The BRCT domain of PARP1 binds intact DNA and mediates intrastrand transfer.

PARP1 is a key player in the response to DNA damage and is the target of clinical inhibitors for the treatment of cancers. Binding of PARP1 to damaged DNA leads to activation wherein PARP1 uses NAD+ to add chains of poly(ADP-ribose) onto itself and other nuclear proteins. PARP1 also binds abundantly to intact DNA and chromatin, where it remains enzymatically inactive. We show that intact DNA makes contacts with the PARP1 BRCT domain, which was not previously recognized as a DNA-binding domain. This binding mode does not result in the concomitant reorganization and activation of the catalytic domain. We visualize the BRCT domain bound to nucleosomal DNA by cryogenic electron microscopy and identify a key motif conserved from ancestral BRCT domains for binding phosphates on DNA and phospho-peptides. Finally, we demonstrate that the DNA-binding properties of the BRCT domain contribute to the "monkey-bar mechanism" that mediates DNA transfer of PARP1.

Histone Acetyltransferase p300 Induces De Novo Super-Enhancers to Drive Cellular Senescence.

Accumulation of senescent cells during aging contributes to chronic inflammation and age-related diseases. While senescence is associated with profound alterations of the epigenome, a systematic view of epigenetic factors in regulating senescence is lacking. Here, we curated a library of short hairpin RNAs for targeted silencing of all known epigenetic proteins and performed a high-throughput screen to identify key candidates whose downregulation can delay replicative senescence of primary human cells. This screen identified multiple new players including the histone acetyltransferase p300 that was found to be a primary driver of the senescent phenotype. p300, but not the paralogous CBP, induces a dynamic hyper-acetylated chromatin state and promotes the formation of active enhancer elements in the non-coding genome, leading to a senescence-specific gene expression program. Our work illustrates a causal role of histone acetyltransferases and acetylation in senescence and suggests p300 as a potential therapeutic target for senescence and age-related diseases.

Condensin-Dependent Chromatin Compaction Represses Transcription Globally during Quiescence.

Quiescence is a stress-resistant state in which cells reversibly exit the cell cycle and suspend most processes. Quiescence is essential for stem cell maintenance, and its misregulation is implicated in tumor formation. One of the hallmarks of quiescent cells is highly condensed chromatin. Because condensed chromatin often correlates with transcriptional silencing, it has been hypothesized that chromatin compaction represses transcription during quiescence. However, the technology to test this model by determining chromatin structure within cells at gene resolution has not previously been available. Here, we use Micro-C XL to map chromatin contacts at single-nucleosome resolution genome-wide in quiescent Saccharomyces cerevisiae cells. We describe chromatin domains on the order of 10-60 kilobases that, only in quiescent cells, are formed by condensin-mediated loops. Condensin depletion prevents the compaction of chromatin within domains and leads to widespread transcriptional de-repression. Finally, we demonstrate that condensin-dependent chromatin compaction is conserved in quiescent human fibroblasts.

Transcriptional Responses to IFN-γ Require Mediator Kinase-Dependent Pause Release and Mechanistically Distinct CDK8 and CDK19 Functions.

Transcriptional responses to external stimuli remain poorly understood. Using global nuclear run-on followed by sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq), we show that CDK8 kinase activity promotes RNA polymerase II pause release in response to interferon-γ (IFN-γ), a universal cytokine involved in immunity and tumor surveillance. The Mediator kinase module contains CDK8 or CDK19, which are presumed to be functionally redundant. We implemented cortistatin A, chemical genetics, transcriptomics, and other methods to decouple their function while assessing enzymatic versus structural roles. Unexpectedly, CDK8 and CDK19 regulated different gene sets via distinct mechanisms. CDK8-dependent regulation required its kinase activity, whereas CDK19 governed IFN-γ responses through its scaffolding function (i.e., it was kinase independent). Accordingly, CDK8, not CDK19, phosphorylates the STAT1 transcription factor (TF) during IFN-γ stimulation, and CDK8 kinase inhibition blocked activation of JAK-STAT pathway TFs. Cytokines such as IFN-γ rapidly mobilize TFs to "reprogram" cellular transcription; our results implicate CDK8 and CDK19 as essential for this transcriptional reprogramming.

BRCA1 Haploinsufficiency Is Masked by RNF168-Mediated Chromatin Ubiquitylation.

