Unperturbed Cells Research Articles

ATR promotes mTORC1 activity via de novo cholesterol synthesis.

DNA damage and cellular metabolism exhibit a complex interplay characterized by bidirectional feedback mechanisms. Key mediators of the DNA damage response and cellular metabolic regulation include Ataxia Telangiectasia and Rad3-related protein (ATR) and the mechanistic Target of Rapamycin Complex 1 (mTORC1), respectively. Previous studies have established ATR as a regulatory upstream factor of mTORC1 during replication stress; however, the precise mechanisms by which mTORC1 is activated in this context remain poorly defined. Additionally, the activity of this signaling axis in unperturbed cells has not been extensively investigated. Here, we demonstrate that ATR promotes mTORC1 activity across various cellular models under basal conditions. This effect is particularly enhanced in cells following the loss of p16, which we have previously associated with hyperactivation of mTORC1 signaling and here found have increased ATR activity. Mechanistically, we found that ATR promotes de novo cholesterol synthesis and mTORC1 activation through the upregulation of lanosterol synthase (LSS), independently of both CHK1 and the TSC complex. Furthermore, the attenuation of mTORC1 activity resulting from ATR inhibition was rescued by supplementation with lanosterol or cholesterol in multiple cellular contexts. This restoration corresponded with enhanced localization of mTOR to the lysosome. Collectively, our findings demonstrate a novel connection linking ATR and mTORC1 signaling through the modulation of cholesterol metabolism.

Multiplexed single-cell imaging reveals diverging subpopulations with distinct senescence phenotypes during long-term senescence induction.

Cellular senescence is a phenotypic state that contributes to the progression of age-related disease through secretion of pro-inflammatory factors known as the senescence associated secretory phenotype (SASP). Understanding the process by which healthy cells become senescent and develop SASP factors is critical for improving the identification of senescent cells and, ultimately, understanding tissue dysfunction. Here, we reveal how the duration of cellular stress modulates the SASP in distinct subpopulations of senescent cells. We used multiplex, single-cell imaging to build a proteomic map of senescence induction in human epithelial cells induced to senescence over the course of 31 days. We map how the expression of SASP proteins increases alongside other known senescence markers such as p53, p21, and p16 INK4a . The aggregated population of cells responded to etoposide with an accumulation of stress response factors over the first 11 days, followed by a plateau in most proteins. At the single-cell level, however, we identified two distinct senescence cell populations, one defined primarily by larger nuclear area and the second by higher protein concentrations. Trajectory inference suggested that cells took one of two discrete molecular paths from unperturbed healthy cells, through a common transitional subpopulation, and ending at the discrete terminal senescence phenotypes. Our results underscore the importance of using single-cell proteomics to identify the mechanistic pathways governing the transition from senescence induction to a mature state of senescence characterized by the SASP.

Gene regulatory network structure informs the distribution of perturbation effects.

Gene regulatory networks (GRNs) govern many core developmental and biological processes underlying human complex traits. Even with broad-scale efforts to characterize the effects of molecular perturbations and interpret gene coexpression, it remains challenging to infer the architecture of gene regulation in a precise and efficient manner. Key properties of GRNs, like hierarchical structure, modular organization, and sparsity, provide both challenges and opportunities for this objective. Here, we seek to better understand properties of GRNs using a new approach to simulate their structure and model their function. We produce realistic network structures with a novel generating algorithm based on insights from small-world network theory, and we model gene expression regulation using stochastic differential equations formulated to accommodate modeling molecular perturbations. With these tools, we systematically describe the effects of gene knockouts within and across GRNs, finding a subset of networks that recapitulate features of a recent genome-scale perturbation study. With deeper analysis of these exemplar networks, we consider future avenues to map the architecture of gene expression regulation using data from cells in perturbed and unperturbed states, finding that while perturbation data are critical to discover specific regulatory interactions, data from unperturbed cells may be sufficient to reveal regulatory programs.

DePARylation is critical for S phase progression and cell survival.

