Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification

We report the identification of 67 previously undescribed histone modifications, increasing the current number of known histone marks by about 70%. We further investigated one of the marks, lysine crotonylation (Kcr), confirming that it represents an evolutionarily-conserved histone posttranslational modification. The unique structure and genomic localization of histone Kcr suggest that it is mechanistically and functionally different from histone lysine acetylation (Kac). Specifically, in both human somatic and mouse male germ cell genomes, histone Kcr marks either active promoters or potential enhancers. In male germinal cells immediately following meiosis, Kcr is enriched on sex chromosomes and specifically marks testis-specific genes, including a significant proportion of X-linked genes that escape sex chromosome inactivation in haploid cells. These results therefore dramatically extend the repertoire of histone PTM sites and designate Kcr as a specific mark of active sex chromosome-linked genes in postmeiotic male germ cells.     

Histone H2A Mono-Ubiquitination Is a Crucial Step to Mediate PRC1-Dependent Repression of Developmental Genes to Maintain ES Cell Identity

Two distinct Polycomb complexes, PRC1 and PRC2, collaborate to maintain epigenetic repression of key developmental loci in embryonic stem cells (ESCs). PRC1 and PRC2 have histone modifying activities, catalyzing mono-ubiquitination of histone H2A (H2AK119u1) and trimethylation of H3 lysine 27 (H3K27me3), respectively. Compared to H3K27me3, localization and the role of H2AK119u1 are not fully understood in ESCs. Here we present genome-wide H2AK119u1 maps in ESCs and identify a group of genes at which H2AK119u1 is deposited in a Ring1-dependent manner. These genes are a distinctive subset of genes with H3K27me3 enrichment and are the central targets of Polycomb silencing that are required to maintain ESC identity. We further show that the H2A ubiquitination activity of PRC1 is dispensable for its target binding and its activity to compact chromatin at Hox loci, but is indispensable for efficient repression of target genes and thereby ESC maintenance. These data demonstrate that multiple effector mechanisms including H2A ubiquitination and chromatin compaction combine to mediate PRC1-dependent repression of genes that are crucial for the maintenance of ESC identity. Utilization of these diverse effector mechanisms might provide a means to maintain a repressive state that is robust yet highly responsive to developmental cues during ES cell self-renewal and differentiation.     

RNF4 controls the extent of replication fork reversal to preserve genome stability

Replication fork reversal occurs via a two-step process that entails reversal initiation and reversal extension. DNA topoisomerase IIalpha (TOP2A) facilitates extensive fork reversal, on one hand through resolving the topological stress generated by the initial reversal, on the other hand via its role in recruiting the SUMO-targeted DNA translocase PICH to stalled forks in a manner that is dependent on its SUMOylation by the SUMO E3 ligase ZATT. However, how TOP2A activities at stalled forks are precisely regulated remains poorly understood. Here we show that, upon replication stress, the SUMO-targeted ubiquitin E3 ligase RNF4 accumulates at stalled forks and targets SUMOylated TOP2A for ubiquitination and degradation. Downregulation of RNF4 resulted in aberrant activation of the ZATT–TOP2A–PICH complex at stalled forks, which in turn led to excessive reversal and elevated frequencies of fork collapse. These results uncover a previously unidentified regulatory mechanism that regulates TOP2A activities at stalled forks and thus the extent of fork reversal.     

SIAH2 regulates DNA end resection and replication fork recovery by promoting CtIP ubiquitination

Human CtIP maintains genomic integrity primarily by promoting 5′ DNA end resection, an initial step of the homologous recombination (HR). A few mechanisms have been suggested as to how CtIP recruitment to damage sites is controlled, but it is likely that we do not yet have full understanding of the process. Here, we provide evidence that CtIP recruitment and functioning are controlled by the SIAH2 E3 ubiquitin ligase. We found that SIAH2 interacts and ubiquitinates CtIP at its N-terminal lysine residues. Mutating the key CtIP lysine residues impaired CtIP recruitment to DSBs and stalled replication forks, DSB end resection, overall HR repair capacity of cells, and recovery of stalled replication forks, suggesting that the SIAH2-induced ubiquitination is important for relocating CtIP to sites of damage. Depleting SIAH2 consistently phenocopied these results. Overall, our work suggests that SIAH2 is a new regulator of CtIP and HR repair, and emphasizes that SIAH2-mediated recruitment of the CtIP is an important step for CtIP’s function during HR repair.     

