Aberrant de novo methylation of the p16 INK4A CpG island is initiated post gene silencing in association with chromatin remodelling and mimics nucleosome positioning

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly enriched in CpG island associated promoter regions. DNA hypermethylation and histone repression correlate with gene silencing, however, the dynamics of this process are still largely unclear. The tumour suppressor gene p16 INK4A is inactivated in association with CpG island methylation during neoplastic progression in a variety of cancers, including breast cancer. Here, we investigated the temporal progression of DNA methylation and histone remodelling in the p16 INK4A CpG island in primary human mammary epithelial cell (HMEC) strains during selection, as a model for early breast cancer. Silencing of p16 INK4A has been previously shown to be necessary before HMECs can escape from selection. Here, we demonstrate that gene silencing occurs prior to de novo methylation and histone remodelling. An increase in DNA methylation was associated with a rapid loss of both histone H3K27 trimethylation and H3K9 acetylation and a gradual gain of H3K9 dimethylation. Interestingly, we found that regional-specific 'seeding' methylation occurs early after post-selection and that the de novo methylation pattern observed in HMECs correlates with the apparent footprint of nucleosomes across the p16 INK4A CpG island. Our results demonstrate for the first time that p16 INK4A gene silencing is a precursor to epigenetic suppression and that subsequent de novo methylation initially occurs in nucleosome-free regions across the p16 INK4A CpG island and this is associated with a dynamic change in histone modifications.