The immediate-early protein 1 of human herpesvirus 6B interacts with NBS1 and inhibits ATM signaling.

Viral infection often trigger an ATM serine/threonine kinase (ATM)-dependent DNA damage response in host cells that suppresses viral replication. Viruses evolved different strategies to counteract this antiviral surveillance system. Here, we report that human herpesvirus 6B (HHV-6B) infection causes genomic instability by suppressing ATM signaling in host cells. Expression of immediate-early protein 1 (IE1) phenocopies this phenotype and blocks homology-directed double-strand break repair. Mechanistically, IE1 interacts with NBS1, and inhibits ATM signaling through two distinct domains. HHV-6B seems to efficiently inhibit ATM signaling as further depletion of either NBS1 or ATM do not significantly boost viral replication in infected cells. Interestingly, viral integration of HHV-6B into the host's telomeres is not strictly dependent on NBS1, challenging current models where integration occurs through homology-directed repair. Given that spontaneous IE1 expression has been detected in cells of subjects with inherited chromosomally-integrated form of HHV-6B (iciHHV-6B), a condition associated with several health conditions, our results raise the possibility of a link between genomic instability and the development of iciHHV-6-associated diseases.

FIRRM cooperates with FIGNL1 to promote RAD51 disassembly during DNA repair.

Interstrand DNA cross-links (ICLs) represent complex lesions that compromise genomic stability. Several pathways have been involved in ICL repair, but the extent of factors involved in the resolution of ICL-induced DNA double-strand breaks (DSBs) remains poorly defined. Using CRISPR-based genomics, we identified FIGNL1 interacting regulator of recombination and mitosis (FIRRM) as a sensitizer of the ICL-inducing agent mafosfamide. Mechanistically, we showed that FIRRM, like its interactor Fidgetin like 1 (FIGNL1), contributes to the resolution of RAD51 foci at ICL-induced DSBs. While the stability of FIGNL1 and FIRRM is interdependent, expression of a mutant of FIRRM (∆WCF), which stabilizes the protein in the absence of FIGNL1, allows the resolution of RAD51 foci and cell survival, suggesting that FIRRM has FIGNL1-independent function during DNA repair. In line with this model, FIRRM binds preferentially single-stranded DNA in vitro, raising the possibility that it directly contributes to RAD51 disassembly by interacting with DNA. Together, our findings establish FIRRM as a promoting factor of ICL repair.

FIRRM/C1orf112 is synthetic lethal with PICH and mediates RAD51 dynamics.

Joint DNA molecules are natural byproducts of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, compromising sister chromatid separation. The DNA translocase PICH (ERCC6L) has a central role in UFB resolution. A genome-wide loss-of-function screen is performed to identify the genetic context of PICH dependency. In addition to genes involved in DNA condensation, centromere stability, and DNA-damage repair, we identify FIGNL1-interacting regulator of recombination and mitosis (FIRRM), formerly known as C1orf112. We find that FIRRM interacts with and stabilizes the AAA+ ATPase FIGNL1. Inactivation of either FIRRM or FIGNL1 results in UFB formation, prolonged accumulation of RAD51 at nuclear foci, and impaired replication fork dynamics and consequently impairs genome maintenance. Combined, our data suggest that inactivation of FIRRM and FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in persistent DNA lesions and a dependency on PICH to preserve cell viability.

The TUDOR domain of SMN is an H3K79me1 histone mark reader.

Spinal muscular atrophy is the leading genetic cause of infant mortality and results from depleted levels of functional survival of motor neuron (SMN) protein by either deletion or mutation of the SMN1 gene. SMN is characterized by a central TUDOR domain, which mediates the association of SMN with arginine methylated (Rme) partners, such as coilin, fibrillarin, and RNA pol II (RNA polymerase II). Herein, we biochemically demonstrate that SMN also associates with histone H3 monomethylated on lysine 79 (H3K79me1), defining SMN as not only the first protein known to associate with the H3K79me1 histone modification but also the first histone mark reader to recognize both methylated arginine and lysine residues. Mutational analyzes provide evidence that SMNTUDOR associates with H3 via an aromatic cage. Importantly, most SMNTUDOR mutants found in spinal muscular atrophy patients fail to associate with H3K79me1.

MORF and MOZ acetyltransferases target unmethylated CpG islands through the winged helix domain.

