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Enzymes of the TET family are methylcytosine dioxygenases that undergo frequent mutational or functional inactivation in human cancers. Recurrent loss-of-function mutations in TET proteins are frequent in human diffuse large B cell lymphoma (DLBCL). Here, we investigate the role of TET proteins in B cell homeostasis and development of B cell lymphomas with features of DLBCL. We show that deletion of Tet2 and Tet3 genes in mature B cells in mice perturbs B cell homeostasis and results in spontaneous development of germinal center (GC)-derived B cell lymphomas with increased G-quadruplexes and R-loops. At a genome-wide level, G-quadruplexes and R-loops were associated with increased DNA double-strand breaks (DSBs) at immunoglobulin switch regions. Deletion of the DNA methyltransferase DNMT1 in TET-deficient B cells prevented expansion of GC B cells, diminished the accumulation of G-quadruplexes and R-loops and delayed B lymphoma development, consistent with the opposing functions of DNMT and TET enzymes in DNA methylation and demethylation. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated depletion of nucleases and helicases that regulate G-quadruplexes and R-loops decreased the viability of TET-deficient B cells. Our studies suggest a molecular mechanism by which TET loss of function might predispose to the development of B cell malignancies.Shukla, Samaniego-Castruita and colleagues show that loss of TET methylcytosine dioxygenases in B cells is associated with increased DNA–RNA hybrids and G-quadruplex DNA structures in parallel with genomic instability and development of germinal center-derived lymphomas.

R-loops are RNA–DNA-hybrid-containing nucleic acids with important cellular roles. Deregulation of R-loop dynamics can lead to DNA damage and genome instability 1 , which has been linked to the action of endonucleases such as XPG 2 – 4 . However, the mechanisms and cellular consequences of such processing have remained unclear. Here we identify a new population of RNA–DNA hybrids in the cytoplasm that are R-loop-processing products. When nuclear R-loops were perturbed by depleting the RNA–DNA helicase senataxin ( SETX ) or the breast cancer gene BRCA1  (refs. 5 – 7 ), we observed XPG- and XPF-dependent cytoplasmic hybrid formation. We identify their source as a subset of stable, overlapping nuclear hybrids with a specific nucleotide signature. Cytoplasmic hybrids bind to the pattern recognition receptors cGAS and TLR3 (ref.  8 ), activating IRF3 and inducing apoptosis. Excised hybrids and an R-loop-induced innate immune response were also observed in SETX -mutated cells from patients with ataxia oculomotor apraxia type 2 (ref.  9 ) and in BRCA1 -mutated cancer cells 10 . These findings establish RNA–DNA hybrids as immunogenic species that aberrantly accumulate in the cytoplasm after R-loop processing, linking R-loop accumulation to cell death through the innate immune response. Aberrant R-loop processing and subsequent innate immune activation may contribute to many diseases, such as neurodegeneration and cancer. RNA–DNA hybrids are immunogenic species that can aberrantly accumulate in the cytoplasm after R-loop processing, linking R-loop accumulation to cell death through the innate immune response.

R-loops are trimeric nucleic acid structures that form when an RNA molecule hybridizes with its complementary DNA strand, displacing the opposite strand. These structures regulate transcription as well as replication, but aberrant R-loops can form, leading to DNA breaks and genomic instability if unresolved. R-loop levels are elevated in many cancers as well as cells that maintain high-risk human papillomaviruses. We investigated how the distribution as well as function of R-loops changed between normal keratinocytes and HPV positive cells derived from a precancerous lesion of the cervix (CIN I). The levels of R-loops associated with cellular genes were found to be up to 10-fold higher in HPV positive cells than in normal keratinocytes while increases at ALU1 elements increased by up to 500-fold. The presence of enhanced R-loops resulted in altered levels of gene transcription, with equal numbers increased as decreased. While no uniform global effects on transcription due to the enhanced levels of R-loops were detected, genes in several pathways were coordinately increased or decreased in expression only in the HPV positive cells. This included the downregulation of genes in the innate immune pathway, such as DDX58, IL-6, STAT1, IFN-β, and NLRP3. All differentially expressed innate immune genes dependent on R-loops were also associated with H3K36me3 modified histones. Genes that were upregulated by the presence of R-loops in HPV positive cells included those in the DNA damage repair such as ATM, ATRX, and members of the Fanconi Anemia pathway. These genes exhibited a linkage between R-loops and H3K36me3 as well as γH2AX histone marks only in HPV positive cells. These studies identify a potential link in HPV positive cells between DNA damage repair as well as innate immune regulatory pathways with R-loops and γH2AX/H3K36me3 histone marks that may contribute to regulating important functions for HPV pathogenesis.

