Genomic patterns of transcription-replication interactions in mouse primary B cells.

Conflicts between transcription and replication machinery are a potent source of replication stress and genome instability; however, no technique currently exists to identify endogenous genomic locations prone to transcription-replication interactions. Here, we report a novel method to identify genomic loci prone to transcription-replication interactions termed transcription-replication immunoprecipitation on nascent DNA sequencing, TRIPn-Seq. TRIPn-Seq employs the sequential immunoprecipitation of RNA polymerase 2 phosphorylated at serine 5 (RNAP2s5) followed by enrichment of nascent DNA previously labeled with bromodeoxyuridine. Using TRIPn-Seq, we mapped 1009 unique transcription-replication interactions (TRIs) in mouse primary B cells characterized by a bimodal pattern of RNAP2s5, bidirectional transcription, an enrichment of RNA:DNA hybrids, and a high probability of forming G-quadruplexes. TRIs are highly enriched at transcription start sites and map to early replicating regions. TRIs exhibit enhanced Replication Protein A association and TRI-associated genes exhibit higher replication fork termination than control transcription start sites, two marks of replication stress. TRIs colocalize with double-strand DNA breaks, are enriched for deletions, and accumulate mutations in tumors. We propose that replication stress at TRIs induces mutations potentially contributing to age-related disease, as well as tumor formation and development.

Mapping R-Loops and RNA:DNA Hybrids with S9.6-Based Immunoprecipitation Methods.

R-loops constitute a prevalent class of transcription-driven non-B DNA structures that occur in all genomes depending on both DNA sequence and topological favorability. In recent years, R-loops have been implicated in a variety of adaptive and maladaptive roles and have been linked to genomic instability in the context of human disorders. As a consequence, the accurate mapping of these structures in genomes is of high interest to many investigators. DRIP-seq (DNA:RNA Immunoprecipitation followed by high throughput sequencing) is described here. It is a robust and reproducible technique that permits accurate and semi-quantitative mapping of R-loops. A recent iteration of the method is also described in which fragmentation is accomplished using sonication (sDRIP-seq), which allows strand-specific and high-resolution mapping of R-loops. sDRIP-seq thus addresses some of the common limitations of the DRIP-seq method in terms of resolution and strandedness, making it a method of choice for R-loop mapping.

PRMT1-dependent regulation of RNA metabolism and DNA damage response sustains pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that has remained clinically challenging to manage. Here we employ an RNAi-based in vivo functional genomics platform to determine epigenetic vulnerabilities across a panel of patient-derived PDAC models. Through this, we identify protein arginine methyltransferase 1 (PRMT1) as a critical dependency required for PDAC maintenance. Genetic and pharmacological studies validate the role of PRMT1 in maintaining PDAC growth. Mechanistically, using proteomic and transcriptomic analyses, we demonstrate that global inhibition of asymmetric arginine methylation impairs RNA metabolism, which includes RNA splicing, alternative polyadenylation, and transcription termination. This triggers a robust downregulation of multiple pathways involved in the DNA damage response, thereby promoting genomic instability and inhibiting tumor growth. Taken together, our data support PRMT1 as a compelling target in PDAC and informs a mechanism-based translational strategy for future therapeutic development.Statement of significancePDAC is a highly lethal cancer with limited therapeutic options. This study identified and characterized PRMT1-dependent regulation of RNA metabolism and coordination of key cellular processes required for PDAC tumor growth, defining a mechanism-based translational hypothesis for PRMT1 inhibitors.

Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids.

The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid-specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.

CARM1 Inhibition Enables Immunotherapy of Resistant Tumors by Dual Action on Tumor Cells and T Cells.

A number of cancer drugs activate innate immune pathways in tumor cells but unfortunately also compromise antitumor immune function. We discovered that inhibition of CARM1, an epigenetic enzyme and cotranscriptional activator, elicited beneficial antitumor activity in both cytotoxic T cells and tumor cells. In T cells, Carm1 inactivation substantially enhanced their antitumor function and preserved memory-like populations required for sustained antitumor immunity. In tumor cells, Carm1 inactivation induced a potent type 1 interferon response that sensitized resistant tumors to cytotoxic T cells. Substantially increased numbers of dendritic cells, CD8 T cells, and natural killer cells were present in Carm1-deficient tumors, and infiltrating CD8 T cells expressed low levels of exhaustion markers. Targeting of CARM1 with a small molecule elicited potent antitumor immunity and sensitized resistant tumors to checkpoint blockade. Targeting of this cotranscriptional regulator thus offers an opportunity to enhance immune function while simultaneously sensitizing resistant tumor cells to immune attack. SIGNIFICANCE: Resistance to cancer immunotherapy remains a major challenge. Targeting of CARM1 enables immunotherapy of resistant tumors by enhancing T-cell functionality and preserving memory-like T-cell populations within tumors. CARM1 inhibition also sensitizes resistant tumor cells to immune attack by inducing a tumor cell-intrinsic type 1 interferon response.This article is highlighted in the In This Issue feature, p. 1861.

