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Fight-or-flight responses involve β-adrenergic-induced increases in heart rate and contractile force. In the present study, we uncover the primary mechanism underlying the heart’s innate contractile reserve. We show that four protein kinase A (PKA)-phosphorylated residues in Rad, a calcium channel inhibitor, are crucial for controlling basal calcium current and essential for β-adrenergic augmentation of calcium influx in cardiomyocytes. Even with intact PKA signaling to other proteins modulating calcium handling, preventing adrenergic activation of calcium channels in Rad-phosphosite-mutant mice (4SA-Rad) has profound physiological effects: reduced heart rate with increased pauses, reduced basal contractility, near-complete attenuation of β-adrenergic contractile response and diminished exercise capacity. Conversely, expression of mutant calcium-channel β-subunits that cannot bind 4SA-Rad is sufficient to enhance basal calcium influx and contractility to adrenergically augmented levels of wild-type mice, rescuing the failing heart phenotype of 4SA-Rad mice. Hence, disruption of interactions between Rad and calcium channels constitutes the foundation toward next-generation therapeutics specifically enhancing cardiac contractility.

Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) are histone-modifying and -binding complexes that mediate the formation of facultative heterochromatin and are required for silencing of developmental genes and maintenance of cell fate1–3. Multiple pathways of RNA decay work together to establish and maintain heterochromatin in fission yeast, including a recently identified role for a conserved RNA-degradation complex known as the rixosome or RIX1 complex4–6. Whether RNA degradation also has a role in the stability of mammalian heterochromatin remains unknown. Here we show that the rixosome contributes to silencing of many Polycomb targets in human cells. The rixosome associates with human PRC complexes and is enriched at promoters of Polycomb target genes. Depletion of either the rixosome or Polycomb results in accumulation of paused and elongating RNA polymerase at Polycomb target genes. We identify point mutations in the RING1B subunit of PRC1 that disrupt the interaction between PRC1 and the rixosome and result in diminished silencing, suggesting that direct recruitment of the rixosome to chromatin is required for silencing. Finally, we show that the RNA endonuclease and kinase activities of the rixosome and the downstream XRN2 exoribonuclease, which degrades RNAs with 5′ monophosphate groups generated by the rixosome, are required for silencing. Our findings suggest that rixosomal degradation of nascent RNA is conserved from fission yeast to human, with a primary role in RNA degradation at facultative heterochromatin in human cells.

Selective breakdown of proteins and aggregates is crucial for maintaining normal cellular activities and is involved in the pathogenesis of diverse diseases. How the cell recognizes and tags these targets in different structural states for degradation by the proteasome and autophagy pathways has not been well understood. Here, we discovered that a HECT-family ubiquitin ligase HUWE1 is broadly required for the efficient degradation of soluble factors and for the clearance of protein aggregates/condensates. Underlying this capacity of HUWE1 is a novel Ubiquitin-Directed ubiquitin Ligase (UDL) activity which recognizes both soluble substrates and aggregates that carry a high density of ubiquitin chains and rapidly expand the ubiquitin modifications on these targets. Ubiquitin signal amplification by HUWE1 recruits the ubiquitin-dependent segregase p97/VCP to process these targets for subsequent degradation or clearance. HUWE1 controls the cytotoxicity of protein aggregates, mediates Targeted Protein Degradation and regulates cell-cycle transitions with its UDL activity.

Fight-or-flight responses involve β-adrenergic-induced increases in heart rate and contractile force. In the present study, we uncover the primary mechanism underlying the heart's innate contractile reserve. We show that four protein kinase A (PKA)-phosphorylated residues in Rad, a calcium channel inhibitor, are crucial for controlling basal calcium current and essential for β-adrenergic augmentation of calcium influx in cardiomyocytes. Even with intact PKA signaling to other proteins modulating calcium handling, preventing adrenergic activation of calcium channels in Rad-phosphosite-mutant mice (4SA-Rad) has profound physiological effects: reduced heart rate with increased pauses, reduced basal contractility, near-complete attenuation of β-adrenergic contractile response and diminished exercise capacity. Conversely, expression of mutant calcium-channel β-subunits that cannot bind 4SA-Rad is sufficient to enhance basal calcium influx and contractility to adrenergically augmented levels of wild-type mice, rescuing the failing heart phenotype of 4SA-Rad mice. Hence, disruption of interactions between Rad and calcium channels constitutes the foundation toward next-generation therapeutics specifically enhancing cardiac contractility.

The B cell receptor (BCR) signals together with a multi-component co-receptor complex to initiate B cell activation in response to antigen binding. This process underlies nearly every aspect of proper B cell function. Here, we take advantage of peroxidase-catalyzed proximity labeling combined with quantitative mass spectrometry to track B cell co-receptor signaling dynamics from 10 seconds to 2 hours after BCR stimulation. This approach enables tracking of 2,814 proximity-labeled proteins and 1,394 quantified phosphosites and provides an unbiased and quantitative molecular map of proteins recruited to the vicinity of CD19, the key signaling subunit of the co-receptor complex. We detail the recruitment kinetics of essential signaling effectors to CD19 following activation, and then identify new mediators of B cell activation. In particular, we show that the glutamate transporter SLC1A1 is responsible for mediating rapid metabolic reprogramming immediately downstream of BCR stimulation and for maintaining redox homeostasis during B cell activation. This study provides a comprehensive map of the BCR signaling pathway and a rich resource for uncovering the complex signaling networks that regulate B cell activation.

Notch signaling relies on ligand-induced proteolysis to liberate a nuclear effector that drives cell fate decisions. The location and timing of individual steps required for proteolysis and movement of Notch from membrane to nucleus, however, remain unclear. Here, we use proximity labeling with quantitative multiplexed mass spectrometry to monitor the microenvironment of endogenous Notch2 after ligand stimulation in the presence of a gamma secretase inhibitor and then as a function of time after inhibitor removal. Our studies show that gamma secretase cleavage of Notch2 occurs in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurs within 45 minutes of inhibitor washout. This work provides a spatiotemporal map of unprecedented detail tracking the itinerary of Notch from membrane to nucleus after activation and identifies molecular events in signal transmission that are potential targets for modulating Notch signaling activity.

