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Human non-CG methylation

Are human stem cells plant-like?

, , , &
Pages 569-572 | Received 18 May 2010, Accepted 16 Jun 2010, Published online: 01 Oct 2010

Abstract

Non-CG methylation is well characterized in plants, where it appears to play a role in gene silencing and genomic imprinting. Although strong evidence for the presence of non-CG methylation in animals has been available for some time, both its origin and function remain elusive. In this review we discuss available evidence on non-CG methylation in animals in light of evidence suggesting that the human stem cell methylome contains significant levels of methylation outside the CG site.

In plant cells non-CG sites are methylated de novo by Chromomethylase 3, DRM1 and DRM2. Chromomethylase 3, along with DRM1 and DRM2 combine in the maintenance of methylation at symmetric CpHpG as well as asymmetric DNA sites where they appear to prevent reactivation of transposons.Citation1 DRM1 and DRM2 modify DNA de novo primarily at asymmetric CpH and CpHpH sequences targeted by siRNA.Citation2

Much less information is available on non-CG methylation in mammals. In fact, studies on mammalian non-CG methylation form a tiny fraction of those on CG methylation, even though data for cytosine methylation in other dinucleotides, CA, CT and CC, have been available since the late 1980s.Citation3 Strong evidence for non-CG methylation was found by examining either exogenous DNA sequences, such as plasmid and viral integrants in mouse and human cell lines,Citation4,Citation5 or transposons and repetitive sequences such as the human L1 retrotransposonCitation6 in a human embryonic fibroblast cell line. In the latter study, non-CG methylation observed in L1 was found to be consistent with the capacity of Dnmt1 to methylate slippage intermediates de novo.Citation6

Non-CG methylation has also been reported at origins of replicationCitation7,Citation8 and a region of the human myogenic gene Myf3.Citation9 The Myf3 gene is silenced in non-muscle cell lines but it is not methylated at CGs. Instead, it carries several methylated cytosines within the sequence CCTGG. Gene-specific non-CG methylation was also reported in a study of lymphoma and myeloma cell lines not expressing many B lineage-specific genes.Citation10 The study focused on one specific gene, B29 and found heavy CG promoter methylation of that gene in most cell lines not expressing it. However, in two other cell lines where the gene was silenced, cytosine methylation was found almost exclusively at CCWGG sites. The authors provided evidence suggesting that CCWGG methylation was sufficient for silencing the B29 promoter and that methylated probes based on B29 sequences had unique gel shift patterns compared to non-methylated but otherwise identical sequences.Citation10 The latter finding suggests that the presence of the non-CG methylation causes changes in the proteins able to bind the promoter, which could be mechanistically related to the silencing seen with this alternate methylation.

Non-CG methylation is rarely seen in DNA isolated from cancer patients. However, the p16 promoter region was reported to contain both CG and non-CG methylation in breast tumor specimens but lacked methylation at these sites in normal breast tissue obtained at mammoplasty.Citation11 Moreover, CWG methylation at the CCWGG sites in the calcitonin gene is not found in normal or leukemic lymphocyte DNA obtained from patients.Citation12 Further, in DNA obtained from breast cancer patients, MspI sites that are refractory to digestion by MspI and thus candidates for CHG methylation were found to carry CpG methylation.Citation13 Their resistance to MspI restriction was found to be caused by an unusual secondary structure in the DNA spanning the MspI site that prevents restriction.Citation13 This latter observation suggests caution in interpreting EcoRII/BstNI or EcoRII/BstOI restriction differences as due to CWG methylation, since in contrast to the 37°C incubation temperature required for full EcoRII activity, BstNI and BstOI require incubation at 60°C for full activity where many secondary structures are unstable.

The recent report by Lister et al.Citation14 confirmed a much earlier report by Ramsahoye et al.Citation15 suggesting that non-CG methylation is prevalent in mammalian stem cell lines. Nearest neighbor analysis was used to detect non-CG methylation in the earlier study on the mouse embryonic stem (ES) cell line,Citation15 thus global methylation patterning was assessed. Lister et al.Citation14 extend these findings to human stem cell lines at single-base resolution with whole-genome bisulfite sequencing. They reportCitation14 that the methylome of the human H1 stem cell line and the methylome of the induced pluripotent IMR90 (iPS) cell line are stippled with non-CG methylation while that of the human IMR90 fetal fibroblast cell line is not. While the results of the two studies are complementary, the human methylome study addresses locus specific non-CG methylation. Based on that data,Citation14 one must conclude that non-CG methylation is not carefully maintained at a given site in the human H1 cell line. The average non-CG site is picked up as methylated in about 25% of the reads whereas the average CG methylation site is picked up in 92% of the reads. Moreover, non-CG methylation is not generally present on both strands and is concentrated in the body of actively transcribed genes.Citation14

