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DNMT1-interacting RNAs block gene-specific DNA methylation

Nature volume 503, pages 371–376 (2013)Cite this article

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Abstract

DNA methylation was first described almost a century ago; however, the rules governing its establishment and maintenance remain elusive. Here we present data demonstrating that active transcription regulates levels of genomic methylation. We identify a novel RNA arising from the CEBPA gene locus that is critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extend the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1–RNA interactions and suggest strategies for gene-selective demethylation of therapeutic targets in human diseases.

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Figure 1: Characterization of ecCEBPA.
Figure 2: Loss- and gain-of-function studies demonstrate that ecCEBPA maintains CEBPA expression by regulating methylation of the CEBPA locus.
Figure 3: ecCEBPA–DNMT1 interactions: DNMT1 binds to RNA with greater affinity than to DNA.
Figure 4: Transcription impedes DNA methylation.
Figure 5: Genome-wide alignment of DNMT1-bound and -unbound transcripts, DNA methylation and gene expression.

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Data deposits

Sequencing and microarray data sets are available for download at Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/projects/geo/) under the following accession numbers: microarray expression, GSE32153; RIPseq, GSE32162; RRBS, GSE32168; and RNAseq, GSE41279. The accession number for the project is GSE32260.

References

  1. Jones, P. A. & Baylin, S. B. The fundamental role of epigenetic events in cancer. Nature Rev. Genet. 3, 415–428 (2002)

    Article  CAS  Google Scholar 

  2. Robertson, K. D. DNA methylation, methyltransferases, and cancer. Oncogene 20, 3139–3155 (2001)

    Article  CAS  Google Scholar 

  3. Illingworth, R. S. et al. Orphan CpG islands identify numerous conserved promoters in the mammalian genome. PLoS Genet. 6, e1001134 (2010)

    Article  Google Scholar 

  4. Saxonov, S., Berg, P. & Brutlag, D. L. A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters. Proc. Natl Acad. Sci. USA 103, 1412–1417 (2006)

    Article  ADS  CAS  Google Scholar 

  5. Tenen, D. G. Disruption of differentiation in human cancer: AML shows the way. Nature Rev. Cancer 3, 89–101 (2003)

    Article  CAS  Google Scholar 

  6. Tada, Y. et al. Epigenetic modulation of tumor suppressor CCAAT/enhancer binding protein α activity in lung cancer. J. Natl. Cancer Inst. 98, 396–406 (2006)

    Article  CAS  Google Scholar 

  7. Hackanson, B. et al. Epigenetic modification of CCAAT/enhancer binding protein α expression in acute myeloid leukemia. Cancer Res. 68, 3142–3151 (2008)

    Article  CAS  Google Scholar 

  8. Gupta, R. A. et al. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464, 1071–1076 (2010)

    Article  ADS  CAS  Google Scholar 

  9. Huarte, M. et al. A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response. Cell 142, 409–419 (2010)

    Article  CAS  Google Scholar 

  10. Rinn, J. L. et al. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129, 1311–1323 (2007)

    Article  CAS  Google Scholar 

  11. Zhao, J. et al. Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol. Cell 40, 939–953 (2010)

    Article  CAS  Google Scholar 

  12. Nagano, T. et al. The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science 322, 1717–1720 (2008)

    Article  ADS  CAS  Google Scholar 

  13. Guttman, M. & Rinn, J. L. Modular regulatory principles of large non-coding RNAs. Nature 482, 339–346 (2012)

    Article  ADS  CAS  Google Scholar 

  14. Ebralidze, A., Wang, Y., Petkova, V., Ebralidse, K. & Junghans, R. P. RNA leaching of transcription factors disrupts transcription in myotonic dystrophy. Science 303, 383–387 (2004)

    Article  ADS  CAS  Google Scholar 

  15. Poliseno, L. et al. A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature 465, 1033–1038 (2010)

    Article  ADS  CAS  Google Scholar 

  16. Tay, Y. et al. Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs. Cell 147, 344–357 (2011)

    Article  CAS  Google Scholar 

  17. Ebisuya, M., Yamamoto, T., Nakajima, M. & Nishida, E. Ripples from neighbouring transcription. Nature Cell Biol. 10, 1106–1113 (2008)

    Article  CAS  Google Scholar 

  18. Preker, P. et al. RNA exosome depletion reveals transcription upstream of active human promoters. Science 322, 1851–1854 (2008)

    Article  ADS  CAS  Google Scholar 

  19. Ebralidze, A. K. et al. PU. 1 expression is modulated by the balance of functional sense and antisense RNAs regulated by a shared cis-regulatory element. Genes Dev. 22, 2085–2092 (2008)

