In eukaryotes, genomic DNA exists in a highly condensed form by being packaged into chromatin. This consists of 147 bp of DNA wrapped around histone octamers (two each of histones H2A, H2B, H3 and H4) and stabilized by linker histones (H1) and other factors. In its condensed form, chromatin makes DNA inaccessible to cellular processes that use DNA as substrates such as transcription, replication and DNA repair. Post-translational modifications of N-terminal tails of the core histones, notably by acetylation, phosphorylation, ubiquitination, sumoylation and methylation, have been implicated in regulating these cellular processes.
An active current area in cancer therapy is the use of agents that modify chromatin, particularly inhibitors of histone deacetylases (HDACs). By inhibiting deacetylation of histone tails, HDAC inhibitors promote the acetylation of these N-terminal tails, which tends to open up chromatin, making it more accessible to transcription. Consistent with this, it is postulated that one of the main mechanisms for the anticancer effects of HDAC inhibitors is the reactivation of silenced checkpoint or tumor suppressor genes such as p21 or p16 (1
). However, in addition to these effects, a number of investigators have shown that HDAC inhibitors sensitize tumor cells to killing by ionizing radiation both in vitro
and in vivo
). The mechanism for this sensitization is not clear, but it is associated with abrogation of the G2
), diminished DNA repair assayed by loss of phosphorylated H2AX (5
), and hyperacetylation of histone H4 (5
). Paradoxically, a number of recent studies, outlined below, have also demonstrated that mutation, deletion or knockdown of genes involved in acetylation of histone H4 in both mammalian and yeast cells sensitizes to killing by ionizing radiation and/or inhibits the repair of DNA double-strand breaks (DSBs).
In the budding yeast S. cerevisiae
, mutations in histone H4 that prohibit its acetylation and defects in components of the NuA4 histone acetyl transferase (HAT) complex, the HAT that predominantly acetylates specific lysine residues on histone H4, sensitize cells to DSB-producing agents such as ionizing radiation (8
). After the induction of DSBs, HAT complexes are recruited to the site of DSBs, resulting in acetylation of histone H4 flanking the break, later followed by deacetylation of histone H4 after break repair (9
). Studies in mammalian cells have shown that deletion of TRRAP, a core component of a number of HAT complexes, causes histone H4 hypoacetylation, a defect in recruitment of a subset of repair factors to the sites of DSBs, and compromised DSB repair (11
). These studies suggest that the role of histone H4 acetylation in mediating DNA DSB repair is to relax a normally closed chromatin structure, allowing for the recruitment of DNA repair proteins and DNA damage signaling proteins to the site of DNA damage (13
In view of these studies suggesting that acetylation of histones, particularly histone H4, protects eukaryotic cells from ionizing radiation, we have explored the possibility of sensitizing cells to radiation using HAT inhibitors. We chose to do this with budding yeast for two reasons: first, because there are as yet no available inhibitors of acetylation of all four H4 lysine residues in mammalian cells, and second, because the availability of mutants in various HATs and DNA repair pathways makes yeast a powerful system to interrogate the specificity of any effects. The two chemicals that have been shown to have HAT-inhibitory activities at nontoxic concentrations in yeast are copper sulfate (CuSO4
) and nickel chloride (NiCl2
), both of which cause a decrease in acetylation at all four of the lysine residues on histone H4 (14
). We hypothesized that wild-type yeast cells treated with HAT inhibitors would behave similarly to mutant cells lacking intact histone H4 acetylation machinery. Our data show that inhibition of HATs under conditions that produce hypoacetylation of H4 sensitizes yeast cells to radiation by inhibiting DNA repair by the homologous recombination pathway.