The role of chromatin at transcription-replication conflicts as a genome safeguard

1 1 2 3 4 5 6 7 The role of chromatin at transcription-replication conflicts as a 8 genome safeguard 9 10 11 Aleix Bayona-Feliu1,2 and Andrés Aguilera1,2, * 12 13 14 1 Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, 15 Universidad de Sevilla-CSIC, Sevilla 41092, Spain 16 2 Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Seville 17 41012, Spain 18 19 20 21 22 23 24 * Corresponding author. E-mail: [email protected] 25 26 27 Word count: 3311 28 29 30 31 32 2 ABSTRACT 33 34 DNA replication ensures the correct copying of the genome and the faithful transfer of 35 the genetic information to the offspring. However, obstacles to replication fork (RF) 36 progression cause RF stalling and compromise efficient genome duplication. Since 37 replication uses the same DNA template as transcription, both transcription and 38 replication must be coordinated to prevent Transcription-Replication Conflicts (TRCs) 39 that could stall RF progression. Several factors contribute to limit the occurrence of 40 such conflicts and their harmful impact on genome integrity. Increasing evidence 41 indicates that chromatin homeostasis plays a key role in the cellular response to TRCs 42 as well as in the preservation of genome integrity. Indeed, chromatin regulating 43 enzymes are frequently mutated in cancer cells, a common characteristic of which is 44 genome instability. Therefore, understanding the role of chromatin in TRC occurrence 45 and resolution may help identify the molecular mechanism by which chromatin protects 46 genome integrity, and the causes and physiological relevance of the high mutation 47 rates of chromatin regulating factors in cancer. Here we review the current knowledge 48 in the field, as well as the perspectives and future applications. 49 50 Abstract: 178 words 51 52 53 54 Keywords: transcription-replication conflicts, R-loops, DNA breaks, genome instability, 55 chromatin, epigenetics, cancer, 56 57 58 59 60 61 62 63 64 65 66 67 68 69 3 Introduction 70 71 Genome stability ensures cell viability, even though certain variability is required for 72 species adaptation and survival. DNA is constantly subjected to either exogenous or 73 endogenous damaging agents that might result in DNA damage and eventually 74 genome instability if not properly addressed. 75 In eukaryotes, DNA replication is bidirectional and initiates at multiple origins to 76 produce a new copy of the whole genome. During this process, replication forks (RFs) 77 have to deal with multiple obstacles to their progression that may compromise accurate 78 genome duplication (1,2). Obstacles include DNA-bound proteins, DNA damage, 79 topological stress, chromatin structure or transcription. Replication and transcription 80 use the same DNA substrate, and several reports have shown the potential of 81 transcription to stall DNA replication, thus compromising genome integrity (3–5). 82 Interestingly, hazardous RF stalling may be further enhanced by the formation of 83 transcription-associated obstacles, including non-B DNA structures. Consequently, 84 cells have developed several mechanisms to prevent and solve transcription- 85 replication conflicts (TRCs). 86 TRCs are actively prevented via different pathways that avoid the formation of 87 transcription-associated obstacles (2). Nevertheless, when occurring, transcription- 88 mediated RF stalling may be solved via a coordinated response involving checkpoint 89 activation, RF stabilization and obstacle removal. Thus, cancer-associated genes as 90 BRCA1/2 and Fanconi Anemia factors have been shown to play a key role during this 91 process (6–9). Recently, however, chromatin remodeling has also emerged as a major 92 player in this response. Indeed, the SWItch/Sucrose Non-Fermentable (SWI/SNF), 93 INOsitol requiring 80 (INO80) or FAcilitates Chromatin Transcription (FACT) chromatin 94 remodeling complexes counteract TRC occurrence (10–13). Interestingly, SWI/SNF 95 components, in particular its main ATPase activity SMARCA4, best known as Brahma- 96 Related Gene 1 (BRG1), are frequently altered in cancer, reaching mutation 97 frequencies only surpassed by Tumor Protein P53 (TP53) (14). 