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 cancer. DNA methylation is commonly associated with TSG silencing, yet mutations in the DNA methylation initiation and recognition machinery in carcinogenesis are unknown. An intriguing possible mechanism for gene regulation involves widespread non-coding RNAs such as microRNA, Piwi-interacting RNA and antisense RNAs. Widespread sense-antisense transcripts have been systematically identified in mammalian cells, 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 gene. 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 patient. 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.

Figure 1. p15 antisense expression in leukaemia cells

a, Native p15AS transcripts were found in leukaemia cell lines. Lanes 1 and 3 are negative controls without reverse transcriptase. Lanes 2 and 4 show 97 bp bands amplified from the cDNA reverse-transcribed from RNA by a strand-specific primer from leukaemia cell lines KG-1 and Kasumi-1. M is a 100 bp ladder marker. b, p15 and p15AS expression in leukaemic (n = 16) and normal lymphocytes (n = 16), analysed by real-time RT-PCR. Leukaemia samples showed higher expression of p15 antisense and lower expression of p15 than normal lymphocytes. Both p15 and its antisense expression levels were normalized with GAPDH. Error bars, s.e.m.; a.u., arbitrary units; *P < 0.05.

Figure 2. p15 promoter is silenced by the expressed p15 antisense transcript in transfected HCT116 cells

a, Maps of constructs. Arrows indicate the direction of transcription. b, Downregulation of GFP (under control of p15 promoter) in cells transfected with pP15-AS and selected for 3 weeks. The transfected cells were analysed by FACS. The percentage of GFP-positive cells with p15 antisense expression was lower than that without the antisense expression (0.3% versus 5.8% and 6.6%) (P < 0.001). c, The second FACS showed downregulation of GFP in expressing cells selected from b and cultured for an additional 3 weeks. The percentage of GFPpositive cells with p15 antisense expression remained lower than that without the antisense expression (11.0% versus 51.1% and 59.3%) (P < 0.0001). d, Downregulation of GFP in cells with a tetracycline-inducible pP15-AS-Tre. Expression was reduced 68% on addition of tetracycline, which persisted after tetracycline withdrawal.

Figure 3. Heterochromatin formation induced by p15AS

a, Antisense expression induced an increase in H3K9 dimethylation and a decrease in H3K4 dimethylation in the exogenous p15 promoter region. ChIP enrichment was measured using real-time PCR, normalized by input DNA. The endogenous and exogenous genes were distinguished with probes specific for sense (pP15′) and antisense (pP15-AS′) vectors transfected after modification by site-directed mutagenesis (see Supplementary Fig. 9). b, Antisense expression induced an increase in H3K9 dimethylation and a decrease in H3K4 dimethylation in the exogenous p15 exon 1 region. c, Antisense expression induced a decrease in H3K4 dimethylation in the proximal endogenous p15 promoter, examined by ChIP followed by realtime PCR at six locations, normalized by input DNA. The numbers on the x axis represent PCR sites relative to the transcription start site of p15. The site (+253) near the transcriptional start site was examined by vectors to distinguish endogenous and exogenous genes, as in a. d, Antisense expression induced an increase in H3K9 dimethylation in a large region (about 6 kb) of the endogenous sequence around the p15 transcriptional start site. e, Reactivation of antisense-silenced p15 promoter by 5-aza-2′-deoxycytidine (AZA) and trichostatin A (TSA). p15 promoter activity was measured by FACS for GFP after 5-aza-2′-deoxycytidine and trichostatin A treatment. The top two panels represent negative controls: HCT116 cells only and untreated HCT116 cells with pP15-AS, respectively. The bottom three panels show the result of treatment of the pP15-AS-transfected cells with either 5-aza-2′-deoxycytidine, or trichostatin A or 5-aza-2′-deoxycytidine + trichostatin A. All treatments reactivated the p15 promoter. Significant differences were tested compared with pP15 (n = 3). Error bars, s.d.; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. p15 silencing, heterochromatin formation and DNA methylation induced by p15 antisense transcript in mouse embryonic stem cells

a, Downregulation of GFP in mouse embryonic stem cells transfected with pP15-AS constructs. The transfected cells were analysed by FACS. The top panel is the negative control, mouse embryonic stem cells without transfection; the middle panel shows mouse embryonic stem cells transfected with pP15; the bottom panel is mouse embryonic stem cells with pP15-AS. Green dots in the R2 area represent GFP-positive cells. The numbers in the brackets show the percentages of GFP-positive cells. b, Antisense expression induced a decrease in H3K4 dimethylation and an increase in H3K9 dimethylation in the exogenous p15 promoter region (blue, pP15 mouse embryonic stem cells; red, pP15-AS mouse embryonic stem cells). c, Alterations in DNA methylation of exogenous p15 promoter region in mouse embryoid bodies analysed by bisulphite pyrosequencing. Top two panels represent mouse embryonic stem cells transfected with pP15-AS and pP15. The bottom two panels represent mouse embryoid bodies differentiated from mouse embryonic stem cells transfected with pP15-AS and pP15. The six examined cytosine-guanosine sites (CpGs) are marked and methylation percentages are indicated on the top. Note that hypermethylation is only observed in mouse embryoid bodies differentiated from mouse embryonic stem cells transfected with the pP15-AS construct. Significant differences were tested compared with mES-pP15 cells (n = 3). Error bars, s.d.; *P < 0.05; **P < 0.01.