Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications

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Abstract

Sequencing-based DNA methylation profiling methods are comprehensive and, as accuracy and affordability improve, will increasingly supplant microarrays for genome-scale analyses. Here, four sequencing-based methodologies were applied to biological replicates of human embryonic stem cells to compare their CpG coverage genome-wide and in transposons, resolution, cost, concordance and its relationship with CpG density and genomic context. The two bisulfite methods reached concordance of 82% for CpG methylation levels and 99% for non-CpG cytosine methylation levels. Using binary methylation calls, two enrichment methods were 99% concordant, while regions assessed by all four methods were 97% concordant. To achieve comprehensive methylome coverage while reducing cost, an approach integrating two complementary methods was examined. The integrative methylome profile along with histone methylation, RNA, and SNP profiles derived from the sequence reads allowed genome-wide assessment of allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression.

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Most cited references 27

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Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning.

Shawn Cokus, Suhua Feng, Xiaoyu Zhang … (2008)

Cytosine DNA methylation is important in regulating gene expression and in silencing transposons and other repetitive sequences. Recent genomic studies in Arabidopsis thaliana have revealed that many endogenous genes are methylated either within their promoters or within their transcribed regions, and that gene methylation is highly correlated with transcription levels. However, plants have different types of methylation controlled by different genetic pathways, and detailed information on the methylation status of each cytosine in any given genome is lacking. To this end, we generated a map at single-base-pair resolution of methylated cytosines for Arabidopsis, by combining bisulphite treatment of genomic DNA with ultra-high-throughput sequencing using the Illumina 1G Genome Analyser and Solexa sequencing technology. This approach, termed BS-Seq, unlike previous microarray-based methods, allows one to sensitively measure cytosine methylation on a genome-wide scale within specific sequence contexts. Here we describe methylation on previously inaccessible components of the genome and analyse the DNA methylation sequence composition and distribution. We also describe the effect of various DNA methylation mutants on genome-wide methylation patterns, and demonstrate that our newly developed library construction and computational methods can be applied to large genomes such as that of mouse.

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Conserved Role of Intragenic DNA Methylation in Regulating Alternative Promoters

Alika Maunakea, Raman P. Nagarajan, Mikhail Bilenky … (2010)

While the methylation of DNA in 5′ promoters suppresses gene expression, the role of DNA methylation in gene bodies is unclear 1–5 . In mammals, tissue- and cell type-specific methylation is present in a small percentage of 5′ CpG island (CGI) promoters, while a far greater proportion occurs across gene bodies, coinciding with highly conserved sequences 5–10 . Tissue-specific intragenic methylation might reduce, 3 or, paradoxically, enhance transcription elongation efficiency 1,2,4,5 . Capped analysis of gene expression (CAGE) experiments also indicate that transcription commonly initiates within and between genes 11–15 . To investigate the role of intragenic methylation, we generated a map of DNA methylation from human brain encompassing 24.7 million of the 28 million CpG sites. From the dense, high-resolution coverage of CpG islands, the majority of methylated CpG islands were revealed to be in intragenic and intergenic regions, while less than 3% of CpG islands in 5′ promoters were methylated. The CpG islands in all three locations overlapped with RNA markers of transcription initiation, and unmethylated CpG islands also overlapped significantly with trimethylation of H3K4, a histone modification enriched at promoters 16 . The general and CpG-island-specific patterns of methylation are conserved in mouse tissues. An in-depth investigation of the human SHANK3 locus 17,18 and its mouse homologue demonstrated that this tissue-specific DNA methylation regulates intragenic promoter activity in vitro and in vivo. These methylation-regulated, alternative transcripts are expressed in a tissue and cell type-specific manner, and are expressed differentially within a single cell type from distinct brain regions. These results support a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies.

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Hypomethylation distinguishes genes of some human cancers from their normal counterparts.

Andrew Feinberg, Bert Vogelstein (1983)

It has been suggested that cancer represents an alteration in DNA, heritable by progeny cells, that leads to abnormally regulated expression of normal cellular genes; DNA alterations such as mutations, rearrangements and changes in methylation have been proposed to have such a role. Because of increasing evidence that DNA methylation is important in gene expression (for review see refs 7, 9-11), several investigators have studied DNA methylation in animal tumours, transformed cells and leukaemia cells in culture. The results of these studies have varied; depending on the techniques and systems used, an increase, decrease, or no change in the degree of methylation has been reported. To our knowledge, however, primary human tumour tissues have not been used in such studies. We have now examined DNA methylation in human cancer with three considerations in mind: (1) the methylation pattern of specific genes, rather than total levels of methylation, was determined; (2) human cancers and adjacent analogous normal tissues, unconditioned by culture media, were analysed; and (3) the cancers were taken from patients who had received neither radiation nor chemotherapy. In four of five patients studied, representing two histological types of cancer, substantial hypomethylation was found in genes of cancer cells compared with their normal counterparts. This hypomethylation was progressive in a metastasis from one of the patients.

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Author and article information

Journal

Journal ID (nlm-journal-id): 9604648

Journal ID (pubmed-jr-id): 20305

Journal ID (nlm-ta): Nat Biotechnol

Title: Nature biotechnology

ISSN (Print): 1087-0156

ISSN (Electronic): 1546-1696

Publication date Nihms-submitted: 27 August 2010

Publication date (Electronic): 19 September 2010

Publication date (Print): October 2010

Publication date PMC-release: 1 April 2011

Volume: 28

Issue: 10

Pages: 1097-1105

Affiliations

[1 ] Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA

[2 ] Center for Genome Sciences and Systems Biology, Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA

[3 ] Brain Tumor Research Center, Department of Neurosurgery, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA

[4 ] Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia, Canada

[5 ] Center for Biomolecular Science and Engineering, University of California, Santa Cruz, CA, USA

[6 ] Department of Pharmacology and the Genome Center, University of California-Davis, Davis, CA, USA

[7 ] Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA

[8 ] Division of Biostatistics, Dan L. Duncan Cancer Center, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA

[9 ] Broad Institute of Harvard and MIT, Cambridge, MA, USA

[10 ] Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

[11 ] Center for Cancer Research, Massachusetts General Hospital, Boston, MA, USA

[12 ] Ludwig Institute for Cancer Research, University of California San Diego, La Jolla, CA, USA

[13 ] Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA

[14 ] Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA

[15 ] Department of Molecular and Cell Biology, Center for Systems Biology, University of Texas at Dallas, Dallas, TX, USA

[16 ] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA

[17 ] Harvard Stem Cell Institute, Cambridge, MA, USA

[18 ] Max Planck Institute for Informatics, Saarbrücken, Germany

[19 ] USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA

Author notes

Correspondence should be addressed to JFC ( jcostello@ 123456cc.ucsf.edu )

Article

Manuscript ID: nihpa230545

DOI: 10.1038/nbt.1682

PMC ID: 2955169

PubMed ID: 20852635

SO-VID: ee778c46-15b1-46e5-8336-95539a4ca726

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