98 Increasing evidence suggests that genes highly mutated in cancer play key 99 roles during tumorigenesis, which may pose an important endogenous instigator of 100 genome instability. Therefore, understanding the underlying mechanisms through 101 which cells prevent TRCs from resulting in genome instability-associated diseases is 102 essential to achieve new therapeutic opportunities against the disease. In this review, 103 we try to gather our current knowledge on how the chromatin network impacts on TRC 104 occurrence and resolution to preserve genome stability. Other reviews have been 105 published on the causes and consequences of TRCs (1,15–17). 106 4 Transcription as a source of replication stress 107 The essential fine-tuned process of transcription uses as template the DNA, which has 108 to be replicated at each cell cycle. Consequently, it is possible that conflict scenarios 109 between transcription and replication raise during S phase at regions in which both 110 processes occur concomitantly. Indeed, numerous reports show that transcription is a 111 potential source of RF stalling and DNA replication stress (1). Thus, the transcription 112 machinery itself and transcription-induced structures such as DNA supercoiling, non-B 113 DNA structures (DNA-RNA hybrids; G4s), DNA damage or closed chromatin states 114 may pose an obstacle to RF progression (Figure 1). 115 116 The transcription machinery 117 Similar to tightly-bound proteins, the transcription machinery may become a roadblock 118 to RF progression. Indeed, yeast mutants undergoing RNA Polymerase II (RNAPII) 119 retention at chromatin result in DNA replication stress (18) and RNAPII has been 120 shown to be released from chromatin after replication stress thru a process involving 121 INO80C and the RNA processing PAF complex in yeast (11). Ongoing RNAPs may 122 also pause, arrest and/or backtrack when facing DNA damage, from which cells take 123 advantage by promoting transcription-coupled repair (TCR) (19). In human cells, the 124 RECQL5 helicase of the RecQ family has been shown to prevent RNAPII backtracking 125 and promote transcription elongation, thus avoiding TRCs (20,21), and supporting the 126 view that backtracked RNAPs may be important obstacles to advancing RFs. 127 Transcription termination factors (TTFs) also prevent RNAPs from becoming a barrier 128 to replication. Thus, yeast transcription termination mutants affecting RNA 5’ and 3’ 129 end processing factors Rna14 and Rna15, Fip1, Usp6/Hrp1, the 5'-3' Exoribonuclease 130 2 Xrn2 or the RNA helicase Sen1 (ortholog of human Senataxin) present inefficient 131 termination and transcription-dependent replication hampering (22–24). Altogether, the 132 data indicate that cells have developed several mechanisms acting at different steps 133 during the transcription process to avoid that the transcription machinery becomes a 134 barrier to RF progression. 135 136 Transcriptional topological stress 137 Positive and negative supercoiling accumulate ahead and behind RNAP, respectively, 138 during transcription elongation (25). While positive supercoiling limits further unwinding 139 of DNA, negative supercoiling can result in DNA alterations making it prone to open 140 and form non-B DNA structures. On the other hand, positive supercoiling accumulated 141 between RNAP and an RF advancing in head-on orientation may stall RF progression 142 without the need of a physical collision between the transcription and replication 143 5 machineries (Figure 1). Nevertheless, topoisomerases are capable of dealing with 144 transcription-induced supercoiled DNA structures ensuring they do not compromise 145 genome integrity (26–29). Therefore, enzymatic activities acting on supercoiled DNA 146 such as topoisomerases plus their interacting partners might play an important role in 147 preventing transcription-associated genome instability. 