The Role of Histone Lysine Methylation in the Response of Mammalian Cells to Ionizing Radiation

The Histone Methylation Landscape at the Damaged Genomic Sites

Damage signaling and repair are operated in the context of chromatin structure with strict requirements for full efficiency (Polo and Jackson, 2011). A number of histone modifications have been associated to specific lesions and their functional role in damage signaling and repair has been postulated (Vidanes et al., 2005; Misteli and Soutoglou, 2009; Wei et al., 2018; Husmann and Gozani, 2019). HMTs and KDMs can directly modify the chromatin at DNA damaged sites to promote damage sensing, to favor the recruitment of DNA repair proteins, to silence transcription during the repair process, to regulate the expression of DDR factors and to change and restore the chromatin state before and after repair. Indeed, lysine methylation is one of the prominent histone modifications at damaged sites. It mainly occurs at lysines on H3 tails, but methylation at H4K20 and K16 and at H2AXK134 are also relevant (Figure 1). In the next sections, we will summarize what it is currently known on the role of the methylation state of specific histone lysines in signaling IR-induced damage and promoting DNA repair, and on the modification machinery involved in this methylation.

H3K4

Strong evidence in the literature shows that lysine methylation in histone H3 tail is implicated in damage signaling. Chromatin changes are required in order to silence transcription in cis to DNA double-strand breaks (Shanbhag et al., 2010). Since H3K4 tri-methylation is associated with active transcription genome-wide (Vakoc et al., 2006), active demethylation of this residue around damaged sites is expected. Indeed, Li and coworkers (Li et al., 2014) found that KDM5B, a HDM specifically involved in H3K4me3 demethylation, is redistributed in the nucleus after exposure to IR and becomes highly enriched at the damaged sites. The mechanism of KDM5B recruitment is quite specific and requires the presence of nucleosomes containing histone variant MacroH2A1.1 and PARylation by PARP1 (Figure 1). KDM5B is required for efficient homologous recombination (HR) and non-homologous end joining (NHEJ) and, specifically, for the recruitment of Ku70 and BRCA1 to IR-induced breaks (Li et al., 2014). KDM5B is upregulated in several tumors and cancer-derived cell lines, which seems to be associated with resistance to genotoxic stress (Xu et al., 2018). Moreover, microRNAs that specifically target KDM5 mRNA are downregulated in tumors and, when overexpressed in cancer cells, increase their radio-sensitivity (Mocavini et al., 2019). KDM5B associates with several other proteins with transcription repressing activities, such as the HDM LSD1, the histone deacetylase HDAC1 and the histone methylase EZH2 (Figure 1). This raises the question of the respective functional roles of the H3K4me3 demethylating activity and of the corepressor recruiting activity of KDM5B. In this respect, it was recently shown that inhibition of KDM5B demethylase activity increases the radio-sensitivity of cancer cell lines upregulating KDM5B, suggesting that the catalytic activity of KDM5 is important in this context (Bayo et al., 2018; Pippa et al., 2019). Two other class 5 HDMs, KDM5A and KDM5C, both specific for H3K4me3, have also been involved in radio-resistance (Hendriks et al., 2015; Gong et al., 2017, Figures 1, 2). Gong and coworkers showed that KDM5A performs H3K4me3 demethylation within chromatin near DSBs and has a crucial role in regulating the ZMYND8-NuRD chromatin remodeler (Gong et al., 2017), which represses transcription and promotes DNA repair (Gong et al., 2015). H3K4me3 must be demethylated to allow repair to proceed, probably because this process is essential for limiting local transcription. The removal of methylation marks is therefore just as important as the addition of new histone modifications (Price, 2017). While KDM5A seems to act in parallel to KDM5B, although with different mechanisms, KDM5C could be recruited in alternative circumstances. Hendriks and coworkers showed that, in response to alkylation damage by methyl methanesulfonate (MMS), SUMOylated JARID1B (KDM5B) is ubiquitylated by the SUMO-targeted ubiquitin ligase RNF4 and degraded by the proteasome, whereas JARID1C (KDM5C) is SUMOylated and recruited to the chromatin to demethylate histone H3K4 (Hendriks et al., 2015). This observation suggests that the redundancy among HDMs could be exploited to differentiate the response pathways triggered by different genotoxic agents (Figure 2). KDM5B is also a physical component of the LSD1/NuRD complex, which functions in transcriptional repression. LSD1 (KDM1A) specifically demethylates H3K4 me1/2 (Shi et al., 2004). Although KDM5B and LSD1 in the complex can act in a sequential and coordinated manner to demethylate H3K4 (Li et al., 2011), the role of LSD1 in DDR may be partially distinct from that of class 5 HDMs. LSD1 can be recruited directly to sites of DNA damage, possibly through the interaction with the E3 ubiquitin ligase RNF168, thus reducing H3K4me2 levels (Mosammaparast et al., 2013). However, since CK2-mediated phosphorylation of LSD1 promotes the molecular interaction between RNF168 and 53BP1, some of LSD1 roles in DDR may be independent of its catalytic activity (Peng et al., 2015). Although the loss of LSD1 does not affect the initial formation of γ-H2AX foci, 53BP1 and BRCA1 complex recruitment appears to be reduced upon LSD1 knockdown (Mosammaparast et al., 2013). This work suggests that LSD1 affects 53BP1 recruitment by promoting H2A ubiquitylation at damaged sites during late S/G2. This could be mediated by the interaction of LSD1 with RNF168 and/or by a possible crosstalk between H3K4 demethylation and H2A ubiquitylation (Figure 3A). Once again, it is likely that one function of LSD1-mediated demethylation in this pathway is to facilitate a repressive chromatin environment near DSBs, which may silence transcription near these sites. Consistent with a role in DDR, knockdown of LSD1 results in moderate hypersensitivity to γ-irradiation, although, paradoxically, the efficiency of HR appears increased (Mosammaparast et al., 2013; Wojtala et al., 2019). To explain this ambivalent effect, it should be considered that LSD1 can also promote p53 demethylation, thereby inhibiting p53 function in DDR activation (Huang et al., 2007).

