Abstract
Disruption of the BRCA1 tumor suppressor can be caused not only by inherited mutations in familial cancers but also by BRCA1 gene silencing in sporadic cancers. Hypoxia, a key feature of the tumor microenvironment, has been shown to downregulate BRCA1 at the transcriptional level via repressive E2F4/p130 complexes. Here we showed that hypoxia also drives epigenetic modification of the BRCA1 promoter, with decreased H3K4 methylation as a key repressive modification produced by the lysine-specific histone demethylase LSD1. We also observed increased H3K9 methylation coupled with decreased H3K9 acetylation. Similar modifications were seen in the RAD51 promoter, which is also downregulated by hypoxia, whereas exactly opposite changes were seen in the promoter of the hypoxia-inducible gene VEGF. In cells containing the BRCA1 promoter driving a selectable HPRT gene, long-term silencing of the promoter was observed following exposure to hypoxic stress. Clones with silenced BRCA1 promoters were detected at frequencies of 2% or more following hypoxia, but at less than 6 × 10(-5) without hypoxia. The silenced clones showed decreased H3K4 methylation and decreased H3K9 acetylation in the BRCA1 promoters, consistent with the acute effects of hypoxic stress. Hypoxia-induced BRCA1 promoter silencing persisted in subsequent normoxic conditions but could be reversed by treatment with a histone deacetylase (HDAC) inhibitor but not with a DNA methylation inhibitor. Interestingly, treatment of cells with inhibitors of poly(ADP-ribose) polymerase (PARP) can cause short-term repression of BRCA1 expression, but such treatment does not produce H3K4 or H3K9 histone modification or BRCA1 promoter silencing. These results suggest that hypoxia is a driving force for long-term silencing of BRCA1, thereby promoting genome instability and tumor progression.