Poly(ADP-ribose)ylation or PARylation by PAR polymerase 1 (PARP1) and dePARylation by poly(ADP-ribose) glycohydrolase (PARG) are equally important for the dynamic regulation of DNA damage response. PARG, the most active dePARylation enzyme, is recruited to sites of DNA damage via pADPr-dependent and PCNA-dependent mechanisms. Targeting dePARylation is considered an alternative strategy to overcome PARP inhibitor resistance. However, precisely how dePARylation functions in normal unperturbed cells remains elusive. To address this challenge, we conducted multiple CRISPR screens and revealed that dePARylation of S phase pADPr by PARG is essential for cell viability. Loss of dePARylation activity initially induced S-phase-specific pADPr signaling, which resulted from unligated Okazaki fragments and eventually led to uncontrolled pADPr accumulation and PARP1/2-dependent cytotoxicity. Moreover, we demonstrated that proteins involved in Okazaki fragment ligation and/or base excision repair regulate pADPr signaling and cell death induced by PARG inhibition. In addition, we determined that PARG expression is critical for cellular sensitivity to PARG inhibition. Additionally, we revealed that PARG is essential for cell survival by suppressing pADPr. Collectively, our data not only identify an essential role for PARG in normal proliferating cells but also provide a potential biomarker for the further development of PARG inhibitors in cancer therapy.

DePARylation is critical for S phase progression and cell survival

Poly(ADP-ribose)ylation or PARylation by PAR polymerase 1 (PARP1) and dePARylation by poly(ADP-ribose) glycohydrolase (PARG) are equally important for the dynamic regulation of DNA damage response. PARG, the most active dePARylation enzyme, is recruited to sites of DNA damage via pADPr-dependent and PCNA-dependent mechanisms. Targeting dePARylation is considered an alternative strategy to overcome PARP inhibitor resistance. However, precisely how dePARylation functions in normal unperturbed cells remains elusive. To address this challenge, we conducted multiple CRISPR screens and revealed that dePARylation of S phase pADPr by PARG is essential for cell viability. Loss of dePARylation activity initially induced S-phase-specific pADPr signaling, which resulted from unligated Okazaki fragments and eventually led to uncontrolled pADPr accumulation and PARP1/2-dependent cytotoxicity. Moreover, we demonstrated that proteins involved in Okazaki fragment ligation and/or base excision repair regulate pADPr signaling and cell death induced by PARG inhibition. In addition, we determined that PARG expression is critical for cellular sensitivity to PARG inhibition. Additionally, we revealed that PARG is essential for cell survival by suppressing pADPr. Collectively, our data not only identify an essential role for PARG in normal proliferating cells but also provide a potential biomarker for the further development of PARG inhibitors in cancer therapy.

Abstract 3523: Self-supervised foundation model captures high-dimensional morphology data from single cell brightfield images

Abstract Our knowledge of cancer biology has advanced with the characterization of distinct cell types and cell states within heterogeneous tumor environments. Many methods for imaging and sorting tumor cells require biomarker labels that alter cell characteristics and create a selection bias. The use of label-free single-cell methods should further improve cancer studies involving viable, unperturbed cells for downstream assays such as RNA-Seq, cell culture expansion, and functional assays. To eliminate biomarkers and enable broader assessment of cells, we developed REM-I, a platform that characterizes and sorts unlabeled single cells based on high-dimensional morphology. Cells are captured with brightfield imaging and processed in real-time by self-supervised deep learning models to generate quantitative AI embeddings representative of cell morphology. A significant technical challenge in building REM-I was developing an AI model based on extracted features from cell images without prior knowledge of cell types, cell preparation, or other application-specific knowledge. Accordingly, we developed the Human Foundation Model (HFM), a hybrid architecture that combines self-supervised learning (SSL) and morphometrics (computer vision) to extract 115 dimensional embeddings representing cell morphology from high-resolution REM-I cell images. SSL produces a foundation model with high generalization capacity that enables hypothesis-free sample exploration and efficient generation of application-specific models. Meanwhile, computer vision extracts features that represent measurable and interpretable concepts (e.g., cell size, shape, texture, intensity). The training process for the HFM self-supervised backbone model utilizes the discriminatory power of supervised tasks. Using synthetic cells and three cancer cell lines, we trained and then validated the reproducibility and generalization capabilities of the resulting model. Results show combining deep learning and morphometrics improve interpretability of data and enable rapid characterization and classification of tumor cells with high accuracy. We also report on the Deepcell® Axon data suite, a tool to analyze data and customize reusable workflows. This includes the ability to store and manage data, visualize high dimensional data as low-dimensional projections, and train classifiers to identify and sort cell populations. To enable compatibility with user-preferred downstream analysis pipelines, Axon provides data export options for images, plots, and embeddings. Our approach allows users of all skill levels to access and interpret AI-enabled morphological profiling. Applications of REM-I include hypothesis-free evaluation of heterogeneous tumor samples, label-free cancer cell enrichment, characterization of distinct cell states, and multi-omic integration. Citation Format: Kiran Saini, Senzeyu Zhang, Ryan Carelli, Kevin B. Jacobs, Amy Wong-Thai, Cris Luengo, Vivian Lu, Anastasia Mavropoulos, Andreja Jovic, Jeanette Mei, Thomas Vollbrecht, Stephane C. Boutet, Mahyar Salek, Maddison Masaeli. Self-supervised foundation model captures high-dimensional morphology data from single cell brightfield images [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3523.