Human acetyltransferases MOZ and MORF are implicated in chromosomal translocations associated with aggressive leukemias. Oncogenic translocations involve the far amino terminus of MOZ/MORF, the function of which remains unclear. Here, we identified and characterized two structured winged helix (WH) domains, WH1 and WH2, in MORF and MOZ. WHs bind DNA in a cooperative manner, with WH1 specifically recognizing unmethylated CpG sequences. Structural and genomic analyses show that the DNA binding function of WHs targets MORF/MOZ to gene promoters, stimulating transcription and H3K23 acetylation, and WH1 recruits oncogenic fusions to HOXA genes that trigger leukemogenesis. Cryo-EM, NMR, mass spectrometry and mutagenesis studies provide mechanistic insight into the DNA-binding mechanism, which includes the association of WH1 with the CpG-containing linker DNA and binding of WH2 to the dyad of the nucleosome. The discovery of WHs in MORF and MOZ and their DNA binding functions could open an avenue in developing therapeutics to treat diseases associated with aberrant MOZ/MORF acetyltransferase activities.

A CRISPR-Cas9 screen identifies EXO1 as a formaldehyde resistance gene.

Fanconi Anemia (FA) is a rare, genome instability-associated disease characterized by a deficiency in repairing DNA crosslinks, which are known to perturb several cellular processes, including DNA transcription, replication, and repair. Formaldehyde, a by-product of metabolism, is thought to drive FA by generating DNA interstrand crosslinks (ICLs) and DNA-protein crosslinks (DPCs). However, the impact of formaldehyde on global cellular pathways has not been investigated thoroughly. Herein, using a pangenomic CRISPR-Cas9 screen, we identify EXO1 as a critical regulator of formaldehyde-induced DNA lesions. We show that EXO1 knockout cell lines exhibit formaldehyde sensitivity leading to the accumulation of replicative stress, DNA double-strand breaks, and quadriradial chromosomes, a typical feature of FA. After formaldehyde exposure, EXO1 is recruited to chromatin, protects DNA replication forks from degradation, and functions in parallel with the FA pathway to promote cell survival. In vitro, EXO1-mediated exonuclease activity is proficient in removing DPCs. Collectively, we show that EXO1 limits replication stress and DNA damage to counteract formaldehyde-induced genome instability.

Oncogenic ZMYND11-MBTD1 fusion protein anchors the NuA4/TIP60 histone acetyltransferase complex to the coding region of active genes.

A recurrent chromosomal translocation found in acute myeloid leukemia leads to an in-frame fusion of the transcription repressor ZMYND11 to MBTD1, a subunit of the NuA4/TIP60 histone acetyltransferase complex. To understand the abnormal molecular events that ZMYND11-MBTD1 expression can create, we perform a biochemical and functional characterization comparison to each individual fusion partner. ZMYND11-MBTD1 is stably incorporated into the endogenous NuA4/TIP60 complex, leading to its mislocalization on the body of genes normally bound by ZMYND11. This can be correlated to increased chromatin acetylation and altered gene transcription, most notably on the MYC oncogene, and alternative splicing. Importantly, ZMYND11-MBTD1 expression favors Myc-driven pluripotency during embryonic stem cell differentiation and self-renewal of hematopoietic stem/progenitor cells. Altogether, these results indicate that the ZMYND11-MBTD1 fusion functions primarily by mistargeting the NuA4/TIP60 complex to the body of genes, altering normal transcription of specific genes, likely driving oncogenesis in part through the Myc regulatory network.

Comprehensive Interactome Mapping of Nuclear Receptors Using Proximity Biotinylation.

Nuclear receptors, including hormone receptors, perform their cellular activities by modulating their protein-protein interactions. They engage with specific ligands and translocate to the nucleus, where they bind the DNA and activate extensive transcriptional programs. Therefore, gaining a comprehensive overview of the protein-protein interactions they establish requires methods that function effectively throughout the cell with fast dynamics and high reproducibility. Focusing on estrogen receptor alpha (ESR1), the founding member of the nuclear receptor family, this chapter describes a new lentiviral system that allows the expression of TurboID-hemagglutinin (HA)-2 × Strep tagged proteins in mammalian cells to perform fast proximity biotinylation assays. Key validation steps for these reagents and their use in interactome mapping experiments in two distinct breast cancer cell lines are described. Our protocol enabled the quantification of ESR1 interactome generated by cellular contexts that were hormone-sensitive or not.

Two redundant ubiquitin-dependent pathways of BRCA1 localization to DNA damage sites.

The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin-dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80- and RING-dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding-deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80-BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways.

CDK1-Mediated Phosphorylation of BAG3 Promotes Mitotic Cell Shape Remodeling and the Molecular Assembly of Mitotic p62 Bodies.