BRCA1 functions at two distinct steps during homologous recombination (HR). Initially, it promotes DNA end resection, and subsequently it recruits the PALB2 and BRCA2 mediator complex, which stabilizes RAD51-DNA nucleoprotein filaments. Loss of 53BP1 rescues the HR defect in BRCA1-deficient cells by increasing resection, suggesting that BRCA1's downstream role in RAD51 loading is dispensable when 53BP1 is absent. Here we show that the E3 ubiquitin ligase RNF168, in addition to its canonical role in inhibiting end resection, acts in a redundant manner with BRCA1 to load PALB2 onto damaged DNA. Loss of RNF168 negates the synthetic rescue of BRCA1 deficiency by 53BP1 deletion, and it predisposes BRCA1 heterozygous mice to cancer. BRCA1+/-RNF168-/- cells lack RAD51 foci and are hypersensitive to PARP inhibitor, whereas forced targeting of PALB2 to DNA breaks in mutant cells circumvents BRCA1 haploinsufficiency. Inhibiting the chromatin ubiquitin pathway may, therefore, be a synthetic lethality strategy for BRCA1-deficient cancers.

Transient Hysteresis in CDK4/6 Activity Underlies Passage of the Restriction Point in G1.

Cells escape the need for mitogens at a restriction point several hours before entering S phase. The restriction point has been proposed to result from CDK4/6 initiating partial Rb phosphorylation to trigger a bistable switch whereby cyclin E-CDK2 and Rb mutually reinforce each other to induce Rb hyperphosphorylation. Here, using single-cell analysis, we unexpectedly found that cyclin E/A-CDK activity can only maintain Rb hyperphosphorylation starting at the onset of S phase and that CDK4/6 activity, but not cyclin E/A-CDK activity, is required to hyperphosphorylate Rb throughout G1 phase. Mitogen removal in G1 results in a gradual loss of CDK4/6 activity with a high likelihood of cells sustaining Rb hyperphosphorylation until S phase, at which point cyclin E/A-CDK activity takes over. Thus, it is short-term memory, or transient hysteresis, in CDK4/6 activity following mitogen removal that sustains Rb hyperphosphorylation, demonstrating a probabilistic rather than an irreversible molecular mechanism underlying the restriction point.

MST1 Negatively Regulates TNFα-Induced NF-κB Signaling through Modulating LUBAC Activity.

The nuclear factor (NF)-κB pathway plays a central role in inflammatory and immune responses, with aberrant activation of NF-κB signaling being implicated in various human disorders. Here, we show that mammalian ste20-like kinase 1 (MST1) is a previously unrecognized component of the tumor necrosis factor α (TNFα) receptor 1 signaling complex (TNF-RSC) and attenuates TNFα-induced NF-κB signaling. Genetic ablation of MST1 in mouse embryonic fibroblasts and bone marrow-derived macrophages potentiated the TNFα-induced increase in IκB kinase (IKK) activity, as well as the expression of NF-κB target genes. TNFα induced the recruitment of MST1 to TNF-RSC and its interaction with HOIP, the catalytic component of the E3 ligase linear ubiquitin assembly complex (LUBAC). Furthermore, MST1 activated in response to TNFα stimulation mediates the phosphorylation of HOIP and thereby inhibited LUBAC-dependent linear ubiquitination of NEMO/IKKγ. Together, our findings suggest that MST1 negatively regulates TNFα-induced NF-κB signaling by targeting LUBAC.

Role of Mitochondria in Ferroptosis.

Ferroptosis is a regulated necrosis process driven by iron-dependent lipid peroxidation. Although ferroptosis and cellular metabolism interplay with one another, whether mitochondria are involved in ferroptosis is under debate. Here, we demonstrate that mitochondria play a crucial role in cysteine-deprivation-induced ferroptosis but not in that induced by inhibiting glutathione peroxidase-4 (GPX4), the most downstream component of the ferroptosis pathway. Mechanistically, cysteine deprivation leads to mitochondrial membrane potential hyperpolarization and lipid peroxide accumulation. Inhibition of mitochondrial TCA cycle or electron transfer chain (ETC) mitigated mitochondrial membrane potential hyperpolarization, lipid peroxide accumulation, and ferroptosis. Blockage of glutaminolysis had the same inhibitory effect, which was counteracted by supplying downstream TCA cycle intermediates. Importantly, loss of function of fumarate hydratase, a tumor suppressor and TCA cycle component, confers resistance to cysteine-deprivation-induced ferroptosis. Collectively, this work demonstrates the crucial role of mitochondria in cysteine-deprivation-induced ferroptosis and implicates ferroptosis in tumor suppression.

