Chromatin context-dependent regulation and epigenetic manipulation of prime editing.

We set out to exhaustively characterize the impact of the cis-chromatin environment on prime editing, a precise genome engineering tool. Using a highly sensitive method for mapping the genomic locations of randomly integrated reporters, we discover massive position effects, exemplified by editing efficiencies ranging from ∼0% to 94% for an identical target site and edit. Position effects on prime editing efficiency are well predicted by chromatin marks, e.g., positively by H3K79me2 and negatively by H3K9me3. Next, we developed a multiplex perturbational framework to assess the interaction of trans-acting factors with the cis-chromatin environment on editing outcomes. Applying this framework to DNA repair factors, we identify HLTF as a context-dependent repressor of prime editing. Finally, several lines of evidence suggest that active transcriptional elongation enhances prime editing. Consistent with this, we show we can robustly decrease or increase the efficiency of prime editing by preceding it with CRISPR-mediated silencing or activation, respectively.

PARP1-DNA co-condensation drives DNA repair site assembly to prevent disjunction of broken DNA ends.

DNA double-strand breaks (DSBs) are repaired at DSB sites. How DSB sites assemble and how broken DNA is prevented from separating is not understood. Here we uncover that the synapsis of broken DNA is mediated by the DSB sensor protein poly(ADP-ribose) (PAR) polymerase 1 (PARP1). Using bottom-up biochemistry, we reconstitute functional DSB sites and show that DSB sites form through co-condensation of PARP1 multimers with DNA. The co-condensates exert mechanical forces to keep DNA ends together and become enzymatically active for PAR synthesis. PARylation promotes release of PARP1 from DNA ends and the recruitment of effectors, such as Fused in Sarcoma, which stabilizes broken DNA ends against separation, revealing a finely orchestrated order of events that primes broken DNA for repair. We provide a comprehensive model for the hierarchical assembly of DSB condensates to explain DNA end synapsis and the recruitment of effector proteins for DNA damage repair.

The BRCA Tumor Suppressor Network in Chromosome Damage Repair by Homologous Recombination.

Mutations in the BRCA1 and BRCA2 genes predispose afflicted individuals to breast, ovarian, and other cancers. The BRCA-encoded products form complexes with other tumor suppressor proteins and with the recombinase enzyme RAD51 to mediate chromosome damage repair by homologous recombination and also to protect stressed DNA replication forks against spurious nucleolytic attrition. Understanding how the BRCA tumor suppressor network executes its biological functions would provide the foundation for developing targeted cancer therapeutics, but progress in this area has been greatly hampered by the challenge of obtaining purified BRCA complexes for mechanistic studies. In this article, we review how recent effort begins to overcome this technical challenge, leading to functional and structural insights into the biochemical attributes of these complexes and the multifaceted roles that they fulfill in genome maintenance. We also highlight the major mechanistic questions that remain.

DNA Repair Network Analysis Reveals Shieldin as a Key Regulator of NHEJ and PARP Inhibitor Sensitivity.

Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.

DNA damage remodels the MITF interactome to increase melanoma genomic instability.

Since genome instability can drive cancer initiation and progression, cells have evolved highly effective and ubiquitous DNA damage response (DDR) programs. However, some cells (for example, in skin) are normally exposed to high levels of DNA-damaging agents. Whether such high-risk cells possess lineage-specific mechanisms that tailor DNA repair to the tissue remains largely unknown. Using melanoma as a model, we show here that the microphthalmia-associated transcription factor MITF, a lineage addition oncogene that coordinates many aspects of melanocyte and melanoma biology, plays a nontranscriptional role in shaping the DDR. On exposure to DNA-damaging agents, MITF is phosphorylated at S325, and its interactome is dramatically remodeled; most transcription cofactors dissociate, and instead MITF interacts with the MRE11-RAD50-NBS1 (MRN) complex. Consequently, cells with high MITF levels accumulate stalled replication forks and display defects in homologous recombination-mediated repair associated with impaired MRN recruitment to DNA damage. In agreement with this, high MITF levels are associated with increased single-nucleotide and copy number variant burdens in melanoma. Significantly, the SUMOylation-defective MITF-E318K melanoma predisposition mutation recapitulates the effects of DNA-PKcs-phosphorylated MITF. Our data suggest that a nontranscriptional function of a lineage-restricted transcription factor contributes to a tissue-specialized modulation of the DDR that can impact cancer initiation.

