No abstract

Radiation therapy is widely used to treat human malignancies, but many tumor types, including gliomas, exhibit significant radioresistance. Radiation therapy creates DNA double-strand breaks (DSBs), and DSB repair is linked to rapid changes in epigenetic modifications, including increased histone methylation. This increased histone methylation recruits DNA repair proteins which can then alter the local chromatin structure and promote repair. Consequently, combining inhibitors of specific histone methyltransferases with radiation therapy may increase tumor radiosensitivity, particularly in tumors with significant therapeutic resistance. Here, we demonstrate that inhibitors of the H4K20 methyltransferase SETD8 (UNC-0379) and the H3K9 methyltransferase G9a (BIX-01294) are effective radiosensitizers of human glioma cells. UNC-0379 blocked H4K20 methylation and reduced recruitment of the 53BP1 protein to DSBs, although this loss of 53BP1 caused only limited changes in radiosensitivity. In contrast, loss of H3K9 methylation through G9a inhibition with BIX-01294 increased radiosensitivity of a panel of glioma cells (SER2Gy range: 1.5 - 2.9). Further, loss of H3K9 methylation reduced DSB signaling dependent on H3K9, including reduced activation of the Tip60 acetyltransferase, loss of ATM signaling and reduced phosphorylation of the KAP-1 repressor. In addition, BIX-0194 inhibited DSB repair through both the homologous recombination and nonhomologous end-joining pathways. Inhibition of G9a and loss of H3K9 methylation is therefore an effective approach for increasing radiosensitivity of glioma cells. These results suggest that combining inhibitors of histone methyltransferases which are critical for DSB repair with radiation therapy may provide a new therapeutic route for sensitizing gliomas and other tumors to radiation therapy.

The n-terminal tail of histone H4 recruits repair proteins, including 53BP1, to DNA double-strand breaks (DSB) and undergoes dynamic acetylation during DSB repair. However, how H4 acetylation (H4Ac) recruits repair proteins and reorganizes chromatin during DNA repair is unclear. Here, we show that the bromodomain protein BRD2 is recruited to DSBs. This recruitment requires binding of BRD2’s tandem bromodomains to H4Ac, which is generated at DSBs by the Tip60/KAT5 acetyltransferase. Binding of BRD2 to H4Ac protects the underlying acetylated chromatin from attack by histone deacetylases and allows acetylation to spread along the flanking chromatin. However, BRD2 recruitment is spatially restricted to a chromatin domain extending only 2 kb either side of the DSB, and BRD2 does not spread into the chromatin domains flanking the break. Instead, BRD2 facilitates recruitment of a second bromodomain protein, ZMYND8, which spreads along the flanking chromatin, but is excluded from the DSB region. This creates a spatially restricted H4Ac/BRD2 domain which reorganizes chromatin at DSBs, limits binding of the L3MBTL1 repressor and promotes 53BP1 binding, while limiting end-resection of DSBs. BRD2 therefore creates a restricted chromatin environment surrounding DSBs which facilitates DSB repair and which is framed by extensive ZMYND8 domains on the flanking chromatin.