RIPK1 and Caspase-8 Ensure Chromosome Stability Independently of Their Role in Cell Death and Inflammation.

Receptor-interacting protein kinase (RIPK) 1 functions as a key mediator of tissue homeostasis via formation of Caspase-8 activating ripoptosome complexes, positively and negatively regulating apoptosis, necroptosis, and inflammation. Here, we report an unanticipated cell-death- and inflammation-independent function of RIPK1 and Caspase-8, promoting faithful chromosome alignment in mitosis and thereby ensuring genome stability. We find that ripoptosome complexes progressively form as cells enter mitosis, peaking at metaphase and disassembling as cells exit mitosis. Genetic deletion and mitosis-specific inhibition of Ripk1 or Caspase-8 results in chromosome alignment defects independently of MLKL. We found that Polo-like kinase 1 (PLK1) is recruited into mitotic ripoptosomes, where PLK1's activity is controlled via RIPK1-dependent recruitment and Caspase-8-mediated cleavage. A fine balance of ripoptosome assembly is required as deregulated ripoptosome activity modulates PLK1-dependent phosphorylation of downstream effectors, such as BUBR1. Our data suggest that ripoptosome-mediated regulation of PLK1 contributes to faithful chromosome segregation during mitosis.

Exuberant fibroblast activity compromises lung function via ADAMTS4.

Severe respiratory infections can result in acute respiratory distress syndrome (ARDS)1. There are no effective pharmacological therapies that have been shown to improve outcomes for patients with ARDS. Although the host inflammatory response limits spread of and eventually clears the pathogen, immunopathology is a major contributor to tissue damage and ARDS1,2. Here we demonstrate that respiratory viral infection induces distinct fibroblast activation states, which we term extracellular matrix (ECM)-synthesizing, damage-responsive and interferon-responsive states. We provide evidence that excess activity of damage-responsive lung fibroblasts drives lethal immunopathology during severe influenza virus infection. By producing ECM-remodelling enzymes-in particular the ECM protease ADAMTS4-and inflammatory cytokines, damage-responsive fibroblasts modify the lung microenvironment to promote robust immune cell infiltration at the expense of lung function. In three cohorts of human participants, the levels of ADAMTS4 in the lower respiratory tract were associated with the severity of infection with seasonal or avian influenza virus. A therapeutic agent that targets the ECM protease activity of damage-responsive lung fibroblasts could provide a promising approach to preserving lung function and improving clinical outcomes following severe respiratory infections.

PELI1 Selectively Targets Kinase-Active RIP3 for Ubiquitylation-Dependent Proteasomal Degradation.

Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is a central protein in necroptosis, but posttranslational processes that regulate RIP3 activity and stability remain poorly understood. Here, we identify pellino E3 ubiquitin protein ligase 1 (PELI1) as an E3 ligase that targets RIP3 for proteasome-dependent degradation. Phosphorylation of RIP3 on T182 leads to interaction with the forkhead-associated (FHA) domain of PELI1 and PELI1-mediated K48-linked polyubiquitylation of RIP3 on K363. This same phosphorylation event is also important for RIP3 kinase activity; thus, PELI1 preferentially targets kinase-active RIP3 for degradation. PELI1-mediated RIP3 degradation effectively prevents cell death triggered by RIP3 hyperactivation. Importantly, upregulated RIP3 expression in keratinocytes from toxic epidermal necrolysis (TEN) patients is correlated with low expression of PELI1, suggesting that loss of PELI1 may play a role in the pathogenesis of TEN. We propose that PELI1 may function to control inadvertent activation of RIP3, thus preventing aberrant cell death and maintaining cellular homeostasis.

Tracing Information Flow from Erk to Target Gene Induction Reveals Mechanisms of Dynamic and Combinatorial Control.

Cell signaling networks coordinate specific patterns of protein expression in response to external cues, yet the logic by which signaling pathway activity determines the eventual abundance of target proteins is complex and poorly understood. Here, we describe an approach for simultaneously controlling the Ras/Erk pathway and monitoring a target gene's transcription and protein accumulation in single live cells. We apply our approach to dissect how Erk activity is decoded by immediate early genes (IEGs). We find that IEG transcription decodes Erk dynamics through a shared band-pass filtering circuit; repeated Erk pulses transcribe IEGs more efficiently than sustained Erk inputs. However, despite highly similar transcriptional responses, each IEG exhibits dramatically different protein-level accumulation, demonstrating a high degree of post-transcriptional regulation by combinations of multiple pathways. Our results demonstrate that the Ras/Erk pathway is decoded by both dynamic filters and logic gates to shape target gene responses in a context-specific manner.

mTOR Inhibition Restores Amino Acid Balance in Cells Dependent on Catabolism of Extracellular Protein.