A Surveillance Mechanism Ensures Repair of DNA Lesions during Zygotic Reprogramming.

Sexual reproduction culminates in a totipotent zygote with the potential to produce a whole organism. Sperm chromatin reorganization and epigenetic reprogramming that alter DNA and histone modifications generate a totipotent embryo. Active DNA demethylation of the paternal genome has been proposed to involve base excision and DNA repair-based mechanisms. The nature and consequence of DNA lesions generated during reprogramming are not known. Using mouse genetics and chemical biology, we discovered that Tet3-dependent zygotic reprogramming generates paternal DNA lesions that are monitored by a surveillance mechanism. In vivo structure-function rescue assays revealed that cohesin-dependent repair of paternal DNA lesions prevents activation of a Chk1-dependent checkpoint that delays mitotic entry. Culturing conditions affect checkpoint stringency, which has implications for human in vitro fertilization. We propose the zygotic checkpoint senses DNA lesions generated during paternal DNA demethylation and ensures reprogrammed loci are repaired before mitosis to prevent chromosome fragmentation, embryo loss, and infertility.

Combining targeted DNA repair inhibition and immune-oncology approaches for enhanced tumor control.

Targeted therapy and immunotherapy have revolutionized cancer treatment. However, the ability of cancer to evade the immune system remains a major barrier for effective treatment. Related to this, several targeted DNA-damage response inhibitors (DDRis) are being tested in the clinic and have been shown to potentiate anti-tumor immune responses. Seminal studies have shown that these agents are highly effective in a pan-cancer class of tumors with genetic defects in key DNA repair genes such as BRCA1/2, BRCA-related genes, ataxia telangiectasia mutated (ATM), and others. Here, we review the molecular consequences of targeted DDR inhibition, from tumor cell death to increased engagement of the anti-tumor immune response. Additionally, we discuss mechanistic and clinical rationale for pairing targeted DDRis with immunotherapy for enhanced tumor control. We also review biomarkers for patient selection and promising new immunotherapy approaches poised to form the foundation of next-generation DDRi and immunotherapy combinations.

Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD.

Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.

IKKα Kinase Regulates the DNA Damage Response and Drives Chemo-resistance in Cancer.

Phosphorylated IKKα(p45) is a nuclear active form of the IKKα kinase that is induced by the MAP kinases BRAF and TAK1 and promotes tumor growth independent of canonical NF-κB signaling. Insights into the sources of IKKα(p45) activation and its downstream substrates in the nucleus remain to be defined. Here, we discover that IKKα(p45) is rapidly activated by DNA damage independent of ATM-ATR, but dependent on BRAF-TAK1-p38-MAPK, and is required for robust ATM activation and efficient DNA repair. Abolishing BRAF or IKKα activity attenuates ATM, Chk1, MDC1, Kap1, and 53BP1 phosphorylation, compromises 53BP1 and RIF1 co-recruitment to sites of DNA lesions, and inhibits 53BP1-dependent fusion of dysfunctional telomeres. Furthermore, IKKα or BRAF inhibition synergistically enhances the therapeutic potential of 5-FU and irinotecan to eradicate chemotherapy-resistant metastatic human tumors in vivo. Our results implicate BRAF and IKKα kinases in the DDR and reveal a combination strategy for cancer treatment.

C1QBP Promotes Homologous Recombination by Stabilizing MRE11 and Controlling the Assembly and Activation of MRE11/RAD50/NBS1 Complex.

MRE11 nuclease forms a trimeric complex (MRN) with RAD50 and NBS1 and plays a central role in preventing genomic instability. When DNA double-strand breaks (DSBs) occur, MRN is quickly recruited to the damage site and initiates DNA end resection; accordingly, MRE11 must be tightly regulated to avoid inefficient repair or nonspecific resection. Here, we show that MRE11 and RAD50 form a complex (MRC) with C1QBP, which stabilizes MRE11/RAD50, while inhibiting MRE11 nuclease activity by preventing its binding to DNA or chromatin. Upon DNA damage, ATM phosphorylates MRE11-S676/S678 to quickly dissociate the MRC complex. Either excess or insufficient C1QBP impedes the recruitment of MRE11 to DSBs and impairs the DNA damage response. C1QBP is highly expressed in breast cancer and positively correlates with MRE11 expression, and the inhibition of C1QBP enhances tumor regression with chemotherapy. By influencing MRE11 at multiple levels, C1QBP is, thus, an important player in the DNA damage response.