Dual effect of heat shock on DNA replication and genome integrity

Heat shock (HS) is one of the better-studied exogenous stress factors. However, little is known about its effects on DNA integrity and the DNA replication process. In this study, we show that in G1 and G2 cells, HS induces a countable number of double-stranded breaks (DSBs) in the DNA that are marked by γH2AX. In contrast, in S-phase cells, HS does not induce DSBs but instead causes an arrest or deceleration of the progression of the replication forks in a temperature-dependent manner. This response also provoked phosphorylation of H2AX, which appeared at the sites of replication. Moreover, the phosphorylation of H2AX at or close to the replication fork rescued the fork from total collapse. Collectively our data suggest that in an asynchronous cell culture, HS might affect DNA integrity both directly and via arrest of replication fork progression and that the phosphorylation of H2AX has a protective effect on the arrested replication forks in addition to its known DNA damage signaling function.     

A role for chromatin remodellers in replication of damaged DNA

In eukaryotic cells, replication past damaged sites in DNA is regulated by the ubiquitination of proliferating cell nuclear antigen (PCNA). Little is known about how this process is affected by chromatin structure. There are two isoforms of the Remodels the Structure of Chromatin (RSC) remodelling complex in yeast. We show that deletion of RSC2 results in a dramatic reduction in the level of PCNA ubiquitination after DNA-damaging treatments, whereas no such effect was observed after deletion of RSC1. Similarly, depletion of the BAF180 component of the corresponding PBAF (Polybromo BRG1 (Brahma-Related Gene 1) Associated Factor) complex in human cells led to a similar reduction in PCNA ubiquitination. Remarkably, we found that depletion of BAF180 resulted after UV-irradiation, in a reduction not only of ubiquitinated PCNA but also of chromatin-associated unmodified PCNA and Rad18 (the E3 ligase that ubiquitinates PCNA). This was accompanied by a modest decrease in fork progression. We propose a model to account for these findings that postulates an involvement of PBAF in repriming of replication downstream from replication forks blocked at sites of DNA damage. In support of this model, chromatin immunoprecipitation data show that the RSC complex in yeast is present in the vicinity of the replication forks, and by extrapolation, this is also likely to be the case for the PBAF complex in human cells.     

Assembly of Slx4 signaling complexes behind DNA replication forks

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress.     

UV-induced ataxia-telangiectasia-mutated and Rad3-related (ATR) activation requires replication stress

Ataxia-telangiectasia-mutated and Rad3-related (ATR) plays an essential role in the maintenance of genome integrity and cell viability. The kinase is activated in response to DNA damage and initiates a checkpoint signaling cascade by phosphorylating a number of downstream substrates including Chk1. Unlike ataxia-telangiectasia-mutated (ATM), which appears to be mainly activated by DNA double-strand breaks, ATR can be activated by a variety of DNA damaging agents. However, it is still unclear what triggers ATR activation in response to such diverse DNA lesions. One model proposes that ATR can directly recognize DNA lesions, while other recent data suggest that ATR is activated by a common single-stranded DNA (ssDNA) intermediate generated during DNA repair. In this study, we show that UV lesions do not directly activate ATR in vivo. In addition, ssDNA lesions created during the repair of UV damage are also not sufficient to activate the ATR-dependent pathway. ATR activation is only observed in replicating cells indicating that replication stress is required to trigger the ATR-mediated checkpoint cascade in response to UV irradiation. Interestingly, H2AX appears to be required for the accumulation of ATR at stalled replication forks. Together our data suggest that ssDNA at arrested replication forks recruits ATR and initiates ATR-mediated phosphorylation of H2AX and Chk1. Phosphorylated H2AX might further facilitate ATR activation by stabilizing ATR at the sites of arrested replication forks.     

HUWE1 interacts with PCNA to alleviate replication stress

Defects in DNA replication, DNA damage response, and DNA repair compromise genomic stability and promote cancer development. In particular, unrepaired DNA lesions can arrest the progression of the DNA replication machinery during S‐phase, causing replication stress, mutations, and DNA breaks. HUWE1 is a HECT‐type ubiquitin ligase that targets proteins involved in cell fate, survival, and differentiation. Here, we report that HUWE1 is essential for genomic stability, by promoting replication of damaged DNA. We show that HUWE1‐knockout cells are unable to mitigate replication stress, resulting in replication defects and DNA breakage. Importantly, we find that this novel role of HUWE1 requires its interaction with the replication factor PCNA, a master regulator of replication fork restart, at stalled replication forks. Finally, we provide evidence that HUWE1 mono‐ubiquitinates H2AX to promote signaling at stalled forks. Altogether, our work identifies HUWE1 as a novel regulator of the replication stress response.     

