dinucleotides. Regions containing a high density of CpG sequences are found in about 72% of promoters in the human genome. 10 Methylation of these regions causes

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