A comprehensive methylome map of lineage commitment from hematopoietic progenitors

Ji, Hong-chen; Ehrlich, Lauren I. R.; Seita, Jun; Murakami, Peter; Doi, Akiko; Lindau, Paul; Lee, Hwajin; Aryee, Martin J; Irizarry, Rafael A.; Kim, Kitai; Rossi, Derrick J; Inlay, Matthew A.; Serwold, Thomas; Karsunky, Holger; Ho, Lena; Daley, George Q.; Weissman, Irving L.; Feinberg, Andrew P

doi:10.1038/nature09367

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A comprehensive methylome map of lineage commitment from hematopoietic progenitors

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Author(s): Hong Ji ¹ , Lauren I. R. Ehrlich ² , Jun Seita ² , Peter Murakami ¹ , Akiko Doi ¹ , Paul Lindau ² , Hwajin Lee ¹ , Martin J. Aryee ⁴ ^, ⁵ , Rafael A. Irizarry ¹ ^, ⁴ , Kitai Kim ⁶ , Derrick J Rossi ² , Matthew A. Inlay ² , Thomas Serwold ² , Holger Karsunky ² , Lena Ho ² , George Q. Daley ⁶ , Irving L. Weissman ² , Andrew P. Feinberg ¹

Publication date (Electronic): 15 August 2010

Journal: Nature

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Abstract

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Hematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. While DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs 1, a comprehensive DNA methylation map of hematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Dramatic epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, suggesting a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome appears to be important in hematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions.

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Most cited references 26

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Although vertebrate DNA is generally depleted in the dinucleotide CpG, it has recently been shown that some vertebrate genes contain CpG islands, regions of DNA with a high G+C content and a high frequency of CpG dinucleotides relative to the bulk genome. In this study, a large number of sequences of vertebrate genes were screened for the presence of CpG islands. Each CpG island was then analysed in terms of length, nucleotide composition, frequency of CpG dinucleotides, and location relative to the transcription unit of the associated gene. CpG islands were associated with the 5' ends of all housekeeping genes and many tissue-specific genes, and with the 3' ends of some tissue-specific genes. A few genes contained both 5' and 3' CpG islands, separated by several thousand base-pairs of CpG-depleted DNA. The 5' CpG islands extended through 5'-flanking DNA, exons and introns, whereas most of the 3' CpG islands appeared to be associated with exons. CpG islands were generally found in the same position relative to the transcription unit of equivalent genes in different species, with some notable exceptions. The locations of G/C boxes, composed of the sequence GGGCGG or its reverse complement CCGCCC, were investigated relative to the location of CpG islands. G/C boxes were found to be rare in CpG-depleted DNA and plentiful in CpG islands, where they occurred in 3' CpG islands, as well as in 5' CpG islands associated with tissue-specific and housekeeping genes. G/C boxes were located both upstream and downstream from the transcription start site of genes with 5' CpG islands. Thus, G/C boxes appeared to be a feature of CpG islands in general, rather than a feature of the promoter region of housekeeping genes. Two theories for the maintenance of a high frequency of CpG dinucleotides in CpG islands were tested: that CpG islands in methylated genomes are maintained, despite a tendency for 5mCpG to mutate by deamination to TpG+CpA, by the structural stability of a high G+C content alone, and that CpG islands associated with exons result from some selective importance of the arginine codon CGX. Neither of these theories could account for the distribution of CpG dinucleotides in the sequences analysed. Possible functions of CpG islands in transcriptional and post-transcriptional regulation of gene expression were discussed, and were related to theories for the maintenance of CpG islands as "methylation-free zones" in germline DNA.

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Evidence for active DNA demethylation in vertebrates is accumulating, but the mechanisms and enzymes remain unclear. Using zebrafish embryos we provide evidence for 5-methylcytosine (5-meC) removal in vivo via the coupling of a 5-meC deaminase (AID, which converts 5-meC to thymine) and a G:T mismatch-specific thymine glycosylase (Mbd4). The injection of methylated DNA into embryos induced a potent DNA demethylation activity, which was attenuated by depletion of AID or the non enzymatic factor Gadd45. Remarkably, overexpression of the deaminase/glycosylase pair AID/Mbd4 in vivo caused demethylation of the bulk genome and injected methylated DNA fragments, likely involving a G:T intermediate. Furthermore, AID or Mbd4 knockdown caused the remethylation of a set of common genes. Finally, Gadd45 promoted demethylation and enhanced functional interactions between deaminase/glycosylase pairs. Our results provide evidence for a coupled mechanism of 5-meC demethylation, whereby AID deaminates 5-meC, followed by thymine base excision by Mbd4, promoted by Gadd45.