The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo1. To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(alnz24) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress. Santoriello, Sporrij et al. show that the progesterone receptor associates with RNA helicase DDX21 during nucleotide depletion, promotes its binding on chromatin and rescues efficient transcription in melanoma cells.

Many of the most effective anti-cancer therapies induce apoptosis in cancer cells by damaging DNA. Even when effective at eradicating primary cancers, DNA-damaging anti-cancer agents cause mutations that can lead to the development of secondary cancers. Furthermore, DNA damage responses are dependent on wild-type p53, which is mutated in over 50% of cancers and causes resistance to therapies. Fortunately, many p53 mutant cancers continue to express high levels of the pro-apoptotic, pore-forming proteins BAX and BAK and are consequently “primed for apoptosis.” Primed cells are highly sensitive to pro-apoptotic signals and this vulnerability can be exploited using inhibitors of pro-survival BCL-2 family proteins such as the BCL-2 inhibitor ABT-199, which has been successful as therapy for chronic lymphocytic and acute myeloid leukemias (AML). Furthermore, most irreplaceable cells within healthy tissues are apoptosis resistant and express low levels of BAX and BAK. We therefore hypothesized that direct activators of BAX or BAK could be effective single-agent therapies for primed cancers and chemo-/radio-sensitizers for unprimed cancers. Using WT versus BAX -/- BAK -/- HeLa cells, we performed a high-throughput screen of nearly 100,000 compounds to identify small molecules that could directly activate BAX or BAK. 196 compounds were identified as hits and multiple orthogonal validation studies identified Apoptosis Inducing Agent 1 (AIA1) as the most specific and potent putative activator of BAX and BAK. Further validation studies demonstrated that AIA1 directly induces cytochrome c release from mitochondria in a BAX/BAK dependent manner and strongly enhances BIM-mediated activation of BAX and BAK and permeabilization of liposomes. Single-agent AIA treatment triggers apoptotic cell death in a broad panel of cancer cell lines and also sensitizes cancer cells to chemotherapeutic agents and especially BH3 mimetics targeting pro-survival BCL-2 family proteins. Importantly, AIA1 sensitizes ovarian cancer cells to BCL-XL inhibitors and AML cells to BCL-2 inhibitors regardless of p53 status. In vivo, AIA1 suppresses tumor growth and prolongs overall survival in both ovarian cancer and AML xenograft models without causing weight loss, thrombocytopenia or leukopenia. Notably, unprimed cancer cells that don’t immediately undergo apoptosis in response to single-agent AIA1 treatment upregulate expression of BAX and BAK, pro-survival proteins (BCL-XL, BCL-2, MCL-1) and pro-apoptotic proteins (BIM, BID, PUMA, Noxa) to produce a more highly primed apoptosis pathway and setting the stage for increased sensitivity to BH3 mimetics. Based on these findings, AIA1 may exploit and induce apoptotic vulnerabilities in cancers in a p53-independent manner and represent a safer strategy to target primed cancer cells while eliminating the potential for secondary malignancies and p53-mediated resistance. Citation Format: Xingping Qin, Cameron Fraser, Adam Presser, Johan KE Spetz, Stacey J. Yu, Gary A. Bradshaw, Marian Kalocsay, Bo R. Rueda, Tudor Moldoveanu, Kristopher A. Sarosiek. Apoptosis Inducing Agent 1 enhances cancer therapy-induced apoptosis by direct interaction with BAX and BAK. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5731.

Rapidly changing and transient protein-protein interactions regulate dynamic cellular processes in the cardiovascular system. Traditional methods, including affinity purification and mass spectrometry, have revealed many macromolecular complexes in cardiomyocytes and the vasculature. Yet these methods often fail to identify in vivo or transient protein-protein interactions. To capture these interactions in living cells and animals with subsequent mass spectrometry identification, enzyme-catalyzed proximity labeling techniques have been developed in the past decade. Although the application of this methodology to cardiovascular research is still in its infancy, the field is developing rapidly, and the promise is substantial. In this review, we outline important concepts and discuss how proximity proteomics has been applied to study physiological and pathophysiological processes relevant to the cardiovascular system.

Increased cardiac contractility during the fight-or-flight response is caused by β-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1–4. However, this augmentation persists in transgenic murine hearts expressing mutant CaV1.2 α1C and β subunits that can no longer be phosphorylated by protein kinase A—an essential downstream mediator of β-adrenergic signalling—suggesting that non-channel factors are also required. Here we identify the mechanism by which β-adrenergic agonists stimulate voltage-gated calcium channels. We express α1C or β2B subunits conjugated to ascorbate peroxidase5 in mouse hearts, and use multiplexed quantitative proteomics6,7 to track hundreds of proteins in the proximity of CaV1.2. We observe that the calcium-channel inhibitor Rad8,9, a monomeric G protein, is enriched in the CaV1.2 microenvironment but is depleted during β-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for β subunits and relieves constitutive inhibition of CaV1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of CaV1.3 and CaV2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels. An in vivo approach to identify proteins whose enrichment near cardiac CaV1.2 channels changes upon β-adrenergic stimulation finds the G protein Rad, which is phosphorylated by protein kinase A, thereby relieving channel inhibition by Rad and causing an increased Ca2+ current.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) cause severe respiratory diseases in infants and elderly adults1. No vaccine or effective antiviral therapy currently exists to control RSV or HMPV infections. During viral genome replication and transcription, the tetrameric phosphoprotein P serves as a crucial adaptor between the ribonucleoprotein template and the L protein, which has RNA-dependent RNA polymerase (RdRp), GDP polyribonucleotidyltransferase and cap-specific methyltransferase activities2,3. How P interacts with L and mediates the association with the free form of N and with the ribonucleoprotein is not clear for HMPV or other major human pathogens, including the viruses that cause measles, Ebola and rabies. Here we report a cryo-electron microscopy reconstruction that shows the ring-shaped structure of the polymerase and capping domains of HMPV-L bound to a tetramer of P. The connector and methyltransferase domains of L are mobile with respect to the core. The putative priming loop that is important for the initiation of RNA synthesis is fully retracted, which leaves space in the active-site cavity for RNA elongation. P interacts extensively with the N-terminal region of L, burying more than 4,016 A2 of the molecular surface area in the interface. Two of the four helices that form the coiled-coil tetramerization domain of P, and long C-terminal extensions projecting from these two helices, wrap around the L protein in a manner similar to tentacles. The structural versatility of the four P protomers—which are largely disordered in their free state—demonstrates an example of a ‘folding-upon-partner-binding’ mechanism for carrying out P adaptor functions. The structure shows that P has the potential to modulate multiple functions of L and these results should accelerate the design of specific antiviral drugs. A cryo-electron microscopy reconstruction shows the ring-shaped structure of the polymerase and capping domains of the L protein bound to a tetramer of phosphoprotein P of the human metapneumovirus.

