EpigenomicsThe many garments of the genome sequence

Winterschool Brisbane, 2014 !

Dr Fabian Buske Garvan Institute of Medical Research

Sequencing has revolutionised life sciences

Epigenetics!ChIP-Seq,

WGBS, HiC,

DNaseHS, Repli-seq,

…

Transcriptomics!RNA-seq, CAGE-seq

Capture-seq, …

Genomics!WGS,

ExonCapture …

Epigenetics

the study of heritable changes that occur without a change in the DNA sequence

Epigenetics

http://www.youtube.com/watch?v=Tj_6DcUTRnM

Outline

• DNA methylation

- Whole Genome Bisulphite Sequencing

• Histone modification

- Chromatin Immunoprecipitation Sequencing

• DNA looping

- Chromosome Conformation Capture (HiC)

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

Shear DNA

Methylated DNA

C GTCT

C GTUT

C GTTT

PCR

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)

Histones

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

DNA-protein complex

DNA extraction

Sample fragmentation

Crosslink proteins and DNA

Immunoprecipitate

ChIP-Seq

Benefits!

• Captures genome-wide tissue-specific protein-DNA interactions !

• Relatively cheap compared to WGBS, HiC!

!Caveats!

• Highly dependent on an available antibody and its specificity !

• ~20-60M reads depending on the fraction of the genome anticipated to be

bound!

• Controls (input) need to be sequenced deeper that actual IP library!

!

ChIP-Seq data analysis

Mapping to the sequence space

Transcribed in cancer cells

Transcribed in normal cells

DNA looping

• The DNA fiber is a flexible polymer

• DNA looping enables genomic regions that are distant in sequence space to come in close physical proximity and thus relay signals (e.g. enhancers and promoters)

1D 3Dvs

Sequencing based 3D assaysCardinality

Resolution

3C 4C

5C

Chia-Pet

HiC

High (bp)

Low (mb)

One-to-one All-to-All

HiC

Chia-Pet

5Csweet spot

Capture-C

Capture-c

$

$$$

quadratic nature of “all versus all” data

3C/HiC protocol

• HiC: Before ligation, the restriction ends are filled in with biotin-labeled nucleotides.

DNACrosslink proteins and DNA Sample fragmentation Ligation PCR amplify ligated junctions

via restriction enzymes

HiC data processing

http://www.bioinformatics.babraham.ac.uk/projects/hicup/

Take Home Messages• Epigenetics: the study of heritable changes that

occur without a change in the DNA sequence

• Variety of assays available for the interrogation of the epigenetic state genome-wide

• Lots of public data available (ENCODE, Epigenome Roadmap, GEO)

• Understand the biological question and the wet-lab protocol… choose your tools accordingly!

• Check out Illumina’s poster http://bit.ly/1kxGdzz

Reading material

Questions?f.buske@garvan.org.au