Abstract

Timely repair of DNA double-strand break (DSB) entails coordination with the local higher order chromatin structure and its transaction activities, including transcription. Recent studies are uncovering how DSBs trigger transient suppression of nearby transcription to permit faithful DNA repair, failing of which leads to elevated chromosomal aberrations and cell hypersensitivity to DNA damage. Here, we summarize the molecular bases for transcriptional control during DSB metabolism, and discuss how the exquisite coordination between the two DNA-templated processes may underlie maintenance of genome stability and cell homeostasis.

DNA double-strand break repair and human health

Preserving genome integrity represents a fundamental process that not only underlies faithful genetic inheritance at the cellular level but is also a key to organismal development. Among the many types of DNA lesions that our cells have adapted to tolerate, DNA double-strand breaks (DSBs) are arguably the most deleterious to genome integrity. Not surprisingly, failing to swiftly mend DSBs contributes to genome instability, which bears causal relationships with a cohort of human diseases. Accordingly, genetic defects that compromise cellular responses to DSBs can be manifested as neurological disorders, as documented in individuals with Ataxia telangiectasia (A-T), Nijmegen breakage syndrome and Seckel syndrome [1,2]. In addition, inability to metabolize programmed DSBs can lead to immunodeficiency [3], such as in cases where individuals with mutations in the exonuclease ARTEMIS develop severe-combined immunodeficiency (SCID) [4]. Perturbed DSB responses are also closely associated with premature aging syndromes, conditions that have been attributed to dysregulated telomere maintenance and persistent activation of DNA damage responses [5]. But perhaps the most direct impact on human health from defective DSB responses comes from findings where genetic inactivation of DSB signaling and repair proteins results in early predisposition to a multitude of human cancers [6]. Multiple lines of evidence have firmly established a role of defective DSB repair in driving human cancer initiation and progression [7]. As such, deciphering the molecular bases of DSB repair control may provide insight toward novel therapeutic opportunities for the treatment and management of a myriad of genome instability associated disorders.

Sources of DNA double-strand breaks

Not only do DSBs result from cell exposure to ionizing radiation and may arise spontaneously during chromatin transaction activities including DNA replication and gene transcription, but programmed DSBs induced in the events of V(D)J recombination and immunoglobulin class-switch recombination necessitate robust and specialized responses that may otherwise lead to undesired genetic alterations (reviewed in [8]). It is now believed that the majority of DSBs our cells encounter during normal cell proliferation results from topoisomerase actions in resolving DNA topological strain, which can exert mechanical stress on chromosomes. In addition, collisions between machineries that mediate DNA-templated processes, coined transcription-replication conflict (TRC), also lead to stalled replication forks and subsequent DSB formation [9]. Paradoxically, while DSB formation may be required for transcription activation and other physiological processes, these naturally occurring DSBs call for prompt responses to suppress the otherwise deleterious effects on genome stability.

Active transcription as an inducer of DSBs

Analysis of translocation frequency and distribution across different chromatin domains previously implicated transcription as an inducer of DSBs, as active chromatin represents hotspots of DSB formation [10–12]. With the advent in next-generation sequencing, genome-wide DSB landscape has recently been determined using high-throughput techniques (reviewed in [13]). Remarkably, using DSB-capture that allows direct capturing and annotation of DSBs in situ, it was shown that spontaneous DSBs are enriched proximal to transcription start sites of highly expressed genes [14]. These findings coincided with those derived by BLESS that also documented profound enrichment of DSBs in active-transcribing chromatin marked by mono-, di-, and tri-methylation of lysine 4 at H3 (H3K4me1/2/3) [15]. Considering the circumstantial evidence where chromosomal translocation and DSB distribution are both enriched on transcribed chromatin, it is reasonable to speculate that transcriptional activity predisposes cells to higher risk of genome instability due to the induction of overwhelming amounts of DSBs.

Cross-talk between DSB repair and transcription

While transcription has been regarded as an inherent source of genome instability [10], only recently have we begun to study how transcription intermediates (a.k.a. R-loops) may be targets of DNA damage. R-loop is a triple-stranded nucleic acid structure that forms when nascent RNA hybridizes with the template DNA strand, leading to the generation of DNA:RNA hybrid duplex that results in the displacement of the non-template ssDNA (reviewed in [16]). Notably, the non-transcribed ssDNA strand is particularly vulnerable to DNA modifying activities or attack by mutagenic agents, such as alkylating agents and activation-induced deaminase (AID)/APOBEC-family cytosine deaminases [10,17]. Unstable ssDNA can also form harmful non-canonical DNA structures including hairpins, loops and G-quadruplexes that obstruct transcription [18]. Furthermore, R-loops have recently been reported to contribute to DSB formation, a process that is mediated by the endonucleases Xeroderma pigmentosum complementation group F (XPF) and Flap structure-specific endonuclease 1 (FEN1) [19]. Considering the highly deleterious nature of co-transcriptional R-loops, cells have evolved complementing mechanisms to suppress or resolve their ectopic formation. For instance, Topoisomerase I (TOPI) resolves torsional strain imposed on ssDNA that results from negative supercoiling accumulated from advancing RNA polymerase II (RNAPII) activity [17]. In addition, nascent mRNA synthesized is packaged into ribonucleoprotein complex by splicing factor ASF/SF2, thus preventing RNA–DNA annealing [20]. DNA:RNA hybrids can also be unwound by helicases such as Senataxin (SETX) and Aquarius or degraded through RNase H enzymes digestion [21].

In the context of DSB responses, R-loops also accumulate on DSB-flanking chromatin domains and are preferentially enriched on the transcribed chromatin, which might be attributed to stalled RNAPII activity upon DSB induction [22,23]. Given the observation that DSB-associated SETX counteracts R-loop formation [22], one would speculate that R-loops are by-products of DSB-induced stalled transcription and may need to be cleared for efficient DSB repair. Importantly, while SETX-mediated R-loop resolution promotes Rad51 docking to damaged chromatin, R-loops at active loci recruit Rad52 to promote Xeroderma pigmentosum complementation group G (XPG)-mediated R-loop processing that initiate transcription-coupled homologous recombination repair [22,24].