MAL expression downregulation through suppressive H3K27me3 marks at the promoter in HPV16-related cervical cancers is prognostically relevant and manifested by the interplay of novel MAL antisense long noncoding RNA AC103563.8, E7 oncoprotein and EZH2

BackgroundMAL (T-lymphocyte maturation-associated protein) is highly downregulated in most cancers, including cervical cancer (CaCx), attributable to promoter hypermethylation. Long noncoding RNA genes (lncGs) play pivotal roles in CaCx pathogenesis, by interacting with human papillomavirus (HPV)-encoded oncoproteins, and epigenetically regulating coding gene expression. Hence, we attempted to decipher the impact and underlying mechanisms of MAL downregulation in HPV16-related CaCx pathogenesis, by interrogating the interactive roles of MAL antisense lncRNA AC103563.8, E7 oncoprotein and PRC2 complex protein, EZH2.ResultsEmploying strand-specific RNA-sequencing, we confirmed the downregulated expression of MAL in association with poor overall survival of CaCx patients bearing HPV16, along with its antisense long noncoding RNA (lncRNA) AC103563.8. The strength of positive correlation between MAL and AC103563.8 was significantly high among patients compared to normal individuals. While downregulated expression of MAL was significantly associated with poor overall survival of CaCx patients bearing HPV16, AC103563.8 did not reveal any such association. We confirmed the enrichment of chromatin suppressive mark, H3K27me3 at MAL promoter, using ChIP-qPCR in HPV16-positive SiHa cells. Subsequent E7 knockdown in such cells significantly increased MAL expression, concomitant with decreased EZH2 expression and H3K27me3 marks at MAL promoter. In silico analysis revealed that both E7 and EZH2 bear the potential of interacting with AC103563.8, at the same binding domain. RNA immunoprecipitation with anti-EZH2 and anti-E7 antibodies, respectively, and subsequent quantitative PCR analysis in E7-silenced and unperturbed SiHa cells confirmed the interaction of AC103563.8 with EZH2 and E7, respectively. Apparently, AC103563.8 seems to preclude EZH2 and bind with E7, failing to block EZH2 function in patients. Thereby, enhanced EZH2 expression in the presence of E7 could potentially inactivate the MAL promoter through H3K27me3 marks, corroborating our previous results of MAL expression downregulation in patients.ConclusionAC103563.8-E7-EZH2 axis, therefore, appears to crucially regulate the expression of MAL, through chromatin inactivation in HPV16-CaCx pathogenesis, warranting therapeutic strategy development.

3D imaging reveals nanodomains of transmembrane proteins at the plasma membrane of live unperturbed cells

3D imaging reveals nanodomains of transmembrane proteins at the plasma membrane of live unperturbed cells

Nucleolar detention of NONO shields DNA double-strand breaks from aberrant transcripts.

RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.

Abstract IA022: Uracil DNA glycosylase activity limits deoxyuridine contamination in genomic DNA and is essential for the type-1 interferon response in cells treated with ATR kinase inhibitors

Abstract Most chemotherapies target DNA replication, and their efficacy depends on the DNA damage response that integrates DNA repair with the cell cycle. Widely used standard-of-care antimetabolites inhibit thymidylate synthase and increase the incorporation of deoxyuridine (dU) into DNA by polymerases. Contamination of dU in DNA is limited by the DNA damage response kinase ATR that induces cell cycle checkpoints and reduces the rate of DNA replication. ATR inhibitors (ATRi) induce origin firing across active replicons and cause ribonuclease reductase degradation in otherwise unperturbed cells. This increases both the amount of DNA replication and the amount of free dUTP in cells. Thus, ATRi increase the incorporation of dU into DNA by polymerases. Since ATRi also inhibit cell cycle checkpoints, ATRi induce more dU contamination than antimetabolites. ATRi-induced dU contamination in DNA is associated with an innate immune response. We showed this with the simple observation that ATRi-induced dU contamination and IFN-α/β expression is reversed by low doses of thymidine. Here we show that ATRi-induced IFN-α/β response is dependent on uracil DNA glycosylase (UNG) which removes dU from DNA. Our data are consistent with a model in which UNG-dependent base excision repair (BER) removes dU from DNA, ultimately generating cytoplasmic dsDNA that induces IFN-α/β. We propose that ATRi-induced dU contamination contributes to dose-limiting leukocytopenia and inflammation in the clinic and CD8+ T cell dependent anti-tumor responses in mouse models of cancer treated with radiation therapy. Citation Format: Pinakin Pandya, Frank P. Vendetti, Joseph A. Ghoubaira, Sudipta Pathak, Joshua J. Deppas, Reyna E. Jones, Yunqi Zhang, Daniel Ivanov, Raquel Buj, Katherine M. Aird, Jan H. Beumer, Robert W. Sobol, Christopher J. Bakkenist. Uracil DNA glycosylase activity limits deoxyuridine contamination in genomic DNA and is essential for the type-1 interferon response in cells treated with ATR kinase inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA022.