Scavenging of extracellular protein via macropinocytosis is an alternative to monomeric amino acid uptake. In pancreatic cancer, macropinocytosis is driven by oncogenic Ras signaling and contributes substantially to amino acid supply. While Ras signaling promotes scavenging, mTOR signaling suppresses it. Here, we present an integrated experimental-computational method that enables quantitative comparison of protein scavenging rates across cell lines and conditions. Using it, we find that, independently of mTORC1, amino acid scarcity induces protein scavenging and that under such conditions the impact of mTOR signaling on protein scavenging rate is minimal. Nevertheless, mTOR inhibition promotes growth of cells reliant on eating extracellular protein. This growth enhancement depends on mTORC1's canonical function in controlling translation rate: mTOR inhibition slows translation, thereby matching protein synthesis to the limited amino acid supply. Thus, paradoxically, in amino acid-poor conditions the pro-anabolic effects of mTORC1 are functionally opposed to growth.

A genome-wide screen reveals that Dyrk1A kinase promotes nucleotide excision repair by preventing aberrant overexpression of cyclin D1 and p21.

Nucleotide excision repair (NER) eliminates highly genotoxic solar UV-induced DNA photoproducts that otherwise stimulate malignant melanoma development. Here, a genome-wide loss-of-function screen, coupling CRISPR/Cas9 technology with a flow cytometry-based DNA repair assay, was used to identify novel genes required for efficient NER in primary human fibroblasts. Interestingly, the screen revealed multiple genes encoding proteins, with no previously known involvement in UV damage repair, that significantly modulate NER uniquely during S phase of the cell cycle. Among these, we further characterized Dyrk1A, a dual specificity kinase that phosphorylates the proto-oncoprotein cyclin D1 on threonine 286 (T286), thereby stimulating its timely cytoplasmic relocalization and proteasomal degradation, which is required for proper regulation of the G1-S phase transition and control of cellular proliferation. We demonstrate that in UV-irradiated HeLa cells, depletion of Dyrk1A leading to overexpression of cyclin D1 causes inhibition of NER uniquely during S phase and reduced cell survival. Consistently, expression/nuclear accumulation of nonphosphorylatable cyclin D1 (T286A) in melanoma cells strongly interferes with S phase NER and enhances cytotoxicity post-UV. Moreover, the negative impact of cyclin D1 (T286A) overexpression on repair is independent of cyclin-dependent kinase activity but requires cyclin D1-dependent upregulation of p21 expression. Our data indicate that inhibition of NER during S phase might represent a previously unappreciated noncanonical mechanism by which oncogenic cyclin D1 fosters melanomagenesis.

Oxidation of caspase-8 by hypothiocyanous acid enables TNF-mediated necroptosis.

Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation.

Short-Chain Acyl-CoA Dehydrogenase as a Therapeutic Target for Cardiac Fibrosis.

ABSTRACT: Cardiac fibrosis is considered as unbalanced extracellular matrix production and degradation, contributing to heart failure. Short-chain acyl-CoA dehydrogenase (SCAD) negatively regulates pathological cardiac hypertrophy. The purpose of this study was to investigate the possible role of SCAD in cardiac fibrosis. In vivo experiments were performed on spontaneously hypertensive rats (SHR) and SCAD-knockout mice. The cardiac tissues of hypertensive patients with cardiac fibrosis were used for the measurement of SCAD expression. In vitro experiments, with angiotensin II (Ang II), SCAD siRNA and adenovirus-SCAD were performed using cardiac fibroblasts (CFs). SCAD expression was significantly decreased in the left ventricles of SHR. Notably, swim training ameliorated cardiac fibrosis in SHR in association with the elevation of SCAD. The decrease in SCAD protein and mRNA expression levels in SHR CFs were in accordance with those in the left ventricular myocardium of SHR. In addition, SCAD expression was downregulated in CFs treated with Ang II in vitro, and SCAD siRNA interference induced the same changes in cardiac fibrosis as Ang II-treated CFs, while adenovirus-SCAD treatment significantly reduced the Ang II-induced CFs proliferation, alpha smooth muscle actin (α-SMA), and collagen expression. In SHR infected with adenovirus-SCAD, the cardiac fibrosis of the left ventricle was significantly decreased. However, cardiac fibrosis occurred in conventional SCAD-knockout mice. SCAD immunofluorescence intensity of cardiac tissue in hypertensive patients with cardiac fibrosis was lower than that of healthy subjects. Altogether, the current experimental outcomes indicate that SCAD has a negative regulatory effect on cardiac fibrosis and support its potential therapeutic target for suppressing cardiac fibrosis.

