Epigenetic modifications are reversible modifications on the DNA that affect gene expression without changing the actual genome sequence. The spectrum of modifications range from DNA methylation, histone modification and nucleosome positioning to DNA packaging and chromatin organization in the three dimensional space. This presentation will highlight different assays and bioinformatic approaches used to query epigenetic modifications genome‐wide as well as how these layers of information can be integrated into meaningful models. First presented at the 2014 Winter School in Mathematical and Computational Biology http://bioinformatics.org.au/ws14/program/
Text of Fabien Buske - Epigenomics - The many garments of the genome sequence
EpigenomicsThe many garments of the genome sequence
Winterschool Brisbane, 2014 !
Dr Fabian Buske Garvan Institute of Medical Research
Sequencing has revolutionised life sciences
the study of heritable changes that occur without a change in the DNA sequence
DNA methylation• Addition of a methyl group to the 5-carbon of
cytosine in DNA (5mC)
• In mammals, almost exclusively occurs at CpG dinucleotides in a strand symmetrical manner
- Strand symmetry allows for stable inheritance through cell divisions via DNMT1 maintenance
- ~28M CpG sites in the human genome
- Majority are methylated
- Except the 3.9M in/adjacent to CpG islands
Why study DNA methylation?
• Has demonstrated roles in!- Cellular programming
- dynamic during development/differentiation - Genomic imprinting/X-inactivation
!• 5mC presence is anti-correlated with “activity” of a DNA sequence!
- Promoters, gene bodies, distal regulatory elements, insulators - MBPs bind 5mC to repress the surrounding chromatin
!• Is stable and relatively easily assayable!
- Covalent modification of the DNA
DNA methylation & cancer• Aberrant promoter methylation in cancer is associated with tumour
suppressor gene silencing!- Occurs at enhancers/insulators as well !
• Alterations in other diseases are relatively poorly studied
How do we study DNA methylation?
• Bisulfite treatment deaminates unmethylated cytosines to uracil!!
- Uracil is converted to thymine via PCR!- 5mC is unaffected, therefore remains as
cytosine after PCR!!
‣ Methylation is then assayable as a SNP
Whole genome bisulphite sequencing
Benefits!• Assays all mappable CpG sites (~27M)!• Get a “free” genome sequence at the same time!!Caveats!• Quantitation ability is proportional to depth of sequencing (count Cs vs Ts)!
- To detect a 10% change in 5mC at a single site, requires lots of coverage!- Pooling possible as adjacent CpG sites are correlated!
• Expensive, low throughput, µgs of DNA needed!• Analysis is not straightforward, few methods are available!!Library preparation is basically the same of WGS but with a bisulfite step and different polymerase (Uracil tolerant proofreader)
Data analysis of methylated regions
• Mapping against an in-‐silico bisulfite-‐treated genome (Bismark) • Discovery of ac>ve regulatory regions de novo (MethylSeekR -‐ HMM)
• Differen>ally Methylated Regions between pa>ent cohorts/treatments/condi>ons (bioconductor bsseq)
the nucleosome is composed of two copies of each of the four core histones (ie, H2A, H2B, H3, and H4), which are wrapped around by 146 bp of DNA
The N-terminal tails of histone polypeptides can be modified by more than 100 different post-translational modifications including methylation, acetylation, phosphorylation, and ubiquitination
Why study Histone modifications?
important epigenetic mechanism in transcriptional regulation through modification of the chromatin structure or through chromatin condensation
interplay between histone modifications and DNA methylation define developmental potential of a cell
chromatin profiling is especially well suited to the characterisation of non-coding portions of the genome in a tissue-specific manner
How do we study Histones?• Chromatin Immunoprecipitation
with subsequent sequencing (ChIP-Seq)!!
- crosslinking of proteins to DNA!- enrichment with specific antibody!- sequencing!!
‣ Analysis of histone mark deposition via read density