DOT1L-mediated RAP80 methylation promotes BRCA1 recruitment to elicit DNA repair.

Breast Cancer Type 1 Susceptibility Protein (BRCA1) is a tumor-suppressor protein that regulates various cellular pathways, including those that are essential for preserving genome stability. One essential mechanism involves a BRCA1-A complex that is recruited to double-strand breaks (DSBs) by RAP80 before initiating DNA damage repair (DDR). How RAP80 itself is recruited to DNA damage sites, however, is unclear. Here, we demonstrate an intrinsic correlation between a methyltransferase DOT1L-mediated RAP80 methylation and BRCA1-A complex chromatin recruitment that occurs during cancer cell radiotherapy resistance. Mechanistically, DOT1L is quickly recruited onto chromatin and methylates RAP80 at multiple lysines in response to DNA damage. Methylated RAP80 is then indispensable for binding to ubiquitinated H2A and subsequently triggering BRCA1-A complex recruitment onto DSBs. Importantly, DOT1L-catalyzed RAP80 methylation and recruitment of BRCA1 have clinical relevance, as inhibition of DOT1L or RAP80 methylation seems to enhance the radiosensitivity of cancer cells both in vivo and in vitro. These data reveal a crucial role for DOT1L in DDR through initiating recruitment of RAP80 and BRCA1 onto chromatin and underscore a therapeutic strategy based on targeting DOT1L to overcome tumor radiotherapy resistance.

BRCA1/BARD1 is a nucleosome reader and writer.

Mutations in BRCA1 and BARD1 predispose carriers to breast and ovarian cancers. The BRCA1 and BARD1 proteins form a heterodimeric complex (BRCA1/BARD1) that regulates many biological processes, including transcription and DNA double-stranded break repair. These functions are mediated by the only known enzymatic activity of BRCA1/BARD1 in its capacity as an E3 ubiquitin ligase and its role as a central hub for many large protein complexes. But the mechanisms by which BRCA1/BARD1 interfaces with chromatin, where it exerts its major functions, have remained unknown. Here, we review recent advancements in structural and cellular biology that have provided critical insights into how BRCA1/BARD1 serves as both a nucleosome reader and writer to facilitate transcriptional regulation and DNA repair by homologous recombination.

Loss of ribonuclease DIS3 hampers genome integrity in myeloma by disrupting DNA:RNA hybrid metabolism.

The ribonuclease DIS3 is one of the most frequently mutated genes in the hematological cancer multiple myeloma, yet the basis of its tumor suppressor function in this disease remains unclear. Herein, exploiting the TCGA dataset, we found that DIS3 plays a prominent role in the DNA damage response. DIS3 inactivation causes genomic instability by increasing mutational load, and a pervasive accumulation of DNA:RNA hybrids that induces genomic DNA double-strand breaks (DSBs). DNA:RNA hybrid accumulation also prevents binding of the homologous recombination (HR) machinery to double-strand breaks, hampering DSB repair. DIS3-inactivated cells become sensitive to PARP inhibitors, suggestive of a defect in homologous recombination repair. Accordingly, multiple myeloma patient cells mutated for DIS3 harbor an increased mutational burden and a pervasive overexpression of pro-inflammatory interferon, correlating with the accumulation of DNA:RNA hybrids. We propose DIS3 loss in myeloma to be a driving force for tumorigenesis via DNA:RNA hybrid-dependent enhanced genome instability and increased mutational rate. At the same time, DIS3 loss represents a liability that might be therapeutically exploited in patients whose cancer cells harbor DIS3 mutations.

Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease.