On the other hand, current evidence suggests that transient DSBs at promoter or enhancer regions underlie activation of gene expression driven by a number of nuclear hormone receptors and transcription factors, such as estrogen receptor (ER)- and androgen receptor (AR)-signaling genes (reviewed in [25]). Induction of these site-specific DSBs is mediated by the action of Topoisomerase IIB (TOPIIB) [26,27], and are subsequently recognized by DSB response machineries including Ataxia–telangiectasia mutated (ATM) kinase, DNA-dependent protein kinase (DNA-PK), poly (ADP-ribose) polymerase (PARP1) and Ku70/80 [25]. Illegitimate repair of these transcription-induced DSBs is undoubtedly a threat to genome stability, as seen in the production of TMPRSS2-ERG gene fusions in prostate cancer that may be largely due to the dysfunction of TOPIIB [27]. Apart from transcription-associated DSBs, TOPIIB-mediated DSBs have recently been implicated in resolving torsional stress when DNA is compacted into higher order chromatin structure, such as chromatin loop anchors bound by CCCTC-binding factor (CTCF) and cohesin [28]. Importantly, while the formation of TOPIIB-induced DSBs at chromatin loop anchors is independent of local transcription activities, their conversion into irreversible DSBs due to trapped TOPII cleavage complexes or incomplete TOPIIB activity is nevertheless enriched at transcribed loci [29,30].

The interplay between DSB metabolism and local transcription has been a subject of growing interest in part owing to the development of methodologies that permitted studying of localized DSB responses. Intriguingly, transcriptional events such as generation of small non-coding RNAs (ncRNAs) are found to regulate DDR response at DSBs [31]. In DICER- and DROSHA-dependent manners, site-specific biogenesis of DNA damage response RNAs (DDRNAs) from damaged-induced long non-coding RNAs (dilncRNAs) has been reported to drive DDR foci formation and maintenance [31,32]. Emerging linkage between RNAPII and critical DSB repair factors such as the major phosphatidylinositol 3-kinase-related kinases (PIKKs), ATM, ATR and DNA-PK suggests that RNAPII-mediated transcriptionally active chromatin mounts a specialized DSB response, namely transcription-coupled DSB repair (reviewed in [33]).

Transcription-coupled double-strand break repair

As DNA repair operates on chromatinized substrates, transient opening of the chromatin at damaged sites is necessary to permit access and loading of DSB response factors. Subsequently, restoration of the repaired chromatin back to its native configuration is a critical event that ensures intact DNA-templated activities [34,35]. Hence, comprehensive understanding of DSB responses at actively transcribed regions has to be addressed at chromatin level that encompasses dynamic processes such as chromatin remodeling, incorporation of histone variants and establishment of post-translational histone modifications. These events must be choreographed during DSB processing and should be coupled with local transcription regulation in order to restore damaged chromatin to its pre-existing active state for faithful gene expression.

DSB induction at active chromatin

RNA polymerase II-dependent actively transcribed loci harbor epigenetic signatures that mark open chromatin configuration and favor transcriptional activities (Figure 1). Specifically, H3K4 tri-methylation (H3K4me3) found at promoter regions of active genes is associated with transcription initiation while enrichment of histone H3 lysine 36 tri-methylation (H3K36me3) at gene bodies correlates with transcription elongation [36]. In addition to the enrichment of H2A histone variant H2A.Z, transcription start sites (TSSs) of active genes are also marked by acetylated H4K16 that is responsible for gene activation [37–39].

DSB induction at active chromatin

Figure 1
DSB induction at active chromatin

RNA polymerase II-dependent transcriptionally active chromatin harbors signature active transcription marks, H3K4me3 at promoter regions and H3K36me3 at gene bodies. Transcription start site (TSS) of active gene is also marked by acetylated H4K16 and enrichment of the H2A.Z histone variant. Induction of DSBs near or at gene bodies mounts specialized DSB responses.

Figure 1
DSB induction at active chromatin

RNA polymerase II-dependent transcriptionally active chromatin harbors signature active transcription marks, H3K4me3 at promoter regions and H3K36me3 at gene bodies. Transcription start site (TSS) of active gene is also marked by acetylated H4K16 and enrichment of the H2A.Z histone variant. Induction of DSBs near or at gene bodies mounts specialized DSB responses.

While a wide array of methods are available to experimentally induce DSBs, a scenario has emerged in which cells mount distinct DSB responses depending on the locus and amount of DSBs [40–42]. Traditionally, global DSB repair responses are studied using ionizing radiation (IR) that is believed to produce DSBs mainly located in intergenic loci, although euchromatin was also proposed to be more susceptible to IR-induced DSB formation when compared with heterochromatin [8,40]. On the other hand, topoisomerase inhibitors such as camptothecin and etoposide induce DSBs by hindering TOPII activities at active genes [8]. Other chemicals that induce DSBs randomly across the genome include radiomimetic compounds such as bleomycin and neocarzinostatin, and cross-linking agents that generate bulky adduct-induced DSBs such as cisplatin and mitomycin C [43]. Contrary to traditional methods in DSB induction, the ability to generate DSBs at site-specific loci has allowed in-depth mechanistic understanding of localized DSB responses. Three common tactics include the engineering of reporter cell line, restriction enzymes with annotated cutting sites distributed across the genome, and genome editing tools such as zinc finger proteins (ZFN), TALENs and CRISPR/Cas9 that induce DNA break at desired location in the genome [42].

DSB detection and signaling

DSBs are recognized by the MRN (MRE11-RAD50-NBS1) complex that promotes the recruitment of ATM kinase (Figure 2) [44]. As the master kinase in early DSB signal transduction, ATM has an established role in initiating local DSB responses by phosphorylation of histone variant H2AX (γ-H2AX) at serine 139, the key histone modification that spreads megabases along chromatin from damaged site [45]. Mediator of DNA-damage checkpoint (MDC1) specifically binds to γ-H2AX and promotes the propagation of DSB signals in concerted efforts with ubiquitination signals catalyzed by E3 ubiquitin ligase RNF8/168 [46,47]. These DSB signaling marks provide docking sites to coordinate sequential recruitment of DSB repair factors.

DSBs detection and signaling

Figure 2
DSBs detection and signaling

(i) Transcription silencing and removal of active histone marks. Demethylation of H3K4me3 is regulated by PARP1-dependent recruitment of KDM5A. This further promotes the recruitment of ZMYND8 that recognizes H4K16ac marks and the NuRD complex that is endowed with deacetylase activity. Hypoacetylation of H4K16 is promoted by transient accumulation of SIRT1 at DSB. (ii) Transcription silencing and loading of repressive histone marks. ATM-dependent transcription repression at DSB-flanking chromatin is marked with ubiquitination at lysine 119 of H2A (H2AK119ub) and stalling of ser2-hypophosphorylated RNAPII. Establishment of H2AK119ub has been observed to be promoted by PRC1 while tri-methylation of H3K27 is mediated by PRC2 subunit EZH2. In addition, recruitment of PcG complex might be regulated by cohesin and PBAF remodeling protein as well as by ATM-dependent phosphorylation of ENL transcription factor. Other than KDM5A, PARylation by PARP1 also promotes the recruitment of NELF complex and CDYL1, with the latter promoting the recruitment of EZH2. On the contrary, DNA-PK-mediated transcription silencing at gene bodies involves the displacement of RNAPII from DSB-flanking chromatin via K48-linked polyubiquitination catalyzed by WWP2. While loading of repressive mark H3K9me3 is promoted by Suv39h1, H3K9me2 deposition upon DSBs induction remains to be determined.