Best practices for the visualization, mapping, and manipulation of R-loops.

R-loops represent an abundant class of large non-B DNA structures in genomes. Even though they form transiently and at modest frequencies, interfering with R-loop formation or dissolution has significant impacts on genome stability. Addressing the mechanism(s) of R-loop-mediated genome destabilization requires a precise characterization of their distribution in genomes. A number of independent methods have been developed to visualize and map R-loops, but their results are at times discordant, leading to confusion. Here, we review the main existing methodologies for R-loop mapping and assess their limitations as well as the robustness of existing datasets. We offer a set of best practices to improve the reproducibility of maps, hoping that such guidelines could be useful for authors and referees alike. Finally, we propose a possible resolution for the apparent contradictions in R-loop mapping outcomes between antibody-based and RNase H1-based mapping approaches.

SETX (senataxin), the helicase mutated in AOA2 and ALS4, functions in autophagy regulation.

SETX (senataxin) is an RNA/DNA helicase that has been implicated in transcriptional regulation and the DNA damage response through resolution of R-loop structures. Mutations in SETX result in either of two distinct neurodegenerative disorders. SETX dominant mutations result in a juvenile form of amyotrophic lateral sclerosis (ALS) called ALS4, whereas recessive mutations are responsible for ataxia called ataxia with oculomotor apraxia type 2 (AOA2). How mutations in the same protein can lead to different phenotypes is still unclear. To elucidate AOA2 disease mechanisms, we first examined gene expression changes following SETX depletion. We observed the effects on both transcription and RNA processing, but surprisingly observed decreased R-loop accumulation in SETX-depleted cells. Importantly, we discovered a strong connection between SETX and the macroautophagy/autophagy pathway, reflecting a direct effect on transcription of autophagy genes. We show that SETX depletion inhibits the progression of autophagy, leading to an accumulation of ubiquitinated proteins, decreased ability to clear protein aggregates, as well as mitochondrial defects. Analysis of AOA2 patient fibroblasts also revealed a perturbation of the autophagy pathway. Our work has thus identified a novel function for SETX in the regulation of autophagy, whose modulation may have a therapeutic impact for AOA2.Abbreviations: 3'READS: 3' region extraction and deep sequencing; ACTB: actin beta; ALS4: amyotrophic lateral sclerosis type 4; AOA2: ataxia with oculomotor apraxia type 2; APA: alternative polyadenylation; AS: alternative splicing; ATG7: autophagy-related 7; ATP6V0D2: ATPase H+ transporting V0 subunit D2; BAF: bafilomycin A1; BECN1: beclin 1; ChIP: chromatin IP; Chloro: chloroquine; CPT: camptothecin; DDR: DNA damage response; DNMT1: DNA methyltransferase 1; DRIP: DNA/RNA IP; DSBs: double strand breaks; EBs: embryoid bodies; FTD: frontotemporal dementia; GABARAP: GABA type A receptor-associated protein; GO: gene ontology; HR: homologous recombination; HTT: huntingtin; IF: immunofluorescence; IP: immunoprecipitation; iPSCs: induced pluripotent stem cells; KD: knockdown; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MN: motor neuron; MTORC1: mechanistic target of rapamycin kinase complex 1; PASS: PolyA Site Supporting; PFA: paraformaldehyde; RNAPII: RNA polymerase II; SCA: spinocerebellar ataxia; SETX: senataxin; SMA: spinal muscular atrophy; SMN1: survival of motor neuron 1, telomeric; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TSS: transcription start site; TTS: transcription termination site; ULK1: unc-51 like autophagy activating kinase 1; WB: western blot; WIPI2: WD repeat domain, phosphoinositide interacting 2; XRN2: 5'-3' exoribonuclease 2.

RNA-DNA strand exchange by the Drosophila Polycomb complex PRC2.

