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      Single-cell, locus-specific bisulfite sequencing (SLBS) for direct detection of epimutations in DNA methylation patterns

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      Author(s): * , , *
      Publication date (Electronic):
      Journal: Nucleic Acids Research
      Publisher: Oxford University Press

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          Abstract

          Stochastic epigenetic changes drive biological processes, such as development, aging and disease. Yet, epigenetic information is typically collected from millions of cells, thereby precluding a more precise understanding of cell-to-cell variability and the pathogenic history of epimutations. Here we present a novel procedure for directly detecting epimutations in DNA methylation patterns using single-cell, locus-specific bisulfite sequencing (SLBS). We show that within gene promoter regions of mouse hepatocytes the epimutation rate is two orders of magnitude higher than the mutation rate.

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          A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.

          M Frommer, L. E. McDonald, D Millar … (1992)
          The modulation of DNA-protein interactions by methylation of protein-binding sites in DNA and the occurrence in genomic imprinting, X chromosome inactivation, and fragile X syndrome of different methylation patterns in DNA of different chromosomal origin have underlined the need to establish methylation patterns in individual strands of particular genomic sequences. We report a genomic sequencing method that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA. The method utilizes bisulfite-induced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive. The sequence under investigation is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues have been amplified as thymine and only 5-methylcytosine residues have been amplified as cytosine. The PCR products can be sequenced directly to provide a strand-specific average sequence for the population of molecules or can be cloned and sequenced to provide methylation maps of single DNA molecules. We tested the method by defining the methylation status within single DNA strands of two closely spaced CpG dinucleotides in the promoter of the human kininogen gene. During the analysis, we encountered in sperm DNA an unusual methylation pattern, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.
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            Spontaneous epigenetic variation in the Arabidopsis thaliana methylome.

            Claude Becker, Jörg Hagmann, Jonas Müller … (2011)
            Heritable epigenetic polymorphisms, such as differential cytosine methylation, can underlie phenotypic variation. Moreover, wild strains of the plant Arabidopsis thaliana differ in many epialleles, and these can influence the expression of nearby genes. However, to understand their role in evolution, it is imperative to ascertain the emergence rate and stability of epialleles, including those that are not due to structural variation. We have compared genome-wide DNA methylation among 10 A. thaliana lines, derived 30 generations ago from a common ancestor. Epimutations at individual positions were easily detected, and close to 30,000 cytosines in each strain were differentially methylated. In contrast, larger regions of contiguous methylation were much more stable, and the frequency of changes was in the same low range as that of DNA mutations. Like individual positions, the same regions were often affected by differential methylation in independent lines, with evidence for recurrent cycles of forward and reverse mutations. Transposable elements and short interfering RNAs have been causally linked to DNA methylation. In agreement, differentially methylated sites were farther from transposable elements and showed less association with short interfering RNA expression than invariant positions. The biased distribution and frequent reversion of epimutations have important implications for the potential contribution of sequence-independent epialleles to plant evolution.
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              Transgenerational epigenetic instability is a source of novel methylation variants.

              Robert Schmitz, Matthew D Schultz, Mathew G. Lewsey … (2011)
              Epigenetic information, which may affect an organism's phenotype, can be stored and stably inherited in the form of cytosine DNA methylation. Changes in DNA methylation can produce meiotically stable epialleles that affect transcription and morphology, but the rates of spontaneous gain or loss of DNA methylation are unknown. We examined spontaneously occurring variation in DNA methylation in Arabidopsis thaliana plants propagated by single-seed descent for 30 generations. We identified 114,287 CG single methylation polymorphisms and 2485 CG differentially methylated regions (DMRs), both of which show patterns of divergence compared with the ancestral state. Thus, transgenerational epigenetic variation in DNA methylation may generate new allelic states that alter transcription, providing a mechanism for phenotypic diversity in the absence of genetic mutation.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Nucleic Acids Res
                Journal ID (iso-abbrev): Nucleic Acids Res
                Journal ID (hwp): nar
                Journal ID (publisher-id): nar
                Title: Nucleic Acids Research
                Publisher: Oxford University Press
                ISSN (Print): 0305-1048
                ISSN (Electronic): 1362-4962
                Publication date (Print): 18 August 2015
                Publication date (Electronic): 19 April 2015
                Publication date PMC-release: 19 April 2015
                Volume: 43
                Issue: 14
                Page: e93
                Affiliations
                Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA
                Author notes
                [* ]To whom correspondence should be addressed. Silvia Gravina, Ph.D. Tel: +1 718 678 1135; Fax: +1 718 430 8878; Email: silvia.gravina@ 123456einstein.yu.edu
                Correspondence may also be addressed to Jan Vijg, Ph.D. Tel: +1 718 678 1151; Fax: +1 718 678 1016; Email: jan.vijg@ 123456einstein.yu.edu
                Article
                DOI: 10.1093/nar/gkv366
                PMC ID: 4538804
                PubMed ID: 25897117
                SO-VID: 5c10bb03-01e4-40be-a8bf-67057e3adbc3
                Copyright © © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date accepted : 07 April 2015
                Date revision received : 20 March 2015
                Date received : 16 December 2014
                Page count
                Pages: 7
                Categories
                Subject: 3
                Subject: Methods Online
                Custom metadata
                cover-date 18 August 2015

                ScienceOpen disciplines: Genetics
                Data availability:
                ScienceOpen disciplines: Genetics

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