Figure 2
DSBs detection and signaling

(i) Transcription silencing and removal of active histone marks. Demethylation of H3K4me3 is regulated by PARP1-dependent recruitment of KDM5A. This further promotes the recruitment of ZMYND8 that recognizes H4K16ac marks and the NuRD complex that is endowed with deacetylase activity. Hypoacetylation of H4K16 is promoted by transient accumulation of SIRT1 at DSB. (ii) Transcription silencing and loading of repressive histone marks. ATM-dependent transcription repression at DSB-flanking chromatin is marked with ubiquitination at lysine 119 of H2A (H2AK119ub) and stalling of ser2-hypophosphorylated RNAPII. Establishment of H2AK119ub has been observed to be promoted by PRC1 while tri-methylation of H3K27 is mediated by PRC2 subunit EZH2. In addition, recruitment of PcG complex might be regulated by cohesin and PBAF remodeling protein as well as by ATM-dependent phosphorylation of ENL transcription factor. Other than KDM5A, PARylation by PARP1 also promotes the recruitment of NELF complex and CDYL1, with the latter promoting the recruitment of EZH2. On the contrary, DNA-PK-mediated transcription silencing at gene bodies involves the displacement of RNAPII from DSB-flanking chromatin via K48-linked polyubiquitination catalyzed by WWP2. While loading of repressive mark H3K9me3 is promoted by Suv39h1, H3K9me2 deposition upon DSBs induction remains to be determined.

Transcription repression

One logical question to ponder upon DSB induction in close proximity to transcribing gene loci is the concurrence of DNA repair and transcriptional activities. Recent discoveries documented that cells respond to DSBs by transiently pausing local transcription. Indeed, upon induction of a cluster of FokI-induced DSBs in a single-cell reporter system, transcriptional activity of the artificial reporter gene in cis was switched off in an ATM-dependent manner [48]. Silencing effect of ATM on DSB-flanking active chromatin is associated with stalling of RNAPII and its hypo-phosphorylated status at serine 2 [48]. Another site-specific DSB induction system that utilizes the I-PpoI meganuclease to target RNAPII-transcribed genes across the genome uncovered a role of DNA-PK in suppressing local transcription but not of neighboring transcriptional units [49]. Mechanistically, DNA-PK activates HECT E3 ubiquitin ligase WWP2 that catalyzes K48-linked polyubiquitination on the largest RNAPII subunit, RPB1. This serves as a signal for proteasome-dependent degradation and eviction of RNAPII from damaged chromatin [49,50]. PARP1 activity is also required for the accumulation of a number of molecular components in DSB-induced transcription suppression. PARP1 catalyses poly(ADP-ribosyl)ation (PARylation) as a post-translational modification on itself and other substrates including RNAPII to serve as a docking site for proteins with PAR-binding motif [51]. For instance, the NELF-E subunit of the negative elongation factor (NELF) complex is recruited to DSBs induced upstream of transcribed regions through its interaction with PARylated RNAPII [52], and suppresses local transcription activity possibly by inhibiting transcription elongation [53,54]. Together, these findings highlight that DSB-induced transcriptional silencing is not simply a direct consequence of physical perturbation to the DNA template but rather represents a specialized protocol during DSB responses. Notably, while ATM-dependent transcription repression might be a signaling event downstream to DSB detection, the requirement of DNA-PK and PARP suggests an earlier silencing event at DSB-flanking active chromatin. It is thus essential to study on the recruitment kinetics of key molecular players in DSB-induced transcription repression that has been summarised in Table 1.

Table 1
Recruitment kinetics of key components in DSB-induced transcription repression
ProteinMethod of DNA damage inductionRecruitment kineticRecruitment dependencyFunction in DDRReferences
PARP1 1. Live cell laser irradiation in MEF (780 nm), GFP-tagged
2. Fixed cell post-laser micro-irradiation (780 nm) 
1. Detectable less than 1 s and half-accumulation of 1.6 s
2. Present at maximal concentration for at least 30 min 
n.d. • PARylation of nucleosomes causes transient chromatin decondensation and histone displacement
• Unmodified PARP1 drives chromatin condensation 
[51,55,56
MRE11 (MRN) 1. Live cell laser irradiation in MEF (780 nm), YFP-tagged
2. Fixed cell post-laser irradiation (337 nm) 
1. Half-accumulation of 13 s
2. Accumulation at 1 h 
PARP • Sensor of DSBs
• Recruitment of ATM and other repair proteins
• Promotes NHEJ and HR 
[55,57,58
ATM 1. Live cell laser irradiation in U2OS (365 nm), YFP-tagged 1. Detectable within seconds where initial localization dependent on MRN and accumulation retained over 2 h MRN • DSB signaling through phosphorylation of H2AX and other substrates in DSB responses
• Promotes chromatin remodeling for access of DSB repair factors 
[59,60
NELF-E (NELF) 1. Live cell laser irradiation in U2OS (405 nm), GFP-tagged 1. Detectable as early as 10 s with peak intensity at about 2–3 min PARP
RNAPII (a-amanitin) 
• Transcriptional repression at active chromatin by inhibiting transcription elongation
• Promotes NHEJ and HDR repair 
[52
WWP2 1. Live cell laser irradiation in U2OS (800 nm), mCherry- and GFP-tagged
2. I-PpoI-induced DSBs at transcribed genes 
1. Maximum level at 50 s, returning to near basal level at 150 s.
2. Maximum level between 30 min and 2 h, returning to near basal level at 6 h. 
RNAPII (DRB) • Targets RNAPII subunit RPB1 for K48-linked ubiquitylation, driving DNA-PK- and proteasome-dependent eviction of RNAPII
• Promotes NHEJ 
[50
RING1B (PRC1) 1. Fixed cell post-laser irradiation (355 nm) in HeLa 1. Fixation after 15 min n.d. • H2A/H2AX ubiquitylation
• Cellular resistance to DNA damage 
[61
BMI1 (PRC1) 1. Fixed cell post-laser irradiation (355 nm) and IR-induced foci in HeLa 1. Fixation after 10 min and retention up to 2 h PARP • Promotes transcriptional repression through enhancing H2A/H2AX ubiquitylation by RING1B
• Promotes HR and checkpoint recovery 
[61–65
EZH2 (PRC2) 1. Live cell laser irradiation in U2OS (750 nm), GFP-tagged 1. Detectable within 2 s and maximum level at 1 min PARP
 
