Hydroxy-Sensitive Cut Counting (HSCC); simultaneous, genome-wide mapping of 5-methylcytosine and 5-hydroxymethylcytosine in mammals

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The human genome contains all the instructions (genes) necessary to create a human being, beginning from a single fertilised egg cell. Although the same genome sequence is present in all cells, there are hundreds of markedly different cell types, such as brain (neurons), kidney, and liver cells, in the human body. Each cell type must 'turn on' a different subset of genes for the correct functioning of each tissue type. A primary mechanism by which cells mark genes to be turned on or off is called, 'epigenetics'. Epigenetics refers to heritable changes in gene expression that are not caused by mutations in the underlying DNA sequence. The most comprehensively studied epigenetic mark in mammals is DNA methylation which involves the attachment of a tag-molecule called a 'methyl group' to cytosines to give 5-methylcytosine (5mC). It typically occurs in a CpG dinucleotide context, although non-CpG methylation occurs in embryonic stem cells, between 60-90% of all CpGs are methylated in mammals. DNA methylation is associated with gene silencing and is essential for normal development. Key processes including genomic imprinting, X-chromosome inactivation, suppression of repetitive elements, stability of gene expression and carcinogenesis either depend or involve dynamic changes in DNA methylation patterns. From this perspective it is important to know where modified DNA resides in the genome.Much of what we know about DNA methylation in mammals is based on a set of techniques which can distinguish between 5mC and an unmethylated cytosine (C). These include the use of methyl-sensitive restriction enzymes (which can cut unmethylated, but not methylated DNA) and bisulfite sequencing, a method that allows accurate quantification of methylation levels at several neighbouring cytosines simultaneously. This analysis has been confounded by the identification of a new type of modified DNA, 5-hydroxymethylcytosine (5hmC) that is present at high levels in mammalian tissues. Indeed, 5hmC is 40% as common as 5mC in mouse brain samples. Despite intense study of DNA methylation for the last 30 years, the presence of 5hmC in mammalian tissues had been missed especially as most the techniques used to identify methylation, do not differentiate between 5mC and 5hmC. We propose a radical, new technique termed Hydroxy Sensitive Cut Counting (HSCC) to simultaneously analyse for 5mC, 5hmC and C at the 1.5 -2.3 million CCGG sequences present throughout the mouse and human genomes respectively. HSCC is based on the observation that the restriction enzyme MspI, can cut its target site, CCGG, if the internal C is 5hydroxymethylcytosine, but not if it is beta-glucosyl-5-hydroxymethylcytosine (ghmC). Using a well characterised and commercially available enzyme T4 Phage beta-glucosyltransferase, we will convert all the 5hmC in the genome to ghmC, and digest the sample before and after treatment with MspI. The sequences surrounding each MspI site will then be sequenced using next generation sequencing. The number of sequences in the treated versus untreated samples for each MspI site is a measure of the amount of 5hydroxymethylcytosine present, allowing for semi-quantification of genome-wide 5hmC levels. The resulting 5hmC profiles will represent tissue 'identifier'; a barcode that will be a read-out of a normal tissue state. HSCC represents a dramatic improvement on the existing 'genome-wide' techniques that use antibodies to harvest DNA containing 5hmC. Such 'affinity' techniques can only assay regions of the genome which contain 5hmC and often exhibit strong sequence-context biases. In contrast, HSCC will assay MspI sites regardless of 5hmC level. This is vital, as knowing where 5hmC is depleted is as important as knowing where it is enriched if we are to understand the role of this exciting new mark in mammalian biology.