A fundamental challenge in chemical biology and medicine is to understand and expand the fraction of the human proteome that can be targeted by small molecules. We recently described a strategy that integrates fragment-based ligand discovery with chemical proteomics to furnish global portraits of reversible small-molecule/protein interactions in human cells. Excavating clear structure-activity relationships from these 'ligandability' maps, however, was confounded by the distinct physicochemical properties and corresponding overall protein-binding potential of individual fragments. Here, we describe a compelling solution to this problem by introducing a next-generation set of fully functionalized fragments differing only in absolute stereochemistry. Using these enantiomeric probe pairs, or 'enantioprobes', we identify numerous stereoselective protein-fragment interactions in cells and show that these interactions occur at functional sites on proteins from diverse classes. Our findings thus indicate that incorporating chirality into fully functionalized fragment libraries provides a robust and streamlined method to discover ligandable proteins in cells.

Recent studies demonstrate that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in human fibrotic kidneys, lungs, kidney organoids, and mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts also induced production of alpha-smooth muscle actin (αSMA) and collagen 1. Knockout of COUP-TFII decreased glycolysis and collagen 1 levels in fibroblasts. Chip-qPCR revealed the binding of COUP-TFII on the promoter of PGC1α. Overexpression of COUP-TFII reduced the cellular level of PGC1α. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potentially fibrosis in other organs.

May et al. combine proximity labeling with quantitative mass spectrometry to profile the proteome of primary cilia in cells responding to Hedgehog. They identify signaling factors that undergo dynamic ciliary enrichment and uncover an unanticipated regulation of ciliary cAMP signaling., The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid, and second messenger compositions. Here, we combine proximity labeling of cilia with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein–coupled receptor GPR161 in response to Hedgehog, and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the phosphatase Paladin as a cell type–specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions.

Vertebrate development from an egg to a complex multi-tissue million-cell organism is supported by multiple phases of genome-scale remodeling of the repertoire of proteins and their post-translational modifications, yet so far we know little about these phases. In this paper we present comprehensive characterization of these processes reflected by eleven time points, approximately eleven thousand proteins, and six thousand phospho-forms in two replicates. We find that the most dramatic changes to the proteome occur during the transition to functional organ systems, which occurs as the embryo becomes a tadpole. At that time the absolute amount of non-yolk protein increases two-fold, and there is a shift in the balance of expression from proteins regulating gene expression to receptors, ligands, and proteins involved in cell-cell and cell-environment interactions. Between the early and late tadpole, the median increase of membrane and secreted proteins is substantially higher than that of nuclear proteins. For the first time, we have included quantitative phosphoproteomic data across the same developmental stages. In contrast to the significant protein changes that are concentrated at the end of the time series, the most significant phosphorylation changes are concentrated in the very early stages of development. A clear exception are phosphorylations of proteins involved in gene expression; these increase just after fertilization, with patterns that are highly correlated with the underlying protein changes. To improve our interpretation of this unique data set, we created a pipeline for identifying homologous human phosphorylations from the measured Xenopus phospho-proteome, and we estimated, where possible, the occupancy of phosphorylation sites. Overall, we detected many profound temporal transitions, which suggest concerted changes in developmental strategies in the embryo that are particularly pronounced once early patterning and specification are complete.

We performed quantitative proteomics on 60 human-derived breast cancer cell lines to a depth of ~13,000 proteins. The resulting high-throughput datasets were assessed for quality and reproducibility. We used the datasets to identify and characterize the subtypes of breast cancer and showed that they conform to known transcriptional subtypes, revealing that molecular subtypes are preserved even in under-sampled protein feature sets. All datasets are freely available as public resources on the LINCS portal. We anticipate that these datasets, either in isolation or in combination with complimentary measurements such as genomics, transcriptomics and phosphoproteomics, can be mined for the purpose of predicting drug response, informing cell line specific context in models of signalling pathways, and identifying markers of sensitivity or resistance to therapeutics.

Background Despite objective responses to PARP inhibition and improvements in progression-free survival compared to standard chemotherapy in patients with BRCA-associated triple-negative breast cancer (TNBC), benefits are transitory. Methods Using high dimensional single-cell profiling of human TNBC, here we demonstrate that macrophages are the predominant infiltrating immune cell type in BRCA-associated TNBC. Through multi-omics profiling we show that PARP inhibitors enhance both anti- and pro-tumor features of macrophages through glucose and lipid metabolic reprogramming driven by the sterol regulatory element-binding protein 1 (SREBP-1) pathway. Results Combined PARP inhibitor therapy with CSF-1R blocking antibodies significantly enhanced innate and adaptive anti-tumor immunity and extends survival in BRCA-deficient tumors in vivo and is mediated by CD8+ T-cells. Conclusions Collectively, our results uncover macrophage-mediated immune suppression as a liability of PARP inhibitor treatment and demonstrate combined PARP inhibition and macrophage targeting therapy induces a durable reprogramming of the tumor microenvironment, thus constituting a promising therapeutic strategy for TNBC.

