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R-loops are three-stranded nucleic acid structures with essential roles in many nuclear processes. However, their unchecked accumulation is associated with genome instability and is observed in neurodevelopmental diseases and cancers. Genome-wide profiling of R-loops in normal and diseased cells can help identify locations of pathogenic R-loops and advance efforts to attenuate them. We present an antibody-independent R-loop detection strategy, BisMapR, that combines nuclease-based R-loop isolation with non-denaturing bisulfite chemistry to produce genome-wide profiles that retain strand information. BisMapR achieves greater resolution and is faster than existing strand-specific R-loop profiling strategies. In mouse embryonic stem cells, we apply BisMapR to find that gene promoters form R-loops in both directions and uncover a subset of active enhancers that, despite being bidirectionally transcribed, form R-loops exclusively on one strand. BisMapR reveals a previously unnoticed feature of active enhancers and provides a tool to systematically examine their mechanisms in gene expression.

R-loops are three-stranded nucleic acid structures that accumulate on chromatin in neurological diseases and cancers and contribute to genome instability. Using a proximity-dependent labeling system, we identified distinct classes of proteins that regulate R-loops in vivo through different mechanisms. We show that ATRX suppresses R-loops by interacting with RNAs and preventing R-loop formation. Our proteomics screen also discovered an unexpected enrichment for proteins containing zinc fingers and homeodomains. One of the most consistently enriched proteins was activity-dependent neuroprotective protein (ADNP), which is frequently mutated in ASD and causal in ADNP syndrome. We find that ADNP resolves R-loops in vitro and that it is necessary to suppress R-loops in vivo at its genomic targets. Furthermore, deletion of the ADNP homeodomain severely diminishes R-loop resolution activity in vitro, results in R-loop accumulation at ADNP targets, and compromises neuronal differentiation. Notably, patient-derived human induced pluripotent stem cells that contain an ADNP syndrome-causing mutation exhibit R-loop and CTCF accumulation at ADNP targets. Our findings point to a specific role for ADNP-mediated R-loop resolution in physiological and pathological neuronal function and, more broadly, to a role for zinc finger and homeodomain proteins in R-loop regulation, with important implications for developmental disorders and cancers. R-loops are three-stranded nucleic acid structures that contribute to genome instability and accumulate in neurological diseases. Here the authors identify R-loop proximal factors, which are enriched for zinc finger and homeodomain proteins, including activity-dependent neuroprotective protein (ADNP). ADNP plays a role in R-loop resolution and loss-of-function leads to R-loop accumulation.

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.

CARM1 is often overexpressed in human cancers including in ovarian cancer. However, therapeutic approaches based on CARM1 expression remain to be an unmet need. Cancer cells exploit adaptive responses such as the endoplasmic reticulum (ER) stress response for their survival through activating pathways such as the IRE1α/XBP1s pathway. Here, we report that CARM1-expressing ovarian cancer cells are selectively sensitive to inhibition of the IRE1α/XBP1s pathway. CARM1 regulates XBP1s target gene expression and directly interacts with XBP1s during ER stress response. Inhibition of the IRE1α/XBP1s pathway was effective against ovarian cancer in a CARM1-dependent manner both in vitro and in vivo in orthotopic and patient-derived xenograft models. In addition, IRE1α inhibitor B-I09 synergizes with immune checkpoint blockade anti-PD1 antibody in an immunocompetent CARM1-expressing ovarian cancer model. Our data show that pharmacological inhibition of the IRE1α/XBP1s pathway alone or in combination with immune checkpoint blockade represents a therapeutic strategy for CARM1-expressing cancers. The unfolded protein response (UPR) promotes cell survival in cancers with hyperactive ER stress response. Here the authors show that CARM1, an arginine methyltransferase, controls the IRE1α/XBP1 pathway of the UPR and the inhibition of this pathway can inhibit growth in CARM1 expressing ovarian cancers.

