About Epigenetics

 

DNA Methylation:

DNA methylation occurs when a methyl group is added to DNA (see figure 1). The methylation of DNA does not alter the DNA sequence itself, but it can alter gene expression (e.g. decreasing gene expression). DNA methylation can be inherited between cell divisions (via the DNA methyltransferase enzyme) and is thought to a crucial part of the development of a large number of cancers.
DNA methylation usually occurs in CG rich regions that are upstream of a gene's promoter region (known as CpG islands). CpG islands are located in the promoter regions of genes that are frequently expressed in the cells. DNA methylation does not usually occur within these housekeeping genes (genes that are essential for general cell functions), but genes that are inactive are usually methylated to express their expression.

DNA methylation patterns are also known to differ between different tissues and cell types, therefore a DNA methylation pattern for an individual within a venous blood sample might not be replicated within a different tissue type such as a buccal swab.

DNA Methylation

 

Epigenetics as a tool:

Epigenetics looks at the methylation patterns that occur through the CpG islands which can act to alter gene expression. Epigenetics adds a layer of understanding on top of the association between genotype and phenotype. Phenotypes can be affected by the methylation patterns that might not be seen by looking at genotype alone, but could be explained by the methylation pattern which can alter the genes expression.

 

Testing methods that will be used in this project:

Methylated DNA immunoprecipitation sequencing (MeDiP-sequencing) – This is to be the main technique that will be used within the EpITwin project. This a genome wide technique that isolates methylated DNA fragments using an antibody raised against 5-methylcytosine. This procedure uses genomic DNA which is subjected to sonication (ultrasound that vibrates the DNA until it fragments) to break the DNA into random fragments. The fragments produced range from 300 base pairs (bp) to 1000bp in length. The DNA fragments are then denatured and incubated with moncloncal 5-methylcytosine antibodies. The resulting suspendion then undergoes a classical immunoprecipitation where DNA that is bound to the 5-methylcytosine antibodies remain and any DNA not bound is then washed off (see figure 2).

MeDIP

Bisulphite sequencing – This will be done on a subset of samples and will be used as verification/replication of the results from the MeDiP sequencing. Genomic DNA is treated with bisulfite that converts unmethylated cytosine to uracil, methylated cytosine remains unchanged. The DNA then undergoes routine sequencing which produces single-nucleotide information on the methylation status of DNA (see figure 3).

Bisulphite Sequencing