There are lots of tools for probing the epigenome
If epigenetics is the study of modifications that aren't coded in the DNA, how can we use DNA sequencing to learn anything about the epigenome?
Epigenetics seeks to understand how non-sequence elements regulate gene expression.
This control occurs as a result of many factors including the external environment, time of day, temperature, stress, location of a particular cell within a tissue, etc.
All of these factors can change what parts of the DNA can be accessed for conversion into functional proteins!
And, this control occurs mainly at two levels:
1. Methylation of Cytosines (5mc) in DNA can make a region unrecognizable.
2. Modification of histones with methylation, phosphorylation or acetylation can result in the formation of heterochromatin (closed access) or euchromatin (open access).
How can we get at this information?
With sequencing!
Cytosine Methylation:
Caveat! PCR amplification of DNA results in the removal of methylated Cytosine - it’s recognized as just plain old cytosine by polymerases, so in most cases you have to pre-treat the DNA to detect it!
1. Bisulfite Conversion - Bisulfite exposure converts unmethylated Cytosines to Uracils. Subsequent PCR amplification during library creation converts those Uracils to Thymine, and in the resulting sequencing data, any position the remains as a Cytosine was methylated in the original DNA strand!
2. Affinity Enrichment - Certain proteins bind specifically to 5mc. You can affinity enrich these proteins using capture techniques. Another alternative is to use a capture based technology where bisulfite converted DNA is bound to RNA probes designed to specifically pick up regions of known methylation.
3. Direct 5mc Sequencing - Both PacBio and Oxford Nanopore can detect 5mc directly, spitting out 5-bases with high accuracy (A, C, T, G, and 5mc)!
Histone Modifications:
1. ChIP-Seq/CUT&(RUN/TAG) - All of these use antibodies that recognize specific histone modifications like acetylation and methylation to map these marks throughout the genome by sequencing the DNA that they're stuck to!
Chromatin Accessibility:
1. DNase-I Hypersensitivity/ATAC-seq - Euchromatin, the open kind, can be chopped up by enzymes that cut DNA while heterochromatin, the closed kind, protects DNA from this treatment. Sequencing the fragments that fall in the 200-500bp size range tells you where there's open chromatin!
2. Hi-C/Looping - Certain proteins, like CTCF, create 'loops' in chromatin that can activate or repress gene expression. These can be mapped using various techniques like Hi-C which ligates together fragments that are 'close' to one another.
But, epigenetics is complicated, and these marks can differ widely across tissues, cell types, and populations of cells!
They can also change frequently...
So, what's the point?
Epigenetic dysfunction, among other things, can cause rare diseases, cancer, or affect how we age.
But that's a discussion for next week!