    Article  CAS  Google Scholar 

  20. Oler, A. J. et al. Human RNA polymerase III transcriptomes and relationships to Pol II promoter chromatin and enhancer-binding factors. Nature Struct. Mol. Biol. 17, 620–628 (2010)

    Article  CAS  Google Scholar 

  21. Listerman, I., Bledau, A. S., Grishina, I. & Neugebauer, K. M. Extragenic accumulation of RNA polymerase II enhances transcription by RNA polymerase III. PLoS Genet. 3, e212 (2007)

    Article  Google Scholar 

  22. Raha, D. et al. Close association of RNA polymerase II and many transcription factors with Pol III genes. Proc. Natl Acad. Sci. USA 107, 3639–3644 (2010)

    Article  ADS  CAS  Google Scholar 

  23. Wouters, B. J. et al. Distinct gene expression profiles of acute myeloid/T-lymphoid leukemia with silenced CEBPA and mutations in NOTCH1. Blood 110, 3706–3714 (2007)

    Article  CAS  Google Scholar 

  24. Meissner, A. et al. Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nucleic Acids Res. 33, 5868–5877 (2005)

    Article  CAS  Google Scholar 

  25. Leonhardt, H., Page, A. W., Weier, H. U. & Bestor, T. H. A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell 71, 865–873 (1992)

    Article  CAS  Google Scholar 

  26. Hofacker, I. L. Vienna RNA secondary structure server. Nucleic Acids Res. 31, 3429–3431 (2003)

    Article  CAS  Google Scholar 

  27. Pradhan, S. & Esteve, P. O. Allosteric activator domain of maintenance human DNA (cytosine-5) methyltransferase and its role in methylation spreading. Biochemistry 42, 5321–5332 (2003)

    Article  CAS  Google Scholar 

  28. Song, J., Rechkoblit, O., Bestor, T. H. & Patel, D. J. Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 331, 1036–1040 (2011)

    Article  ADS  CAS  Google Scholar 

  29. Fuerst, T. R., Niles, E. G., Studier, F. W. & Moss, B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl Acad. Sci. USA 83, 8122–8126 (1986)

    Article  ADS  CAS  Google Scholar 

  30. Svedruzić, Z. M. Mammalian cytosine DNA methyltransferase Dnmt1: enzymatic mechanism, novel mechanism-based inhibitors, and RNA-directed DNA methylation. Curr. Med. Chem. 15, 92–106 (2008)

    Article  Google Scholar 

  31. Bolden, A., Ward, C., Siedlecki, J. A. & Weissbach, A. DNA methylation. Inhibition of de novo and maintenance methylation in vitro by RNA and synthetic polynucleotides. J. Biol. Chem. 259, 12437–12443 (1984)

    CAS  PubMed  Google Scholar 

  32. Bolden, A. H., Nalin, C. M., Ward, C. A., Poonian, M. S. & Weissbach, A. Primary DNA sequence determines sites of maintenance and de novo methylation by mammalian DNA methyltransferases. Mol. Cell. Biol. 6, 1135–1140 (1986)

    Article  CAS  Google Scholar 

  33. Shin, H., Liu, T., Manrai, A. K. & Liu, X. S. CEAS: cis-regulatory element annotation system. Bioinformatics 25, 2605–2606 (2009)

    Article  CAS  Google Scholar 

  34. Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010)

    Article  CAS  Google Scholar 

  35. Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009)

    Article  CAS  Google Scholar 

  36. Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nature Protocols 7, 562–578 (2012)

    Article  CAS  Google Scholar 

  37. Tsirigos, A. & Rigoutsos, I. Human and mouse introns are linked to the same processes and functions through each genome’s most frequent non-conserved motifs. Nucleic Acids Res. 36, 3484–3493 (2008)

    Article  CAS  Google Scholar 

  38. Bock, C. et al. Quantitative comparison of genome-wide DNA methylation mapping technologies. Nature Biotechnol. 28, 1106–1114 (2010)

    Article  CAS  Google Scholar 

  39. Gill, G. & Ptashne, M. Negative effect of the transcriptional activator GAL4. Nature 334, 721–724 (1988)

    Article  ADS  CAS  Google Scholar 

  40. Frank-Kamenetskii, M. D. & Mirkin, S. M. Triplex DNA structures. Annu. Rev. Biochem. 64, 65–95 (1995)

    Article  CAS  Google Scholar 

  41. Karreth, F. A. et al. In vivo identification of tumor-suppressive PTEN ceRNAs in an oncogenic BRAF-induced mouse model of melanoma. Cell 147, 382–395 (2011)