Polycomb Group (PcG) proteins form memory of transient transcriptional repression that is necessary for development. In Drosophila, DNA elements termed Polycomb Response Elements (PREs) recruit PcG proteins. How PcG activities are targeted to PREs to maintain repressed states only in appropriate developmental contexts has been difficult to elucidate. PcG complexes modify chromatin, but also interact with both RNA and DNA, and RNA is implicated in PcG targeting and function. Here we show that R-loops form at many PREs in Drosophila embryos, and correlate with repressive states. In vitro, both PRC1 and PRC2 can recognize R-loops and open DNA bubbles. Unexpectedly, we find that PRC2 drives formation of RNA-DNA hybrids, the key component of R-loops, from RNA and dsDNA. Our results identify R-loop formation as a feature of Drosophila PREs that can be recognized by PcG complexes, and RNA-DNA strand exchange as a PRC2 activity that could contribute to R-loop formation.

Ultra-deep Coverage Single-molecule R-loop Footprinting Reveals Principles of R-loop Formation.

R-loops are a prevalent class of non-B DNA structures that have been associated with both positive and negative cellular outcomes. DNA:RNA immunoprecipitation (DRIP) approaches based on the anti-DNA:RNA hybrid S9.6 antibody revealed that R-loops form dynamically over conserved genic hotspots. We have developed an orthogonal approach that queries R-loops via the presence of long stretches of single-stranded DNA on their looped-out strand. Nondenaturing sodium bisulfite treatment catalyzes the conversion of unpaired cytosines to uracils, creating permanent genetic tags for the position of an R-loop. Long-read, single-molecule PacBio sequencing allows the identification of R-loop 'footprints' at near nucleotide resolution in a strand-specific manner on long single DNA molecules and at ultra-deep coverage. Single-molecule R-loop footprinting coupled with PacBio sequencing (SMRF-seq) revealed a strong agreement between S9.6-based and bisulfite-based R-loop mapping and confirmed that R-loops form over genic hotspots, including gene bodies and terminal gene regions. Based on the largest single-molecule R-loop dataset to date, we show that individual R-loops form nonrandomly, defining discrete sets of overlapping molecular clusters that pileup through larger R-loop zones. R-loops most often map to intronic regions and their individual start and stop positions do not match with intron-exon boundaries, reinforcing the model that they form cotranscriptionally from unspliced transcripts. SMRF-seq further established that R-loop distribution patterns are not simply driven by intrinsic DNA sequence features but most likely also reflect DNA topological constraints. Overall, DRIP-based and SMRF-based approaches independently provide a complementary and congruent view of R-loop distribution, consolidating our understanding of the principles underlying R-loop formation.

High-resolution, strand-specific R-loop mapping via S9.6-based DNA-RNA immunoprecipitation and high-throughput sequencing.

R-loops are prevalent three-stranded non-B DNA structures composed of an RNA-DNA hybrid and a single strand of DNA. R-loops are implicated in various basic nuclear processes, such as class-switch recombination, transcription termination and chromatin patterning. Perturbations in R-loop metabolism have been linked to genomic instability and have been implicated in human disorders, including cancer. As a consequence, the accurate mapping of these structures has been of increasing interest in recent years. Here, we describe two related immunoprecipitation-based methods for mapping R-loop structures: basic DRIP-seq (DNA-RNA immunoprecipitation followed by high-throughput DNA sequencing), an easy, robust, but resolution-limited technique; and DRIPc-seq (DNA-RNA immunoprecipitation followed by cDNA conversion coupled to high-throughput sequencing), a high-resolution and strand-specific iteration of the method that permits accurate R-loop mapping genome wide. Briefly, after gentle DNA extraction and restriction digestion with a cocktail of enzymes, R-loop structures are immunoprecipitated with the anti-RNA-DNA hybrid S9.6 antibody. Compared with DRIP-seq, in which the immunoprecipitated DNA is directly sequenced, DRIPc-seq permits the recovery of the RNA moiety of R-loops, and these RNA strands are subjected to strand-specific RNA sequencing (RNA-seq) analysis. DRIPc-seq can be performed in 5 d and can be applied to any cell type, provided sufficient starting material can be collected. Accurately mapping R-loop distribution in various cell lines and under varied conditions is essential to understanding the formation, roles and dynamic resolution of these important structures.

DNA Topoisomerase I differentially modulates R-loops across the human genome.