• Methylates H3K27me3
• Promotes DSB repair 
[66
CDYL1 1. Live cell laser irradiation in U2OS and MCF7 (405 nm), EGFP-tagged 1. Detectable within 20 s and retained after 10 min PARP • Promotes recruitment of EZH2
• Promotes transcription repression
• Promotes HDR 
[67
BAF180 (PBAF) 1. Live cell laser irradiation in U2OS (405 nm), EGFP-tagged 1. Detectable within 20 s, maximum accumulation at 1 min but dissipates to basal level within 5 min n.d. • Promotes transcription repression
• Promotes NHEJ 
[68,69
SA2 (Cohesin) 1. Live cell laser irradiation in U2OS (405 nm), EGFP-tagged 1. Detectable within 20 s, maximum accumulation at 1.5 min and retains after 5 min n.d. • Mediates DNA repair through sister chromatid cohesion
• Promotes transcription repression
• Promotes HDR 
[69
KDM5A/ JARID1A 1. Live cell laser irradiation in U2OS (405 nm), GFP-tagged
2. Fixed cell post-laser irradiation (405 nm) 
1. Maximum accumulation at 3 min and retains after 6 min.
2. Accumulation at 20, 40 and 90 min 
PARP
RNAPII (DRB) 
• Demethylates H3K4me3
• Promotes transcriptional repression at active chromatin
• Mediates localization of ZMYND8-NuRD to damaged sites
• Promotes HR repair 
[70,71
ZMYND8 1. Live cell laser irradiation in U2OS (405 nm), GFP-tagged. 1. Maximum accumulation at 3 min, diminishing after 20 min PARP
RNAPII (DRB) 
• Recruits NuRD to damaged site
• Promotes HR repair 
[70,72,73
CHD4 (NuRD) 1. Fixed cell post-laser irradiation (355 nm) in U2OS
2. Live cell laser irradiation (405 nm), GFP-tagged 
1. Fixation after 10 min
2. Detectable within 30 s, half-maximum at 40 s and steady-state levels at 3 min 
PARP
RNAPII (DRB) 
• Promotes RNF8/RNF168-dependent ubiquitination
• Promotes HR repair
• Promotes checkpoint activation and p53/p21 apoptotic responses 
[61,72–74
ProteinMethod of DNA damage inductionRecruitment kineticRecruitment dependencyFunction in DDRReferences
PARP1 1. Live cell laser irradiation in MEF (780 nm), GFP-tagged
2. Fixed cell post-laser micro-irradiation (780 nm) 
1. Detectable less than 1 s and half-accumulation of 1.6 s
2. Present at maximal concentration for at least 30 min 
n.d. • PARylation of nucleosomes causes transient chromatin decondensation and histone displacement
• Unmodified PARP1 drives chromatin condensation 
[51,55,56
MRE11 (MRN) 1. Live cell laser irradiation in MEF (780 nm), YFP-tagged
2. Fixed cell post-laser irradiation (337 nm) 
1. Half-accumulation of 13 s
2. Accumulation at 1 h 
PARP • Sensor of DSBs
• Recruitment of ATM and other repair proteins
• Promotes NHEJ and HR 
[55,57,58
ATM 1. Live cell laser irradiation in U2OS (365 nm), YFP-tagged 1. Detectable within seconds where initial localization dependent on MRN and accumulation retained over 2 h MRN • DSB signaling through phosphorylation of H2AX and other substrates in DSB responses
• Promotes chromatin remodeling for access of DSB repair factors 
[59,60
NELF-E (NELF) 1. Live cell laser irradiation in U2OS (405 nm), GFP-tagged 1. Detectable as early as 10 s with peak intensity at about 2–3 min PARP
RNAPII (a-amanitin) 
• Transcriptional repression at active chromatin by inhibiting transcription elongation
• Promotes NHEJ and HDR repair 
[52
WWP2 1. Live cell laser irradiation in U2OS (800 nm), mCherry- and GFP-tagged
2. I-PpoI-induced DSBs at transcribed genes 
1. Maximum level at 50 s, returning to near basal level at 150 s.
2. Maximum level between 30 min and 2 h, returning to near basal level at 6 h. 
RNAPII (DRB) • Targets RNAPII subunit RPB1 for K48-linked ubiquitylation, driving DNA-PK- and proteasome-dependent eviction of RNAPII
• Promotes NHEJ 
[50
RING1B (PRC1) 1. Fixed cell post-laser irradiation (355 nm) in HeLa 1. Fixation after 15 min n.d. • H2A/H2AX ubiquitylation
• Cellular resistance to DNA damage 
[61
BMI1 (PRC1) 1. Fixed cell post-laser irradiation (355 nm) and IR-induced foci in HeLa 1. Fixation after 10 min and retention up to 2 h PARP • Promotes transcriptional repression through enhancing H2A/H2AX ubiquitylation by RING1B
• Promotes HR and checkpoint recovery 
[61–65
EZH2 (PRC2) 1. Live cell laser irradiation in U2OS (750 nm), GFP-tagged 1. Detectable within 2 s and maximum level at 1 min PARP
 
• Methylates H3K27me3
• Promotes DSB repair 
[66
CDYL1 1. Live cell laser irradiation in U2OS and MCF7 (405 nm), EGFP-tagged 1. Detectable within 20 s and retained after 10 min PARP • Promotes recruitment of EZH2
• Promotes transcription repression
• Promotes HDR 
[67
BAF180 (PBAF) 1. Live cell laser irradiation in U2OS (405 nm), EGFP-tagged 1. Detectable within 20 s, maximum accumulation at 1 min but dissipates to basal level within 5 min n.d. • Promotes transcription repression
• Promotes NHEJ 
[68,69
SA2 (Cohesin) 1. Live cell laser irradiation in U2OS (405 nm), EGFP-tagged 1. Detectable within 20 s, maximum accumulation at 1.5 min and retains after 5 min n.d. • Mediates DNA repair through sister chromatid cohesion
• Promotes transcription repression
• Promotes HDR 
[69
KDM5A/ JARID1A 1. Live cell laser irradiation in U2OS (405 nm), GFP-tagged
2. Fixed cell post-laser irradiation (405 nm) 
1. Maximum accumulation at 3 min and retains after 6 min.
2. Accumulation at 20, 40 and 90 min 
PARP
RNAPII (DRB) 
• Demethylates H3K4me3
• Promotes transcriptional repression at active chromatin
• Mediates localization of ZMYND8-NuRD to damaged sites
• Promotes HR repair 
[70,71
ZMYND8 1. Live cell laser irradiation in U2OS (405 nm), GFP-tagged. 1. Maximum accumulation at 3 min, diminishing after 20 min PARP
RNAPII (DRB) 
• Recruits NuRD to damaged site
• Promotes HR repair 
[70,72,73
CHD4 (NuRD) 1. Fixed cell post-laser irradiation (355 nm) in U2OS
2. Live cell laser irradiation (405 nm), GFP-tagged 
1. Fixation after 10 min
2. Detectable within 30 s, half-maximum at 40 s and steady-state levels at 3 min 
PARP
RNAPII (DRB) 
• Promotes RNF8/RNF168-dependent ubiquitination
• Promotes HR repair
• Promotes checkpoint activation and p53/p21 apoptotic responses 
[61,72–74

