Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA

Abstract

Widespread sense-antisense transcripts have been identified in mammalian cells. Many tumour suppressor genes have nearby antisense RNAs, and an antisense RNA to the cyclin-dependent kinase inhibitor p15 can silence the p15 gene by inducing heterochromatin formation. 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.