Normal tissue physiology and repair depends on communication with the immune system. Understanding this communication at the molecular level in intact tissue requires new methods. The consequences of SARS-CoV-2 infection, which can result in acute respiratory distress, thrombosis and death, has been studied primarily in accessible liquid specimens such as blood, sputum and bronchoalveolar lavage, all of which are peripheral to the primary site of infection in the lung. Here, we describe the combined use of multiplexed deep proteomics with multiplexed imaging to profile infection and its sequelae directly in fixed lung tissue specimens obtained from necropsy of infected animals and autopsy of human decedents. We characterize multiple steps in disease response from cytokine accumulation and protein phosphorylation to activation of receptors, changes in signaling pathways, and crosslinking of fibrin to form clots. Our data reveal significant differences between naturally resolving SARS-CoV-2 infection in rhesus macaques and lethal COVID-19 in humans. The approach we describe is broadly applicable to other tissues and diseases.SummaryProteomics of infected tissue reveals differences in inflammatory and thrombotic responses between resolving and lethal COVID-19.

Author(s): May, Elena; Kalocsay, Marian; D’Auriac, Ines Galtier; Gygi, Steven; Nachury, Maxence; Mick, David | Abstract: ABSTRACT The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid and second messenger compositions. Here, we combine proximity labeling of cilia with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein-coupled receptor GPR161 in response to Hedgehog; and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the putative phosphatase Paladin as a cell type-specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions.

Recent studies demonstrated that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in kidneys of patients with chronic kidney disease, fibrotic lungs of patients with idiopathic pulmonary fibrosis, fibrotic human kidney organoids, and fibrotic mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts was sufficient to enhance glycolysis and increase alpha smooth muscle actin (αSMA) and collagen1 levels. Knockout of COUP-TFII decreased glycolysis and collagen1 levels in fibroblasts. Chip-qPCR assays revealed the binding of COUP-TFII on the promoter of PGC1α, a critical regulator of mitochondrial genesis and oxidative metabolism. Overexpression of COUP-TFII reduced the cellular level of PGC1α. In conclusion, COUP-TFII mediates fibrosis by serving as a key regulator of the shift in cellular metabolism of interstitial pericytes/fibroblasts from oxidative respiration to aerobic glycolysis. The fibrogenic response may share a common pathway in different organ injury and failure. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potential other organ fibrosis.

Heterochromatic domains containing histone H3 lysine 9 methylation (H3K9me) can be epigenetically inherited independently of underlying DNA sequence. To gain insight into the mechanisms that mediate epigenetic inheritance, we used a Schizosaccharomyces pombe inducible heterochromatin formation system to perform a genetic screen for mutations that abolish heterochromatin inheritance without affecting its establishment. We identified mutations in several pathways, including the conserved and essential Rix1-associated complex (henceforth the rixosome), which contains RNA endonuclease and polynucleotide kinase activities with known roles in ribosomal RNA processing. We show that the rixosome is required for spreading and epigenetic inheritance of heterochromatin in fission yeast. Viable rixosome mutations that disrupt its association with Swi6/HP1 fail to localize to heterochromatin, lead to accumulation of heterochromatic RNAs, and block spreading of H3K9me and silencing into actively transcribed regions. These findings reveal a new pathway for degradation of heterochromatic RNAs with essential roles in heterochromatin spreading and inheritance.

Background The developing field of osteoimmunology supports importance of an interferon (IFN) response pathway in osteoblasts. Clarifying osteoblast-IFN interactions is important because IFN is used as salvage anti-tumor therapy but systemic toxicity is high with variable clinical results. In addition, osteoblast response to systemic bursts and disruptions of IFN pathways induced by viral infection may influence bone remodeling. ZIKA virus (ZIKV) infection impacts bone development in humans and IFN response in vitro. Consistently, initial evidence of permissivity to ZIKV has been reported in human osteoblasts. Hypothesis Osteoblast-like Saos-2 cells are permissive to ZIKV and responsive to IFN. Methods Multiple approaches were used to assess whether Saos-2 cells are permissive to ZIKV infection and exhibit IFN-mediated ZIKV suppression. Proteomic methods were used to evaluate impact of ZIKV and IFN on Saos-2 cells. Results Evidence is presented confirming Saos-2 cells are permissive to ZIKV and support IFN-mediated suppression of ZIKV. ZIKV and IFN differentially impact the Saos-2 proteome, exemplified by HELZ2 protein which is upregulated by IFN but non responsive to ZIKV. Both ZIKV and IFN suppress proteins associated with microcephaly/pseudo-TORCH syndrome (BI1, KI20A and UBP18), and ZIKV induces potential entry factor PLVAP. Conclusions Transient ZIKV infection influences osteoimmune state, and IFN and ZIKV activate distinct proteomes in Saos-2 cells, which could inform therapeutic, engineered, disruptions.

Cyclin-dependent kinase 12 (CDK12) is an emerging therapeutic target due to its role in regulating transcription of DNA-damage response (DDR) genes. However, development of selective small molecules targeting CDK12 has been challenging due to the high degree of homology between kinase domains of CDK12 and other transcriptional CDKs, most notably CDK13. In the present study, we report the rational design and characterization of a CDK12-specific degrader, BSJ-4-116. BSJ-4-116 selectively degraded CDK12 as assessed through quantitative proteomics. Selective degradation of CDK12 resulted in premature cleavage and poly(adenylation) of DDR genes. Moreover, BSJ-4-116 exhibited potent antiproliferative effects, alone and in combination with the poly(ADP-ribose) polymerase inhibitor olaparib, as well as when used as a single agent against cell lines resistant to covalent CDK12 inhibitors. Two point mutations in CDK12 were identified that confer resistance to BSJ-4-116, demonstrating a potential mechanism that tumor cells can use to evade bivalent degrader molecules. The discovery of a specific CDK12 bivalent degrader, BSJ-4-116, reveals that chronic exposure of MOLT-4 and Jurkat cells to BSJ-4-116 leads to acquired resistance to the compound via point mutations in the CDK12 kinase domain.

