Coordinated transcription of ANRIL and P16 genes is silenced by P16 DNA methylation

Objective: To investigate the relationship between the transcription of ANRIL, P15, P14 and P16 at the same locus and the regulation mechanism of ANRIL.Methods: Publicly available database of Cancer Cell Line Encyclopedia(CCLE) was used in bioinformatic analyses. Methylation of Cp G islands was detected by denaturing high performance liquid chromatography(DHPLC). Gene transcript levels were determined using quantitative real-time polymerase chain reaction(q RTPCR) assays. An engineered P16-specific transcription factor and DNA methyltransferase were used to induce P16-specific DNA demethylation and methylation.Results: The expression level of ANRIL was positively and significantly correlated with that of P16 but not with that of P15 in the CCLE database. This was confirmed in human cell lines and patient colon tissue samples. In addition, ANRIL was significantly upregulated in colon cancer tissues. Transcription of ANRIL and P16 was observed only in cell lines in which the P16 alleles were unmethylated and not in cell lines with fully methylated P16 alleles.Notably, P16-specific methylation significantly decreased transcription of P16 and ANRIL in BGC823 and GES1 cells. In contrast, P16-specific demethylation re-activated transcription of ANRIL and P16 in H1299 cells(P<0.001).Alteration of ANRIL expression was not induced by P16 expression changes.Conclusions: ANRIL and P16 are coordinately transcribed in human cells and regulated by the methylation status of the P16 Cp G islands around the transcription start site.