148 149 Co-transcriptional DNA-RNA hybrids 150 Current evidence indicates that nascent transcripts can hybridize with the template 151 DNA resulting in the formation of a DNA-RNA hybrid, which may further interfere with 152 the DNA replication process (1) (Figure 1). Hybrids may form during transcription in the 153 form of an R-loop containing in addition the displaced ssDNA identical to the RNA 154 moiety of the hybrid. R-loops can also form at the vicinity of double strand breaks 155 (DSBs) and evidence has also been provided that TRCs may lead to R loops (30,31), 156 In addition, the cell cycle phase is a major determinant of the type of molecular event 157 resulting in an R-loop (32,33). R-loops may occur naturally with a physiological role, as 158 in the S regions of the Immunoglobulin genes. Nevertheless, unscheduled R-loop 159 formation compromises genome integrity (2). Current data supports the view that 160 persistent unscheduled R-loop accumulation results in DNA damage mainly as a 161 consequence of replication blockage, even though other mechanisms, such as the 162 action of nucleotide excision repair (NER) nucleases XPG or XPF can also cause such 163 DNA breaks (34). Consequently, cells have developed mechanisms to prevent 164 unscheduled R-loop accumulation (Figure 1). These strategies include proper 165 assembly of the messenger ribonucleoprotein (mRNP), activities to resolve the R-loops 166 as DNA-RNA helicases or ribonuclease H (RNH), which degrades the RNA moiety of 167 the hybrids, and the DNA Damage Response (DDR), as recently reviewed (35). 168 169 A role of chromatin in the coordination of transcription and replication 170 The involvement of chromatin in regulation of gene expression has been largely 171 explored and several epigenetic mechanisms have been described to help regulate 172 transcription (36). In the last years, growing evidence indicates that chromatin 173 homeostasis must also be properly preserved to prevent transcription-associated 174 genomic instability. DNA methylation, histone post-translational modifications, ATP- 175 dependent chromatin remodeling and even RNA modifications have been described to 176 influence TRC-mediated DNA damage (Figure 2). 177 178 179 180 6 DNA methylation 181 Initial genome-wide analysis of DNA-RNA hybrids unveiled that they are enhanced at 182 CpG island (CGI)-promoters and its occurrence correlates with unmethylated states of 183 CGIs. Indeed, R-loops protect CGI from the methyltransferase 3B1 (DNMT3B1) 184 activity, a major de novo DNMT in early development (37) (Figure 2a). Interestingly, 185 low R-loop levels lead to high DNA methylation and gene silencing in Amyotrophic 186 Lateral Sclerosis (ALS) 4 patient cells, as suggested by the observation that DNMTs 187 bind much more efficiently to dsDNA than to DNA-RNA hybrid-prone sequences (38). 188 A different study also points to a role for the GADD45 factor in this process (39). 189 Notably, GADD45A was found to bind R-loop-prone regions next to promoters and 190 trigger DNA demethylation thru recruitment of Ten-Eleven Translocation 1 (TET1) 191 (Figure 2a), suggesting that GADD45A might work as an epigenetic reader that 192 induces promoter CGI demethylation in response to R-loop formation. Thus, R-loops 193 formed at CGI-promoters seems to favor gene transcription by reducing DNMTs’ 194 affinity to genomic DNA containing DNA-RNA hybrids thus leading to promoter 195 demethylation. 196 197 Histones and their post-translational modifications 198 Physiological R-loops occurring at promoter regions have been found enriched in 199 histone post-translational modifications (PTMs) associated with active transcription 200 (40). In particular, high levels of histone H3 lysine 4 di/trimethylation (H3K4me2/me3), 201 lysine 9/27 acetylation (H3K9/K27ac) and of certain H3.3 histone variants, but low 202 histone H3 lysine 9 trimethylation (H3K9me3) are observed close to TSSs at R-loop- 203 prone promoters. Instead, histone H3 lysine 4 mono-methylation (H3K4me1) and lysine 204 36 methylation (H3K36me) are enhanced at the R-loop-accumulating sites of such 205 promoters. On the other hand, R-loops emerging at transcription termination 206 sequences show an association with increased H3K4me1 levels. Interestingly, 207 H3K4me has been shown to play a key role ensuring S-phase checkpoint activity and 208 reliable DNA duplication under replication stress as seen in highly transcribed yeast 209 genes (41). Histone H3 lysine 9 dimethylation (H3K9me2) has also been reported to 210 promote efficient transcription termination in mammalian protein-coding genes prone to 211 R-loop formation at transcription termination sites (TTSs) (42). 