Even so, the consistent finding that non-CG methylation appears to be confined to stem cell lines,Citation14,Citation15 raises the possibility that cancer stem cellsCitation16 carry non-CG methylation while their nonstem progeny in the tumor carry only CG methylation. Given the expected paucity of cancer stem cells in a tumor cell population, it is unlikely that bisulfite sequencing would detect non-CG methylation in DNA isolated from tumor cells since the stem cell population is expected to be only a very minor component of tumor DNA. Published sequences obtained by bisulfite sequencing generally report only CG methylation, and to the best of our knowledge bisulfite sequenced tumor DNA specimens have not reported non-CG methylation. On the other hand, when sequences from cell lines have been reported, bisulfite-mediated genomic sequencingCitation8 or ligation mediated PCRCitation17 methylcytosine signals outside the CG site have been observed. In a more recent study plasmid DNAs carrying the Bcl2-major breakpoint clusterCitation18 or human breast cancer DNACitation13 treated with bisulfite under non-denaturing conditions, cytosines outside the CG side were only partially converted on only one strandCitation18 or at a symmetrical CWG site.Citation13 In the breast cancer DNA study the apparent CWG methylation was not detected when the DNA was fully denatured before bisulfite treatment.Citation13

In both stem cell studies, non-CG methylation was attributed to the Dnmt3a,Citation14,Citation15 a DNA methyltransferase with similarities to the plant DRM methyltransferase familyCitation19 and having the capacity to methylate non-CG sites when expressed in Drosophila melanogaster.Citation15 DRM proteins however, possess a unique permuted domain structure found exclusively in plantsCitation19 and the associated RNA-directed non-CG DNA methylation has not been reproducibly observed in mammals despite considerable publishedCitation20Citation23 and unpublished efforts in that area. Moreover, reports where methylation was studied often infer methylation changes from 5AzaC reactivation studiesCitation24 or find that CG methylation seen in plants but not non-CG methylation is detected.Citation21,Citation22,Citation25,Citation26 In this regard, it is of interest that the level of non-CG methylation reported in stem cells corresponds to background non-CG methylation observed in vitro with human DNA methyltransferase I,Citation27 and is consistent with the recent report that cultured stem cells are epigenetically unstable.Citation28

The function of non-CG methylation remains elusive. A role in gene expression has not been ruled out, as the studies above on Myf3 and B29 suggest.Citation9,Citation10 However, transgene expression of the bacterial methyltransferase M.EcoRII in a human cell line (HK293), did not affect the CG methylation state at the APC and SerpinB5 genesCitation29 even though the promoters were symmetrically de novo methylated at mCWGs within each CCWGG sequence in each promoter. This demonstrated that CG and non-CG methylation are not mutually exclusive as had been suggested by earlier reports.Citation9,Citation10 That observation is now extended to the human stem cell line methylome where CG and non-CG methylation co-exist.Citation14 Gene expression at the APC locus was likewise unaffected by transgene expression of M.EcoRII. In those experiments genome wide methylation of the CCWGG site was detected by restriction analysis and bisulfite sequencing,Citation29 however stem cell characteristics were not studied.

Many alternative functions can be envisioned for non-CG methylation, but the existing data now constrains them to functions that involve low levels of methylation that are primarily asymmetric. Moreover, inheritance of such methylation patterns requires low fidelity methylation. If methylation were maintained with high fidelity at particular CHG sites one would expect that the spontaneous deamination of 5-methylcytosine would diminish the number of such sites, so as to confine the remaining sites to those positions performing an essential function, as is seen in CG methylation.Citation30Citation33 However, depletion of CWG sites is not observed in the human genome.Citation34 Since CWG sites account for only about 50% of the non-CG methylation observed in the stem cell methylomeCitation14 where methylated non-CG sites carry only about 25% methylation, the probability of deamination would be about 13% of that for CWG sites that are subject to maintenance methylation in the germ line. Since mutational depletion of methylated cytosines has to have its primary effect on the germ line, if the maintenance of non-CG methylation were more accurate and more widespread, one would have had to argue that stem cells in the human germ lines lack CWG methylation. As it is the data suggests that whatever function non-CG methylation may have in stem cells, it does not involve accurate somatic inheritance in the germ line.