Histone H3 lysine 9 methylation (H3K9me) mediates heterochromatic gene silencing and is important for genome stability and the regulation of gene expression1–4. The establishment and epigenetic maintenance of heterochromatin involve the recruitment of H3K9 methyltransferases to specific sites on DNA, followed by the recognition of pre-existing H3K9me by the methyltransferase and methylation of proximal histone H35–11. This positive feedback loop must be tightly regulated to prevent deleterious epigenetic gene silencing. Extrinsic anti-silencing mechanisms involving histone demethylation or boundary elements help to limit the spread of inappropriate H3K9me12–15. However, how H3K9 methyltransferase activity is locally restricted or prevented from initiating random H3K9me—which would lead to aberrant gene silencing and epigenetic instability—is not fully understood. Here we reveal an autoinhibited conformation in the conserved H3K9 methyltransferase Clr4 (also known as Suv39h) of the fission yeast Schizosaccharomyces pombe that has a critical role in preventing aberrant heterochromatin formation. Biochemical and X-ray crystallographic data show that an internal loop in Clr4 inhibits the catalytic activity of this enzyme by blocking the histone H3K9 substrate-binding pocket, and that automethylation of specific lysines in this loop promotes a conformational switch that enhances the H3K9me activity of Clr4. Mutations that are predicted to disrupt this regulation lead to aberrant H3K9me, loss of heterochromatin domains and inhibition of growth, demonstrating the importance of the intrinsic inhibition and auto-activation of Clr4 in regulating the deposition of H3K9me and in preventing epigenetic instability. Conservation of the Clr4 autoregulatory loop in other H3K9 methyltransferases and the automethylation of a corresponding lysine in the human SUV39H2 homologue16 suggest that the mechanism described here is broadly conserved. An autoinhibitory conformation of the histone H3K9 methyltransferase Clr4 of Schizosaccharomyces pombe helps to prevent aberrant heterochromatin formation and maintains epigenetic stability.

The Hippo pathway regulates organ size, regeneration, and cell growth by controlling the stability of the transcription factor, YAP (Yorkie in Drosophila). When there is tissue damage, YAP is activated allowing the restoration of homeostatic tissue size. The exact signals by which YAP is activated are still not fully understood, but its activation is known to affect both cell size and cell number. Here we used cultured cells to examine the coordinated regulation of cell size and number under the control of YAP. Our experiments in isogenic HEK293 cells reveal that YAP can affect cell size and number by independent circuits. Some of these effects are cell autonomous, such as proliferation, while others are mediated by secreted signals. In particular CYR61, a known secreted YAP target, is a non-cell autonomous mediator of cell survival, while another unidentified secreted factor controls cell size.

Heterochromatin assembly requires the methylation of histone H3 lysine 9 by the Clr4(Suv39h) methyltransferase and both the spreading and epigenetic inheritance of heterochromatin involve the recognition of H3K9me-containing nucleosomes by Clr4 and catalysis of H3K9me on adjacent nucleosomes. How this read-write mechanism overcomes obstacles posed by RNA polymerase II and nascent RNA in its path is not fully understood. Here we identify a role for the highly conserved and essential Rix1-containing complex (here referred to as the rixosome), with known RNA endonuclease and polynucleotide kinase activities required for ribosomal RNA (rRNA) processing, in spreading and epigenetic inheritance of heterochromatin. Viable mutations in rixosome subunits that disrupt its association with Swi6/HP1 fail to localize to heterochromatin, lead to accumulation of heterochromatic RNAs, and block spreading of H3K9me and silencing away from nucleation sites into actively transcribed regions. These findings reveal a new pathway for degradation of heterochromatic RNAs with essential roles in heterochromatin spreading and inheritance.

Peroxidase-catalyzed proximity labeling is a powerful technique for defining the molecular environment of proteins in vivo. Expressing a protein of interest fused to a modified plant peroxidase (APEX2) allows labeling of nearby polypeptides. Addition of hydrogen peroxide (H

Proteins are essential agents of biological processes. To date, large-scale profiling of cell line collections including the Cancer Cell Line Encyclopedia (CCLE) has focused primarily on genetic information whereas deep interrogation of the proteome has remained out of reach. Here, we expand the CCLE through quantitative profiling of thousands of proteins by mass spectrometry across 375 cell lines from diverse lineages to reveal information undiscovered by DNA and RNA methods. We observe unexpected correlations within and between pathways that are largely absent from RNA. An analysis of microsatellite instable (MSI) cell lines reveals the dysregulation of specific protein complexes associated with surveillance of mutation and translation. These and other protein complexes were associated with sensitivity to knockdown of several different genes. These data in conjunction with the wider CCLE are a broad resource to explore cellular behavior and facilitate cancer research.

SummaryWe present an unprecedentedly comprehensive characterization of protein dynamics across early development inXenopus laevis, available immediately via a convenient Web portal. This resource allows interrogation of the protein expression data in conjunction with other data modalities such as genome wide mRNA expression. This study provides detailed data for absolute levels of ∼14K uniqueXenopusproteins representing homologues of ∼9K unique human genes – a rich resource for developmental biologists. The purpose of this manuscript is limited to presenting and releasing the data browser.HighlightsRelative protein expression from stage IV oocyte, blastula, gastrula, neurula, and early organogenesisBiological triplicates with confidence intervals on protein expression reflect certainty in dynamic patternsConvenient time-series Web-browser integrated with the multi-media Xenbase portalGene-symbol search and multi-gene protein/mRNA juxtaposition capabilities

A fundamental challenge in chemical biology and medicine is to understand and expand the fraction of the human proteome that can be targeted by small molecules. We recently described a strategy that integrates fragment-based ligand discovery with chemical proteomics to furnish global portraits of reversible small molecule-protein interactions in human cells.Excavating clear structure-activity relationships from these “ligandability” maps, however, was confounded by the distinct physicochemical properties and corresponding overall protein-binding potential of individual fragments. Here, we describe a compelling solution to this problem by introducing a next-generation set of fully functionalized fragments (FFFs) differing only in absolute stereochemistry. Using these enantiomeric probe pairs, or “enantioprobes”, we identify numerous stereoselective protein-fragment interactions in cells and show that these interactions occur at functional sites on proteins from diverse classes. Our findings thus indicate that incorporating chirality into FFF libraries provides a robust and streamlined method to discover ligandable proteins in cells.