H3K9

Coherently with the requirement of a repressive chromatin environment, DNA damage promotes methylation of histone H3 on lysine 9 (Sun et al., 2009; Ayrapetov et al., 2014; Khurana et al., 2014). Tri-methylated H3K9 is strongly associated with transcriptional repression (Campos and Reinberg, 2009; Bannister and Kouzarides, 2011). X-ray-induced repositioning of γ-H2AX overlaps with histone H3K9me3 heterochromatin marks in the nuclei of irradiated cells (Hausmann et al., 2018). A complex containing the H3K9 methyltransferase KMT1A (SUV39H1) is rapidly loaded onto the chromatin at DSBs induced by laser micro-irradiation, spreading tens of kilobases away from DSBs (Hausmann et al., 2018). The role of H3K9 methylation at the site of damage goes beyond transcriptional repression, since it is required to activate the Tip60 acetyltransferase, allowing Tip60 to acetylate both ataxia- telangiectasia mutated (ATM) kinase and histones H4 and H2A (Sun et al., 2009; Ayrapetov et al., 2014). Histone acetylation creates local regions of open and relaxed chromatin, which facilitate DSB repair and compete with the binding of 53BP1 to damaged chromatin. Since 53BP1 inhibits DNA ends resection and promotes NHEJ-mediated repair, H3K9 methylation can favor HR over NHEJ (Tang et al., 2013; Jacquet et al., 2016; Gursoy-Yuzugullu et al., 2017; Figures 3A,B). Cells lacking KMT1A, which was previously implicated in genome stability (Peters et al., 2001; Peng and Karpen, 2009), display defective activation of Tip60 and ATM, decreased DSB repair, and increased radio-sensitivity (Sun et al., 2009; Ayrapetov et al., 2014). Other H3K9 HMTs have been implicated in DDR (Figure 1). KMT8 (PRDM2) is recruited to damage chromatin sites through histone MacroH2A1.2 and contributes to H3K9 methylation (Khurana et al., 2014). KMT1E (SETDB1) is also enriched at damaged sites, where it promotes 53BP1 repositioning and HR (Alagoz et al., 2015). In contrast, KMT1C (G9A), which can interact with KDM5B-LSD1, may have an inhibitory role on DDR. This protein is specifically degraded by the proteasome in response to DSBs (Takahashi et al., 2012) and its downregulation was recently shown to trigger DDR in colorectal cancer cells (Zhang et al., 2015). KMT1C is not entirely specific for H3K9, since it can methylate H3K27 and has some non-histone substrates related to DDR, such as p53 (Huang et al., 2010). In agreement with a key role of H3K9 methylation in DDR, the G9A inhibitors UNC0638 and A-366 hypersintetized tumor cells to low doses of DSB-inducing agents (Agarwal and Jackson, 2016); moreover, Price and coworkers showed that the G9A specific inhibitor BIX-01294 can significantly increase tumor cell sensitivity to ionizing radiation (Gursoy-Yuzugullu et al., 2017). H3K9-specific HDMs were also associated with DDR, suggesting that H3K9 methylation could play a dynamic role in DDR (Young et al., 2013; Khoury-Haddad et al., 2014; Figure 1). In particular, KDM4D is rapidly recruited to laser micro-irradiated genomic regions through a PARP-mediated process and is required for efficient formation of IR-induced foci of Rad51 and 53BP1 and for ATM recruitment. A catalytic deficient KDM4D cannot rescue the phenotypes caused by KDM4D knockdown (Khoury-Haddad et al., 2014), showing that the demethylase activity of KDM4D is required for DDR regulation. This was confirmed by using 8-hydroxyquinoline (8-HQ), a catalytic inhibitor of KDM4D (Khoury-Haddad et al., 2014). KDM4B is also selectively recruited via PARP1 to sites of IR-dependent damage, and its overexpression increases the kinetics of γ-H2AX foci resolution and confers a survival advantage following γ-irradiation (Young et al., 2013). Conversely, Fan et al. (2020) found that the histone demethylase KDM3A (JMJD1A) positively regulates the expression of DDR genes by promoting both the expression of c-Myc and its chromatin recruitment through H3K9 demethylation, suggesting a new possible target to sensitize prostate cancer to radiotherapy (Fan et al., 2020). KDM7C (PHF2) is a new H3K9 demethylase, which positively regulates HR-mediated repair through the transcriptional control of CtIP and BRCA1 by direct demethylation of H3K9me2 at their promoters (Alonso-de Vega et al., 2020).