212 Unscheduled R-loops have been associated with increases in repressive 213 epigenetic marks such as histone H3 serine 10 phosphorylation (H3S10P) and 214 H3K9me2/3 (Figure 2b). R-loop-dependent H3S10P accumulation was found in R- 215 loop-prone mutants in yeast, C. elegans and human cells, suggesting the effect is 216 conserved among species (43). Further investigation in yeast unveiled that such a 217 7 modification was causative of the observed genomic instability, as yeast mutants 218 impaired in histone H3S10P formation result in R-loop accumulation not associated 219 with increased DNA damage (44). Interestingly, Aurora Kinase A (AURKA) was 220 recently revealed to mediate R-loop-dependent H3S10P deposition during S phase 221 and its inhibition results in TRCs and checkpoint activation in MYCN-amplified 222 neuroblastoma cells (45). Thus, H3S10P and AURKA, might play key roles preventing 223 transcription-dependent RF stalling and its deleterious consequences in S phase. 224 Aberrant R-loops have also been reported in triplet-repeat expansions, a feature of 225 Friedrich's ataxia and Fragile X syndrome that are associated with ectopic repressive 226 H3K9me2/3 that impedes RNAPII progression and results in gene silencing (46). 227 Linker histone H1, which has been related to chromatin compaction and 228 heterochromatin, prevents R-loop-mediated DNA damage as well (Figure 2b). Histone 229 H1 depletion in Drosophila results in R-loop-dependent genome instability in 230 heterochromatin (47), and accumulation of transcription-dependent stalled forks and 231 DNA damage are observed in histone H1 triple knock-out (TKO) human cells (48). 232 Therefore, linker histones might help coordinate transcription and DNA replication to 233 prevent transcription-induced DNA damage. 234 235 Chromatin modifiers 236 Genome-wide analyses have revealed that components of COMPASS (RBBP5), 237 PAF1C (PAF1), SIN3 complex (SIN3A; SAP30; HDAC2), p300 acetyltransferase, 238 EZH2 methyltransferase and KDM4A and PHF8 histone demethylases are found at 239 high frequency at R-loop-prone promoters, and higher abundance of PAF1, CTCF and 240 cohesin components ZNF143 and RAD21 are detected at R-loop-prone TTSs (40). 241 However, deficiencies in several chromatin-modifying activities have been 242 observed to promote R-loop-dependent genome instability. Regulators of histone 243 acetylation/deacetylation might play a key role in this process, as several reports have 244 connected deficiencies in such activities with unscheduled R-loop formation and DNA 245 damage. Thus, depletion of Sin3A histone deacetylase (HDAC) complex factors (SIN3; 246 SAP130) as well as histone deacetylation inhibition produced by trichostatin A (TSA), 247 suberoylanilide hydroxamic acid (SAHA) results in an accumulation of R-loops and R- 248 loop-dependent DNA damage in human cells (49) (Figure 2c). Similarly, R-loop- 249 dependent genome instability phenotypes are also induced by deficiencies in sirtuins 250 (NAD+-dependent deacetylases). R-loop-dependent DSBs arise in hst3 and hst4 yeast 251 mutants of the hSIRT6 homologs (50) and in SIRT7-deficient human cells (51). In 252 human cells the HDAC inhibitor romidepsin also causes R-loop-mediated ssDNA 253 breaks (52). On the other hand, the Tip60-400 histone acetyltransferase complex 254 8 associates with genes harboring promoter-proximal R-loops and influence genome- 255 wide occupancy of polycomb repressor complex (PRC)-2 (PRC2) histone 256 methyltransferase (53). Deficiency of Bromodomain-containing protein 4 (BRD4), a 257 reader that recognizes and binds acetylated histones, was also shown to cause an 258 increase in R-loops, TRCs and DNA damage, consistent with a major role for histone 259 acetylation state on R-loop homeostasis (54). 