Dynamic regulation of histone post-translational modifications (PTM) represents key processes in both transcription regulation and DSB signaling (Table 2) [41]. Transition into transcriptionally repressed state involves down-regulation of canonical active chromatin signatures in concordance with up-regulation or deposition of repressive histone marks to establish a conducive micro-environment for efficient DSB repair (Figure 2) [75]. Indeed, ATM-dependent silencing of transcribing chromatin regions is accompanied by histone H2A mono-ubiquitination at lysine 119 (H2AK119ub) [48]. H2AK119ub is associated with repressed chromatin indicated in Polycomb-mediated silencing where its deposition is catalyzed by E3 ubiquitin ligase RING1B subunits of Polycomb repressive complex 1 (PRC1) [39,76]. Notably, aside the fact that RING1B is recruited to DSB-flanking chromatin [61], a role of Polycomb-group proteins (PcG) in facilitating transcription silencing is also evident from the global loss of H2AK119ub that results from the inactivation of the BMII subunit of PRC1 [68].

Table 2
Histone modifications, its writers or erasers and their roles at transcription-coupled DSB repair
Histone modificationType of modificationWriter/ EraserFunctionReference
γ-H2AX S139 Phosphorylation ATM/ DNA-PK DSB signaling: Propagation of DSB signaling for accumulation of DSB repair factors [45,104
 Dephosphorylation PP2A DSB repair: Regulation of DSB repair pathways through dephosphorylation of ATM, Ku and DNA-PK
Chromatin restoration and transcription resumption: Unknown that might be responsible for γ-H2AX removal 
[93,94,105
H3K4me3 Methylation – Transcription activation: Active transcription mark at promoter and enhancer regions for transcription initiation [36
 Demethylation KDM5A Transcription repression: Binding of downstream transcription silencing factors such as EZH2 and ZMYND8-NuRD [70,71,106,107
H3K36me3 Methylation SETD2 Transcription activation: Active transcription mark at gene bodies for transcription elongation
DSB repair: Promoting HR as a binding site for LEDGF favouring DNA end resection 
[36,81,90,108
H3K27me3 Methylation EZH2 (PRC2) Transcription repression: Repressive transcription mark with chromatin condensation activity [66,68
 Demethylation KDM6A DSB repair: Promotes chromatin relaxation and loading of DSB repair factors [103
H3K9me3 Methylation Suv39h1 Transcription repression: Transient repression upon DSBs by KAP-1/HP1/Suv39h1
DSB repair: Activation of Tip60-mediated repair 
[88,103
 Demethylation KDM4B DSB repair: Promotes global chromatin relaxation and loading of DSB repair factors [102
H4K20me2 Methylation SETD8 DSB repair: Promoting NHEJ by acting as binding site for 53BP1 [109
H2AK13/15ub Ubiquitination RNF168 DSB signaling: Facilitates the recruitment of DSB repair proteins such as 53BP1 [110
 De-ubiquitination USP51 DSB signaling: Regulates the dynamic assembly/disassembly of 53BP1 and BRCA1 [111
H2AK119ub Ubiquitination RING1B-BMI1 (PRC1) Transcription repression: Repressive transcription mark which promotes the recruitment of silencing factors [48,68
 De-ubiquitination USP16 Transcription repression: Regulation of H2AK119 ubiquitination
Transcription activation: unknown where de-ubiquitination of H2AK119 upon DSB repair might be regulated by other DUBs such as BAP1 and USP11 
[48,99,100
H4K16ac Acetylation Tip60 Transcription activation: Maintaining open chromatin configuration for transcription activity.
DSB repair: Promotes chromatin relaxation for loading of DSB repair factors 
[37,112,113
 Deacetylation SIRT1 Transcription repression: Unknown where transient chromatin compaction in repressing transcription activity might be promoted by other HDACs
DSB repair: Regulating chromatin structure for assembly/disassembly of DSB repair factors 
[82
Histone modificationType of modificationWriter/ EraserFunctionReference
γ-H2AX S139 Phosphorylation ATM/ DNA-PK DSB signaling: Propagation of DSB signaling for accumulation of DSB repair factors [45,104
 Dephosphorylation PP2A DSB repair: Regulation of DSB repair pathways through dephosphorylation of ATM, Ku and DNA-PK
Chromatin restoration and transcription resumption: Unknown that might be responsible for γ-H2AX removal 
[93,94,105
H3K4me3 Methylation – Transcription activation: Active transcription mark at promoter and enhancer regions for transcription initiation [36
 Demethylation KDM5A Transcription repression: Binding of downstream transcription silencing factors such as EZH2 and ZMYND8-NuRD [70,71,106,107
H3K36me3 Methylation SETD2 Transcription activation: Active transcription mark at gene bodies for transcription elongation
DSB repair: Promoting HR as a binding site for LEDGF favouring DNA end resection 
[36,81,90,108
H3K27me3 Methylation EZH2 (PRC2) Transcription repression: Repressive transcription mark with chromatin condensation activity [66,68
 Demethylation KDM6A DSB repair: Promotes chromatin relaxation and loading of DSB repair factors [103
H3K9me3 Methylation Suv39h1 Transcription repression: Transient repression upon DSBs by KAP-1/HP1/Suv39h1
DSB repair: Activation of Tip60-mediated repair 
[88,103
 Demethylation KDM4B DSB repair: Promotes global chromatin relaxation and loading of DSB repair factors [102
H4K20me2 Methylation SETD8 DSB repair: Promoting NHEJ by acting as binding site for 53BP1 [109
H2AK13/15ub Ubiquitination RNF168 DSB signaling: Facilitates the recruitment of DSB repair proteins such as 53BP1 [110
 De-ubiquitination USP51 DSB signaling: Regulates the dynamic assembly/disassembly of 53BP1 and BRCA1 [111
H2AK119ub Ubiquitination RING1B-BMI1 (PRC1) Transcription repression: Repressive transcription mark which promotes the recruitment of silencing factors [48,68
 De-ubiquitination USP16 Transcription repression: Regulation of H2AK119 ubiquitination
Transcription activation: unknown where de-ubiquitination of H2AK119 upon DSB repair might be regulated by other DUBs such as BAP1 and USP11 
[48,99,100
H4K16ac Acetylation Tip60 Transcription activation: Maintaining open chromatin configuration for transcription activity.
DSB repair: Promotes chromatin relaxation for loading of DSB repair factors 
[37,112,113
 Deacetylation SIRT1 Transcription repression: Unknown where transient chromatin compaction in repressing transcription activity might be promoted by other HDACs
DSB repair: Regulating chromatin structure for assembly/disassembly of DSB repair factors 
[82