DExD/H-box RNA helicases (DDX/DHX) are encoded by a large paralogous gene family; in a subset of these human helicase genes, pathogenic variation causes neurodevelopmental disorder (NDD) traits and cancer. DHX9 encodes a BRCA1-interacting nuclear helicase regulating transcription, R-loops, and homologous recombination and exhibits the highest mutational constraint of all DDX/DHX paralogs but remains unassociated with disease traits in OMIM. Using exome sequencing and family-based rare-variant analyses, we identified 20 individuals with de novo, ultra-rare, heterozygous missense or loss-of-function (LoF) DHX9 variant alleles. Phenotypes ranged from NDDs to the distal symmetric polyneuropathy axonal Charcot-Marie-Tooth disease (CMT2). Quantitative Human Phenotype Ontology (HPO) analysis demonstrated genotype-phenotype correlations with LoF variants causing mild NDD phenotypes and nuclear localization signal (NLS) missense variants causing severe NDD. We investigated DHX9 variant-associated cellular phenotypes in human cell lines. Whereas wild-type DHX9 was restricted to the nucleus, NLS missense variants abnormally accumulated in the cytoplasm. Fibroblasts from an individual with an NLS variant also showed abnormal cytoplasmic DHX9 accumulation. CMT2-associated missense variants caused aberrant nucleolar DHX9 accumulation, a phenomenon previously associated with cellular stress. Two NDD-associated variants, p.Gly411Glu and p.Arg761Gln, altered DHX9 ATPase activity. The severe NDD-associated variant p.Arg141Gln did not affect DHX9 localization but instead increased R-loop levels and double-stranded DNA breaks. Dhx9-/- mice exhibited hypoactivity in novel environments, tremor, and sensorineural hearing loss. All together, these results establish DHX9 as a critical regulator of mammalian neurodevelopment and neuronal homeostasis.

SPIN1 facilitates chemoresistance and HR repair by promoting Tip60 binding to H3K9me3.

The tandem Tudor-like domain-containing protein Spindlin1 (SPIN1) is a transcriptional coactivator with critical functions in embryonic development and emerging roles in cancer. However, the involvement of SPIN1 in DNA damage repair has remained unclear. Our study shows that SPIN1 is recruited to DNA lesions through its N-terminal disordered region that binds to Poly-ADP-ribose (PAR), and facilitates homologous recombination (HR)-mediated DNA damage repair. SPIN1 promotes H3K9me3 accumulation at DNA damage sites and enhances the interaction between H3K9me3 and Tip60, thereby promoting the activation of ATM and HR repair. We also show that SPIN1 increases chemoresistance. These findings reveal a novel role for SPIN1 in the activation of H3K9me3-dependent DNA repair pathways, and suggest that SPIN1 may contribute to cancer chemoresistance by modulating the efficiency of double-strand break (DSB) repair.

The interplay of homology-directed repair pathways in the repair of zebularine-induced DNA-protein crosslinks in Arabidopsis.

DNA-protein crosslinks (DPCs) are highly toxic DNA lesions represented by proteins covalently bound to the DNA. Persisting DPCs interfere with fundamental genetic processes such as DNA replication and transcription. Cytidine analog zebularine (ZEB) has been shown to crosslink DNA METHYLTRANSFERASE1 (MET1). Recently, we uncovered a critical role of the SMC5/6-mediated SUMOylation in the repair of DPCs. In an ongoing genetic screen, we identified two additional candidates, HYPERSENSITIVE TO ZEBULARINE 2 and 3, that were mapped to REGULATOR OF TELOMERE ELONGATION 1 (RTEL1) and polymerase TEBICHI (TEB), respectively. By monitoring the growth of hze2 and hze3 plants in response to zebularine, we show the importance of homologous recombination (HR) factor RTEL1 and microhomology-mediated end-joining (MMEJ) polymerase TEB in the repair of MET1-DPCs. Moreover, genetic interaction and sensitivity assays showed the interdependency of SMC5/6 complex, HR, and MMEJ in the homology-directed repair of MET1-DPCs in Arabidopsis. Altogether, we provide evidence that MET1-DPC repair in plants is more complex than originally expected.

NSE5 subunit interacts with distant regions of the SMC arms in the Physcomitrium patens SMC5/6 complex.

