Quantifying the Relationship between DNA Methylation and Gene Expression in Human Colon Cancer Matthias Lienhard Max Planck Institute for Molecular Genetics, Berlin IMPRS Colloquium July 9th 2014 Epigenetics CH Waddington, 1942: “epigenetic landscape” Epigenetics Athur Riggs: "the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence." Riggs AD, Russo VEA, Martienssen RA (1996, Cold Spring Harbor Monograph): Epigenetic mechanisms of gene regulation. DNA Methylation ● Methyl modification at Cytosine ● In CpG context ● Represses gene activity Gene silencing ● Promoter methylation→gene repressed Colon Cancer rd ● 3 most common cancer ● 5 y survival ~40% - 60% ● Risk factors: age, lifestyle ● Epigenetic alteration – Relatively frequent – Epigenetic markers Human Colon Cancer Samples ● 14 Colon Cancer Patients ● Normal Colon and Tumor Tissue ● MeDIP-Seq ● Low coverage input sequencing (0.2 x) ● RNA-Seq (12 Patients) ● High coverage WGS (4 Patients, 80 x, CG) MEDIPS package A: IP-seq DNA fragments Immunopreciptiation Epigenetic mark Sequencing Enriched fragments rpkm Pearson correlation MeDIP reads in genomic window MeDIP read density Estimated linear fit for MeDIP Input read density Saturation Estimated saturation 0 Short reads 5M 10M 15M 20M Number of reads C: Quality control CpG Coupling Factor Fraction of differentially methylated regions [%] LogFC r = 0.74 Lower quantile 25%-75% CF Upper quantile MeDIP-seq rms value D: Normalization 1.0 B: Alignment Bisulfite [% methylated] MEDIPS package E: Validation hypomethylated hypermethylated 0.8 0.6 0.4 0.2 0.0 All regions Introns Exons Promoter CpG Islands CGI Promoter Log( Ad x No ) F: Statistical test for differential coverage http://www.bioconductor.org/MEDIPS G: Annotation and functional interpretation M. Lienhard et al. (Bioinformatics, 2014) MEDIPS: genome wide differential coverage analysis of sequencing data derived from DNA enrichment experiments MeDIP-seq MeDIP-seq ImmunoSequencing preciptiation Enriched DNA Short reads methylated fragments fragments cytosine Alignment MEDIPS: Preprocessing Alignment Counting 2 1 2 7 6 2 250 bases → Count matrix: 11.524.123 windows x 28 samples 1 MEDIPS: Normalization Coverage logFC Library Size CpG content 1 0 -1 avg. log coverage M Robinson et al., Genome Biology (2010): A scaling normalization method for differential expression analysis of RNA-seq data Number of CpG L Chavez et al., Genome Research (2010): Computational analysis of genome-wide DNA methylation during the differentiation of human embryonic stem cells along the endodermal lineage. MEDIPS: Differential coverage analysis edgeR: ● ● Developed for RNA-seq Negative binomial distribution Over-dispersion estimation LogFC ● Log( Ad x No ) M. D. Robinson et al., Bioinformatics (2010): edgeR: a bioconductor package for dierential expression analysis of digital gene expression data CNV deletion ● ● amplification Present in most Tumors Tumor ● CNA free Accumulation at genomic locations Normal Profiles define clusters Tumor Chr 1 2 3 4 5 6 7 8 910 … 22 CNV and MeDIP signal CNA-free amplifications methylation logFC deletions methylation logFC avg. log methylation level Mean CNV logFC of CNV state M. Robinson et al. (Genome Research, 2012): Copy-numberaware differential analysis of quantitative DNA sequencing data. Explorative analysis PC 2 PCA PC 1 Differentially methylated Regions Log Ratio T/N 27711 windows with gain of methylation (0.22%) 16193 windows with loss of methylation (0.13%) avg log abundance DNA Methylation Gain of methylation Fraction differentially methylated regions[%] 7 Loss of methylation 6 5 4 3 2 1 at BS r s TF mote pro ter mo Pro BS TF ne Ge y bod All gions Re 0 MAL WNT2 No DMR in TFBS Promoter 5.5% 88.5% 6% Hypermethylated promoters 13.4% 81.4% 5.3% 13.4% 5.5% Hypomethylated promoters 4.4% 81.4% 88.5% 5.3% 6% 15.7% 79.9% Enrichment of TFBS in DMRs Fraction with loss of methylation 2% 1.5% 1% 0.5% 0% Enrichment of TFBS in DMRs Fraction with loss of methylation Enrichment of TFBS in DMRs Fraction with loss of methylation 2% 1.5% 1% 0.5% 0% 35% 30% 25% 20% 15% 10% 5% 0% Fraction with gain of methylation Enrichment of TFBS in DMRs 2% 1.5% 1% 0.5% 0% Fraction with gain of methylation EZH2 binding sites 40% 30% 20% 10% 0% Genome Promoter wide Gene body intergenic Promoter methylation logFC Relationship of methylation and gene expression Expression logFC Relationship of methylation and gene expression CHD1 binding sites Acknowledgement Max Planck Institute for Molecular Genetics Bernd Timmermann Hans Lehrach Christina Grimm Michal Schweiger Lukas Chavez Ralf Herwig Medical University Graz Kurt Zatloukal Universität des Saarlandes Jörn Walter
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