Current evidence suggests that PRC1 recruitment to DNA damage sites may be facilitated by a number of distinct mechanisms. The chromodomain-containing chromobox 1 (CBX) subunit in PRC1 can potentially facilitate its DSB docking by recognizing lysine methyl marks on histones, particularly H3K27me3 [77]. Tri-methylation on lysine 27 of H3 is well-annotated as a repressive histone mark deposited by the methyltransferase subunit EZH2 that resides in the Polycomb Repressive Complex 2 (PRC2). While rapid accumulation of EZH2 as well as H3K27me3 mark at DSB-flanking chromatin dissipate with time [61,66], its recruitment has recently been suggested to be promoted by chromodomain Y-like (CDYL1) protein. CDYL1 directly binds to PAR moieties and promotes DSB-induced transcription repression and homology-dependent repair (HDR) at active chromatin [67]. On the other hand, via a PARP- and PRC2-independent mechanism, recruitment of PRC1 may also be regulated by the establishment of cohesin in the vicinity of DSBs through chromatin remodeling activity of Polybromo-associated BAF (PBAF) from the SWI/SNF family. Depletion of PBAF subunit BAF180 and BRG1 as well as centromere-specific cohesin subunits (SA2 and PDS5B) compromise transcription silencing in cis to DSBs [68,69]. Of note, while PBAF-mediated transcription repression is dependent on ATM, and that BAF180 harbors conserved ATM target SQ sites, localization of BAF180 to DSBs was unaffected by ATM inhibition [68]. On the contrary, SQ site in the transcription elongation factor ENL is phosphorylated by ATM that subsequently promotes the recruitment of PRC1 [78].

While the underlying mechanism of DSB-induced transcription repression mediated by H2AK119ub remains elusive, mono-ubiquitination of H2A has been reported to inhibit transcription initiation by trans-histone down-regulation of H3K4me2/3 [79]. Coincidently, demethylation of H3K4me3 was observed at DSBs that inversely correlated with γ-H2AX foci [71]. Removal of H3K4me3 is regulated by histone demethylase KDM5A that is recruited to DSBs in PARP-dependent manner [70]. Demethylating activity of KDM5A at DSBs is necessary for downstream recruitment of ZMYND8-NuRD complex [70]. In concordance as a ‘reader’ of TIP60-induced H4 acetylation (H4K16ac) at active chromatin, the NuRD complex is targeted to DSBs via the bromodomain protein ZMYND8, where its ATPase subunit CHD4 and HDAC deacetylase activity mediates transcriptional repression and HR repair [72,73].

In contrast with H3K4me3, removal of the active transcription mark H3K36me3 at transcribed regions flanking DSBs has remained controversial. Down-regulation of H3K36me3 compromises DNA repair efficiency due to the multilevel regulatory roles of H3K36me3 in both transcription elongation and DSB repair [80]. Notably, during transcription elongation, 3′ distribution of H3K36 methylation across transcribed chromatin from TSSs confers repressive effect in preventing aberrant local transcription initiation from elongating RNAPII and cryptic gene promoters [80]. Hence, retention of pre-existing tri-methylation of H3K36 at active chromatin may promote immediate transcription repression in response to localized DSBs. In the regulation of HR, SETD2-mediated H3K36 tri-methylation serves as a docking site for LEDGF/p75 which enhances its binding with CtIP and mediates end resection [81].

Transcription arrest upon DSB induction on CpG island of an exogenous promoter coincides with the appearance of hypoacetylated H4K16 and repressive di- and tri-methylation on lysine 9 and 27 of histone 3 in SIRT1-dependent manners [35,82,83]. Both di- and tri-methylation of H3K9 (H3K9me2/3) have been implicated as hallmarks of constitutive and facultative heterochromatin [36]. ATM-dependent H3K9me2 accumulation was observed following laser micro-irradiation and I-SceI-induced DSBs [84]. Interestingly, H3K9 tri-methylation has a similar distribution profile as H3K36me3 across 3′ transcribed regions of euchromatin [85]. Pre-existing H3K9me3 at DSB-flanking active chromatin may modulate transcription repression through transient establishment of facultative heterochromatin. As the major methyltransferase for H3K9 tri-methylation, deacetylation of Suv39h1 at its SET domain by SIRT1 enhances its methyltransferase activity in promoting the up-regulation of H3K9me3 [86]. In addition to their roles in transcription silencing, recruitment of Suv39h1 at DSBs is required for the activation of both TIP60 and ATM, while SIRT1 has also recently been implicated in promoting chromatin de-compaction and HDR by deacetylating chromatin remodeler BRG1 in a PARP-dependent manner [87,88].

In conclusion, local recruitment of an expanding lists of molecular players at DSB-flanking active chromatin might directly tether downstream DDR factors or indirectly by priming a conducive chromatin platform for DDR players loading to drive subsequent DSB repair events.

Regulation of DSB repair pathways

While transcription repression at DSB-flanking active chromatin is vital for rapid repair of damaged DNA, inactivation of this silencing event does not completely abrogate DSB repair but results in its delay [89]. Impairment in transcription silencing due to the loss of BMI1 causes persistence of γ-H2AX foci and impedes the repair of late DNA-damage-induced foci [62]. In addition to boosting repair kinetics, molecular players that participate in transcription suppression have direct roles in regulating DSB repair pathway choice. Notably, the choice of DSB repair pathways at active chromatin, homologous recombination (HR) or non-homologous end joining (NHEJ), has utmost importance in dictating the fate of transcribing genes expression (Figure 3). In addition to cell cycle stages and age of the cells, recent discoveries indicate that pre-existing chromatin context at DSBs represents a critical determinant of DSB repair pathway choice. Distinct DSB repair has also been reported between heterochromatin and euchromatin [90].

Regulation of DSB repair pathways

Figure 3
Regulation of DSB repair pathways

(i) HR is reported to be the preferential DSB repair pathway at actively transcribing regions. During HR, LEDGF recognizes H3K36me3 transcription elongation marks and interacting with CtIP for DNA proximal resection while extensive resection is facilitated by EXO1 and BLM. Resected ssDNA is coated by RPA. With the help of mediator proteins such as BRCA2 complexes, RAD51 recombinase replaces RPA to perform homology search and strand invasion for templated DNA repair. (ii) NHEJ that entails direct ligation of DNA broken ends is active throughout cell cycle but is error prone. As a double reader of both H2AK15ub and H4K20me1/2 histone marks, retention of 53BP1 at DSB further recruits RIF1 and Shieldin to protect DSB from resection. Broken DNA ends are then held in proximity by Ku70-Ku80 heterodimer and promotes the recruitment of activated DNA-PKcs. Phosphorylation of Artemis nuclease trims the DNA ends for ligation by DNA ligase IV-XRCC5-XLF complex.