R-loops are ubiquitous, dynamic nucleic-acid structures that play fundamental roles in DNA replication and repair, chromatin and transcription regulation, as well as telomere maintenance. The DNA-RNA hybrid–specific S9.6 monoclonal antibody is widely used to map R-loops. Here, we report crystal structures of a S9.6 antigen-binding fragment (Fab) free and bound to a 13-bp hybrid duplex. We demonstrate that S9.6 exhibits robust selectivity in binding hybrids over double-stranded (ds) RNA and in categorically rejecting dsDNA. S9.6 asymmetrically recognizes a compact epitope of two consecutive RNA nucleotides via their 2′-hydroxyl groups and six consecutive DNA nucleotides via their backbone phosphate and deoxyribose groups. Recognition is mediated principally by aromatic and basic residues of the S9.6 heavy chain, which closely track the curvature of the hybrid minor groove. These findings reveal the molecular basis for S9.6 recognition of R-loops, detail its binding specificity, identify a new hybrid-recognition strategy, and provide a framework for S9.6 protein engineering. The S9.6 monoclonal antibody is widely used to map R-loops genome wide. Here, Bou-Nader et al., define the nucleic acid-binding specificity of S9.6 and report its crystal structures free and bound to a hybrid, which reveal the asymmetric recognition of the RNA and DNA strands and its A-form conformation.

Centromeres are defined by chromatin containing the histone H3 variant CENP-A assembled onto repetitive α-satellite sequences, which are actively transcribed throughout the cell cycle. Centromeres play an essential role in chromosome inheritance and genome stability through coordinating kinetochores assembly during mitosis. Structural and functional alterations of the centromeres cause aneuploidy and chromosome aberrations which can induce cell death. In human cells, the tumor suppressor BRCA1 associates with centromeric chromatin in the absence of exogenous damage. While we previously reported that BRCA1 contributes to proper centromere homeostasis, the mechanism underlying its centromeric function and recruitment was not fully understood. Here, we show that BRCA1 association with centromeric chromatin depends on the presence of R-loops, which are non-canonical three-stranded structures harboring a DNA:RNA hybrid and are frequently formed during transcription. Subsequently, BRCA1 counteracts the accumulation of R-loops at centromeric α-satellite repeats. Strikingly, BRCA1-deficient cells show impaired localization of CENP-A, higher transcription of centromeric RNA, increased breakage at centromeres and formation of acentric micronuclei, all these features being R-loop-dependent. Finally, BRCA1 depletion reveals a Rad52-dependent hyper-recombination process between centromeric satellite repeats, associated with centromere instability and missegregation. Altogether, our findings provide molecular insights into the key function of BRCA1 in maintaining centromere stability and identity.

The cytidine deaminase, Apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B, herein termed A3B), is a critical mutation driver that induces genomic instability in cancer by catalyzing cytosine-to-thymine (C-to-T) conversion and promoting replication stress (RS). However, the detailed function of A3B in RS is not fully determined and it is not known whether the mechanism of A3B action can be exploited for cancer therapy. Here, we conducted an immunoprecipitation-mass spectrometry (IP-MS) study and identified A3B to be a novel binding component of R-loops, which are RNA:DNA hybrid structures. Mechanistically, overexpression of A3B exacerbated RS by promoting R-loop formation and altering the distribution of R-loops in the genome. This was rescued by the R-loop gatekeeper, Ribonuclease H1 (RNASEH1, herein termed RNH1). In addition, a high level of A3B conferred sensitivity to ATR/Chk1 inhibitors (ATRi/Chk1i) in melanoma cells, which was dependent on R-loop status. Together, our results provide novel insights into the mechanistic link between A3B and R-loops in the promotion of RS in cancer. This will inform the development of markers to predict the response of patients to ATRi/Chk1i.

R-loops, which are noncanonical three-stranded nucleic acid structures formed when RNA hybridizes with complementary DNA strand while displacing the other DNA strand, have emerged as crucial players in cellular homeostasis and cancer pathogenesis. Here we explore the intricate relationship between R-loops and inflammation in the context of cancer development and progression. R-loops can trigger inflammatory responses through various mechanisms, including DNA damage induction, genome instability and activation of innate immune pathways, particularly in cancer cells, where R-loop regulation is frequently dysregulated. In the tumor microenvironment, R-loop-mediated genomic instability contributes to inflammatory signaling cascades, affecting both cancer cells and the surrounding tumor microenvironment. We discuss how aberrant R-loop formation influences key inflammatory pathways, including the cGAS–STING axis and NF-κB signaling, and their subsequent effects on tumor progression. Furthermore, we explored how cancer cells manipulate R-loops to modify their inflammatory microenvironment, potentially affecting their therapeutic responses. Understanding the complex interplay between R-loops and cancer-associated inflammation provides novel insights into tumor biology and opens new avenues for therapeutic intervention. This Review summarizes the current knowledge on R-loop biology in cancer, its inflammatory consequences and potential strategies for targeting R-loop-mediated inflammation in cancer treatment, underscoring the importance of this emerging field in cancer medicine. R-loops drive inflammation and cancer progression insights Cells constantly undergo changes, and one such change involves R-loops. R-loops are three-stranded structures formed when RNA binds to DNA, leaving one DNA strand single. While R-loops are normal and help in various cell functions, their imbalance can cause problems such as cancer. This Review explores how R-loops can both promote and fight cancer. Researchers explored how R-loops affect cancer by either causing instability in the genome or triggering immune responses. They used various methods to study these effects, focusing on how R-loops interact with immune pathways such as cGAS–STING, which helps the body in fighting tumors. The study found that, while excessive R-loops can lead to cancer by causing DNA damage, they can also activate immune responses that help to fight cancer. The researchers suggest that targeting R-loop pathways could be a new way to treat cancer. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