H3K27

Methylation of H3K27 has been observed at DSB sites (O’Hagan et al., 2008; Chou et al., 2010) or following oxidative damage (Campbell et al., 2013). H3K27 methylation may contribute to PARP-dependent transcriptional repression by removing elongating RNA polymerase II from sites adjacent to DNA lesions and promoting recruitment of subunits of the Polycomb repressive complexes PRC1 (Chou et al., 2010) and PRC2 (O’Hagan et al., 2008, 2011; Chou et al., 2010; Campbell et al., 2013) to damaged sites. PRC2 produces H3K27me2 and H3K27me3 via EZH1 and EZH2 (Simon and Kingston, 2009). Enrichment of EZH2 (KMT6) has been observed near DSBs induced by I-SceI endonuclease in epithelial cells (O’Hagan et al., 2008) or DNA lesions caused by UV laser micro-irradiation (Chou et al., 2010; Campbell et al., 2013) and H₂O₂ treatment, but not at IR-induced foci (O’Hagan et al., 2011). EZH2 could also be indirectly involved in DDR by transcriptional regulation of specific genes after DNA damage (Wu et al., 2011). Moreover, EZH2 localizes at stalled replication forks, where it increases H3K27me3 levels, mediating recruitment of the MUS81 nuclease to promote replication fork restart (Rondinelli et al., 2017). Indeed, low EZH2 levels reduce H3K27 methylation and prevent MUS81 recruitment at stalled forks, causing fork stabilization (Rondinelli et al., 2017). Although shRNAs targeting EZH2 or its chemical inhibitor GSK126 significantly promoted genotoxicity and increased cell chemosensitivity (Gao et al., 2017), GSK126 treatments did not enhance the sensitivity of glioma cells to γ-irradiation (Gursoy-Yuzugullu et al., 2017), suggesting that the role of EZH2 could be dependent on DNA damage quality. As in the case of H3K9, H3K27-specific HDMs have also a role in DDR, suggesting that a dynamic equilibrium between methylation and demethylation is required. Irradiation of tumor cells was shown to result in a rapid loss of H3K27me3, which was prevented by the siRNA-mediated knockdown of the H3K27 demethylase KDM6A (UTX) (Rath et al., 2018). Knockdown of UTX enhanced the radio-sensitivity of tumor cell lines. Treatment of tumor cells with the UTX demethylase inhibitor GSKJ4 shortly before irradiation prevented the IR-induced reduction in H3K27me3, enhancing radio-sensitivity (Rath et al., 2018; Katagi et al., 2019). As determined by comet assay and γH2AX expression levels, this GSKJ4 treatment protocol inhibited the repair of IR-induced DSBs. Consistently with in vitro results, treatments of mice bearing leg tumor xenografts with GSKJ4 enhanced IR-induced tumor growth delay (Rath et al., 2018). In contrast with results obtained in tumor cell lines, IR had no effects on H3K27me3 levels in normal fibroblast cell lines and GSKJ4 treatments did not increase their radio-sensitivity. Overall, these data suggest that H3K27me3 demethylation plays a role in DSB repair in tumor cells and that the UTX demethylase may be a target for the selective radio-sensitization of tumor cells (Rath et al., 2018). As in the case of EZH2, the effects of H3K27-HDMs on radio-sensitivity could be indirect and mediated by the transcriptional regulation of target genes. Indeed, upon exposure to IR, KDM6A (UTX) in Drosophila and KDM6B (JMJD3) in mammalian cells are recruited to promote the expression of specific genes involved in DNA repair, including Ku80, in a p53-dependent manner (Zhang et al., 2013; Williams et al., 2014; Katagi et al., 2019).