The cochaperone BCL2-associated athanogene 3 (BAG3), in complex with the heat shock protein HSPB8, facilitates mitotic rounding, spindle orientation, and proper abscission of daughter cells. BAG3 and HSPB8 mitotic functions implicate the sequestosome p62/SQSTM1, suggesting a role for protein quality control. However, the interplay between this chaperone-assisted pathway and the mitotic machinery is not known. Here, we show that BAG3 phosphorylation at the conserved T285 is regulated by CDK1 and activates its function in mitotic cell shape remodeling. BAG3 phosphorylation exhibited a high dynamic at mitotic entry and both a non-phosphorylatable BAG3T285A and a phosphomimetic BAG3T285D protein were unable to correct the mitotic defects in BAG3-depleted HeLa cells. We also demonstrate that BAG3 phosphorylation, HSPB8, and CDK1 activity modulate the molecular assembly of p62/SQSTM1 into mitotic bodies containing K63 polyubiquitinated chains. These findings suggest the existence of a mitotically regulated spatial quality control mechanism for the fidelity of cell shape remodeling in highly dividing cells.

Approaches to Study Native Chromatin-Modifying Complex Activities and Functions.

The modification of histones-the structural components of chromatin-is a central topic in research efforts to understand the mechanisms regulating genome expression and stability. These modifications frequently occur through associations with multisubunit complexes, which contain active enzymes and additional components that orient their specificity and read the histone modifications that comprise epigenetic signatures. To understand the functions of these modifications it is critical to study the enzymes and substrates involved in their native contexts. Here, we describe experimental approaches to purify native chromatin modifiers complexes from mammalian cells and to produce recombinant nucleosomes that are used as substrates to determine the activity of the complex. In addition, we present a novel approach, similar to the yeast anchor-away system, to study the functions of essential chromatin modifiers by quickly inducing their depletion from the nucleus. The step-by-step protocols included will help standardize these approaches in the research community, enabling convincing conclusions about the specificities and functions of these crucial regulators of the eukaryotic genome.

ActiveDriverDB: Interpreting Genetic Variation in Human and Cancer Genomes Using Post-translational Modification Sites and Signaling Networks (2021 Update).

Deciphering the functional impact of genetic variation is required to understand phenotypic diversity and the molecular mechanisms of inherited disease and cancer. While millions of genetic variants are now mapped in genome sequencing projects, distinguishing functional variants remains a major challenge. Protein-coding variation can be interpreted using post-translational modification (PTM) sites that are core components of cellular signaling networks controlling molecular processes and pathways. ActiveDriverDB is an interactive proteo-genomics database that uses more than 260,000 experimentally detected PTM sites to predict the functional impact of genetic variation in disease, cancer and the human population. Using machine learning tools, we prioritize proteins and pathways with enriched PTM-specific amino acid substitutions that potentially rewire signaling networks via induced or disrupted short linear motifs of kinase binding. We then map these effects to site-specific protein interaction networks and drug targets. In the 2021 update, we increased the PTM datasets by nearly 50%, included glycosylation, sumoylation and succinylation as new types of PTMs, and updated the workflows to interpret inherited disease mutations. We added a recent phosphoproteomics dataset reflecting the cellular response to SARS-CoV-2 to predict the impact of human genetic variation on COVID-19 infection and disease course. Overall, we estimate that 16-21% of known amino acid substitutions affect PTM sites among pathogenic disease mutations, somatic mutations in cancer genomes and germline variants in the human population. These data underline the potential of interpreting genetic variation through the lens of PTMs and signaling networks. The open-source database is freely available at www.ActiveDriverDB.org.

Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response.

High-risk human papillomaviruses (HR-HPVs) promote cervical cancer as well as a subset of anogenital and head and neck cancers. Due to their limited coding capacity, HPVs hijack the host cell's DNA replication and repair machineries to replicate their own genomes. How this host-pathogen interaction contributes to genomic instability is unknown. Here, we report that HPV-infected cancer cells express high levels of RNF168, an E3 ubiquitin ligase that is critical for proper DNA repair following DNA double-strand breaks, and accumulate high numbers of 53BP1 nuclear bodies, a marker of genomic instability induced by replication stress. We describe a mechanism by which HPV E7 subverts the function of RNF168 at DNA double-strand breaks, providing a rationale for increased homology-directed recombination in E6/E7-expressing cervical cancer cells. By targeting a new regulatory domain of RNF168, E7 binds directly to the E3 ligase without affecting its enzymatic activity. As RNF168 knockdown impairs viral genome amplification in differentiated keratinocytes, we propose that E7 hijacks the E3 ligase to promote the viral replicative cycle. This study reveals a mechanism by which tumor viruses reshape the cellular response to DNA damage by manipulating RNF168-dependent ubiquitin signaling. Importantly, our findings reveal a pathway by which HPV may promote the genomic instability that drives oncogenesis.

