SARS-CoV-2 restructures host chromatin architecture.

Some viruses restructure host chromatin, influencing gene expression, with implications for disease outcome. Whether this occurs for SARS-CoV-2, the virus causing COVID-19, is largely unknown. Here we characterized the 3D genome and epigenome of human cells after SARS-CoV-2 infection, finding widespread host chromatin restructuring that features widespread compartment A weakening, A-B mixing, reduced intra-TAD contacts and decreased H3K27ac euchromatin modification levels. Such changes were not found following common-cold-virus HCoV-OC43 infection. Intriguingly, the cohesin complex was notably depleted from intra-TAD regions, indicating that SARS-CoV-2 disrupts cohesin loop extrusion. These altered 3D genome/epigenome structures correlated with transcriptional suppression of interferon response genes by the virus, while increased H3K4me3 was found in the promoters of pro-inflammatory genes highly induced during severe COVID-19. These findings show that SARS-CoV-2 acutely rewires host chromatin, facilitating future studies of the long-term epigenomic impacts of its infection.

Acute depletion of human core nucleoporin reveals direct roles in transcription control but dispensability for 3D genome organization.

The nuclear pore complex (NPC) comprises more than 30 nucleoporins (NUPs) and is a hallmark of eukaryotes. NUPs have been suggested to be important in regulating gene transcription and 3D genome organization. However, evidence in support of their direct roles remains limited. Here, by Cut&Run, we find that core NUPs display broad but also cell-type-specific association with active promoters and enhancers in human cells. Auxin-mediated rapid depletion of two NUPs demonstrates that NUP93, but not NUP35, directly and specifically controls gene transcription. NUP93 directly activates genes with high levels of RNA polymerase II loading and transcriptional elongation by facilitating full BRD4 recruitment to their active enhancers. dCas9-based tethering confirms a direct and causal role of NUP93 in gene transcriptional activation. Unexpectedly, in situ Hi-C and H3K27ac or H3K4me1 HiChIP results upon acute NUP93 depletion show negligible changesS2211-1247(22)01437-1 of 3D genome organization ranging from A/B compartments and topologically associating domains (TADs) to enhancer-promoter contacts.

Glutamine synthetase licenses APC/C-mediated mitotic progression to drive cell growth.

Tumors can reprogram the functions of metabolic enzymes to fuel malignant growth; however, beyond their conventional functions, key metabolic enzymes have not been found to directly govern cell mitosis. Here, we report that glutamine synthetase (GS) promotes cell proliferation by licensing mitotic progression independently of its metabolic function. GS depletion, but not impairment of its enzymatic activity, results in mitotic arrest and multinucleation across multiple lung and liver cancer cell lines, patient-derived organoids and xenografted tumors. Mechanistically, GS directly interacts with the nuclear pore protein NUP88 to prevent its binding to CDC20. Such interaction licenses activation of the CDC20-mediated anaphase-promoting complex or cyclosome to ensure proper metaphase-to-anaphase transition. In addition, GS is overexpressed in human non-small cell lung cancer and its depletion reduces tumor growth in mice and increases the efficacy of microtubule-targeted chemotherapy. Our findings highlight a moonlighting function of GS in governing mitosis and illustrate how an essential metabolic enzyme promotes cell proliferation and tumor development, beyond its main metabolic function.

Family Tree for Aqueous Organic Redox Couples for Redox Flow Battery Electrolytes: A Conceptual Review.

Redox flow batteries (RFBs) are an increasingly attractive option for renewable energy storage, thus providing flexibility for the supply of electrical energy. In recent years, research in this type of battery storage has been shifted from metal-ion based electrolytes to soluble organic redox-active compounds. Aqueous-based organic electrolytes are considered as more promising electrolytes to achieve "green", safe, and low-cost energy storage. Many organic compounds and their derivatives have recently been intensively examined for application to redox flow batteries. This work presents an up-to-date overview of the redox organic compound groups tested for application in aqueous RFB. In the initial part, the most relevant requirements for technical electrolytes are described and discussed. The importance of supporting electrolytes selection, the limits for the aqueous system, and potential synthetic strategies for redox molecules are highlighted. The different organic redox couples described in the literature are grouped in a "family tree" for organic redox couples. This article is designed to be an introduction to the field of organic redox flow batteries and aims to provide an overview of current achievements as well as helping synthetic chemists to understand the basic concepts of the technical requirements for next-generation energy storage materials.

Influence of stabilizing osmolytes on hen egg white lysozyme fibrillation.

Communicated by Ramaswamy H. Sarma.

Enhancer RNA m6A methylation facilitates transcriptional condensate formation and gene activation.

The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control.

SARS-CoV-2 Restructures the Host Chromatin Architecture.

SARS-CoV-2 has made >190-million infections worldwide, thus it is pivotal to understand the viral impacts on host cells. Many viruses can significantly alter host chromatin 1 , but such roles of SARS-CoV-2 are largely unknown. Here, we characterized the three-dimensional (3D) genome architecture and epigenome landscapes in human cells after SARS-CoV-2 infection, revealing remarkable restructuring of host chromatin architecture. High-resolution Hi-C 3.0 uncovered widespread A compartmental weakening and A-B mixing, together with a global reduction of intra-TAD chromatin contacts. The cohesin complex, a central organizer of the 3D genome, was significantly depleted from intra-TAD regions, supporting that SARS-CoV-2 disrupts cohesin loop extrusion. Calibrated ChIP-Seq verified chromatin restructuring by SARS-CoV-2 that is particularly manifested by a pervasive reduction of euchromatin modifications. Built on the rewired 3D genome/epigenome maps, a modified activity-by-contact model 2 highlights the transcriptional weakening of antiviral interferon response genes or virus sensors (e.g., DDX58 ) incurred by SARS-CoV-2. In contrast, pro-inflammatory genes (e.g. IL-6 ) high in severe infections were uniquely regulated by augmented H3K4me3 at their promoters. These findings illustrate how SARS-CoV-2 rewires host chromatin architecture to confer immunological gene deregulation, laying a foundation to characterize the long-term epigenomic impacts of this virus.