BACKGROUND: Co-transcriptional R-loops are abundant non-B DNA structures in mammalian genomes. DNA Topoisomerase I (Top1) is often thought to regulate R-loop formation owing to its ability to resolve both positive and negative supercoils. How Top1 regulates R-loop structures at a global level is unknown. RESULTS: Here, we perform high-resolution strand-specific R-loop mapping in human cells depleted for Top1 and find that Top1 depletion results in both R-loop gains and losses at thousands of transcribed loci, delineating two distinct gene classes. R-loop gains are characteristic for long, highly transcribed, genes located in gene-poor regions anchored to Lamin B1 domains and in proximity to H3K9me3-marked heterochromatic patches. R-loop losses, by contrast, occur in gene-rich regions overlapping H3K27me3-marked active replication initiation regions. Interestingly, Top1 depletion coincides with a block of the cell cycle in G0/G1 phase and a trend towards replication delay. CONCLUSIONS: Our findings reveal new properties of Top1 in regulating R-loop homeostasis in a context-dependent manner and suggest a potential role for Top1 in modulating the replication process via R-loop formation.

Co-transcriptional R-loops are the main cause of estrogen-induced DNA damage.

The hormone estrogen (E2) binds the estrogen receptor to promote transcription of E2-responsive genes in the breast and other tissues. E2 also has links to genomic instability, and elevated E2 levels are tied to breast cancer. Here, we show that E2 stimulation causes a rapid, global increase in the formation of R-loops, co-transcriptional RNA-DNA products, which in some instances have been linked to DNA damage. We show that E2-dependent R-loop formation and breast cancer rearrangements are highly enriched at E2-responsive genomic loci and that E2 induces DNA replication-dependent double-strand breaks (DSBs). Strikingly, many DSBs that accumulate in response to E2 are R-loop dependent. Thus, R-loops resulting from the E2 transcriptional response are a significant source of DNA damage. This work reveals a novel mechanism by which E2 stimulation leads to genomic instability and highlights how transcriptional programs play an important role in shaping the genomic landscape of DNA damage susceptibility.

Prevalent, Dynamic, and Conserved R-Loop Structures Associate with Specific Epigenomic Signatures in Mammals.

R-loops are three-stranded nucleic acid structures formed upon annealing of an RNA strand to one strand of duplex DNA. We profiled R-loops using a high-resolution, strand-specific methodology in human and mouse cell types. R-loops are prevalent, collectively occupying up to 5% of mammalian genomes. R-loop formation occurs over conserved genic hotspots such as promoter and terminator regions of poly(A)-dependent genes. In most cases, R-loops occur co-transcriptionally and undergo dynamic turnover. Detailed epigenomic profiling revealed that R-loops associate with specific chromatin signatures. At promoters, R-loops associate with a hyper-accessible state characteristic of unmethylated CpG island promoters. By contrast, terminal R-loops associate with an enhancer- and insulator-like state and define a broad class of transcription terminators. Together, this suggests that the retention of nascent RNA transcripts at their site of expression represents an abundant, dynamic, and programmed component of the mammalian chromatin that affects chromatin patterning and the control of gene expression.

EHMT2 directs DNA methylation for efficient gene silencing in mouse embryos.

The extent to which histone modifying enzymes contribute to DNA methylation in mammals remains unclear. Previous studies suggested a link between the lysine methyltransferase EHMT2 (also known as G9A and KMT1C) and DNA methylation in the mouse. Here, we used a model of knockout mice to explore the role of EHMT2 in DNA methylation during mouse embryogenesis. The Ehmt2 gene is expressed in epiblast cells but is dispensable for global DNA methylation in embryogenesis. In contrast, EHMT2 regulates DNA methylation at specific sequences that include CpG-rich promoters of germline-specific genes. These loci are bound by EHMT2 in embryonic cells, are marked by H3K9 dimethylation, and have strongly reduced DNA methylation in Ehmt2(-/-) embryos. EHMT2 also plays a role in the maintenance of germline-derived DNA methylation at one imprinted locus, the Slc38a4 gene. Finally, we show that DNA methylation is instrumental for EHMT2-mediated gene silencing in embryogenesis. Our findings identify EHMT2 as a critical factor that facilitates repressive DNA methylation at specific genomic loci during mammalian development.

Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE). AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid-degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4), and SAMHD1 (AGS5). However, the identity and source of such immunogenic nucleic acid species remain undefined. Using genome-wide approaches, we show that fibroblasts from AGS patients with AGS1-5 mutations are burdened by excessive loads of RNA:DNA hybrids. Using MethylC-seq, we show that AGS fibroblasts display pronounced and global loss of DNA methylation and demonstrate that AGS-specific RNA:DNA hybrids often occur within DNA hypomethylated regions. Altogether, our data suggest that RNA:DNA hybrids may represent a common immunogenic form of nucleic acids in AGS and provide the first evidence of epigenetic perturbations in AGS, furthering the links between AGS and SLE.