Figure 3
Regulation of DSB repair pathways

(i) HR is reported to be the preferential DSB repair pathway at actively transcribing regions. During HR, LEDGF recognizes H3K36me3 transcription elongation marks and interacting with CtIP for DNA proximal resection while extensive resection is facilitated by EXO1 and BLM. Resected ssDNA is coated by RPA. With the help of mediator proteins such as BRCA2 complexes, RAD51 recombinase replaces RPA to perform homology search and strand invasion for templated DNA repair. (ii) NHEJ that entails direct ligation of DNA broken ends is active throughout cell cycle but is error prone. As a double reader of both H2AK15ub and H4K20me1/2 histone marks, retention of 53BP1 at DSB further recruits RIF1 and Shieldin to protect DSB from resection. Broken DNA ends are then held in proximity by Ku70-Ku80 heterodimer and promotes the recruitment of activated DNA-PKcs. Phosphorylation of Artemis nuclease trims the DNA ends for ligation by DNA ligase IV-XRCC5-XLF complex.

By ChIP-seq analyses of AsiSI-induced DSBs across the genome, HR was found to be the preferential DSB repair pathway at active chromatin enriched by trimethylation of histone H3 lysine 36 (H3K36me3) [90]. In support to this, TIP60-mediated H4 acetylation at active chromatin favors the occupancy of BRCA1 at DSBs and promotes HR [91]. Preferential repair by HR underlies production of faithful DNA template for subsequent transcription resumption and preserves transcriptome integrity. However, HR operates only in S and G2 stages of the cell cycles due to the need of sister chromatid as repair templates. On the other hand, NHEJ is generally regarded as the predominant DSB repair pathway that functions throughout the cell cycle. Mechanistic insight into how the repair direction at DSB-flanking active chromatin is wheeled toward HR in G1 phase remains elusive but delayed repair via DSB clustering has been suggested and requires further studies.

Chromatin restoration and transcription resumption

Following DSB repair, reorganization of chromatin structure and establishment of pre-existing histone marks are necessary to preserve the chromatin landscape for fully functional gene expression [34,35]. While molecular determinants in promoting DSB-induced transcription recovery are only beginning to emerge, it is reasonable to suggest that histone chaperones and chromatin assembling factors are needed to restructure chromatin back to its original state (Figure 4) [92]. Upon completion of DSB repair or in parallel with repair activity, phosphorylation of H2AX as the damage signal across DSB landscape has to be reversed or down-regulated in a timely manner that probably involves direct dephosphorylation and/or H2A histone variant exchange. In this regard, direct binding of protein phosphatase 2A (PP2A) to γ-H2AX modulates the dynamics of γ-H2AX foci formation at DSB sites [93]. While PP2A has been reported to activate NHEJ through direct dephosphorylation of DNA-PKcs and Ku, its subunit PPP2R2A modulates ATM phosphorylation and promotes HR [94,95]. To a lesser extent, type 2C protein phosphatase (PP2Cγ) is also involved in dephosphorylating γ-H2AX and acts as a histone chaperone in H2A-H2B incorporation into chromatin that may promote full recovery from DNA damage [96].

Chromatin restoration and transcription resumption

Figure 4
Chromatin restoration and transcription resumption

Upon completion of DSB repair, re-establishment of nucleosome involves the incorporation of histones H2A-H2B dimers and H3-H4 tetradimers. In the DDR, FACT plays a role in H2A-H2B exchange at DNA damage site where its activity is inhibited through ubiquitination by UBR5. Exchange of histone H2A variants through H2AX eviction to establish histones equilibrium has also been found to be facilitated by FACT and PP2Cγ. Removal of DSB-triggered histone marks might involve dephosphorylation of H2AX mediated by protein phosphatase PP2A, USP16-dependent deubiquitination of H2AK119 or other DUBs such as USP11 and BAP11. Demethylation at H3K9 and H3K27 may also be promoted by lysine demethylases KDM4 and KDM6A, respectively. Clearance of repressive histone marks may be accompanied with re-deposition of active transcription marks including H4K16ac, H3K4me3 and H3K36me3.

Figure 4
Chromatin restoration and transcription resumption

Upon completion of DSB repair, re-establishment of nucleosome involves the incorporation of histones H2A-H2B dimers and H3-H4 tetradimers. In the DDR, FACT plays a role in H2A-H2B exchange at DNA damage site where its activity is inhibited through ubiquitination by UBR5. Exchange of histone H2A variants through H2AX eviction to establish histones equilibrium has also been found to be facilitated by FACT and PP2Cγ. Removal of DSB-triggered histone marks might involve dephosphorylation of H2AX mediated by protein phosphatase PP2A, USP16-dependent deubiquitination of H2AK119 or other DUBs such as USP11 and BAP11. Demethylation at H3K9 and H3K27 may also be promoted by lysine demethylases KDM4 and KDM6A, respectively. Clearance of repressive histone marks may be accompanied with re-deposition of active transcription marks including H4K16ac, H3K4me3 and H3K36me3.

During DSB repair, eviction of H2A-H2B heterodimers and H3-H4 tetradimers from the breakage site results in naked DNA that requires deposition of new histones or possibly recycling of parental histones [97]. FACT histone chaperone is the major player in mobilizing H2A-H2B exchange upon UV micro-irradiation damage [34]. Interestingly, it has also been reported in transcription repression at UV micro-irradiated damage site by regulating RNAPII elongation activity [98]. Aligned with PRC1-dependent transcriptional silencing at DSB-induced active chromatin, E3 ligase UBR5 as the downstream factor of BMI1 subunit of PRC1 shuts down transcription by ubiquitinating SPT16 of FACT complex that negatively regulates FACT activity on RNAPII [65]. However, the role of FACT in chromatin recovery from DSB repair has to be further verified in transcription-dependent manner.

Notably, while reversal of histone repressive mark H2AK119ub appears to depend on ubiquitin-specific peptidase 16 (USP16) upon local transcription silencing at active chromatin, other deubiquitinating enzymes (DUBs) might play a role in promoting full recovery from DSB repair, including USP11 and BAP1, both of which have been reported in DSB repair [48,99,100]. USP11 as a DUB for H2AK119 and H2BK120 is involved in stabilizing the BRCA1-PALB2-BRCA2 complex and promotes HR in G1 cells [99]. Depletion of BAP1 impairs HR and elevated PARP inhibition sensitivity [100,101].