Peroxidase-catalyzed proximity labeling is a powerful technique for defining the molecular environment of proteins in vivo. Expressing a protein of interest fused to a modified plant peroxidase (APEX2) allows labeling of nearby polypeptides. Addition of hydrogen peroxide (H2O2) and biotin-tyramide (biotin-phenol) generates short-lived radicals around the peroxidase. Labeling is thus restricted to proteins in close proximity, providing a snapshot of the local environment around the APEX2 fusion protein. Combined with an initial perturbation, progressive changes in interaction partners can be tracked, e.g., after drug treatment. Multiplexed quantitative mass spectrometry permits the parallel analysis of several experimental replicates or of up to 11 time points. Here we describe the denaturing purification of biotin-labeled proteins with magnetic streptavidin beads, and subsequent sample preparation for multiplexed quantitative mass spectrometry. Proximity-labeled proteins are enriched under strong denaturing conditions. Tryptic on-bead digest of purified proteins is combined with tandem mass tag peptide labeling (TMT), alkaline reversed-phase peptide fractionation, and SPS MS3 mass spectrometry. This analysis pipeline enables studies of complex protein environment changes in perturbed biological systems, as well as comparative studies of functional protein proximity in different cell lines. Through multiplexing, hundreds of proteins can be quantified in each experimental condition in parallel.

Patients with BRCA-associated triple-negative breast cancer (TNBC) have few effective treatment options. PARP inhibitors are promising, and we recently showed they induce an influx of white blood cells, including CD8+ T cells and macrophages into the tumor. The influx of CD8+ cells, mediated by activation of the STING pathway in tumor cells, contributes substantially to efficacy of PARP inhibition in mice. Strikingly, in these studies the greatest infiltration of immune cells into the tumor was macrophages. Given that objective responses to PARP inhibition have been observed in clinical trials but the benefits are transitory, we hypothesized that this was due to a suppressive tumor microenvironment, driven by tumor macrophages. To better understand the molecular basis of resistance to PARP inhibitors, we used high-dimensional single-cell immune profiling on human TNBC. We observed a ≥10-fold increase in TAMs in BRCA-associated TNBC compared to BRCA-wild-type TNBC. Using a preclinical model of BRCA1-deficient triple-negative breast cancer, we found that PARP inhibitors not only further increased TAM abundance but also induced functional and phenotypic changes associated with STING pathway activation, antigen presentation, and chemokine and cytokine signaling. PARP inhibitors increased the frequency of TAMs expressing costimulatory molecules CD80 and CD86 as well as the activation and maturation marker CD40, which are indicative of an antitumor phenotype. We also identified a novel negative feedback mechanism that limits the functionality of the anti-tumor TAMs and is consistent with induction of an immune-suppressive macrophage population. Utilizing transcriptomic, proteomic, and metabolic profiling of ex vivo cultured human myeloid cells, we identified multiple biologic processes associated with PARP inhibition, showing that these drugs directly affect macrophage states and phenotypes. Remarkably, in the preclinical BRCA1-deficient TNBC model, the novel combination of PARP inhibition with macrophage modulation significantly extended remissions obtained with PARP inhibitor therapy only, and this advantage persisted when treatment was discontinued, suggestive of a durable reprogramming of the tumor microenvironment. Moreover, CD8+ cells were required for the extension of PARP inhibitor-induced remissions, suggesting that targeting macrophages lifted the constraints imposed by protumor macrophages on CD8+ T cell-mediated tumor cell killing. We identify mechanisms related to macrophage and T-cell activation that increase PFS and provide evidence that TAMs may serve as targets for new therapeutic interventions designed to overcome PARP inhibitor resistance in BRCA-associated TNBC. Citation Format: Anita K. Mehta, Emily M. Cheney, Jessica A. Castrillon, Jia-Ren Lin, Mateus de Oliveira Taveira, Christina A. Hartl, Nathan T. Johnson, William M. Oldham, Marian Kalocsay, Sarah A. Boswell, Olmo Sonzogni, Constantia Pantelidou, Brett P. Gross, Shawn Johnson, Deborah A. Dillon, Sandro Santagata, Judy E. Garber, Nadine Tung, Elizabeth A. Mittendorf, Gerburg M. Wulf, Geoffrey I. Shapiro, Peter K. Sorger, Jennifer L. Guerriero. PARP inhibition modulates the infiltration, phenotype, and function of tumor-associated macrophages (TAMs) in BRCA-associated breast cancer and can be augmented by harnessing the antitumor potential of TAMs [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A105.

SummaryThe Hippo pathway, in which changes at the cell surface and in the extracellular environment control the activity of a downstream transcription factor, known as YAP in mammalian cells and Yorkie in Drosophila, has recently taken center-stage as perhaps the most important pathway in metazoans for controlling organ size. In intact tissues YAP activity is inhibited and the organ does not overgrow. When the organ is damaged, YAP is active and necessary for growth and regeneration to occur. The exact process by which YAP drives organ and tissue growth is not fully understood, although it is known to affect both cell size and cell number. Since cell size and proliferation are highly interdependent in many cultured cell studies, we investigated the role of YAP in the simultaneous regulation of both cell size and number. Our experiments reveal that YAP controls both cell size and cell proliferation by independent circuits, and that it affects each process non-cell autonomously via extracellular mediators. We identify that CYR61, a known secreted YAP target, is the major regulator of the non-cell autonomous increase in cell number, but does not affect cell size. The molecular identity of the non-cell autonomously acting mediator of cell size is yet to be identified.