The extensive detail on non-CG methylation in the H1 methylomeCitation14 raises interesting questions about the nature of this form of methylation in human cell lines. A key finding in this report is the contrast between the presence of non-CG methylation in the H1 stem cell line and its absence in the IMR90 human fetal lung fibroblast cell line.Citation14 This suggests that it may have a role in the origin and maintenance of the pluripotent lineage.Citation14

By analogy with the well known methylated DNA binding proteins specific for CG methylation,Citation35 methylated DNA binding proteins that selectively bind sites of non-CG methylation are expected to exist in stem cells. Currently the only protein reported to have this binding specificity is human Dnmt1.Citation36Citation38 While Dnmt1 has been proposed to function stoichiometricallyCitation39 and could serve a non-CG binding role in stem cells, this possibility and the possibility that other stem-cell specific non-CG binding proteins might exist remain to be been explored.

Finally, the nature of the non-CG methylation patterns in human stem cell lines present potentially difficult technical problems in methylation analysis. First, based on the data in the H1 stem cell methylome,Citation40 a standard MS-qPCR for non-CG methylation would be impractical because non-CG sites are infrequent, rarely clustered and are generally characterized by partial asymmetric methylation. This means that a PCR primer that senses the 3 adjacent methylation sites usually recommended for MS-qPCR primer designCitation41,Citation42 cannot be reliably found. For example in the region near Oct4 (Chr6:31,246,431), a potential MS-qPCR site exists with a suboptimal set of two adjacent CHG sites both methylated on the + strand at Chr6:31,252,225 and 31,252,237.Citation14,Citation40 However these sites were methylated only in 13/45 and 30/52 reads. Thus the probability that they would both be methylated on the same strand is about 17%. Moreover, reverse primer locations containing non-CG methylation sites are generally too far away for practical bisulfite mediated PCR. Considering the losses associated with bisulfite mediated PCRCitation43 the likelihood that such an MS-qPCR system would detect non-CG methylation in the H1 cell line or stem cells present in a cancer stem cell nicheCitation44,Citation45 is very low.

The second difficulty is that methods based on the specificity of MeCP2 and similar methylated DNA binding proteins for enriching methylated DNA (e.g., MIRA,Citation46 COMPARE-MSCitation47) will discard sequences containing non-CG methylation since they require cooperative binding afforded by runs of adjacent methylated CG sites for DNA capture. This latter property of the methylated cytosine capture techniques makes it also unlikely that methods based on 5-methylcytosine antibodies (e.g., meDIPCitation48) will capture non-CG methylation patterns accurately since the stem cell methylome shows that adjacent methylated non-CG sites are rare in comparison to methylated CG sites.Citation14

In summary, whether or not mammalian stem cells in general or human stem cells in particular possess functional plant-like methylation patterns is likely to continue to be an interesting and challenging question. At this point we can conclude that the non-CG patterns reported in human cells appear to differ significantly from the non-CG patterns seen in plants, suggesting that they do not have a common origin or function.

Acknowledgements

This work was supported in part by grants from the Congressionally Directed Medical Research Program of the US ARMY (W81XWH-08-1-0517) and the US National Cancer Institute of the National Institutes of Health (CA102521 and CA 136055) to S.S.S. and by grant 09-08-00687 to O.D. and 10-04-00037 to Y.B. from the Russian Foundation of Fundamental Research.