Transcription-associated R-loop formation across the human FMR1 CGG-repeat region.

Expansion of a trinucleotide (CGG) repeat element within the 5' untranslated region (5'UTR) of the human FMR1 gene is responsible for a number of heritable disorders operating through distinct pathogenic mechanisms: gene silencing for fragile X syndrome (>200 CGG) and RNA toxic gain-of-function for FXTAS (∼ 55-200 CGG). Existing models have focused almost exclusively on post-transcriptional mechanisms, but co-transcriptional processes could also contribute to the molecular dysfunction of FMR1. We have observed that transcription through the GC-rich FMR1 5'UTR region favors R-loop formation, with the nascent (G-rich) RNA forming a stable RNA:DNA hybrid with the template DNA strand, thereby displacing the non-template DNA strand. Using DNA:RNA (hybrid) immunoprecipitation (DRIP) of genomic DNA from cultured human dermal fibroblasts with both normal (∼ 30 CGG repeats) and premutation (55

Genome-wide DNA demethylation in mammals.

The cytidine deaminase AID and elongator-complex proteins contribute to the extensive removal of DNA methylation in mammalian primordial germ cells and in the paternal pronucleus of the zygote.

Histone methylation is mechanistically linked to DNA methylation at imprinting control regions in mammals.

Mono-allelic expression of imprinted genes from either the paternal or the maternal allele is mediated by imprinting control regions (ICRs), which are epigenetically marked in an allele-specific fashion. Although, in somatic cells, these epigenetic marks comprise both DNA methylation and histone methylation, the relationship between these two modifications in imprint acquisition and maintenance remains unclear. To address this important question, we analyzed histone modifications at ICRs in mid-gestation embryos that were obtained from Dnmt3L(-/-) females, in which DNA methylation imprints at ICRs are not established during oogenesis. The absence of maternal DNA methylation imprints in these conceptuses led to a marked decrease and loss of allele-specificity of the repressive H3K9me3, H4K20me3 and H2A/H4R3me2 histone modifications, providing the first evidence of a mechanistic link between DNA and histone methylation at ICRs. The existence of this relationship was strengthened by the observation that when DNA methylation was still present at the Snrpn and Peg3 ICRs in some of the progeny of Dnmt3L(-/-) females, these ICRs were associated with the usual patterns of histone methylation. Combined, our data establish that DNA methylation is involved in the acquisition and/or maintenance of histone methylation at ICRs.

The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin.

A number of large noncoding RNAs (ncRNAs) epigenetically silence genes through unknown mechanisms. The Air ncRNA is imprinted--monoallelically expressed from the paternal allele. Air is required for allele-specific silencing of the cis-linked Slc22a3, Slc22a2, and Igf2r genes in mouse placenta. We show that Air interacts with the Slc22a3 promoter chromatin and the H3K9 histone methyltransferase G9a in placenta. Air accumulates at the Slc22a3 promoter in correlation with localized H3K9 methylation and transcriptional repression. Genetic ablation of G9a results in nonimprinted, biallelic transcription of Slc22a3. Truncated Air fails to accumulate at the Slc22a3 promoter, which results in reduced G9a recruitment and biallelic transcription. Our results suggest that Air, and potentially other large ncRNAs, target repressive histone-modifying activities through molecular interaction with specific chromatin domains to epigenetically silence transcription.

A mono-allelic bivalent chromatin domain controls tissue-specific imprinting at Grb10.

Genomic imprinting is a developmental mechanism that mediates parent-of-origin-specific expression in a subset of genes. How the tissue specificity of imprinted gene expression is controlled remains poorly understood. As a model to address this question, we studied Grb10, a gene that displays brain-specific expression from the paternal chromosome. Here, we show in the mouse that the paternal promoter region is marked by allelic bivalent chromatin enriched in both H3K4me2 and H3K27me3, from early embryonic stages onwards. This is maintained in all somatic tissues, but brain. The bivalent domain is resolved upon neural commitment, during the developmental window in which paternal expression is activated. Our data indicate that bivalent chromatin, in combination with neuronal factors, controls the paternal expression of Grb10 in brain. This finding highlights a novel mechanism to control tissue-specific imprinting.