Epigenetic modifications in double-strand break DNA damage signaling and repair

Abstract

Factors involved in the cellular response to double-strand break (DSB) DNA damage have been identified as potential therapeutic targets that would greatly sensitize cancer cells to radiotherapy and genotoxic chemotherapy. These targets could disable the repair machinery and/or reinstate normal cell-cycle checkpoint leading to growth arrest, senescence, and apoptosis. It is now clear that a major aspect of the DNA damage response occurs through specific interactions with chromatin structure and its modulation. It implicates highly dynamic posttranslational modifications of histones that are critical for DNA damage recognition and/or signaling, repair of the lesion, and release of cell-cycle arrest. Therefore, drugs that target the enzymes responsible for these modifications, or the protein modules reading them, have very high therapeutic potential. This review presents the current state of knowledge on the different chromatin modifications and their roles in each step of eukaryotic DSB DNA damage response.

Figure 1

Model of histone modifications and chromatin remodeling during DNA DSB repair, Step 1: Recognition and signaling of a DSB. g-H2AX plays a key role in DNA-damage signaling, acting as a platform of assembly for the repair factors as well as for checkpoint proteins. Immediately following the apparition of a DSB, the MRN complex binds DNA-ends and participates in ATM kinase recruitment. ATM then rapidly phosphorylates H2AX histone variant at the site of the break. Phospho-H2AX, also called γ-H2AX allows the binding, retention and accumulation at the break of the complexes involved in the DDR. The simultaneous presence of the RSC remodeling complex at the break may facilitate the access of the recruited repair factors. Indeed, the mediator protein MDC1 is recruited to the DSB and binds γ-H2AX, where it promotes further ATM and MRN accumulation. As a consequence, γ-H2AX bi-directionally spreads out from the DSB (approximately 2Mb), thus increasing the accumulation of repair factors. MDC1 also recruits RNF8/UBC13 ubiquitin-ligase which ubiquitinates H2A and H2AX, which in turn, is recognized by RNF168-UBC13 H2AX-ubiquitin-ligase complex, resulting in the amplification of γ-H2AX poly-ubiquitination near the DSB. In parallel, γ-H2AX also permits TIP60 HAT recruitment at the break, followed by the acetylation of H2A and H4 histones and destabilization of the nucleosomes. In addition, phosphorylation of H2AX could induce conformational changes in the nucleosome, resulting in the exposition of H4K20me and H3K79me, recognized by the checkpoint protein 53BP1.

Figure 2

Model of histone modifications and chromatin remodeling during DNA DSB repair, Step 2: Opening of chromatin to repair the break. Once the DSB has been recognized and signaled, it is time to repair the break. Histones need to be removed from chromatin in the vicinity of the break to allow access to the DNA to the repair factors. Chromatin-remodelers are then recruited to the DSB. TIP60 complex recruited at the DSB comprises HAT activity as well as histone exchange ability. Following acetylation-dependant nucleosome destabilization, TIP60 complex can remove H2A(X)-H2B histone dimers from chromatin at the break. INO80 is also recruited at the break where it helps removing histones close to the DSB. The SWI/SNF/RSC/BRG-1 remodeling complex is also present at the break where it can associate with γ-H2AX and promote histone eviction or exchange. Such histone eviction allows association of the ssDNA-binding protein RPA with resected DNA and subsequent recruitment of repair factors such as Rad51. Moreover, BRCA1-A repair complex accumulates at the break through direct interaction of its RAP80 subunit with poly-UbH2A(X).

Figure 3

Model of histone modifications and chromatin remodeling during DNA DSB repair, Step 3: Chromatin restoration after DNA-break repair. When repair of the DSB is completed, the chromatin needs to be restored and the repair specific histone marks need to be removed in order to release repair factors and cell cycle checkpoints. Thus, γ-H2AX has to disappear from the repaired site. Phosphatases such as PP2A and PP4C dephosphorylate γ-H2AX and allow release of checkpoint factors like 53BP1. In order to restore chromatin, new histones are deposited onto the DNA. Histone chaperones such as FACT and CAF1 have been implicated in this process. Moreover, H3-H4 histones deposited by CAF1 are first acetylated by Hat1, and then by CBP/p300/Rtt109-Asf1, as marks of new synthesized histones. This incorporation of new histones is though to occur at the site of the repaired DNA. More distal to the site, repair marks are removed from nucleosomal histones in the chromatin context. Acetyl-marks associated with chromatin “opening” are eliminated by HDACs. ySin3/Rpd3 HDAC associates with Casein Kinase 2 (CK2) that is responsible for subsequent phosphorylation of H4S1, reinforcing nucleosome stability by blocking re-acetylation.