References

  • Lindroth AM, Cao X, Jackson JP, Zilberman D, McCallum CM, Henikoff S, et al. Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation. Science 2001; 292:30 - 33
  • Cao X, Aufsatz W, Zilberman D, Mette MF, Huang MS, Matzke M, et al. Role of the DRM and CMT3 methyltransferases in RNA-directed DNA methylation. Curr Biol 2003; 13:2212 - 2217
  • Woodcock DM, Crowther PJ, Diver WP. The majority of methylated deoxycytidines in human DNA are not in the CpG dinucleotide. Biochem Biophys Res Commun 1987; 145:888 - 894
  • Toth M, Muller U, Doerfler W. Establishment of de novo DNA methylation patterns. Transcription factor binding and deoxycytidine methylation at CpG and non-CpG sequences in an integrated adenovirus promoter. J Mol Biol 1990; 214:673 - 683
  • Clark SJ, Harrison J, Frommer M. CpNpG methylation in mammalian cells. Nat Genet 1995; 10:20 - 27
  • Woodcock DM, Lawler CB, Linsenmeyer ME, Doherty JP, Warren WD. Asymmetric methylation in the hypermethylated CpG promoter region of the human L1 retrotransposon. J Biol Chem 1997; 272:7810 - 7816
  • Tasheva ES, Roufa DJ. A mammalian origin of bidirectional DNA replication within the Chinese hamster RPS14 locus. Mol Cell Biol 1994; 14:5628 - 5635
  • Tasheva ES, Roufa DJ. Densely methylated DNA islands in mammalian chromosomal replication origins. Mol Cell Biol 1994; 14:5636 - 5644
  • Franchina M, Kay PH. Evidence that cytosine residues within 5′-CCTGG-3′ pentanucleotides can be methylated in human DNA independently of the methylating system that modifies 5′-CG-3′ dinucleotides. DNA Cell Biol 2000; 19:521 - 526
  • Malone CS, Miner MD, Doerr JR, Jackson JP, Jacobsen SE, Wall R, et al. CmC(A/T)GG DNA methylation in mature B cell lymphoma gene silencing. Proc Natl Acad Sci USA 2001; 98:10404 - 10409
  • Woodcock DM, Linsenmeyer ME, Doherty JP, Warren WD. DNA methylation in the promoter region of the p16 (CDKN2/MTS-1/INK4A) gene in human breast tumours. Br J Cancer 1999; 79:251 - 256
  • Buryanov YI, Shevchuk TV, Zakharchenko NS, D'yachenko OV, Marinich DV, Vorob'ev IA. The absence of the CpNpG methylation at the 5′-terminal region of the human calcitonin gene in norm and leukemias. Russ J Bioorg Chem 2000; 26:358 - 360
  • Clark J, Smith SS. Secondary structure at a hot spot for DNA methylation in DNA from human breast cancers. Cancer Genomics Proteomics 2008; 5:241 - 251
  • Lister R, Pelizzola M, Dowen RH, Hawkins RD, Hon G, Tonti-Filippini J, et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 2009; 462:315 - 322
  • Ramsahoye BH, Biniszkiewicz D, Lyko F, Clark V, Bird AP, Jaenisch R. Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proc Natl Acad Sci USA 2000; 97:5237 - 5242
  • Gupta PB, Chaffer CL, Weinberg RA. Cancer stem cells: mirage or reality?. Nat Med 2009; 15:1010 - 1012
  • Pfeifer GP, Steigerwald SD, Mueller PR, Wold B, Riggs AD. Genomic sequencing and methylation analysis by ligation mediated PCR. Science 1989; 246:810 - 813
  • Raghavan SC, Chastain P, Lee JS, Hegde BG, Houston S, Langen R, et al. Evidence for a triplex DNA conformation at the bcl-2 major breakpoint region of the t(14;18) translocation. J Biol Chem 2005; 280:22749 - 22760
  • Cao X, Springer NM, Muszynski MG, Phillips RL, Kaeppler S, Jacobsen SE. Conserved plant genes with similarity to mammalian de novo DNA methyltransferases. Proc Natl Acad Sci USA 2000; 97:4979 - 4984
  • Svoboda P, Stein P, Filipowicz W, Schultz RM. Lack of homologous sequence-specific DNA methylation in response to stable dsRNA expression in mouse oocytes. Nucleic Acids Res 2004; 32:3601 - 3606
  • Taira K. Induction of DNA methylation and gene silencing by short interfering RNAs in human cells. Nature 2006; 441:1176
  • Castanotto D, Tommasi S, Li M, Li H, Yanow S, Pfeifer GP, et al. Short hairpin RNA-directed cytosine (CpG) methylation of the RASSF1A gene promoter in HeLa cells. Mol Ther 2005; 12:179 - 183
  • Morris KV, Chan SW, Jacobsen SE, Looney DJ. Small interfering RNA-induced transcriptional gene silencing in human cells. Science 2004; 305:1289 - 1292