Cyclin-dependent kinase 14 (CDK14) and other TAIRE family kinases (CDKs 15-18) are proteins that lack functional annotation but are frequent off-targets of clinical kinase inhibitors. In this study we develop and characterize FMF-04-159-2, a tool compound that specifically targets CDK14 covalently and possesses a TAIRE kinase-biased selectivity profile. This tool compound and its reversible analog were used to characterize the cellular consequences of covalent CDK14 inhibition, including an unbiased investigation using phospho-proteomics. To reduce confounding off-target activity, washout conditions were used to deconvolute CDK14-specific effects. This investigation suggested that CDK14 plays a supporting role in cell-cycle regulation, particularly mitotic progression, and identified putative CDK14 substrates. Together, these results represent an important step forward in understanding the cellular consequences of inhibiting CDK14 kinase activity.

G-protein-coupled receptors (GPCRs) play critical roles in regulating physiological processes ranging from neurotransmission to cardiovascular function. Current methods for tracking GPCR signaling suffer from low throughput, modification or overexpression of effector proteins, and low temporal resolution. Here, we show that peroxidase-catalyzed proximity labeling can be combined with isobaric tagging and mass spectrometry to enable quantitative, time-resolved measurement of GPCR agonist response in living cells. Using this technique, termed "GPCR-APEX," we track activation and internalization of the angiotensin II type 1 receptor and the β2 adrenoceptor. These receptors co-localize with a variety of G proteins even before receptor activation, and activated receptors are largely sequestered from G proteins upon internalization. Additionally, the two receptors show differing internalization kinetics, and we identify the membrane protein LMBRD2 as a potential regulator of β2 adrenoceptor signaling, underscoring the value of a dynamic view of receptor function.

Patients with BRCA-associated triple negative breast cancer (TNBC) have few effective treatment options. PARP inhibitors are promising, and we recently showed they induce an influx of white blood cells, including CD8+ T-cells and macrophages into the tumor. The influx of CD8+ cells, mediated by activation of the STING pathway in tumor cells, contributes substantially to efficacy of PARP inhibition in mice. Strikingly, in these studies, the greatest infiltration of immune cells into the tumor was macrophages. Given objective responses to PARP inhibition have been observed in clinical trials but the benefits are transitory, we hypothesized that this was presumably due to a suppressive tumor microenvironment, driven by tumor macrophages. To better understand the molecular basis of resistance to PARP inhibitors, we used high dimensional single-cell immune profiling on human TNBC. We observed a ≥10-fold increase in TAMs in BRCA-associated TNBC compared to BRCA-wildtype TNBC. Using a pre-clinical model of BRCA1-deficient triple-negative breast cancer, we found that PARP inhibitors not only further increased TAM abundance but also induced functional and phenotypic changes associated with STING pathway activation, antigen presentation, and chemokine and cytokine signaling. PARP inhibitors increased the frequency of TAMs expressing co-stimulatory molecules CD80 and CD86 as well as the activation and maturation marker CD40, which are indicative of an anti-tumor phenotype. We also identified a novel negative feedback mechanism which limits the functionality of the anti-tumor TAMs, and is consistent with induction of an immune suppressive macrophage population. Utilizing transcriptomic, proteomic and metabolic profiling of ex vivo cultured human myeloid cells, we identified multiple biological processes associate with PARP inhibition, showing that these drugs directly affect macrophage states and phenotypes. Remarkably, in the pre-clinical BRCA1-deficient TNBC model, the novel combination of PARP inhibition with macrophage modulation significantly extended remissions obtained with PARP inhibitor therapy only, and this advantage persisted when treatment was discontinued, suggestive of a durable reprogramming of the tumor microenvironment. Moreover, CD8+ cells were required for the extension of PARP inhibitor-induced remissions, suggesting that targeting macrophages lifted the constraints imposed by pro-tumor macrophages on CD8+ T cell-mediated tumor cell killing. We identify mechanisms related to macrophage and T-cell activation that increase PFS and provide evidence that TAMs may serve as targets for new therapeutic interventions designed to overcome PARP inhibitor resistance in BRCA-associated TNBC. Citation Format: Jennifer L Guerriero, Anita K Mehta, Emily M Cheney, Jessica A. Castrillon, Jia-Ren Lin, Mateus de Oliveira Taveira, Olmo Sonzogni, Constantia Pantelidou, Christina A Hartl, William M Oldham, Nathan T Johnson, Sarah A Boswell, Marian Kalocsay, Matthew J Berberich, Sholin Mei, Dan Wang, Shawn Johnson, Brett Gross, Deborah A Dillon, Mikel Lipschitz, Evisa Gjini, Scott Rodig, Sandro Santagata, Judy E Garber, Nadine Tung, Peter Sorger, Geoffrey I Shapiro, Gerburg M Wulf, Elizabeth A Mittendorf. PARP inhibition modulates the infiltration, phenotype and function of tumor-associated macrophages (TAMs) in BRCA-associated breast cancer and can be augmented by harnessing the anti-tumor potential of TAMs [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-01.

Homologous recombination (HR) is crucial for genetic exchange and accurate repair of DNA double-strand breaks and is pivotal for genome integrity. HR uses homologous sequences for repair, but how homology search, the exploration of the genome for homologous DNA sequences, is conducted in the nucleus remains poorly understood. Here, we use time-resolved chromatin immunoprecipitations of repair proteins to monitor homology search in vivo. We found that homology search proceeds by a probing mechanism, which commences around the break and samples preferentially on the broken chromosome. However, elements thought to instruct chromosome loops mediate homology search shortcuts, and centromeres, which cluster within the nucleus, may facilitate homology search on other chromosomes. Our study thus reveals crucial parameters for homology search in vivo and emphasizes the importance of linear distance, chromosome architecture, and proximity for recombination efficiency.