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Comprehensive high-throughput arrays for relative methylation (CHARM).

Benilton Carvalho, Hao Wu, Andrew Feinberg … (2008)

This study was originally conceived to test in a rigorous way the specificity of three major approaches to high-throughput array-based DNA methylation analysis: (1) MeDIP, or methylated DNA immunoprecipitation, an example of antibody-mediated methyl-specific fractionation; (2) HELP, or HpaII tiny fragment enrichment by ligation-mediated PCR, an example of differential amplification of methylated DNA; and (3) fractionation by McrBC, an enzyme that cuts most methylated DNA. These results were validated using 1466 Illumina methylation probes on the GoldenGate methylation assay and further resolved discrepancies among the methods through quantitative methylation pyrosequencing analysis. While all three methods provide useful information, there were significant limitations to each, specifically bias toward CpG islands in MeDIP, relatively incomplete coverage in HELP, and location imprecision in McrBC. However, we found that with an original array design strategy using tiling arrays and statistical procedures that average information from neighboring genomic locations, much improved specificity and sensitivity could be achieved, e.g., approximately 100% sensitivity at 90% specificity with McrBC. We term this approach "comprehensive high-throughput arrays for relative methylation" (CHARM). While this approach was applied to McrBC analysis, the array design and computational algorithms are fractionation method-independent and make this a simple, general, relatively inexpensive tool suitable for genome-wide analysis, and in which individual samples can be assayed reliably at very high density, allowing locus-level genome-wide epigenetic discrimination of individuals, not just groups of samples. Furthermore, unlike the other approaches, CHARM is highly quantitative, a substantial advantage in application to the study of human disease.

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Author and article information

Journal

Journal ID (nlm-journal-id): 0410462

Journal ID (pubmed-jr-id): 6011

Journal ID (nlm-ta): Nature

Title: Nature

ISSN (Print): 0028-0836

ISSN (Electronic): 1476-4687

Publication date Nihms-submitted: 2 August 2010

Publication date (Electronic): 15 August 2010

Publication date (Print): 16 September 2010

Publication date PMC-release: 16 March 2011

Volume: 467

Issue: 7313

Pages: 338-342

Affiliations

[1 ] Center for Epigenetics, Johns Hopkins University School of Medicine, 570 Rangos, 725 N. Wolfe St., Baltimore, MD 21205

[2 ] Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA

[4 ] Department of Biostatistics, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA

[5 ] Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland, USA

[6 ] Stem Cell Transplantation Program, Division of Pediatric Hematology/Oncology, Manton Center for Orphan Disease Research, Howard Hughes Medical Institute, Children’s Hospital Boston and Dana Farber Cancer Institute; Division of Hematology, Brigham and Women’s Hospital; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School; Harvard Stem Cell Institute; Boston, MA 02115, USA

Author notes

Address correspondence to: Andrew Feinberg, Phone:(410) 614-3489, Fax: (410) 614-9819, afeinberg@ 123456jhu.edu

[3]

Current address: Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78712, USA

[7]

Current address: Immune Disease Institute, Harvard Stem Cell Institute Department of Pathology, Harvard Medical School Boston, MA 02115, USA;

[8]

Current address: Joslin Diabetes Center, Department of Medicine, Harvard Medical School, Boston, MA 02215, USA;

[9]

Current address: Cellant Therapeutics, San Carlos, CA 94070, USA

[*]

These authors contributed equally to this work

Article

Manuscript ID: nihpa225022

DOI: 10.1038/nature09367

PMC ID: 2956609

PubMed ID: 20720541

SO-VID: a36da717-2927-4ae9-89bc-3c3e9aae2f7d

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History

Funding

Funded by: National Cancer Institute : NCI

Funded by: National Institute of General Medical Sciences : NIGMS

Funded by: National Institute of Allergy and Infectious Diseases Extramural Activities : NIAID

Funded by: National Institute on Aging : NIA