PTEN regulates RPA1 and protects DNA replication forks

Tumor suppressor PTEN regulates cellular activities and controls genome stability through multiple mechanisms. In this study, we report that PTEN is necessary for the protection of DNA replication forks against replication stress. We show that deletion of PTEN leads to replication fork collapse and chromosomal instability upon fork stalling following nucleotide depletion induced by hydroxyurea. PTEN is physically associated with replication protein A 1 (RPA1) via the RPA1 C-terminal domain. STORM and iPOND reveal that PTEN is localized at replication sites and promotes RPA1 accumulation on replication forks. PTEN recruits the deubiquitinase OTUB1 to mediate RPA1 deubiquitination. RPA1 deletion confers a phenotype like that observed in PTEN knockout cells with stalling of replication forks. Expression of PTEN and RPA1 shows strong correlation in colorectal cancer. Heterozygous disruption of RPA1 promotes tumorigenesis in mice. These results demonstrate that PTEN is essential for DNA replication fork protection. We propose that RPA1 is a target of PTEN function in fork protection and that PTEN maintains genome stability through regulation of DNA replication.     

USP3 counteracts RNF168 via deubiquitinating H2A and γH2AX at lysine 13 and 15

Histone ubiquitination plays a vital role in DNA damage response (DDR), which is important for maintaining genomic integrity in eukaryotic cells. In DDR, ubiquitination of histone H2A and γH2AX by the concerted action of ubiquitin (Ub) ligases, RNF168 and RNF8, generates a cascade of ubiquitination signaling. However, little is known about deubiquitinating enzymes (DUBs) that may catalyze the removal of Ub from these histones. This study demonstrated that USP3, an apparent DUB for mono-ubiquitinated H2A, is indeed the enzyme for deubiquitinating Ub conjugates of γH2AX and H2A from lysine sites, where the ubiquitination is initiated by RNF168. Here, we showed that ectopic expression of USP3 led to the deubiquitination of both H2A and γH2AX in response to UV-induced DNA damage. Moreover, ectopic USP3 expression abrogated FK2 antibody-reactive Ub-conjugate foci, which co-localize with damage-induced γH2AX foci. In addition, USP3 overexpression impaired the accumulation of downstream repair factors BRCA1 and 53BP1 at the damage sites in response to both UV and γ-irradiation. We further identified that the USP3 removes Ub at lysine 13 and 15 of H2A and γH2AX, as well as lysine 118 and 119 of H2AX in response to DNA damage. Taken together, the results suggested that USP3 is a negative regulator of ubiquitination signaling, counteracting RNF168- and RNF8-mediated ubiquitination.     

Extended DNA-binding interfaces beyond the canonical SAP domain contribute to the function of replication stress regulator SDE2 at DNA replication forks

Elevated DNA replication stress causes instability of the DNA replication fork and increased DNA mutations, which underlies tumorigenesis. The DNA replication stress regulator silencing-defective 2 (SDE2) is known to bind to TIMELESS (TIM), a protein of the fork protection complex, and enhances its stability, thereby supporting replisome activity at DNA replication forks. However, the DNA-binding activity of SDE2 is not well defined. Here, we structurally and functionally characterize a new conserved DNA-binding motif related to the SAP (SAF-A/B, Acinus, PIAS) domain in human SDE2 and establish its preference for ssDNA. Our NMR solution structure of the SDE2^SAP domain reveals a helix-extended loop-helix core with the helices aligned parallel to each other, consistent with known canonical SAP folds. Notably, we have shown that the DNA interaction of this SAP domain extends beyond the core SAP domain and is augmented by two lysine residues in the C-terminal tail, which is uniquely positioned adjacent to the SAP motif and conserved in the pre-mRNA splicing factor SF3A3. Furthermore, we found that mutation in the SAP domain and extended C terminus not only disrupts ssDNA binding but also impairs TIM localization at replication forks, thus inhibiting efficient fork progression. Taken together, our results establish SDE2^SAP as an essential element for SDE2 to exert its role in preserving replication fork integrity via fork protection complex regulation and highlight the structural diversity of the DNA–protein interactions achieved by a specialized DNA-binding motif.     

DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination

DNMT1 is recruited by PCNA and UHRF1 to maintain DNA methylation after replication. UHRF1 recognizes hemimethylated DNA substrates via the SRA domain, but also repressive H3K9me3 histone marks with its TTD. With systematic mutagenesis and functional assays, we could show that chromatin binding further involved UHRF1 PHD binding to unmodified H3R2. These complementation assays clearly demonstrated that the ubiquitin ligase activity of the UHRF1 RING domain is required for maintenance DNA methylation. Mass spectrometry of UHRF1-deficient cells revealed H3K18 as a novel ubiquitination target of UHRF1 in mammalian cells. With bioinformatics and mutational analyses, we identified a ubiquitin interacting motif (UIM) in the N-terminal regulatory domain of DNMT1 that binds to ubiquitinated H3 tails and is essential for DNA methylation in vivo. H3 ubiquitination and subsequent DNA methylation required UHRF1 PHD binding to H3R2. These results show the manifold regulatory mechanisms controlling DNMT1 activity that require the reading and writing of epigenetic marks by UHRF1 and illustrate the multifaceted interplay between DNA and histone modifications. The identification and functional characterization of the DNMT1 UIM suggests a novel regulatory principle and we speculate that histone H2AK119 ubiquitination might also lead to UIM-dependent recruitment of DNMT1 and DNA methylation beyond classic maintenance.     

Brc1 links replication stress response and centromere function

Protection of genome integrity depends on the coordinated activities of DNA replication, DNA repair, chromatin assembly and chromosome segregation mechanisms. DNA lesions are detected by the master checkpoint kinases ATM (Tel1) and ATR (Rad3/Mec1), which phosphorylate multiple substrates, including a C-terminal SQ motif in histone H2A or H2AX. The 6-BRCT domain protein Brc1, which is required for efficient recovery from replication fork arrest and collapse in fission yeast, binds phospho-histone H2A (γH2A)-coated chromatin at stalled and damaged replication forks. We recently found that Brc1 co-localizes with γH2A that appears in pericentromeric heterochromatin during S-phase. Our studies indicate that Brc1 contributes to the maintenance of pericentromeric heterochromatin, which is required for efficient chromosome segregation during mitosis. Here, we review these studies and present additional results that establish the functional requirements for the N-terminal BRCT domains of Brc1 in the replication stress response and resistance to the microtubule destabilizing drug thiabendazole (TBZ). We also identify the nuclear localization signal (NLS) in Brc1, which closely abuts the C-terminal pair of BRCT domains that form the γH2A-binding pocket. This compact arrangement of localization domains may be a shared feature of other γH2A-binding proteins, including Rtt107, PTIP and Mdc1.     

The DNA repair component Metnase regulates Chk1 stability

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylation of downstream effectors. Metnase (also termed SETMAR) is a SET histone methylase and transposase nuclease protein that promotes both DNA double strand break (DSB) repair and re-start of stalled replication forks. We previously found that Chk1 phosphorylation of Metnase on S495 enhanced its DNA DSB repair activity but decreased its ability to re-start stalled replication forks. Here we show that phosphorylated Metnase feeds back to increase the half-life of Chk1. Chk1 half-life is regulated by DDB1 targeting it to Cul4A for ubiquitination and destruction. Metnase decreases Chk1 interaction with DDB1, and decreases Chk1 ubiquitination. These data define a novel pathway for Chk1 regulation, whereby a target of Chk1, Metnase, feeds back to amplify Chk1 stability, and therefore enhance replication fork arrest.     

Rtt105 functions as a chaperone for replication protein A to preserve genome stability

Generation of single‐stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA‐binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA–ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA–importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA–ssDNA complex. Single‐molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA. These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks.     

E3 ligase RFWD3 participates in replication checkpoint control

RFWD3 has E3 ligase activity in vitro, but its in vivo function remains unknown. In this study we identified RFWD3 as a novel replication protein A (RPA)-associated protein. Using purified proteins, we observed a direct interaction between RPA2 and RFWD3. Further analysis showed that RFWD3 is recruited to stalled replication forks and co-localizes with RPA2 in response to replication stress. Moreover, RFWD3 is important for ATR-dependent Chk1 activation in response to replication stress. Upon replication stress, deletion of RPA2 binding region on RFWD3 impairs its localization to stalled replication forks and decreases Chk1 activation. Taken together, our results suggest that RFWD3 and RPA2 functionally interact and participate in replication checkpoint control.