  • Kim JW, Zhang YH, Zern MA, Rossi JJ, Wu J. Short hairpin RNA causes the methylation of transforming growth factor-beta receptor II promoter and silencing of the target gene in rat hepatic stellate cells. Biochem Biophys Res Commun 2007; 359:292 - 297
  • Ting AH, Schuebel KE, Herman JG, Baylin SB. Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation. Nat Genet 2005; 37:906 - 910
  • Tan Y, Zhang B, Wu T, Skogerbo G, Zhu X, Guo X, et al. Transcriptional inhibiton of Hoxd4 expression by miRNA-10a in human breast cancer cells. BMC Mol Biol 2009; 10:12
  • Smith SS. Biological implications of the mechanism of action of human DNA (cytosine-5)methyltransferase. Prog Nucleic Acid Res Mol Biol 1994; 49:65 - 111
  • Tanasijevic B, Dai B, Ezashi T, Livingston K, Roberts RM, Rasmussen TP. Progressive accumulation of epigenetic heterogeneity during human ES cell culture. Epigenetics 2009; 4:330 - 338
  • Shevchuk T, Kretzner L, Munson K, Axume J, Clark J, Dyachenko OV, et al. Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells. Nucleic Acids Res 2005; 33:6124 - 6136
  • McClelland M, Ivarie R. Asymmetrical distribution of CpG in an ‘average’ mammalian gene. Nucleic Acids Res 1982; 10:7865 - 7877
  • Adams RL, Davis T, Rinaldi A, Eason R. CpG deficiency, dinucleotide distributions and nucleosome positioning. Eur J Biochem 1987; 165:107 - 115
  • Cooper DN, Gerber-Huber S. DNA methylation and CpG suppression. Cell Differ 1985; 17:199 - 205
  • Cooper DN, Youssoufian H. The CpG dinucleotide and human genetic disease. Hum Genet 1988; 78:151 - 155
  • Watson B, Munson K, Clark J, Shevchuk T, Smith SS. Distribution of CWG and CCWGG in the human genome. Epigenetics 2007; 2:151 - 154
  • Free A, Wakefield RI, Smith BO, Dryden DT, Barlow PN, Bird AP. DNA recognition by the methyl-CpG binding domain of MeCP2. J Biol Chem 2001; 276:3353 - 3360
  • Smith SS, Baker DJ. Stalling of human methyltransferase at single-strand conformers from the Huntington's locus. Biochem Biophys Res Commun 1997; 234:73 - 78
  • Kho MR, Baker DJ, Laayoun A, Smith SS. Stalling of human DNA (cytosine-5) methyltransferase at single-strand conformers from a site of dynamic mutation. J Mol Biol 1998; 275:67 - 79
  • Clark J, Shevchuk T, Kho MR, Smith SS. Methods for the design and analysis of oligodeoxynucleotide-based DNA (cytosine-5) methyltransferase inhibitors. Anal Biochem 2003; 321:50 - 64
  • Smith SS. Gilbert's conjecture: the search for DNA (cytosine-5) demethylases and the emergence of new functions for eukaryotic DNA (cytosine-5) methyltransferases. J Mol Biol 2000; 302:1 - 7
  • The Human DNA Methylone 2010; 2 5 [Internet]. San Diego Epigenome Center Available from: http://neomorph.salk.edu/human_methylome/
  • Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specificPCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996; 93:9821 - 9826
  • Boyd VL, Moody KI, Karger AE, Livak KJ, Zon G, Burns JW. Methylation-dependent fragment separation: Direct detection of DNA methylation by capillary electrophoresis of PCR products from bisulfite-converted genomic DNA. Anal Biochem 2006; 354:266 - 273
  • Munson K, Clark J, Lamparska-Kupsik K, Smith SS. Recovery of bisulfite-converted genomic sequences in the methylation-sensitive QPCR. Nucleic Acids Research 2007; 35:2893 - 2903
  • Sneddon JB, Werb Z. Location, location, location: the cancer stem cell niche. Cell Stem Cell 2007; 1:607 - 611
  • McGovern M, Voutev R, Maciejowski J, Corsi AK, Hubbard EJ. A “latent niche” mechanism for tumor initiation. Proc Natl Acad Sci USA 2009; 106:11617 - 11622
  • Rauch TA, Pfeifer GP. The MIRA method for DNA methylation analysis. Methods Mol Biol 2009; 507:65 - 75
  • Yegnasubramanian S, Lin X, Haffner MC, DeMarzo AM, Nelson WG. Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation. Nucleic Acids Res 2006; 34:19
  • Weber M, Davies JJ, Wittig D, Oakeley EJ, Haase M, Lam WL, et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 2005; 37:853 - 862

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