Hydroxymethylated-P16 Allele Is Transcription-Inactive

Abstract

Background5-Methylcytosine can be oxidized into 5-hydroxymethylcytosine (5hmC) in the genome. Methylated-P16 (P16M) can be oxidized into completely hydroxymethylated-P16 (P16H) in human cancer and precancer cells. The aim of this study is to investigate the biological function of P16H.\n\nMethodsTrue P16M and P16H were analyzed using bisulfite/TAB-based assays. A ZFP-based P16-specific dioxygenase (P16-TET) was constructed and used to induce P16H. Cell proliferation and migration were determined with a series of biological analyses.\n\nResults(A) The 5hmCs were enriched in the antisense-strand of the P16 exon-1 in HCT116 and AGS cells containing methylated-P16 alleles (P16M). (B) P16-TET induced both P16H and P16 demethylation in H1299 and AGS cells and reactivated P16 expression. Notably, P16H was only detectable in the sorted P16-TET H1299 and AGS cells that did not show P16 expression. (C) P16-TET significantly inhibited the xenograft growth derived from H1299 cells in NOD-SCID mice, but did not inhibit the growth of P16-deleted A549 control cells. P16-siRNA knockdown could rescue P16-TET-inhibited cell migration.\n\nConclusionHydroxymethylated P16 alleles are transcriptionally inactive.\n\nAUTHOR SUMMARYIt is well known that 5-methylcytosine (5mC) in genomic DNA of mammalian cells can be oxidized into 5-hydroxymethylcytosine (5hmC) and other derivates by DNA dioxygenase TETs. While conversion of 5mC to 5hmC plays an important role in active DNA demethylation through further oxidations, a certain proportion of 5hmCs remain in the genome. Although it is supposed that occurrence of 5hmCs may contribute to the flexibility of chromatin and the protection of the bivalent promoters from hypermethylation, the direct effect of 5hmCs on gene transcription is unknown. In the present study, we engineered a zinc-finger protein-based P16-specific DNA dioxygenase and used it to induce P16 hydroxymethylation and demethylation in cancer cells. Our results demonstrate, for the first time, that the hydroxymethylated P16 alleles retain transcriptionally inactive. This is supported by our recent findings that mRNAs are always transcribed only from the unmethylated P16 strands, but not from the hydroxymethylated/methylated strands in HCT116 cells, and that the risks for malignant transformation are similar for patients with the P16 methylation-positive oral epithelial dysplasia with and without P16 hydroxymethylation in a prospective study.