Histone demethylases for the two major repressive methyl marks on lysine 9 and 27 of histone H3 at DSB-flanking active chromatin remain to be determined. Lysine demethylase KDM4 subfamily, in particular KDM4B is rapidly recruited to damage sites upon laser micro-irradiation to promote H3K9 demethylation [102]. Rapid loss of H3K27me3 by lysine demethylase KDM6A upon DSB induction has also been reported that might promote chromatin relaxation important for the loading of DSB repair factors [103]. Clearance of DSB repair and transcription silencing factors promotes the restoration of chromatin architecture that favors transcription restart through the deposition of active transcription epigenetic signatures.

Concluding remarks

In recent years a lot of attention has been fixated in targeting DSB repair as anti-cancer interventions [44]. Our understanding that cancerous cells are genomically unstable due to misregulated DSB repair have rationalized utility of radiotherapy and chemotherapy as frontline cancer therapeutics, and is guiding combinatorial approaches in which application of small molecules that modulate DSB responses may enhance cancer cell sensitivity to genotoxic agents [43]. Considering the prevalent impact of transcription activity in influencing DSB repair, in-depth understanding into the interplay between them may aid the development of functional therapeutic application in modulating DSB responses.

More research efforts are needed to identify key molecular determinants that facilitate transcription resumption at DSB-flanking active chromatin in order to provide relevant insights into queries like: How transcription resumption at DSBs is regulated tempo-spatially? Can repaired DSBs recover to its canonical state and perform fully? Do NHEJ and HR lead to distinct transcription activation pathway as a checkpoint to safeguard the integrity of DDR?

Summary

  • Preserving integrity of transcribed chromatin entails specialized DNA damage responses.

  • DSB repair on transcribed chromatin encompasses tempo-spatial regulations of chromatin dynamics, histone post-translational modifications, local transcription and choice of DNA repair pathways.

  • DSB detection and metabolism is coupled to transient suppression of local transcriptional activities.

  • Re-establishment of chromatin signature and resumption of local transcription following DSB repair are key to maintaining cell identity and function.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work is supported in part by fund from Research Grants Council to MSYH [grant numbers C7007-17GF and 17104618]. X.Y.T. is a recipient of the Hong Kong PhD Fellowship.

Acknowledgements

The authors thank the anonymous reviewers for their insightful and constructive comments.

Abbreviations

     
  • APOBEC

    apolipoprotein B editing complex

  •  
  • ASF1/SF2

    alternative splicing factor 1/pre-mRNA-splicing factor 2

  •  
  • ATM

    ataxia-telangiectasia mutated

  •  
  • BAF180

    BRG1-associated factor 180

  •  
  • BAP1

    BRCA1-associated protein-1

  •  
  • BLESS

    breaks labeling, enrichment on streptavidin and sequencing

  •  
  • BMI1

    BMI1 proto-oncogene, polycomb ring finger

  •  
  • BRCA1

    breast cancer type 1 susceptibility protein

  •  
  • BRCA2

    breast cancer type 2 susceptibility protein

  •  
  • BRG1

    Brahma-related gene 1

  •  
  • CDYL1

    chromodomain Y-like 1

  •  
  • CHD4

    chromodomain helicase dna-binding protein 4

  •  
  • DRB

    5,6-Dichloro-1-β- d-ribofuranosylbenzimidazole

  •  
  • DSB

    double-strand break

  •  
  • EZH2

    enhancer of zeste 2, polycomb repressive complex 2 subunit

  •  
  • FACT

    facilitates chromatin transcription

  •  
  • γ-H2AX

    phosphorylation of histone variant H2AX

  •  
  • H2AK119ub

    histone H2A mono-ubiquitination at lysine 119

  •  
  • H3K4me1/2/3

    histone H3 mono-, di-, and tri-methylation at lysine 4

  •  
  • H3K9me2/3

    histone H3 di-, and tri-methylation at lysine 9

  •  
  • H3K27me3

    histone H3 tri-methylation at lysine 27

  •  
  • H3K36me3

    histone H3 tri-methylation at lysine 36

  •  
  • HDAC

    Histone deacetylase

  •  
  • HDR

    homology-dependent repair

  •  
  • HeLa

    human cervical cancer cell line

  •  
  • HR

    homologous recombination

  •  
  • IR

    ionizing radiation

  •  
  • KDM4B

    lysine demethylase 4B

  •  
  • KDM5A

    lysine demethylase 5A

  •  
  • KDM6A

    lysine demethylase 6A

  •  
  • Ku70(XRCC6)

    X-ray repair cross complementing 6

  •  
  • Ku80(XRCC5)

    X-ray repair cross complementing 5

  •  
  • LEDGF/p75

    lens epithelium-derived growth factor

  •  
  • MCF7

    human breast cancer cell line

  •  
  • MEF

    mouse embryonic fibroblasts

  •  
  • n.d.

    not determined

  •  
  • NHEJ

    non-homologous end joining

  •  
  • NuRD

    nucleosome remodelling and deacetylase complex

  •  
  • PALB2

    Partner and localizer of BRCA2

  •  
  • PARP1

    poly (ADP-ribose) polymerase

  •  
  • PcG

    polycomb-group proteins

  •  
  • PDS5B

    PDS5 cohesin associated factor B

  •  
  • PP2A

    protein phosphatase 2A

  •  
  • PRC1/2

    polycomb repressive complex 1/2

  •  
  • PTM

    post-translational modifications

  •  
  • RING1B

    Ring finger protein 2

  •  
  • RNAPII

    RNA polymerase II

  •  
  • RNF8/168

    RING finger 8 and 168

  •  
  • SA2

    STAG2 stromal antigen 2

  •  
  • SETD2

    SET domain-containing protein 2

  •  
  • SETD8

    SET domain-containing protein 8

  •  
  • SIRT1

    sirtuin 1

  •  
  • ssDNA

    single-stranded deoxyribonucleic acid

  •  
  • Suv39h1

    suppressor of variegation 3-9 homolog 1

  •  
  • SWI/SNF

    switch/sucrose non-fermentable

  •  
  • TIP60

    histone acetyltransferase KAT5

  •  
  • TMPRSS2-ERG

    transmembrane serine protease 2-ETS transcription factor ERG

  •  
  • TOPIIB

    topoisomerase IIb

  •  
  • U2OS

    human osteosarcoma epithelial cell line

  •  
  • USP

    ubiquitin-specific peptidase

  •  
  • WWP2

    WW domain-containing protein 2

  •  
  • ZMYND8

    zinc finger and MYND [myeloid, nervy, and DEAF-1] domain containing 8

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