    Article  CAS  Google Scholar 

  42. Keene, J. D. RNA regulons: coordination of post-transcriptional events. Nature Rev. Genet. 8, 533–543 (2007)

    Article  ADS  CAS  Google Scholar 

  43. Figueroa, M. E. et al. DNA methylation signatures identify biologically distinct subtypes in acute myeloid leukemia. Cancer Cell 17, 13–27 (2010)

    Article  CAS  Google Scholar 

  44. Smith, Z. D., Gu, H., Bock, C., Gnirke, A. & Meissner, A. High-throughput bisulfite sequencing in mammalian genomes. Methods 48, 226–232 (2009)

    Article  CAS  Google Scholar 

  45. Xi, Y. & Li, W. BSMAP: whole genome bisulfite sequence MAPping program. BMC Bioinformatics 10, 232 (2009)

    Article  Google Scholar 

  46. Benoukraf, T., Wongphayak, S., Hadi, L. H., Wu, M. & Soong, R. GBSA: a comprehensive software for analysing whole genome bisulfite sequencing data. Nucleic Acids Res. 41, e55 (2013)

    Article  CAS  Google Scholar 

  47. Akalin, A. et al. methylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles. Genome Biol. 13, R87 (2012)

    Article  Google Scholar 

  48. Jothi, R., Cuddapah, S., Barski, A., Cui, K. & Zhao, K. Genome-wide identification of in vivo protein-DNA binding sites from ChIP-Seq data. Nucleic Acids Res. 36, 5221–5231 (2008)

    Article  CAS  Google Scholar 

  49. Maniatis, T., Fritsch, E. F. & Sambrook, J. Molecular Cloning: a Laboratory Manual (Cold Spring Harbor Laboratory Press, 1982)

    Google Scholar 

  50. Blobel, G. & Potter, V. R. Nuclei from rat liver: isolation method that combines purity with high yield. Science 154, 1662–1665 (1966)

    Article  ADS  CAS  Google Scholar 

  51. Sambrook, J. & Russell, D. W. Molecular Cloning: a Laboratory Manual (Cold Spring Harbor Laboratory Press, 2001)

    Google Scholar 

  52. Mitchell, J. A. & Fraser, P. Transcription factories are nuclear subcompartments that remain in the absence of transcription. Genes Dev. 22, 20–25 (2008)

    Article  CAS  Google Scholar 

  53. Dieci, G., Fiorino, G., Castelnuovo, M., Teichmann, M. & Pagano, A. The expanding RNA polymerase III transcriptome. Trends Genet. 23, 614–622 (2007)

    Article  CAS  Google Scholar 

  54. Pagano, A. et al. New small nuclear RNA gene-like transcriptional units as sources of regulatory transcripts. PLoS Genet. 3, e1 (2007)

    Article  Google Scholar 

  55. Stewart, S. A. et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9, 493–501 (2003)

    Article  CAS  Google Scholar 

  56. Frommer, M. et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl Acad. Sci. USA 89, 1827–1831 (1992)

    Article  ADS  CAS  Google Scholar 

  57. Bock, C. et al. BiQ Analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing. Bioinformatics 21, 4067–4068 (2005)

    Article  CAS  Google Scholar 

  58. Christman, J. K. 5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 21, 5483–5495 (2002)

    Article  CAS  Google Scholar 

  59. Dunn, J. J., Krippl, B., Bernstein, K. E., Westphal, H. & Studier, F. W. Targeting bacteriophage T7 RNA polymerase to the mammalian cell nucleus. Gene 68, 259–266 (1988)

    Article  CAS  Google Scholar 

  60. Estève, P. O. et al. Direct interaction between DNMT1 and G9a coordinates DNA and histone methylation during replication. Genes Dev. 20, 3089–3103 (2006)

    Article  Google Scholar 

  61. Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods 5, 621–628 (2008)

    Article  CAS  Google Scholar 

  62. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009)

    Article  CAS  Google Scholar 

  63. Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnol. 28, 511–515 (2010)