H3K36

Histone H3K36 methylation is well-known for its role in transcriptional regulation (Chen et al., 2020). H3K36me2 and me3 are hallmarks of active transcription preferentially located in the 5′ and 3′ portion of gene bodies, respectively. H3K36 methylation has also been linked to multiple biological processes, including transcriptional silencing, alternative splicing, DNA replication, as well as DDR and repair (Chen et al., 2020). H3K36me2 deposition is rapidly induced globally and locally after IR and DSB induction (Fnu et al., 2011; Aymard et al., 2014). Di-methylation of H3K36 improves the association of early DNA repair components, including Ku70/Ku80 heterodimers, with the IR-induced DSBs, and enhances DSB repair. The DNA repair protein METNASE (SETMAR), which has a SET histone methylase domain, was thought to be required for NHEJ, by localizing at DSBs and directly mediating the formation of H3K36me2 near them (Fnu et al., 2011; Figures 1, 3A). A recent study, however, suggests that the SET domain in full length SETMAR protein has lost its function in NHEJ DNA repair, possibly because its activity may be inhibited by the dimerization of the protein mediated by the MAR domain (Tellier and Chalmers, 2019).

Several studies revealed the function of H3K36me3 in recruiting RAD51 in active transcription-associated HR (Daugaard et al., 2012; Pfister et al., 2014), although H3K36me3-enriched heterochromatin is also mainly repaired by HR (Beucher et al., 2009). Interestingly, no increase of H3K36me3 levels is detected at IR-induced DSBs, indicating that pre-existing H3K36me3 is involved in RAD51 recruitment (Aymard et al., 2014; Pfister et al., 2014). In the absence of DNA damage factors, Lens epithelium-derived growth factor (LEDGF) masks H3K36me3 marks (Daugaard et al., 2012). Following IR, LEDGF promotes the recruitment of CtIP at DSBs to drive the end processing for RAD51 loading, which is necessary for HR repair (Daugaard et al., 2012; Figure 3B). HR appears as the preferred pathway for repairing DSBs located in transcriptionally active genes and H3K36me3-enriched loci, whereas inactive genes and H3K36me3-depleted regions mostly undergo NHEJ-mediated repair (Aymard et al., 2014). Coherently, depletion of SETD2 (KMT3A), the main H3K36me3-specific HMT, severely impedes HR at DSBs (Aymard et al., 2014). Furthermore, SETD2 depletion in HT1080 fibrosarcoma cells caused similar effects to RAD51 depletion, impairing DNA repair at I-SceI-induced DSBs and increasing the frequency of deletions arising from microhomology-mediated end-joining (Pfister et al., 2014). SETD2 is also required for activation of the p53-mediated checkpoint (Carvalho et al., 2014). Several studies have described the functions of H3K36-specific KDMs, such as KDM2A and KDM4A, in regulating DDR and repair (Figure 3). In particular, an ambivalent function of KDM2A in DNA damage repair was described. On the one hand, KDM2A reduces the accumulation of H3K36me2, which promotes MRE11 complex recruitment at the DSBs (Fnu et al., 2011); indeed, ATM-mediated KDM2A phosphorylation, in response to DSBs, abrogates its chromatin-binding capacity, promoting H3K36me2 accumulation near DNA damage sites and NHEJ (Figure 3A; Cao et al., 2016). Coherently, KDM2A overexpression decreases the association of early NHEJ repair components with DSBs and their repair (Fnu et al., 2011). On the other hand, KDM2A is recruited to DSBs through its demethylase activity and its zinc finger domain and stimulates 53BP1 ubiquitylation and recruitment at damaged sites (Bueno et al., 2018); indeed, KDM2A depletion or disruption of its zinc finger domain causes micronuclei’s accumulation following IR treatment. Moreover, irradiated KDM2A-deficient cells show premature exit from the G2/M checkpoint (Bueno et al., 2018). Unlike KDM2A, KDM4A negatively regulates both NHEJ and HR, through two different mechanisms. By acting as a reader of H4K20me2 (Huang et al., 2006), an important docking site for 53BP1, KDM4A regulates DNA repair by restraining 53BP1 recruitment at damaged sites (Mallette et al., 2012; Figure 3A). By acting as an eraser of H3K36me3, KDM4A impairs a proper HR (Pfister et al., 2014; Figure 3B). RNF8-dependent degradation of KDM4A reduces its binding to H4K20me2, allowing the exposure of this mark and facilitating the recruitment of 53BP1 to DSBs (Huang et al., 2006; Mallette et al., 2012). 53BP1 is a key protein in DDR (Panier and Boulton, 2014). It acts as a molecular scaffold which recruits additional DSB responsive proteins to damaged chromatin, promoting checkpoint signaling and the context-dependent selection of HR or NHEJ repair pathways (Panier and Boulton, 2014). 53BP1 and BRCA1 opposing activities influence the choice between these pathways for DBS repair (Densham et al., 2016; Figure 3).