Nek1 Regulates Rad54 to Orchestrate Homologous Recombination and Replication Fork Stability.

Never-in-mitosis A-related kinase 1 (Nek1) has established roles in apoptosis and cell cycle regulation. We show that human Nek1 regulates homologous recombination (HR) by phosphorylating Rad54 at Ser572 in late G2 phase. Nek1 deficiency as well as expression of unphosphorylatable Rad54 (Rad54-S572A) cause unresolved Rad51 foci and confer a defect in HR. Phospho-mimic Rad54 (Rad54-S572E), in contrast, promotes HR and rescues the HR defect associated with Nek1 loss. Although expression of phospho-mimic Rad54 is beneficial for HR, it causes Rad51 removal from chromatin and degradation of stalled replication forks in S phase. Thus, G2-specific phosphorylation of Rad54 by Nek1 promotes Rad51 chromatin removal during HR in G2 phase, and its absence in S phase is required for replication fork stability. In summary, Nek1 regulates Rad51 removal to orchestrate HR and replication fork stability.

The Noncanonical Role of ULK/ATG1 in ER-to-Golgi Trafficking Is Essential for Cellular Homeostasis.

ULK1 and ULK2 are thought to be essential for initiating autophagy, and Ulk1/2-deficient mice die perinatally of autophagy-related defects. Therefore, we used a conditional knockout approach to investigate the roles of ULK1/2 in the brain. Although the mice showed neuronal degeneration, the neurons showed no accumulation of P62(+)/ubiquitin(+) inclusions or abnormal membranous structures, which are observed in mice lacking other autophagy genes. Rather, neuronal death was associated with activation of the unfolded protein response (UPR) pathway. An unbiased proteomics approach identified SEC16A as an ULK1/2 interaction partner. ULK-mediated phosphorylation of SEC16A regulated the assembly of endoplasmic reticulum (ER) exit sites and ER-to-Golgi trafficking of specific cargo, and did not require other autophagy proteins (e.g., ATG13). The defect in ER-to-Golgi trafficking activated the UPR pathway in ULK-deficient cells; both processes were reversed upon expression of SEC16A with a phosphomimetic substitution. Thus, the regulation of ER-to-Golgi trafficking by ULK1/2 is essential for cellular homeostasis.

Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN.

Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair mechanisms until the recent identification of a DPC-processing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred from data in Xenopus laevis egg extracts, but its identity remains elusive. Here we identify the metalloprotease SPRTN as the DPC protease acting in metazoans. Loss of SPRTN results in failure to repair DPCs and hypersensitivity to DPC-inducing agents. SPRTN accomplishes DPC processing through a unique DNA-induced protease activity, which is controlled by several sophisticated regulatory mechanisms. Cellular, biochemical, and structural studies define a DNA switch triggering its protease activity, a ubiquitin switch controlling SPRTN chromatin accessibility, and regulatory autocatalytic cleavage. Our data also provide a molecular explanation on how SPRTN deficiency causes the premature aging and cancer predisposition disorder Ruijs-Aalfs syndrome.

Viral pseudo-enzymes activate RIG-I via deamidation to evade cytokine production.

RIG-I is a pattern recognition receptor that senses viral RNA and is crucial for host innate immune defense. Here, we describe a mechanism of RIG-I activation through amidotransferase-mediated deamidation. We show that viral homologs of phosphoribosylformylglycinamidine synthetase (PFAS), although lacking intrinsic enzyme activity, recruit cellular PFAS to deamidate and activate RIG-I. Accordingly, depletion and biochemical inhibition of PFAS impair RIG-I deamidation and concomitant activation. Purified PFAS and viral homolog thereof deamidate RIG-I in vitro. Ultimately, herpesvirus hijacks activated RIG-I to avoid antiviral cytokine production; loss of RIG-I or inhibition of RIG-I deamidation results in elevated cytokine production. Together, these findings demonstrate a surprising mechanism of RIG-I activation that is mediated by an enzyme.