Genome-wide mapping of nucleosome positioning and DNA methylation within individual DNA molecules

DNA methylation and nucleosome positioning work together to generate chromatin structures that regulate gene expression. Nucleosomes are typically mapped using nuclease digestion requiring significant amounts of material and varying enzyme concentrations. We have developed a method (NOMe-seq) that uses a GpC methyltransferase (M.CviPI) and next generation sequencing to generate a high resolution footprint of nucleosome positioning genome-wide using less than 1 million cells while retaining endogenous DNA methylation information from the same DNA strand. Using a novel bioinformatics pipeline, we show a striking anti-correlation between nucleosome occupancy and DNA methylation at CTCF regions that is not present at promoters. We further show that the extent of nucleosome depletion at promoters is directly correlated to expression level and can accommodate multiple nucleosomes and provide genome-wide evidence that expressed non-CpG island promoters are nucleosome-depleted. Importantly, NOMe-seq obtains DNA methylation and nucleosome positioning information from the same DNA molecule, giving the first genome-wide DNA methylation and nucleosome positioning correlation at the single molecule, and thus, single cell level, that can be used to monitor disease progression and response to therapy.

[1]  Peter A. Jones,et al.  Role of nucleosomal occupancy in the epigenetic silencing of the MLH1 CpG island. , 2007, Cancer cell.

[2]  V. Iyer,et al.  FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin. , 2007, Genome research.

[3]  Ryan A. Flynn,et al.  A unique chromatin signature uncovers early developmental enhancers in humans , 2011, Nature.

[4]  C. Roberts,et al.  SWI/SNF nucleosome remodellers and cancer , 2011, Nature Reviews Cancer.

[5]  Archana Dhasarathy,et al.  Mapping chromatin structure in vivo using DNA methyltransferases. , 2004, Methods.

[6]  Gangning Liang,et al.  Polycomb-Repressed Genes Have Permissive Enhancers that Initiate Reprogramming , 2011, Cell.

[7]  Peter A. Jones,et al.  Dynamic Nucleosome-Depleted Regions at Androgen Receptor Enhancers in the Absence of Ligand in Prostate Cancer Cells , 2011, Molecular and Cellular Biology.

[8]  Dustin E. Schones,et al.  Global analysis of the insulator binding protein CTCF in chromatin barrier regions reveals demarcation of active and repressive domains. , 2008, Genome research.

[9]  P. Laird,et al.  Bis-SNP: Combined DNA methylation and SNP calling for Bisulfite-seq data , 2012, Genome Biology.

[10]  J. V. Van Etten,et al.  Cloning, characterization and expression of the gene coding for a cytosine-5-DNA methyltransferase recognizing GpC. , 1998, Nucleic acids research.

[11]  Ian M. Carr,et al.  MethylViewer: computational analysis and editing for bisulfite sequencing and methyltransferase accessibility protocol for individual templates (MAPit) projects , 2010, Nucleic Acids Res..

[12]  Daiya Takai,et al.  Comprehensive analysis of CpG islands in human chromosomes 21 and 22 , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[13]  Peter A. Jones,et al.  Hypomethylation of a LINE-1 Promoter Activates an Alternate Transcript of the MET Oncogene in Bladders with Cancer , 2010, PLoS genetics.

[14]  Héctor Corrada Bravo.  Increased Methylation Variation in Epigenetic Domains across Cancer Types , 2012, RECOMB.

[15]  Dustin E. Schones,et al.  High-Resolution Profiling of Histone Methylations in the Human Genome , 2007, Cell.

[16]  R. Durbin,et al.  Mapping Quality Scores Mapping Short Dna Sequencing Reads and Calling Variants Using P

, 2022 .

[17]  H. Aburatani,et al.  Methylation screening of reciprocal genome-wide UPDs identifies novel human-specific imprinted genes. , 2011, Human molecular genetics.

[18]  Lee E. Edsall,et al.  Distinct epigenomic landscapes of pluripotent and lineage-committed human cells. , 2010, Cell stem cell.

[19]  S. Horvath,et al.  Neurosphere Formation Is an Independent Predictor of Clinical Outcome in Malignant Glioma , 2009, Stem cells.

[20]  D. Pinto,et al.  A novel approach identifies new differentially methylated regions (DMRs) associated with imprinted genes. , 2011, Genome research.

[21]  Peter A. Jones,et al.  Analysis of individual remodeled nucleosomes reveals decreased histone–DNA contacts created by hSWI/SNF , 2009, Nucleic acids research.

[22]  Peter A. Jones,et al.  H2A.Z maintenance during mitosis reveals nucleosome shifting on mitotically silenced genes. , 2010, Molecular cell.

[23]  T. Mikkelsen,et al.  The NIH Roadmap Epigenomics Mapping Consortium , 2010, Nature Biotechnology.

[24]  M. Pellegrini,et al.  Relationship between nucleosome positioning and DNA methylation , 2010, Nature.

[25]  D. Price,et al.  Formaldehyde‐assisted isolation of regulatory elements , 2009, Wiley interdisciplinary reviews. Systems biology and medicine.

[26]  Wei Li,et al.  BSMAP: whole genome bisulfite sequence MAPping program , 2009, BMC Bioinformatics.

[27]  Helga Thorvaldsdóttir,et al.  Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration , 2012, Briefings Bioinform..

[28]  Michael Q. Zhang,et al.  Analysis of the Vertebrate Insulator Protein CTCF-Binding Sites in the Human Genome , 2007, Cell.

[29]  A. Franke,et al.  DNA methylome analysis using short bisulfite sequencing data , 2012, Nature Methods.

[30]  T. Kelly,et al.  Methylation‐Sensitive Single‐Molecule Analysis of Chromatin Structure , 2010, Current protocols in molecular biology.

[31]  Amos Tanay,et al.  Constitutive Nucleosome Depletion and Ordered Factor Assembly at the GRP78 Promoter Revealed by Single Molecule Footprinting , 2006, PLoS genetics.

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

[33]  P. Laird,et al.  Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina–associated domains , 2011, Nature Genetics.

[34]  Scott A. Hoose,et al.  Single-molecule and population probing of chromatin structure using DNA methyltransferases. , 2007, Methods.

[35]  Dustin E. Schones,et al.  Dynamic Regulation of Nucleosome Positioning in the Human Genome , 2008, Cell.

[36]  Peter A. Jones,et al.  OCT4 establishes and maintains nucleosome-depleted regions that provide additional layers of epigenetic regulation of its target genes , 2011, Proceedings of the National Academy of Sciences.

[37]  T. Mikkelsen,et al.  Systematic discovery of regulatory motifs in conserved regions of the human genome, including thousands of CTCF insulator sites , 2007, Proceedings of the National Academy of Sciences.

[38]  Scott A. Hoose,et al.  Active PHO5 chromatin encompasses variable numbers of nucleosomes at individual promoters , 2006, Nature Structural &Molecular Biology.