BCL-XL regulates the timing of mitotic apoptosis independently of BCL2 and MCL1 compensation

Mitotic catastrophe induced by prolonged mitotic arrest is a major anticancer strategy. Although antiapoptotic BCL2-like proteins, including BCL-XL, are known to regulate apoptosis during mitotic arrest, adaptive changes in their expression can complicate loss-of-function studies. Our studies revealed compensatory alterations in the expression of BCL2 and MCL1 when BCL-XL is either downregulated or overexpressed. To circumvent their reciprocal regulation, we utilized a degron-mediated system to acutely silence BCL-XL just before mitosis. Our results show that in epithelial cell lines including HeLa and RPE1, BCL-XL and BCL2 acted collaboratively to suppress apoptosis during both unperturbed cell cycle and mitotic arrest. By tagging BCL-XL and BCL2 with a common epitope, we estimated that BCL-XL was less abundant than BCL2 in the cell. Nonetheless, BCL-XL played a more prominent antiapoptotic function than BCL2 during interphase and mitotic arrest. Loss of BCL-XL led to mitotic cell death primarily through a BAX-dependent process. Furthermore, silencing of BCL-XL led to the stabilization of MCL1, which played a significant role in buffering apoptosis during mitotic arrest. Nevertheless, even in a MCL1-deficient background, depletion of BCL-XL accelerated mitotic apoptosis. These findings underscore the pivotal involvement of BCL-XL in controlling timely apoptosis during mitotic arrest, despite adaptive changes in the expression of other BCL2-like proteins.

Using the yeast vacuole as a system to test the lipidic drivers of membrane heterogeneity in living cells.

The biophysical drivers of membrane lateral heterogeneity, often termed lipid rafts, have been largely explored using synthetic liposomes or mammalian plasma membrane-derived giant vesicles. Yeast vacuoles, an organelle comparable to mammalian lysosomes, is the only in vivo system that shows stable micrometer scale phase separation in unperturbed cells. The ease of manipulating lipid metabolism in yeast makes this a powerful system for identifying lipids involved in the onset of vacuole membrane heterogeneity. Vacuole domains are induced by stationary stage growth and nutritional starvation, during which they serve as a docking and internalization site for lipid droplet energy stores. Here we describe methods for characterizing vacuole phase separation, its physiological function, and its lipidic drivers. First, we detail methodologies for robustly inducing vacuole domain formation and quantitatively characterizing during live cell imaging experiments. Second, we detail a new protocol for biochemical isolation of stationary stage vacuoles, which allows for lipidomic dissection of membrane phase separation. Third, we describe biochemical techniques for analyzing lipid droplet internalization in vacuole domains. When combined with genetic or chemical perturbations to lipid metabolism, these methods allow for systematic dissection of lipid composition in the structure and function of ordered membrane domains in living cells.

Abstract B178: Proteomics platform identifies vulnerabilities for specific DNA damage repair drugs