260 Chromatin-modifying enzymes regulating other epigenetic marks different from 261 histone acetylation participate either in this process. PRC1 was reported to act in 262 parallel with Mdm2, a chromatin modifier modulating PRC-driven histone modifications, 263 suppressing R-loop formation and promoting productive DNA replication via a direct 264 impact on histone H2A lysine 118/119 (K118/K119) ubiquitination (55). Indeed, R-loops 265 drive Polycomb repression at a subgroup of developmental genes (56) (Figure 2c). At 266 these genes, decreased PRC1 and PRC2 abundance, RNAPII activation and 267 productive transcript elongation were observed upon R-loop removal. Furthermore, a 268 connection between R-loop formation and Euchromatic Histone Lysine 269 Methyltransferase 2 (EHMT2), also known as G9a, has also been described at TTSs 270 (42) (Figure 2c). At these sites, R-loop formation was suggested to drive G9a 271 recruitment and results in histone H3K9me2, promoting RNAPII pausing and facilitating 272 termination. 273 274 Histone chaperones 275 In agreement with a major impact of the content of histones and their PTMs, histone 276 turnover also mediates transcription-associated RF stalling. The histone chaperone 277 FACT was observed to prevent transcription-mediated genome instability, since its 278 deficiency results in transcription-associated DNA damage and RF progression 279 impairment in yeast and human cells (13) (Figure 2d). The observation that the MCM2- 280 7 helicase dissociates from chromatin in FACT-deficient cells causing loss of ssDNA- 281 RPA binding and checkpoint activation (57) may be behind the replication deficiency, 282 even though it needs experimental evidence. 283 FACT and Chromatin Assembly Factor-1 (CAF1) histone chaperones have 284 been described to be specifically recruited at transcribing loci to facilitate RF 285 progression (58) (Figure 2d). Notably, CAF-1 depletion was shown to slow down DNA 286 replication and promote CHK1 phosphorylation at serine 317, a mark associated with 287 DNA replication stress (59). Similarly, the Anti-Silencing Function 1 (ASF1) factor has 288 also been implicated in promoting RF progression by driving recycling of H3-H4 289 tetramers in conjunction with CAF-1 (60). Indeed, ASF1 deficiency promotes 290 replication-dependent genome instability and sensitizes cells to replication stress- 291 9 inducing compounds (61,62). The results suggest that histone turnover must be 292 properly regulated to ensure efficient RF progression, especially at regions enriched in 293 transcription-associated obstacles. 294 295 ATP-dependent chromatin remodeling 296 Nucleosome positioning on chromatin depends directly on the coordinated action of 297 histone chaperons and ATP-dependent chromatin remodelers. Consistent with the idea 298 of a major contribution of chromatin to the resolution of TRCs, remodeling activities are 299 also emerging as required to prevent transcription-associated genome instability. 300 Indeed, members of different chromatin remodeling families (SWI/SNF, INO80, ISWI) 301 have been shown to protect against transcription-dependent DNA damage. 302 The SWI/SNF complex, the ATP-dependent chromatin remodeling complex 303 most frequently altered in cancer (63), has recently being shown to control TRCs (10). 304 Depletion of BRG1, the main SWI/SNF ATPase, is epistatic to FANCD2 deficiency in 305 its capacity to help solve TRCs, especially those occurring in a head-on orientation 306 (Figure 2e). Consistently, BRG1 co-localizes with DNA replication factors and promote 307 RF progression. In addition, AT-Rich Interaction Domain 1A (ARID1A) and Polybromo 308 1 (PBRM1), members of the canonical BRG1-associated factor (cBAF) and polybromo 309 BRG1-associated factor (PBAF) SWI/SNF complex subtypes, respectively, were also 310 reported to protect from transcription-associated DNA damage. Similalry, ARID1A and 311 PBRM1 deficiencies also induce R-loop-dependent DNA damage. Additionally, a 312 recently observed connection between ARID1A and topoisomerase IIa (TOP2A) at 313 TRCs (64) and high levels of replication stress, micronuclei and R-loops in PBRM1- 314 deficient human cells (65), further supports the involvement of SWI/SNF at TRCs. 315 Interestingly, another member of the SWI/SNF family, Alpha Thalassemia/Mental 316 Retardation Syndrome X-Linked (ATRX), suppress R-loop formation in telomeric 317 repeats (66). All these factors present high mutation frequencies in malignant cells, 318 suggesting a possible relation with the high mutation rates observed in cancer. 319 INO80C has been implicated in RNAPII release from chromatin together with 320 the PAF RNA processing complex thus limiting TRCs in budding yeast (11). 