Stimulation of hematopoietic stem and progenitor cells (HSPCs) with the inflammatory mediator Prostaglandin E2 (PGE2) enhances self-renewal and stem cell engraftment following transplantation. Currently, the long-acting derivative of PGE2, 16,16-dimethyl-PGE2 (dmPGE2) is in its fourth clinical trial to improve HSC engraftment and reduce graft versus host disease. To understand the effect of dmPGE2, we assessed genome-wide chromatin reorganization and gene expression changes in human CD34+ HSPCs after 2 hours of dmPGE2 treatment, the time period of treatment in the clinical trials. Enhancers are known to regulate gene expression changes in specific environmental contexts such as stress or inflammation, however the regulatory principles by which subsets of enhancers become activated are poorly understood. Here, we mapped active enhancers by ChIP-seq for H2K27ac and found that dmPGE2 activates a discrete set of enhancers in HSPCs. To investigate enhancer chromatin remodeling, we performed micrococcal nuclease digestion followed by high-throughput sequencing (MNase-seq) to map the occupancy and position of nucleosomes. We found that, contrary to the predominant assumption that open chromatin structures are essentially nucleosome-free, MNase-accessible nucleosomes are retained at inducible enhancers following dmPGE2 stimulation. Through ATAC-seq analysis we mapped changes in open chromatin and found that induced enhancers gain chromatin accessibility following stimulation while maintaining their nucleosome configuration. Surprisingly, this indicates that nucleosomes present at the center of dmPGE2-responsive enhancers play an important function in enhancer accessibility and activity. We then correlated enhancers with gene expression changes by performing RNA-seq and found that genes associated with dmPGE2-induced enhancers display higher gene expression changes after stimulation compared to genes associated with non-responsive enhancers. Transcripts upregulated after dmPGE2 treatment include previously identified regulators of self-renewal and migration such as NR4A2, EGR1 and CXCR4. Moreover, inflammatory chemokines including CXCL2 and CXCL8 as well as members of the activating protein 1 (AP-1) transcription factor gene family such as FOS, FOSL2 and JUNB are increasingly expressed upon stimulation. The gene expression profile included a signature implying CREB as the main transcription factor responsible for the acute dmPGE2 response. Western blot revealed dmPGE2-mediated activation of the signaling transcription factor CREB through phosphorylation in HSPCs. Using ChIP-seq, we found increased genomic binding of phospho-CREB (pCREB) after dmPGE2 treatment in the enhancers. Surprisingly, the binding of pCREB coincided directly with variant histone H2A.Z containing labile nucleosomes in enhancers. We validated the interaction between pCREB and H2A.Z on chromatin in dmPGE2-responsive U937 cells through chromatin fractionation followed by complex immunoprecipitation. This suggests that labile nucleosomes provide sufficient DNA access to allow for binding of pCREB at enhancers. Taken together, our study proposes a novel model for stimulus-mediated activation of enhancers by the inflammatory mediator dmPGE2. dmPGE2 induces the phosphorylation of CREB and subsequently leads to a specific interaction of pCREB with previously deposited H2A.Z-rich nucleosomes at inducible enhancers who regulate genes that promote HSPC fate. This new mechanism of variant histone deposition followed by the interaction with a signaling transcription factor at enhancers supports a rapid inducible response from the environment. Disclosures No relevant conflicts of interest to declare.

Yeast RNA polymerase (Pol) II consists of a 10-subunit core enzyme and the Rpb4/7 subcomplex, which is dispensable for catalytic activity and dissociates in vitro. To investigate whether Rpb4/7 is an integral part of DNA-associated Pol II in vivo, we used chromatin immunoprecipitation coupled to high resolution tiling microarray analysis. We show that the genome-wide occupancy profiles for Rpb7 and the core subunit Rpb3 are essentially identical. Thus, the complete Pol II associates with DNA in vivo, consistent with functional roles of Rpb4/7 throughout the transcription cycle.

Cell fate transitions involve rapid gene expression changes and global chromatin remodeling, yet the underlying regulatory pathways remain incompletely understood. Here, we identified the RNA-processing factor Nudt21 as a novel regulator of cell fate change using transcription-factor-induced reprogramming as a screening assay. Suppression of Nudt21 enhanced the generation of induced pluripotent stem cells, facilitated transdifferentiation into trophoblast stem cells, and impaired differentiation of myeloid precursors and embryonic stem cells, suggesting a broader role for Nudt21 in cell fate change. We show that Nudt21 directs differential polyadenylation of over 1,500 transcripts in cells acquiring pluripotency, although only a fraction changed protein levels. Remarkably, these proteins were strongly enriched for chromatin regulators, and their suppression neutralized the effect of Nudt21 during reprogramming. Collectively, our data uncover Nudt21 as a novel post-transcriptional regulator of cell fate and establish a direct, previously unappreciated link between alternative polyadenylation and chromatin signaling.

DNA double-strand breaks (DSBs) are acutely hazardous for cells, as they can cause genome instability. DSB repair involves the sequential recruitment of repair factors to the DSBs, followed by Rad51-mediated homology probing, DNA synthesis, and ligation. However, little is known about how cells react if no homology is found and DSBs persist. Here, by monitoring a single persistent DNA break, we show that, following DNA resection and RPA recruitment, Rad51 spreads chromosome-wide bidirectionally from the DSB but selectively only on the broken chromosome. Remarkably, the persistent DSB is later fixed to the nuclear periphery in a process that requires Rad51, the histone variant H2A.Z, its SUMO modification, and the DNA-damage checkpoint. Indeed, H2A.Z is deposited close to the break early but transiently and directs DNA resection, single DSB-induced checkpoint activation, and DSB anchoring. Thus, a persistent DSB induces a multifaceted response, which is linked to a specific chromatin mark.