    Article  CAS  Google Scholar 

  64. Harrow, J. et al. GENCODE: producing a reference annotation for ENCODE. Genome Biol. 7, (suppl. 1)S4.1–9 (2006)

    Article  Google Scholar 

  65. Irizarry, R. A. et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249–264 (2003)

    Article  Google Scholar 

  66. Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants CA118316, CA66996 and HL56745 from the National Institutes of Health (NIH) to D.G.T., the Italian Foundation for Cancer Research (FIRC) ‘Leonino Fontana e Maria Lionello’ fellowship, the NIH T32 HL007917-11A1 and the Società Italiana di Ematologia Sperimentale (SIES) ‘Dr.Tito Bastianello’ fellowship to A.D.R.; FAMRI CIA (103063) grant to A.K.E.; Fondazione Roma ‘Progetto cellule staminali’ to G.L. and F.D’A.; the American Italian Cancer Foundation Fellowship (AICF) to G.A.; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)—grant no. 2011/11822-6—to L.L.D.F.P.; the National Research Foundation and the Singapore Ministry of Education under its Centres of Excellence initiative to M.W. and T.B.; the MSMT Navrat grant LK21307 to M.A.-J. S.P. and J.T. were supported by New England Biolabs. We thank R. White, D. Johnson, M. Frank-Kamenetskii, S. M. Mirkin, D. Gautheret, B. Tazon-Vega, C. Bonifer, C. Bock, M. T. Voso, J. J. Dunn (deceased) and R. A. M. Fouchier for helpful advice and reagents; I. Rigoutsos for providing the latest released pyknons database; all the members of the Tenen Laboratory; P. Tan and T. S. Ting from the Genome Institute of Singapore; R. Soong from the Cancer Science Institute Translational Interface; J. LaVecchio and G. Buruzula from the Harvard Stem Cell Institute/Joslin Diabetes Center flow cytometry facility; and F. Hyde from Epicentre-Illumina. This research is supported by the Singapore Ministry of Health’s National Medical Research Council under its Singapore Translational Research (STaR) Investigator Award (D.G.T.).

Author information

Author notes

  1. Annalisa Di Ruscio and Alexander K. Ebralidze: These authors contributed equally to this work.

Authors and Affiliations

  1. Harvard Stem Cell Institute, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Annalisa Di Ruscio, Alexander K. Ebralidze, Giovanni Amabile, Lorena Lobo De Figueiredo Pontes, Meritxell Alberich-Jorda, Pu Zhang, John L. Rinn & Daniel G. Tenen

  2. Beth Israel Deaconess Medical Center, Boston, 02115, Massachusetts, USA

    Annalisa Di Ruscio, Alexander K. Ebralidze, Giovanni Amabile, Lorena Lobo De Figueiredo Pontes, Meritxell Alberich-Jorda, Konstantin K. Ebralidze & John L. Rinn

  3. Università Cattolica del Sacro Cuore, Institute of Hematology, L.go A. Gemelli 8, Rome 00168, Italy,

    Annalisa Di Ruscio, Francesco D’Alò & Giuseppe Leone

  4. Cancer Science Institute, National University of Singapore, 117599, Singapore,

    Touati Benoukraf, Mengchu Wu & Daniel G. Tenen

  5. Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA,

    Loyal A. Goff & John L. Rinn

  6. Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, 02142, Massachusetts, USA

    Loyal A. Goff & John L. Rinn

  7. Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, 32 Vassar Street, Cambridge, Massachusetts 02139, USA,

    Loyal A. Goff

  8. New England Biolabs, 240 County Road, Ipswich, 01938-2723, Massachusetts, USA

    Jolyon Terragni & Sriharsa Pradhan

  9. Department of Pathology, University of Michigan, Ann Arbor, 48109-2200, Michigan, USA

    Maria Eugenia Figueroa

  10. Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic,

    Meritxell Alberich-Jorda

  11. Department of Medicine, Hematology-Oncology, C-620 Weill Cornell Medical College, 1300 York Avenue, New York, New York 10021, USA,

    Ari Melnick

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  1. Annalisa Di Ruscio
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Contributions

D.G.T. supervised the project; A.D.R., A.K.E. and D.G.T. conceived and designed the study; A.D.R., A.K.E., G.A., P.Z., M.A.-J., F.D’A., S.P., L.L.D.F.P. and J.T. performed experiments; M.W. performed sequencing and microarray experiments; T.B. and L.A.G. analysed RIP-seq, RNA-seq, RRBS and microarray data; M.E.F. and A.M. performed the MassARRAY experiment and assisted in the analysis; A.D.R., A.K.E., G.L., K.K.E., J.L.R. and D.G.T. wrote the paper.

Corresponding author

Correspondence to Daniel G. Tenen.

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Supplementary information

Supplementary Information

This file contains Supplementary Results, Supplementary Methods, Supplementary References and Supplementary Figures 1-6. (PDF 4040 kb)

Supplementary Data 1

This file contains the MassARRAY primer sets. (XLS 46 kb)

Supplementary Data 2

This file contains a list of gene loci belonging to DNMT1-bound and unbound groups along with respective expression and DNA methylation levels. (XLS 2680 kb)

Supplementary Data 3

This file contains a full list of Biological Process Gene Ontology terms significantly enriched in cluster C. (XLS 250 kb)

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Di Ruscio, A., Ebralidze, A., Benoukraf, T. et al. DNMT1-interacting RNAs block gene-specific DNA methylation. Nature 503, 371–376 (2013). https://doi.org/10.1038/nature12598

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