Resources for methylome analysis suitable for gene knockout studies of potential epigenome modifiers

BackgroundMethylated DNA immunoprecipitation (MeDIP) is a popular enrichment based method and can be combined with sequencing (termed MeDIP-seq) to interrogate the methylation status of cytosines across entire genomes. However, quality control and analysis of MeDIP-seq data have remained to be a challenge.ResultsWe report genome-wide DNA methylation profiles of wild type (wt) and mutant mouse cells, comprising 3 biological replicates of Thymine DNA glycosylase (Tdg) knockout (KO) embryonic stem cells (ESCs), in vitro differentiated neural precursor cells (NPCs) and embryonic fibroblasts (MEFs). The resulting 18 methylomes were analysed with MeDUSA (Methylated DNA Utility for Sequence Analysis), a novel MeDIP-seq computational analysis pipeline for the identification of differentially methylated regions (DMRs). The observed increase of hypermethylation in MEF promoter-associated CpG islands supports a previously proposed role for Tdg in the protection of regulatory regions from epigenetic silencing. Further analysis of genes and regions associated with the DMRs by gene ontology, pathway, and ChIP analyses revealed further insights into Tdg function, including an association of TDG with low-methylated distal regulatory regions.ConclusionsWe demonstrate that MeDUSA is able to detect both large-scale changes between cells from different stages of differentiation and also small but significant changes between the methylomes of cells that only differ in the KO of a single gene. These changes were validated utilising publicly available datasets and confirm TDG's function in the protection of regulatory regions from epigenetic silencing.

[1]  W. Reik,et al.  Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation , 2011, Nature.

[2]  Jingde Zhu,et al.  Whole-genome DNA methylation profiling using MethylCap-seq. , 2010, Methods.

[3]  Vijay K. Tiwari,et al.  DNA-binding factors shape the mouse methylome at distal regulatory regions , 2011, Nature.

[4]  W. Huber,et al.  which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MAnorm: a robust model for quantitative comparison of ChIP-Seq data sets , 2011 .

[5]  M. Robinson,et al.  A scaling normalization method for differential expression analysis of RNA-seq data , 2010, Genome Biology.

[6]  Ralf Herwig,et al.  Computational analysis of genome-wide DNA methylation during the differentiation of human embryonic stem cells along the endodermal lineage. , 2010, Genome research.

[7]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[8]  Tom H. Pringle,et al.  The human genome browser at UCSC. , 2002, Genome research.

[9]  W. Lam,et al.  Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells , 2005, Nature Genetics.

[10]  Gang Li,et al.  Jarid2 and PRC2, partners in regulating gene expression. , 2010, Genes & development.

[11]  Chuan He,et al.  Tet Proteins Can Convert 5-Methylcytosine to 5-Formylcytosine and 5-Carboxylcytosine , 2011, Science.

[12]  D. Reinberg,et al.  The Polycomb complex PRC2 and its mark in life , 2011, Nature.

[13]  Yang Wang,et al.  Tet-Mediated Formation of 5-Carboxylcytosine and Its Excision by TDG in Mammalian DNA , 2011, Science.

[14]  S. Orkin,et al.  Jumonji Modulates Polycomb Activity and Self-Renewal versus Differentiation of Stem Cells , 2009, Cell.

[15]  S. Michel,et al.  MeQA: a pipeline for MeDIP-seq data quality assessment and analysis , 2012, Bioinform..

[16]  A. Bird.  DNA methylation patterns and epigenetic memory. , 2002, Genes & development.

[17]  Jean YH Yang,et al.  Bioconductor: open software development for computational biology and bioinformatics , 2004, Genome Biology.

[18]  W. Ouwehand,et al.  Combinatorial transcriptional control in blood stem/progenitor cells: genome-wide analysis of ten major transcriptional regulators. , 2010, Cell stem cell.

[19]  Guo-Cheng Yuan,et al.  EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. , 2008, Molecular cell.

[20]  Lu Zhang,et al.  Comparative methylome analysis of benign and malignant peripheral nerve sheath tumors. , 2011, Genome research.

[21]  D. Reinberg,et al.  Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms. , 2008, Molecular cell.

[22]  A. Urban,et al.  MEDME: an experimental and analytical methodology for the estimation of DNA methylation levels based on microarray derived MeDIP-enrichment. , 2008, Genome research.

[23]  A. Klein-Szanto,et al.  Thymine DNA Glycosylase Is Essential for Active DNA Demethylation by Linked Deamination-Base Excision Repair , 2011, Cell.

[24]  P. Laird.  Principles and challenges of genome-wide DNA methylation analysis , 2010, Nature Reviews Genetics.

[25]  Cory Y. McLean,et al.  GREAT improves functional interpretation of cis-regulatory regions , 2010, Nature Biotechnology.

[26]  R. Young,et al.  SetDB1 contributes to repression of genes encoding developmental regulators and maintenance of ES cell state. , 2009, Genes & development.

[27]  Steven S. Smith,et al.  Human non-CG methylation , 2010, Epigenetics.

[28]  A. Bird,et al.  Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability , 2011, Nature.

[29]  Chia-Lin Wei,et al.  Dynamic changes in the human methylome during differentiation. , 2010, Genome research.

[30]  M. Pellegrini,et al.  A comparative analysis of DNA methylation across human embryonic stem cell lines , 2011, Genome Biology.

[31]  Aaron R. Quinlan,et al.  Bioinformatics Applications Note Genome Analysis Bedtools: a Flexible Suite of Utilities for Comparing Genomic Features , 2022 .

[32]  David A. Nix,et al.  Empirical methods for controlling false positives and estimating confidence in ChIP-Seq peaks , 2008, BMC Bioinformatics.

[33]  T. Mikkelsen,et al.  Genome-scale DNA methylation maps of pluripotent and differentiated cells , 2008, Nature.

[34]  C. Kunz,et al.  The enigmatic thymine DNA glycosylase. , 2007, DNA repair.

[35]  Li Chen,et al.  hmChIP: a database and web server for exploring publicly available human and mouse ChIP-seq and ChIP-chip data , 2011, Bioinform..

[36]  R. Durbin,et al.  A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis , 2008, Nature Biotechnology.

[37]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[38]  Lee E. Edsall,et al.  Human DNA methylomes at base resolution show widespread epigenomic differences , 2009, Nature.

[39]  Stephan Beck,et al.  The methylome: approaches for global DNA methylation profiling. , 2008, Trends in genetics : TIG.

[40]  Megan F. Cole,et al.  Connecting microRNA Genes to the Core Transcriptional Regulatory Circuitry of Embryonic Stem Cells , 2008, Cell.

[41]  S. Orkin,et al.  Supplemental Data EZH 1 Mediates Methylation on Histone H 3 Lysine 27 and Complements EZH 2 in Maintaining Stem Cell Identity and Executing Pluripotency , 2008 .

[42]  H. Clevers,et al.  Wnt signalling in stem cells and cancer , 2005, Nature.

[43]  Daniel Rios,et al.  Ensembl 2011 , 2010, Nucleic Acids Res..