(P04-060-24) Distinct Mechanisms of Action of Phytoestrogens in SIRT1-Mediated Effects on DNA Methylation and Silencing of Aging-Related Genes in Mammary Epithelial Cells

Objectives: Using our previous genome-wide DNA methylation data in human mammary epithelial cells exposed to berry polyphenols with estrogenic activity (i.e., phytoestrogens), we have identified hypermethylated genes enriched with functional categories that disrupt homeostasis and health maintenance, e.g., by limiting DNA break repair (RNF169) or promoting ribosomal biogenesis (rDNA). Existing evidence suggests that DNA methylation may be governed by chromatin regulatory proteins, withsirtuin 1 (SIRT1) deacetylase as an example. SIRT1 may interact with DNA methylating enzymes (i.e., DNMT3B) to repress transcription. Since SIRT1 was reported as a possible target of phytoestrogens, we test whether phytoestrogens suppress aging-related genes via SIRT1-mediated effects on DNA methylation.

Methods: Human MCF10A mammary epithelial cells were treated with phytoestrogens, pterostilbene (PTS) and genistein (GEN), followed by the analysis of cell growth (trypan blue exclusion test), DNA methylation (pyrosequencing) and expression (qRT-PCR) of target genes, and SIRT1 and DNMT3B occupancy at target regions (chromatin immunoprecipitation, ChIP).

Results: PTS and GEN decreased MCF10A cell growth with 50% inhibition and less than 10% dead cells at 11µM and 22µM concentrations respectively, as compared with vehicle-treated cells (control). SIRT1 occupancy at the selected hypermethylated phytoestrogen-target genes, RNF169 and rDNA, was accompanied by consistent promoter hypermethylation and gene downregulation in response to GEN, but not PTS. GEN-mediated DNA hypermethylation and SIRT1 occupancy were linked to a robust DNMT3B binding at both RNF169 and rDNA promoters. This was not observed in cells exposed to PTS, suggesting a distinct mechanism of action. Although both SIRT1 and DNMT3B bind to RNF169 and rDNA promoters upon GEN, the two proteins do not co-occupy the regions, as confirmed by double ChIP. Upon siRNA SIRT1 depletion, GEN does not decrease expression of rDNA anymore, suggesting that SIRT1 is required for epigenetic suppression of rDNA in response to GEN.

Conclusions: The findings further our understanding of SIRT1 role in epigenetic mechanisms mediating the impact of the environment-diet on cell biology.

Funding Sources: The project was supported by NSERC Discovery granted to B.S.