During mammalian X-chromosome inactivation, the Xist long noncoding RNA coats the future inactive X chromosome and recruits polycomb repressive complex 2 to a nucleation site, but how Xist spreads silencing across the entire X chromosome is unclear; here high-resolution maps of Xist binding sites across the X chromosome are generated and show that Xist does not spread across the inactive X chromosome uniformly but in two steps, initially targeting gene-rich islands before later spreading to intervening gene-poor domains. Mapping Xist long noncoding RNA During mammalian X-chromosome inactivation (XCI), the Xist long noncoding RNA coats the future inactive X (Xi) and recruits polycomb repressive complex 2 (PRC2) to a nucleation site, but how Xist spreads silencing across the entire X chromosome is unclear. Here, Jeannie Lee and colleagues generate high-resolution maps of Xist binding sites across the X chromosome using a technique termed CHART-seq. By following four early developmental stages in female mouse cells undergoing XCI, they show that Xist does not spread across the Xi uniformly but in two steps — initially targeting gene-rich islands and later spreading to intervening gene-poor domains. The Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation (XCI), the process by which mammals compensate for unequal numbers of sex chromosomes 1 , 2 , 3 . During XCI, Xist coats the future inactive X chromosome (Xi) 4 and recruits Polycomb repressive complex 2 (PRC2) to the X-inactivation centre ( Xic ) 5 . How Xist spreads silencing on a 150-megabases scale is unclear. Here we generate high-resolution maps of Xist binding on the X chromosome across a developmental time course using CHART-seq. In female cells undergoing XCI de novo , Xist follows a two-step mechanism, initially targeting gene-rich islands before spreading to intervening gene-poor domains. Xist is depleted from genes that escape XCI but may concentrate near escapee boundaries. Xist binding is linearly proportional to PRC2 density and H3 lysine 27 trimethylation (H3K27me3), indicating co-migration of Xist and PRC2. Interestingly, when Xist is acutely stripped off from the Xi in post-XCI cells, Xist recovers quickly within both gene-rich and gene-poor domains on a timescale of hours instead of days, indicating a previously primed Xi chromatin state. We conclude that Xist spreading takes distinct stage-specific forms. During initial establishment, Xist follows a two-step mechanism, but during maintenance, Xist spreads rapidly to both gene-rich and gene-poor regions.

Heterochromatin in the eukaryotic genome is rigorously controlled by the concerted action of protein factors and RNAs. Here, we investigate the RNA binding function of ATRX, a chromatin remodeler with roles in silencing of repetitive regions of the genome and in recruitment of the polycomb repressive complex 2 (PRC2). We identify ATRX RNA binding regions (RBRs) and discover that the major ATRX RBR lies within the N-terminal region of the protein, distinct from its PHD and helicase domains. Deletion of this ATRX RBR (ATRXΔRBR) compromises ATRX interactions with RNAs in vitro and in vivo and alters its chromatin binding properties. Genome-wide studies reveal that loss of RNA interactions results in a redistribution of ATRX on chromatin. Finally, our studies identify a role for ATRX-RNA interactions in regulating PRC2 localization to a subset of polycomb target genes. ATRX is an RNA binding protein that mediates targeting of polycomb repressive complex 2 (PRC2) to genomic sites. Here the authors identify the RNA binding region and show that the RNA binding is required for ATRX localization and for its recruitment of PRC2 to a subset of polycomb targets.

R-loops are three-stranded nucleic acid structures that are abundant and widespread across the genome and that have important physiological roles in many nuclear processes. Their accumulation is observed in cancers and neurodegenerative disorders. Recent studies have implicated a function for R-loops and G-quadruplex (G4) structures, which can form on the displaced single strand of R-loops, in three-dimensional genome organization in both physiological and pathological contexts. Here we discuss the interconnected functions of DNA:RNA hybrids and G4s within R-loops, their impact on DNA repair and gene regulatory networks, and their emerging roles in genome organization during development and disease. R-loops and G-quadruplexes are non-canonical nucleic acid structures with known roles in genome organization. Here, Wulfridge and Sarma highlight emerging roles in DNA repair and transcriptional and epigenetic gene regulation.