H3K79

H3K79 methylation has been involved in the regulation of telomeric silencing, cellular development, cell-cycle checkpoint, DNA repair, and transcriptional regulation (Farooq et al., 2016). A unique evolutionarily conserved enzyme, KMT4 (DOT1, DOT1L), is responsible for all forms of H3K79 methylation (mono-, di- and tri-methylation) in eukaryotes. H3K79 methylation is accurately regulated by a crosstalk with other histone modifications, namely H3K4 methylation and H2B ubiquitylation (Farooq et al., 2016). Although it has been suggested that KDM2B is a histone H3K79 demethylase (Kang et al., 2018), the turnover rate of this modification on a global scale is slow and likely to be dependent on the cell cycle. Indeed, H3K79 demethylation by KDM2B contributes to timely cell cycle progression by facilitating correct DNA replication through the regulation of PCNA dissociation from chromatin (Kang et al., 2020). Moreover, the early recruitment of KDM2B into the ternary complex FBXL10/RNF2/RNF68 promotes DDR (Rona et al., 2018), but the detailed mechanism of this effect remains to be investigated, also considering that there are different protein isoforms of KDM2B and of its paralog KDM2A, which could be related to different biological functions (Vacík et al., 2018). So far, the role of H3K79 methylation in DDR has been mainly related to its ability to target 53BP1 to DSBs (Huyen et al., 2004; Figure 3A). In vitro, 53BP1 tandem Tudor domain specifically binds to methylated H3K79 and is required for 53BP1 recruitment to DSBs, which is inhibited by the suppression of KMT4 function. Since the methylation levels of H3K79 remained unchanged after the induction of DNA damage, 53BP1 may detect DSBs indirectly through changes in higher order chromatin structure, which expose 53BP1 binding sites (Huyen et al., 2004). Docked 53BP1 at damage sites may, in turn, recruit additional proteins to activate the checkpoint response. Studies in yeast suggest that the interaction between methylated H3K79 and Rad9 (the human ortholog of 53BP1) may inhibit single-strand DNA production, representing a functional and physical barrier to end processing both at DSBs sites and uncapped telomeres (Lazzaro et al., 2008). H3K79me2 is also involved in Rad51 foci formation. Indeed, Bre1 deficiency impairs HR-mediated repair and cell cycle checkpoint response to IR-dependent damage by specifically reducing H3K79 dimethylation in mammalian cells, possibly as a consequence of the downregulation of HR-related genes (Chernikova et al., 2010). In colorectal cancer cells, KMT4 depletion or its catalytic inhibition modulate chemotherapy responsiveness, due to its role in DDR and HR pathways (Kari et al., 2019). KMT4-mediated H3K79 methylation also plays a critical role in repairing ultraviolet (UV)-induced DNA damage in yeast (Bostelman et al., 2007; Boudoures et al., 2017) and mammalian cells (Zhu et al., 2018).