321 Interestingly, INO80 prevents R-loop-dependent DNA damage in prostate cancer PC3 322 human cells (12) (Figure 2e), and R-loops promote recruitment of INO80 protein to 323 chromatin. In agreement, yeast Ino80, the ATPase component of the INO80 complex, 324 was reported to function in parallel with Isw2, the catalytic component of the ISW2 325 complex, promoting RF progression (67). Transcription-dependent hyper- 326 recombination was shown to increase also in yeast cells lacking Isw1, the catalytic 327 subunit of the yeast ISW1 complex (68). Similar mechanisms might also exist in human 328 16 48. 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Nature Communications. 638 12(1):5512. 639 640 641 642 19 FIGURE LEGENDS 643 644 Figure 1. Transcription-associated obstacles to DNA replication. 645 Transcription occurs at the same template as DNA replication, posing an obstacle to 646 RF progression that needs to be surpassed to proceed with efficient DNA duplication. 647 The transcription machinery itself is tightly bound to DNA and this may impede RF 648 progression. In addition, transcription induces the occurrence of additional structures 649 such as DNA supercoiling, non-B DNA structures (R-loops; G4s), DNA damage or 650 closed chromatin states that can further hinder DNA replication. Coordinated action of 651 several cellular activities (messenger ribonucleoprotein (mRNP) biogenesis factors, 652 RNA helicases, nucleases or topoisomerases) prevents the accumulation of such 653 structures, and the DNA Damage Response (DDR) helps solve transcription-replication 654 conflicts (TRCs). 655 656 Figure 2. Epigenetic mechanisms at transcription-replication collisions. 657 Multiple chromatin factors contribute to the prevention of TRCs to warrant genome 658 integrity. a, Promoter proximal R-loops prevent DNMTs and promote DNA 659 demethylation of CpG islands (CGI) and gene activation. b, Linker histones prevent 660 unscheduled R-loops, which induce repressive epigenetic marks that may block RF 661 progression. Aurora-A phosphorylate histone H3 serine 10 in S phase in response to 662 R-loop formation. G9a and PRC are well-known interphase methyltransferase 663 complexes that could be involved in histone H3 di/tri-methylation of lysine 9 in 664 response to unscheduled R-loop accumulation. c, FACT and CAF-1 histone chaperons 665 promote RF progression at transcribing loci. Evidence also indicates that ASF-1 could 666 have a role in this process. d, Histone deacetylation complexes (Sin3A, Sirtuins) 667 protect against R-loop-mediated genome instability. BRD4, which binds histone 668 acetylated residues through its bromodomain, prevent R-loop-dependent genome 669 instablity. Polycomb-repressive complexes 1 and 2 (PRC1, PRC2) and the G9a 670 complex are also connected to R-loop metabolism. e, ATP-dependent chromatin 671 remodelers have a major impact on TRCs. The SWI/SNF complex would act together 672 with FANCD2 preventing TRCs, while INO80 complex prevent unscheduled R-loop 673 formation and promote RNAPII release in response to TRCs. f, RNA modifications also 674 influences R-loop occurrence. METTL3 methylates N6 position of adenosine 675 ribonucleotides that have been suggested to drive cell cycle regulation of R-loop 676 homeostasis through YTHDF2. TonEBP binds R-loops and recruits METTL3. 677 Methylation of N5 position of cytosine ribonucleotide by TRDMT1 was shown to 678 increase RAD52 affinity for DNA-RNA hybrids. 679 RNAPII USSB G4 RNAPII 5’ CAP RNA helicases RNA nucleases R loop Supercoiling (+) Replisome mRNP biogenesis FANCONI BRCA2 BRCA1 DNA repair ANEMIA TOP1 DNA topoisomerases Figure 1 DNMTs methylated CpG RNAPII R loop Replisome GADD45TET1 GC content GC skew CpG islands DNA methylation Histones and their PTMs RNAPII R loop P m P m P m P m P m Aurora-A Replisome G9a/PRC ? H1 H1 P m H3S10 phosphorylation H3K9 di-/tri-methylation ab BRD4 Histone chaperons Chormatin modifiers RNAPII R loop Replisome FACT CAF-1 ASF-1? FACT CAF-1 ASF-1? histones m ub m ub RNAPII R loop Replisome PRC 1/2 ac Sin3A ac ac m ub histone acetylation histone methylation histone ubiquitination cd sirtuins ef R loop Replisome RNAPII R loop SWI/SNF (BRG1) FANCONI ANEMIA INO80 ISWI? ATP-dependent chromatin remodellers RNA modification Replisome RNAPII TonEBP METTL3 TRDMT1 YTHDF2 m6A m5C ADAR1 RNAPII Figure 2