In the previous post on epigenetics, I introduced the topic of promoter CpG island methylation and how that could silence gene expression. In effect, by looking at whether the promoters of genes are methylated or not, one could go some way towards predicting whether those genes are expressed or not, and building genome wide methylation profiles is a very good way of trying to figure out what expression changes happen.There is a variety of approaches that may be employed to look at methylation profiles, and with the advent of genomics and the publication of the human genome, it has become possible to do so on a large scale basis, both of which are based on Methylated DNA Precipitation (MeDIP) and a subsequent set of processes.
In MeDIP, DNA is isolated from the tissue that is being profiled, is denatured and separated into methylated and non-methylated components. In order to do this, first, DNA is sonicated into small fragments that are 100-1000 base pairs long. It is then incubated overnight with magnetic beads and an antibody that only binds to methylcytosine (this is how DNA is methylated). The magnetic beads contain an antibody to the antibody that holds methylated DNA, and this enables methylated DNA to be collected using a magnetic field that aggregates all these magnetic beads and by extension methylated DNA together. Whatever is not attached to the beads is then discarded.
The antibody is then digested away using Proteinase K, which is an enzyme, and this frees up DNA to go into the proteinase K solution, where it stays unharmed.
This DNA is then isolated using ethanol extraction and purification, and once it is isolated, further processing can take place.Since we’re looking across the whole genome, the same DNA must be amplified, this is done using commercially available whole genome amplification kits that work in ways which only the manufacturers know. Once this DNA is amplified and purified, it can be stored away for the next phase, which involves the actual data gathering and analysis. MeDIP is cheap but array analysis can be expensive, which is why there are quality control features built in, and after the DNA is prepared, it is subjected to quantitative PCR and more electrophoresis to ensure that the fragments are still in the originally sonicated range and to confirm that immunoprecipitation has enriched the methylated fraction compared to unmethylated DNA.If a processed sample meets requirements, array analysis (Chip) can go ahead.
The immunoprecipitated DNA and the unprocessed input (which is also whole genome amplified) are then labelled with different fluorescent dyes and hybridized to an array with specific gene probes. A readout of the array will then yield an address of all the regions of the genome that have a probe on the array and will have been hypermethylated compared to the control. Doing this across multiple samples enables those samples to be compared, for instance, cancerous tissue versus normal tissue, or two different types of cancer, or those that respond to a certain kind of therapy versus those that do not.
Such analysis is carried out computationally, and the results are used to identify differentially methylated regions (DMRs) which can serve as a molecular signature of a certain tissue state. Again, the possibilities are endless.The data generated is often complex, but further bioinformatics analysis can simplify things a great deal.
Nina Pälmke et al, Comprehensive analysis of DNA-methylation in mammalian tissues using MeDIP-chip, Methods, Volume 53, Issue 2,
February 2011, Pages 175-184, ISSN 1046-2023, 10.1016/j.ymeth.2010.07.006.
Weber et al, Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells
Nature Genetics – 37, 853 – 862 (2005)
Stephan Beck, Vardhman K. Rakyan, The methylome: approaches for global DNA methylation profiling, Trends in Genetics, Volume 24, Issue 5, May 2008, Pages 231-237, ISSN 0168-9525, 10.1016/j.tig.2008.01.006.