Aberrant Epigenetic Silencing Is Triggered by a Transient Reduction in Gene Expression

Abstract

BackgroundAberrant epigenetic silencing plays a major role in cancer formation by inactivating tumor suppressor genes. While the endpoints of aberrant silencing are known, i.e., promoter region DNA methylation and altered histone modifications, the triggers of silencing are not known. We used the tet-off system to test the hypothesis that a transient reduction in gene expression will sensitize a promoter to undergo epigenetic silencing.Methodology/Principal FindingsThe tet responsive promoter (PTRE) was used to drive expression of the selectable human HPRT cDNA in independent transfectants of an Hprt deficient mouse cell line. In this system, high basal HPRT expression is greatly reduced when doxycycline (Dox) is added to the culture medium. Exposure of the PTRE-HPRT transfectants to Dox induced HPRT deficient clones in a time dependent manner. A molecular analysis demonstrated promoter region DNA methylation, loss of histone modifications associated with expression (i.e., H3 lysine 9 and 14 acetylation and lysine 4 methylation), and acquisition of the repressive histone modification H3 lysine 9 methylation. These changes, which are consistent with aberrant epigenetic silencing, were not present in the Dox-treated cultures, with the exception of reduced H3 lysine 14 acetylation. Silenced alleles readily reactivated spontaneously or after treatment of cells with inhibitors of histone deacetylation and/or DNA methylation, but re-silencing of reactivated alleles did not require a new round of Dox exposure. Inhibition of histone deacetylation inhibited both the induction of silencing and re-silencing, whereas inhibition of DNA methylation had no such effect.Conclusions/SignificanceThis study demonstrates that a transient reduction in gene expression triggers a pathway for aberrant silencing in mammalian cells and identifies histone deacetylation as a critical early step in this process. DNA methylation, in contrast, is a secondary step in the silencing pathway under study. A model to explain these observations is offered.

Epimutations and cancer predisposition: importance and mechanisms

“…Spread of methylation may be influenced by competition between the transcriptional machinery and DNA methyltransferases and active transcription may provide protection from de novo methylation [17]. This hypothesis seems to be supported by recent studies showing that a decrease in transcriptional output predisposes to de novo methylation [18,19]. The gradual spread in DNA methylation during the ageing process coupled with the stochastic manner by which methylation is initiated may play a role in the predisposition to sporadic cancers that are typically associated with ageing.…”

“…Reductions in transcriptional output may predispose a promoter to DNA methylation [18]. A genetic alteration that affects transcription factor binding, alters an enhancer region or results in the recruitment of a repressor may either protect or predispose a promoter to methylation.…”

CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs

“…We also used this approach to knockdown the HERG potassium channel to mimic an LQT2-type phenotype in iPS-CM. We found that the inducible TetO promoter is partially silenced during the cardiac differentiation process, and this is reported to be due to methylation at CpG dinucleotides (Oyer et al, 2009). This silencing is independent of integration at the AAVS1 locus, as CAG-driven transgenes integrated at the AAVS1 locus remain active after differentiation.…”

Modulation Of Gene Expression In Ovarian Cancer By Active And Repressive Histone Marks

“…This outlines a new model in which extended domains of DNA methylation and histone marks are coordinately established to achieve specific gene expression [55]. A recent study suggests that transient alterations in gene expression can trigger a pathway for aberrant silencing of mammalian gene promoters in which histone deacetylation is a critical early step, while DNA methylation surprisingly, follows in the silencing pathway [56]. The effects of these mechanisms are often evident or can be derived from the response of cells to treatments with various epigenetic drugs.…”