Abstract While poly-ADP-ribosylation (PAR) polymerase inhibitors (PARPi) are the only DNA damage repair (DDR) agent currently approved for clinical use, there are numerous ongoing clinical and preclinical drug development efforts of many other DDR-related targets, including ATR, ATM, CHK1 and others. One of the major challenges of these efforts is the ability to identify responsive populations and assign the right population for the right drug or drug combination. Patients are routinely assigned to PARPi or to on-going DDRi clinical trials based on the presence of BRCA mutation, or BRCAness profile encompassing various homologous recombination deficiencies (HRD). However, these biomarkers only partially represent the responsive populations, resulting in low response rates as well as misidentification of potential patient populations outside the genomic label. Since DNA replication and DNA repair mechanisms are dominated by signaling events, we hypothesized that HRD extends beyond the genomic status, and is manifested also in the level of protein expression and protein modification.In the current work, we compare the differences between cell lines that are either sensitive or resistant to a variety of DDR agents and highlight potential predictive biomarkers that are independent of HRD mutation status. We combined publicly available proteomic profiles of unperturbed cell lines, with both existing and newly generated proteomic and phosphoproteomic data of multiple cell lines before and after treatment of multiple DDR drugs.Analysis of HRD mutations showed that drug sensitivity is only partially explained by mutations. In addition, we found DNA replication and spliceosome as pathways that are consistently related to response regardless of drug and mutation status. While there is a general agreement between drugs in the pathway level, we identify smaller groups of proteins that are either drug or target-specific. We present here a proteogenomic approach to tackle the challenge of predictive biomarkers to DDR drugs. While DNA repair vulnerability is essential for DDR drug response, it is often not defined by mutations alone. We provide a proteomics-based framework that would enable future patient selection in DDR clinical trials. Citation Format: Gali Arad, Avital Hay-Koren, David Futorian, Oren Fischman, Dina Daitchman, Ofer Givton, Iris Alchanati, Yonatan Katzenelenbogen, Eran Seger, Kirill Pevzner. Proteomics platform identifies vulnerabilities for specific DNA damage repair drugs [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B178.

A cyclin-dependent kinase-mediated phosphorylation switch of disordered protein condensation

Cell cycle transitions result from global changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs). To understand how this complexity produces an ordered and rapid cellular reorganisation, we generated a high-resolution map of changing phosphosites throughout unperturbed early cell cycles in single Xenopus embryos, derived the emergent principles through systems biology analysis, and tested them by biophysical modelling and biochemical experiments. We found that most dynamic phosphosites share two key characteristics: they occur on highly disordered proteins that localise to membraneless organelles, and are CDK targets. Furthermore, CDK-mediated multisite phosphorylation can switch homotypic interactions of such proteins between favourable and inhibitory modes for biomolecular condensate formation. These results provide insight into the molecular mechanisms and kinetics of mitotic cellular reorganisation.

RNAPII-dependent ATM signaling at collisions with replication forks

Deregulation of RNA Polymerase II (RNAPII) by oncogenic signaling leads to collisions of RNAPII with DNA synthesis machinery (transcription-replication conflicts, TRCs). TRCs can result in DNA damage and are thought to underlie genomic instability in tumor cells. Here we provide evidence that elongating RNAPII nucleates activation of the ATM kinase at TRCs to stimulate DNA repair. We show the ATPase WRNIP1 associates with RNAPII and limits ATM activation during unperturbed cell cycle. WRNIP1 binding to elongating RNAPII requires catalytic activity of the ubiquitin ligase HUWE1. Mutation of HUWE1 induces TRCs, promotes WRNIP1 dissociation from RNAPII and binding to the replisome, stimulating ATM recruitment and activation at RNAPII. TRCs and translocation of WRNIP1 are rapidly induced in response to hydroxyurea treatment to activate ATM and facilitate subsequent DNA repair. We propose that TRCs can provide a controlled mechanism for stalling of replication forks and ATM activation, instrumental in cellular response to replicative stress.

Trim33 masks a non-transcriptional function of E2f4 in replication fork progression

Replicative stress promotes genomic instability and tumorigenesis but also presents an effective therapeutic endpoint, rationalizing detailed analysis of pathways that control DNA replication. We show here that the transcription factor E2f4 recruits the DNA helicase Recql to facilitate progression of DNA replication forks upon drug- or oncogene-induced replicative stress. In unperturbed cells, the Trim33 ubiquitin ligase targets E2f4 for degradation, limiting its genomic binding and interactions with Recql. Replicative stress blunts Trim33-dependent ubiquitination of E2f4, which stimulates transient Recql recruitment to chromatin and facilitates recovery of DNA synthesis. In contrast, deletion of Trim33 induces chronic genome-wide recruitment of Recql and strongly accelerates DNA replication under stress, compromising checkpoint signaling and DNA repair. Depletion of Trim33 in Myc-overexpressing cells leads to accumulation of replication-associated DNA damage and delays Myc-driven tumorigenesis. We propose that the Trim33-E2f4-Recql axis controls progression of DNA replication forks along transcriptionally active chromatin to maintain genome integrity.

