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Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA

Abstract

Tumour suppressor genes (TSGs) inhibiting normal cellular growth are frequently silenced epigenetically in cancer1. DNA methylation is commonly associated with TSG silencing1, yet mutations in the DNA methylation initiation and recognition machinery in carcinogenesis are unknown2. An intriguing possible mechanism for gene regulation involves widespread non-coding RNAs such as microRNA, Piwi-interacting RNA and antisense RNAs3,4,5. Widespread sense–antisense transcripts have been systematically identified in mammalian cells6, and global transcriptome analysis shows that up to 70% of transcripts have antisense partners and that perturbation of antisense RNA can alter the expression of the sense gene7. For example, it has been shown that an antisense transcript not naturally occurring but induced by genetic mutation leads to gene silencing and DNA methylation, causing thalassaemia in a patient8. Here we show that many TSGs have nearby antisense RNAs, and we focus on the role of one RNA in silencing p15, a cyclin-dependent kinase inhibitor implicated in leukaemia. We found an inverse relation between p15 antisense (p15AS) and p15 sense expression in leukaemia. A p15AS expression construct induced p15 silencing in cis and in trans through heterochromatin formation but not DNA methylation; the silencing persisted after p15AS was turned off, although methylation and heterochromatin inhibitors reversed this process. The p15AS-induced silencing was Dicer-independent. Expression of exogenous p15AS in mouse embryonic stem cells caused p15 silencing and increased growth, through heterochromatin formation, as well as DNA methylation after differentiation of the embryonic stem cells. Thus, natural antisense RNA may be a trigger for heterochromatin formation and DNA methylation in TSG silencing in tumorigenesis.

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Figure 1: p15 antisense expression in leukaemia cells.
Figure 2: p15 promoter is silenced by the expressed p15 antisense transcript in transfected HCT116 cells.
Figure 3: Heterochromatin formation induced by p15AS.
Figure 4: p15 silencing, heterochromatin formation and DNA methylation induced by p15 antisense transcript in mouse embryonic stem cells.

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Acknowledgements

We thank G. Hannon for the Dicer knockout line, R. Ambinder for providing leukaemia specimens from the Johns Hopkins SPORE lymphoma tissue bank, M. Gao for help in detection of DNA methylation, and I. Cui for assistance with the manuscript. This work was supported by a grant from the National Institutes of Health.

Author Contributions W.Y. performed most of the experiments; D.G., K.M.-J. and P.O. performed some vector design; J.K. provided clinical samples and expertise; A.P.F. and H.C. performed the experimental design, supervised the research project and wrote the manuscript.

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Correspondence to Andrew P. Feinberg or Hengmi Cui.

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The file contains Supplementary Figures S1-S19 with Legends, Supplementary Tables 1-5 and Supplementary Methods. (PDF 839 kb)

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Yu, W., Gius, D., Onyango, P. et al. Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA. Nature 451, 202–206 (2008). https://doi.org/10.1038/nature06468

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