A large fraction of the mammalian genome is transcribed into long noncoding RNAs. The RNAs remain largely uncharacterized as the field awaits new technologies to aid functional analysis. Here, we describe a unique use of locked nucleic acids (LNAs) for studying nuclear long noncoding RNA, an RNA subclass that has been less amenable to traditional knockdown techniques. We target LNAs at Xist RNA and show displacement from the X chromosome with fast kinetics. Xist transcript stability is not affected. By targeting different Xist regions, we identify a localization domain and show that polycomb repressive complex 2 (PRC2) is displaced together with Xist. Thus, PRC2 depends on RNA for both initial targeting to and stable association with chromatin. H3K27-trimethyl marks and gene silencing remain stable. Time-course analysis of RNA relocalization suggests that Xist and PRC2 bind to different regions of the X at the same time but do not reach saturating levels immediately. Thus, LNAs provide a tool for studying an emerging class of regulatory RNA and offer a window of opportunity to target epigenetic modifications with possible therapeutic applications.

Key Points Chromatin is organized into specialized domains, some of which contain specialized histones called variant histones. Variant histones have evolved to carry out functions that are distinct from those of the major core histones. Histones are deposited onto DNA by chaperones. Recent studies have shown that some chaperones recruit specific histone variants for deposition. The chaperones HIRA and SWR1 specifically recruit and exchange the histone variants H3.3 and H2AZ, respectively, whereas the chromatin assembly factor-1 (CAF1) mediates the deposition of the H3.1 major core histone in a process that is coupled to DNA replication. The implications of two modes of deposition of the histones H3 and H4 (that is, tetramers versus dimers) are discussed, as they are important for the transmission of epigenetic information from the mother to the daughter cells. The functions and possible modes of deposition of other variant histones such as CENPA, H2AX and others are also discussed. Both H3.3 and H2AZ have been suggested to function in activated transcription on the basis of their localization to euchromatic loci. Models are proposed whereby histone exchangers coordinate with FACT — a chaperone that has a crucial role in facilitating transcription elongation on chromatin — to allow the incorporation of histone variants during the process of transcription. A model is also presented that addresses CAF1-mediated deposition of histone H3 that is methylated at Lys9, through CAF1 interaction with the histone methyltransferase SUV39H1, which results in the formation of repressive chromatin. A fascinating aspect of how chromatin structure impacts on gene expression and cellular identity is the transmission of information from mother to daughter cells, independently of the primary DNA sequence. This epigenetic information seems to be contained within the covalent modifications of histone polypeptides and the distinctive characteristics of variant histone subspecies. There are specific deposition pathways for some histone variants, which provide invaluable mechanistic insights into processes whereby the major histones are exchanged for their more specialized counterparts.

The CRL4-DCAF15 E3 ubiquitin ligase complex is targeted by the aryl-sulfonamide molecular glues, leading to neo-substrate recruitment, ubiquitination, and proteasomal degradation. However, the physiological function of DCAF15 remains unknown. Using a domain-focused genetic screening approach, we reveal DCAF15 as an acute myeloid leukemia (AML)-biased dependency. Loss of DCAF15 results in suppression of AML through compromised replication fork integrity and consequent accumulation of DNA damage. Accordingly, DCAF15 loss sensitizes AML to replication stress-inducing therapeutics. Mechanistically, we discover that DCAF15 directly interacts with the SMC1A protein of the cohesin complex and destabilizes the cohesin regulatory factors PDS5A and CDCA5. Loss of PDS5A and CDCA5 removal precludes cohesin acetylation on chromatin, resulting in uncontrolled chromatin loop extrusion, defective DNA replication, and apoptosis. Collectively, our findings uncover an endogenous, cell autonomous function of DCAF15 in sustaining AML proliferation through post-translational control of cohesin dynamics. The DCAF15 E3 ubiquitin ligase is targeted by aryl-sulfonamide molecular glues leading to neo-substrate proteasomal degradation. Here, the authors reveal DCAF15 as a cell autonomous acute myeloid leukemia dependency sustaining proliferation through control of cohesin complex recycling dynamics.