The Epstein-Barr Virus BMRF1 Protein Activates Transcription and Inhibits the DNA Damage Response by Binding NuRD.

The BMRF1 protein of Epstein-Barr virus (EBV) has multiple roles in viral lytic infection, including serving as the DNA polymerase processivity factor, activating transcription from several EBV promoters and inhibiting the host DNA damage response to double-stranded DNA breaks (DSBs). Using affinity purification coupled to mass spectrometry, we identified the nucleosome remodeling and deacetylation (NuRD) complex as the top interactor of BMRF1. We further found that NuRD components localize with BMRF1 at viral replication compartments and that this interaction occurs through the BMRF1 C-terminal region previously shown to mediate transcriptional activation. We identified an RBBP4 binding motif within this region that can interact with both RBBP4 and MTA2 components of the NuRD complex and showed that point mutation of this motif abrogates NuRD binding as well as the ability of BMRF1 to activate transcription from the BDLF3 and BLLF1 EBV promoters. In addition to its role in transcriptional regulation, NuRD has been shown to contribute to DSB signaling in enabling recruitment of RNF168 ubiquitin ligase and subsequent ubiquitylation at the break. We showed that BMRF1 inhibited RNF168 recruitment and ubiquitylation at DSBs and that this inhibition was at least partly relieved by loss of the NuRD interaction. The results reveal a mechanism by which BMRF1 activates transcription and inhibits DSB signaling and a novel role for NuRD in transcriptional activation in EBV.IMPORTANCE The Epstein-Barr virus (EBV) BMRF1 protein is critical for EBV infection, playing key roles in viral genome replication, activation of EBV genes, and inhibition of host DNA damage responses (DDRs). Here we show that BMRF1 targets the cellular nucleosome remodeling and deacetylation (NuRD) complex, using a motif in the BMRF1 transcriptional activation sequence. Mutation of this motif disrupts the ability of BMRF1 to activate transcription and interfere with DDRs, showing the importance of the NuRD interaction for BMRF1 functions. BMRF1 was shown to act at the same step in the DDR as NuRD, suggesting that it interferes with NuRD function.

Virus DNA Replication and the Host DNA Damage Response.

Viral DNA genomes have limited coding capacity and therefore harness cellular factors to facilitate replication of their genomes and generate progeny virions. Studies of viruses and how they interact with cellular processes have historically provided seminal insights into basic biology and disease mechanisms. The replicative life cycles of many DNA viruses have been shown to engage components of the host DNA damage and repair machinery. Viruses have evolved numerous strategies to navigate the cellular DNA damage response. By hijacking and manipulating cellular replication and repair processes, DNA viruses can selectively harness or abrogate distinct components of the cellular machinery to complete their life cycles. Here, we highlight consequences for viral replication and host genome integrity during the dynamic interactions between virus and host.

A Screen for Epstein-Barr Virus Proteins That Inhibit the DNA Damage Response Reveals a Novel Histone Binding Protein.

To replicate and persist in human cells, linear double-stranded DNA (dsDNA) viruses, such as Epstein-Barr virus (EBV), must overcome the host DNA damage response (DDR) that is triggered by the viral genomes. Since this response is necessary to maintain cellular genome integrity, its inhibition by EBV is likely an important factor in the development of cancers associated with EBV infection, including gastric carcinoma. Here we present the first extensive screen of EBV proteins that inhibit dsDNA break signaling. We identify the BKRF4 tegument protein as a DDR inhibitor that interferes with histone ubiquitylation at dsDNA breaks and recruitment of the RNF168 histone ubiquitin ligase. We further show that BKRF4 binds directly to histones through an acidic domain that targets BKRF4 to cellular chromatin and is sufficient to inhibit dsDNA break signaling. BKRF4 transcripts were detected in EBV-positive gastric carcinoma cells (AGS-EBV), and these increased in lytic infection. Silencing of BKRF4 in both latent and lytic AGS-EBV cells (but not in EBV-negative AGS cells) resulted in increased dsDNA break signaling, confirming a role for BKRF4 in DDR inhibition in the context of EBV infection and suggesting that BKRF4 is expressed in latent cells. BKRF4 was also found to be consistently expressed in EBV-positive gastric tumors in the absence of a full lytic infection. The results suggest that BKRF4 plays a role in inhibiting the cellular DDR in latent and lytic EBV infection and that the resulting accumulation of DNA damage might contribute to development of gastric carcinoma.IMPORTANCE Epstein-Barr virus (EBV) infects most people worldwide and is causatively associated with several types of cancer, including ∼10% of gastric carcinomas. EBV encodes ∼80 proteins, many of which are believed to manipulate cellular regulatory pathways but are poorly characterized. The DNA damage response (DDR) is one such pathway that is critical for maintaining genome integrity and preventing cancer-associated mutations. In this study, a screen for EBV proteins that inhibit the DDR identified BKRF4 as a DDR inhibitor that binds histones and blocks their ubiquitylation at the DNA damage sites. We also present evidence that BKRF4 is expressed in both latent and lytic forms of EBV infection, where it downregulates the DDR, as well as in EBV-positive gastric tumors. The results suggest that BKRF4 could contribute to the development of gastric carcinoma through its ability to inhibit the DDR.