Replicative stress (RS) is emerging as a promising therapeutic target in oncology, yet full exploitation of its potential requires a detailed understanding of the mechanisms and genes involved. Here, we investigated the RNA helicase Senataxin (SETX), an enzyme that resolves RNA-DNA hybrids and R-loops, to address its role in preventing RS by oncogenic Myc. Upon Myc activation, silencing of SETX led to selective engagement of the DNA damage response (DDR) and robust cytotoxicity. Pharmacological dissection of the upstream kinases regulating the DDR uncovered a protective role of the ATR pathway, that once inhibited, boosted SETX driven-DDR. While SETX loss did not lead to a genome-wide increase of R-loops, mechanistic analyses revealed enhanced R-loops localized at DDR-foci and newly replicated genomic loci, compatible with a selective role of SETX in resolving RNA-DNA hybrids to alleviate Myc-induced RS. Genome-wide mapping of DNA double-strand breaks confirmed that SETX silencing exacerbated DNA damage at transcription-replication conflict (TRC) regions at early replicated sites. We propose that SETX prevents Myc-induced TRCs by resolving transcription-associated R-loops that encounter the replisome. The identification of SETX as a genetic liability of oncogenic Myc opens up new therapeutic options against aggressive Myc-driven tumors.

This study used DNA methyltransferase 3b (DNMT3b) knockout cells and the functional loss of DNMT3b mutation in immunodeficiency-centromeric instability-facial anomalies syndrome (ICF) cells to understand how DNMT3b dysfunction causes genome instability. We demonstrated that R-loops contribute to DNA damages in DNMT3b knockout and ICF cells. More prominent DNA damage signal in DNMT3b knockout cells was due to the loss of DNMT3b expression and the acquirement of p53 mutation. Genome-wide ChIP-sequencing mapped DNA damage sites at satellite repetitive DNA sequences including (peri-)centromere regions. However, the steady-state levels of (peri-)centromeric R-loops were reduced in DNMT3b knockout and ICF cells. Our analysis indicates that XPG and XPF endonucleases-mediated cleavages remove (peri-)centromeric R-loops to generate DNA beaks, causing chromosome instability. DNMT3b dysfunctions clearly increase R-loops susceptibility to the cleavage process. Finally, we showed that DNA double-strand breaks (DSBs) in centromere are probably repaired by error-prone end-joining pathway in ICF cells. Thus, DNMT3 dysfunctions undermine the integrity of centromere by R-loop-mediated DNA damages and repair.

Genomic instability is a hallmark of cancer, encompassing both sequence and structural alterations that drive tumor evolution and heterogeneity. The APOBEC3 family of deoxycytidine deaminases has emerged as a major source of mutagenic activity in cancers. R-loops are RNA-DNA hybrids and structural barriers that interfere with replication and transcription. Among the APOBEC3 family, APOBEC3C (A3C) is particularly worthy of attention for its upregulation, driving the DNA replication stress tolerance in response to replication stress-inducing drug gemcitabine. However, the molecular mechanisms of gemcitabine resistance and regulatory circuitries mediated by A3C remain largely unknown, especially in checkpoint-deficient tumors. Initially, we screened that A3C was a putative transcriptional target of p53, and p53-deficient H1299 cells harboring A3C elicited a chemoresistant phenotype upon gemcitabine treatment both in vitro and in vivo. A3C expression enhanced Chk1-dependent S-phase checkpoint activation, thus slowing down replication fork progression and facilitating DNA repair. Pull-down assay and proteomic analysis identified that A3C had a specific interaction with the RNA helicase DDX5, which coordinately played critical roles in R-loop resolution. In contrast to A3C, DDX5 expression attenuated Chk1-dependent S-phase checkpoint activation. Knockdown of DDX5 in A3C-proficient H1299 cells attenuated gemcitabine-induced Chk1 activation and enhanced the therapeutic index of gemcitabine by promoting R-loop accumulation. Therefore, we conclude that A3C/DDX5/R-loop complex may impair the sensitivity of gemcitabine by modulating Chk1 dynamics and DNA replication/damage response machinery.