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DNMT1-interacting RNAs block gene-specific DNA methylation


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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Gene Expression Omnibus

Data deposits

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


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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




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.

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Correspondence to Daniel G. Tenen.

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The authors declare no competing financial interests.

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).

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