TRAIP resolves DNA replication-transcription conflicts during the S-phase of unperturbed cells

Cell division is the basis for the propagation of life and requires accurate duplication of all genetic information. DNA damage created during replication (replication stress) is a major cause of cancer, premature aging and a spectrum of other human disorders. Over the years, TRAIP E3 ubiquitin ligase has been shown to play a role in various cellular processes that govern genome integrity and faultless segregation. TRAIP is essential for cell viability, and mutations in TRAIP ubiquitin ligase activity lead to primordial dwarfism in patients. Here, we have determined the mechanism of inhibition of cell proliferation in TRAIP-depleted cells. We have taken advantage of the auxin induced degron system to rapidly degrade TRAIP within cells and to dissect the importance of various functions of TRAIP in different stages of the cell cycle. We conclude that upon rapid TRAIP degradation, specifically in S-phase, cells cease to proliferate, arrest in G2 stage of the cell cycle and undergo senescence. Our findings reveal that TRAIP works in S-phase to prevent DNA damage at transcription start sites, caused by replication-transcription conflicts.

Nucleosome retention by histone chaperones and remodelers occludes pervasive DNA-protein binding.

DNA packaging within chromatin depends on histone chaperones and remodelers that form and position nucleosomes. Cells express multiple such chromatin regulators with overlapping in-vitro activities. Defining specific in-vivo activities requires monitoring histone dynamics during regulator depletion, which has been technically challenging. We have recently generated histone-exchange sensors in Saccharomyces cerevisiae, which we now use to define the contributions of 15 regulators to histone dynamics genome-wide. While replication-independent exchange in unperturbed cells maps to promoters, regulator depletions primarily affected gene bodies. Depletion of Spt6, Spt16 or Chd1 sharply increased nucleosome replacement sequentially at the beginning, middle or end of highly expressed gene bodies. They further triggered re-localization of chaperones to affected gene body regions, which compensated for nucleosome loss during transcription complex passage, but concurred with extensive TF binding in gene bodies. We provide a unified quantitative screen highlighting regulator roles in retaining nucleosome binding during transcription and preserving genomic packaging.

ATR protects centromere identity by promoting DAXX association with PML nuclear bodies

Centromere protein A (CENP-A) defines centromere identity and nucleates kinetochore formation for mitotic chromosome segregation. Here, we show that ataxia telangiectasia and Rad3-related (ATR) kinase, a master regulator of the DNA damage response, protects CENP-A occupancy at interphase centromeres in a DNA damage-independent manner. In unperturbed cells, ATR localizes to promyelocytic leukemia nuclear bodies (PML NBs), which house the histone H3.3 chaperone DAXX (death domain-associated protein 6). We find that ATR inhibition reduces DAXX association with PML NBs, resulting in the DAXX-dependent loss of CENP-A and an aberrant increase in H3.3 at interphase centromeres. Additionally, we show that ATR-dependent phosphorylation within the C terminus of DAXX regulates CENP-A occupancy at centromeres and DAXX localization. Lastly, we demonstrate that acute ATR inhibition during interphase leads to kinetochore formation defects and an increased rate of lagging chromosomes. These findings highlight a mechanism by which ATR protects centromere identity and genome stability.

The BRCA1 BRCT promotes antisense RNA production and double-stranded RNA formation to suppress ribosomal R-loops

R-loops, or RNA:DNA hybrids, can induce DNA damage, which requires DNA repair factors including breast cancer type 1 susceptibility protein (BRCA1) to restore genomic integrity. To date, several pathogenic mutations have been found within the tandem BRCA1 carboxyl-terminal (BRCT) domains that mediate BRCA1 interactions with proteins and DNA in response to DNA damage. Here, we describe a nonrepair role of BRCA1 BRCT in suppressing ribosomal R-loops via two mechanisms. Through its RNA binding and annealing activities, BRCA1 BRCT facilitates the formation of double-stranded RNA between ribosomal RNA (rRNA) and antisense-rRNA (as-rRNA), hereby minimizing rRNA hybridization to ribosomal DNA to form R-loops. BRCA1 BRCT also promotes RNA polymerase I-dependent transcription of as-rRNA to enhance double-stranded rRNA (ds-rRNA) formation. In addition, BRCA1 BRCT-mediated as-rRNA production restricts rRNA maturation in unperturbed cells. Hence, impairing as-rRNA transcription and ds-rRNA formation due to BRCA1 BRCT deficiency deregulates rRNA processing and increases ribosomal R-loops and DNA breaks. Our results link ribosomal biogenesis dysfunction to BRCA1-associated genomic instability.