Structural maintenance of chromosome (SMC) complexes play roles in cohesion, condensation, replication, transcription, and DNA repair. Their cores are composed of SMC proteins with a unique structure consisting of an ATPase head, long arm, and hinge. SMC complexes form long rod-like structures, which can change to ring-like and elbow-bent conformations upon binding ATP, DNA, and other regulatory factors. These SMC dynamic conformational changes are involved in their loading, translocation, and DNA loop extrusion. Here, we examined the binding and role of the PpNSE5 regulatory factor of Physcomitrium patens PpSMC5/6 complex. We found that the PpNSE5 C-terminal half (aa230-505) is required for binding to its PpNSE6 partner, while the N-terminal half (aa1-230) binds PpSMC subunits. Specifically, the first 71 amino acids of PpNSE5 were required for binding to PpSMC6. Interestingly, the PpNSE5 binding required the PpSMC6 head-proximal joint region and PpSMC5 hinge-proximal arm, suggesting a long distance between binding sites on PpSMC5 and PpSMC6 arms. Therefore, we hypothesize that PpNSE5 either links two antiparallel SMC5/6 complexes or binds one SMC5/6 in elbow-bent conformation, the later model being consistent with the role of NSE5/NSE6 dimer as SMC5/6 loading factor to DNA lesions. In addition, we generated the P. patens Ppnse5KO1 mutant line with an N-terminally truncated version of PpNSE5, which exhibited DNA repair defects while keeping a normal number of rDNA repeats. As the first 71 amino acids of PpNSE5 are required for PpSMC6 binding, our results suggest the role of PpNSE5-PpSMC6 interaction in SMC5/6 loading to DNA lesions.

Co-phase separation of Y14 and RNA in vitro and its implication for DNA repair.

The multifunctional RNA recognition motif-containing protein Y14/RBM8A participates in mRNA metabolism and is essential for the efficient repair of DNA double-strand breaks (DSBs). Y14 contains highly charged, low-complexity sequences in both the amino- and carboxy-terminal domains. The feature of charge segregation suggests that Y14 may undergo liquid-liquid phase separation (LLPS). Recombinant Y14 formed phase-separated droplets, which were sensitive to pH and salt concentration. Domain mapping suggested that LLPS of Y14 involves multivalent electrostatic interactions and is partly determined by the net charge of its low-complexity regions. Phospho-mimicry of the carboxy-terminal arginine-serine dipeptides of Y14 suppressed phase separation. Moreover, RNA could phase separate into Y14 droplets and modulate Y14 LLPS in a concentration-dependent manner. Finally, the capacity of Y14 in LLPS and coacervation with RNA in vitro correlated with its activity in DSB repair. These results reveal a molecular rule for LLPS of Y14 in vitro and an implication for its co-condensation with RNA in genome stability.

Systematic investigation of the homology sequences around the human fusion gene breakpoints in pan-cancer - bioinformatics study for a potential link to MMEJ.

Microhomology-mediated end joining (MMEJ), an error-prone DNA damage repair mechanism, frequently leads to chromosomal rearrangements due to its ability to engage in promiscuous end joining of genomic instability and also leads to increasing mutational load at the sequences flanking the breakpoints (BPs). In this study, we systematically investigated the homology sequences around the genomic breakpoint area of human fusion genes, which were formed by the chromosomal rearrangements initiated by DNA double-strand breakage. Since the RNA-seq data is the typical data set to check the fusion genes, for the known exon junction fusion breakpoints identified from RNA-seq data, we have to infer the high chance of genomic breakpoint regions. For this, we utilized the high feature importance score area calculated from our recently developed fusion BP prediction model, FusionAI and identified 151 K microhomologies among ~24 K fusion BPs in 20 K fusion genes. From our multiple bioinformatics studies, we found a relationship between sequence homologies and the immune system. This in-silico study will provide novel knowledge on the sequence homologies around the coded structural variants.

FBL promotes cancer cell resistance to DNA damage and BRCA1 transcription via YBX1.

Fibrillarin (FBL) is a highly conserved nucleolar methyltransferase responsible for methylation of ribosomal RNA and proteins. Here, we reveal a role for FBL in DNA damage response and its impact on cancer proliferation and sensitivity to DNA-damaging agents. FBL is highly expressed in various cancers and correlates with poor survival outcomes in cancer patients. Knockdown of FBL sensitizes tumor cells and xenografts to DNA crosslinking agents, and leads to homologous recombination-mediated DNA repair defects. We identify Y-box-binding protein-1 (YBX1) as a key interacting partner of FBL, and FBL increases the nuclear accumulation of YBX1 in response to DNA damage. We show that FBL promotes the expression of BRCA1 by increasing the binding of YBX1 to the BRCA1 promoter. Our study sheds light on the regulatory mechanism of FBL in tumorigenesis and DNA damage response, providing potential therapeutic targets to overcome chemoresistance in cancer.