ActiveDriverDB: human disease mutations and genome variation in post-translational modification sites of proteins.

Interpretation of genetic variation is needed for deciphering genotype-phenotype associations, mechanisms of inherited disease, and cancer driver mutations. Millions of single nucleotide variants (SNVs) in human genomes are known and thousands are associated with disease. An estimated 21% of disease-associated amino acid substitutions corresponding to missense SNVs are located in protein sites of post-translational modifications (PTMs), chemical modifications of amino acids that extend protein function. ActiveDriverDB is a comprehensive human proteo-genomics database that annotates disease mutations and population variants through the lens of PTMs. We integrated >385,000 published PTM sites with ∼3.6 million substitutions from The Cancer Genome Atlas (TCGA), the ClinVar database of disease genes, and human genome sequencing projects. The database includes site-specific interaction networks of proteins, upstream enzymes such as kinases, and drugs targeting these enzymes. We also predicted network-rewiring impact of mutations by analyzing gains and losses of kinase-bound sequence motifs. ActiveDriverDB provides detailed visualization, filtering, browsing and searching options for studying PTM-associated mutations. Users can upload mutation datasets interactively and use our application programming interface in pipelines. Integrative analysis of mutations and PTMs may help decipher molecular mechanisms of phenotypes and disease, as exemplified by case studies of TP53, BRCA2 and VHL. The open-source database is available at https://www.ActiveDriverDB.org.

Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency.

Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR). However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ). We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.

The RNF168 paralog RNF169 defines a new class of ubiquitylated histone reader involved in the response to DNA damage.

Site-specific histone ubiquitylation plays a central role in orchestrating the response to DNA double-strand breaks (DSBs). DSBs elicit a cascade of events controlled by the ubiquitin ligase RNF168, which promotes the accumulation of repair factors such as 53BP1 and BRCA1 on the chromatin flanking the break site. RNF168 also promotes its own accumulation, and that of its paralog RNF169, but how they recognize ubiquitylated chromatin is unknown. Using methyl-TROSY solution NMR spectroscopy and molecular dynamics simulations, we present an atomic resolution model of human RNF169 binding to a ubiquitylated nucleosome, and validate it by electron cryomicroscopy. We establish that RNF169 binds to ubiquitylated H2A-Lys13/Lys15 in a manner that involves its canonical ubiquitin-binding helix and a pair of arginine-rich motifs that interact with the nucleosome acidic patch. This three-pronged interaction mechanism is distinct from that by which 53BP1 binds to ubiquitylated H2A-Lys15 highlighting the diversity in site-specific recognition of ubiquitylated nucleosomes.

BRCA2 functions: from DNA repair to replication fork stabilization.

Maintaining genomic integrity is essential to preserve normal cellular physiology and to prevent the emergence of several human pathologies including cancer. The breast cancer susceptibility gene 2 (BRCA2, also known as the Fanconi anemia (FA) complementation group D1 (FANCD1)) is a potent tumor suppressor that has been extensively studied in DNA double-stranded break (DSB) repair by homologous recombination (HR). However, BRCA2 participates in numerous other processes central to maintaining genome stability, including DNA replication, telomere homeostasis and cell cycle progression. Consequently, inherited mutations in BRCA2 are associated with an increased risk of breast, ovarian and pancreatic cancers. Furthermore, bi-allelic mutations in BRCA2 are linked to FA, a rare chromosome instability syndrome characterized by aplastic anemia in children as well as susceptibility to leukemia and cancer. Here, we discuss the recent developments underlying the functions of BRCA2 in the maintenance of genomic integrity. The current model places BRCA2 as a central regulator of genome stability by repairing DSBs and limiting replication stress. These findings have direct implications for the development of novel anticancer therapeutic approaches.