Fabien Buske - Epigenomics - The many garments of the genome sequence

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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

  • Epigenomics The 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 modication - 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 modication 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? Bisulte 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 Benets! 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, gs of DNA needed! Analysis is not straightforward, few methods are available! ! Library preparation is basically the same of WGS but with a bisulte step and different polymerase (Uracil tolerant proofreader)
  • Data analysis of methylated regions Mapping against an in-silico bisulte-treated genome (Bismark) Discovery of ac>ve regulatory regions de novo (MethylSeekR - HMM) ! ! ! ! ! Dieren>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 modied by more than 100 different post- translational modications including methylation, acetylation, phosphorylation, and ubiquitination
  • Why study Histone modifications? important epigenetic mechanism in transcriptional regulation through modication of the chromatin structure or through chromatin condensation interplay between histone modications and DNA methylation dene developmental potential of a cell chromatin proling is especially well suited to the characterisation of non-coding portions of the genome in a tissue-specic manner
  • How do we study Histones? Chromatin Immunoprecipitation with subsequent sequencing (ChIP-Seq)! ! - crosslinking of proteins to DNA! - enrichment with specic antibody! - sequencing! ! Analysis of histone mark deposition via read density DNA-protein complex DNA extraction Sample fragmentation Crosslink proteins and DNA Immunoprecipitate
  • ChIP-Seq Benets! Captures genome-wide tissue-specic protein-DNA interactions ! Relatively cheap compared to WGBS, HiC! ! Caveats! Highly dependent on an available antibody and its specicity ! ~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 ber is a exible 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 assays Cardinality Resolution 3C 4C 5C Chia- Pet HiC High (bp) Low (mb) One-to-one All-to-All HiC Chia- Pet 5C sweet spot Captu re-C Captu re-c $ $$$ quadratic nature of all versus all data
  • 3C/HiC protocol HiC: Before ligation, the restriction ends are lled 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 Illuminas poster http://bit.ly/1kxGdzz
  • Reading material Questions? f.buske@garvan.org.au