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Aberrant de novo methylation of the p16INK4A CpG island is initiated post gene silencing in association with chromatin remodeling 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 is still largely unclear. One of the problems in addressing this question is that epigenetic changes often occur early in oncogenesis and are therefore difficult to dissect in clinical samples. The tumour suppressor gene p16INK4A 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 p16INK4A CpG island in primary human mammary epithelial cell (HMEC) strains during escape from growth arrest. Silencing of p16INK4A has been previously shown to be necessary before HMECs can escape from selection. We now 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 individual post-selection HMEC strains all share a common pattern of regional-specific ?seeding? methylation within the p16INK4A CpG island that then spreads progressively to adjacent regions with continued passaging. Using a high-resolution foot printing technique we found that the regional-specific ?seeding? methylation pattern correlated with the position of nucleosomes in the HMECs. Our results demonstrate for the first time that p16INK4A gene silencing is a precursor to epigenetic suppression and that subsequent de novo methylation initially occurs in nucleosome-free regions across the p16INK4A CpG island and this is associated with a dynamic change in histone modifications.

Type Journal
ISBN 1460-2083 (Electronic)
Authors Hinshelwood, R.A.; Melki, J.R.; Huschtscha, L.I.; Paul, C.; Song, J.Z.; Stirzaker, C.; Reddel, R.R.; Clark, S.J.
Published Date 2009-08-15
Published Volume 18
Published Issue 16
Published Pages 3098-3109
Status Published in-print
DOI 10.1093/hmg/ddp251
URL link to publisher's version
OpenAccess link to author's accepted manuscript version