RNA m6A modification orchestrates a LINE-1-host interaction that facilitates retrotransposition and contributes to long gene vulnerability.

The molecular basis underlying the interaction between retrotransposable elements (RTEs) and the human genome remains poorly understood. Here, we profiled N6-methyladenosine (m6A) deposition on nascent RNAs in human cells by developing a new method MINT-Seq, which revealed that many classes of RTE RNAs, particularly intronic LINE-1s (L1s), are strongly methylated. These m6A-marked intronic L1s (MILs) are evolutionarily young, sense-oriented to hosting genes, and are bound by a dozen RNA binding proteins (RBPs) that are putative novel readers of m6A-modified RNAs, including a nuclear matrix protein SAFB. Notably, m6A positively controls the expression of both autonomous L1s and co-transcribed L1 relics, promoting L1 retrotransposition. We showed that MILs preferentially reside in long genes with critical roles in DNA damage repair and sometimes in L1 suppression per se, where they act as transcriptional "roadblocks" to impede the hosting gene expression, revealing a novel host-weakening strategy by the L1s. In counteraction, the host uses the SAFB reader complex to bind m6A-L1s to reduce their levels, and to safeguard hosting gene transcription. Remarkably, our analysis identified thousands of MILs in multiple human fetal tissues, enlisting them as a novel category of cell-type-specific regulatory elements that often compromise transcription of long genes and confer their vulnerability in neurodevelopmental disorders. We propose that this m6A-orchestrated L1-host interaction plays widespread roles in gene regulation, genome integrity, human development and diseases.

Subcellular localization of the J-protein Sis1 regulates the heat shock response.

Cells exposed to heat shock induce a conserved gene expression program, the heat shock response (HSR), encoding protein homeostasis (proteostasis) factors. Heat shock also triggers proteostasis factors to form subcellular quality control bodies, but the relationship between these spatial structures and the HSR is unclear. Here we show that localization of the J-protein Sis1, a cofactor for the chaperone Hsp70, controls HSR activation in yeast. Under nonstress conditions, Sis1 is concentrated in the nucleoplasm, where it promotes Hsp70 binding to the transcription factor Hsf1, repressing the HSR. Upon heat shock, Sis1 forms an interconnected network with other proteostasis factors that spans the nucleolus and the surface of the endoplasmic reticulum. We propose that localization of Sis1 to this network directs Hsp70 activity away from Hsf1 in the nucleoplasm, leaving Hsf1 free to induce the HSR. In this manner, Sis1 couples HSR activation to the spatial organization of the proteostasis network.

Enforced Activation of Enhancer RNAs In Situ through the dCas9 Synergistic Activation Mediator System.

Enhancers are pivotal genomic elements scattered through the mammalian genome and dictate tissue-specific gene expression programs. Increasing evidence has shown that enhancers not only provide DNA binding motifs for transcription factors (TFs) but also generate non-coding RNAs that are referred to as eRNAs. Studies have demonstrated that eRNA transcripts can play significant roles in gene regulation in both physiology and disease. Commonly used methods to investigate the function of eRNAs are constrained to "loss-of-function" approaches by knockdown of eRNAs, or by chemical inhibition of the enhancer transcription. There has not been a robust method to conduct "gain-of-function" studies of eRNAs to mimic specific disease conditions such as human cancer, where eRNAs are often overexpressed. Here, we introduce a method for precisely and robustly activating eRNAs for functional interrogation of their roles by applying the dCas9 mediated Synergistic Activation Mediators (SAM) system. We present the entire workflow of eRNA activation, from the selection of eRNAs, the design of gRNAs to the validation of eRNA activation by RT-qPCR. This method represents a unique approach to study the roles of a particular eRNA in gene regulation and disease development. In addition, this system can be employed for unbiased CRISPR screening to identify phenotype-driving eRNA targets in the context of a specific disease.

Multi-kinase control of environmental stress responsive transcription.

Cells respond to changes in environmental conditions by activating signal transduction pathways and gene expression programs. Here we present a dataset to explore the relationship between environmental stresses, kinases, and global gene expression in yeast. We subjected 28 drug-sensitive kinase mutants to 10 environmental conditions in the presence of inhibitor and performed mRNA deep sequencing. With these data, we reconstructed canonical stress pathways and identified examples of crosstalk among pathways. The data also implicated numerous kinases in novel environment-specific roles. However, rather than regulating dedicated sets of target genes, individual kinases tuned the magnitude of induction of the environmental stress response (ESR)-a gene expression signature shared across the set of perturbations-in environment-specific ways. This suggests that the ESR integrates inputs from multiple sensory kinases to modulate gene expression and growth control. As an example, we provide experimental evidence that the high osmolarity glycerol pathway is an upstream negative regulator of protein kinase A, a known inhibitor of the ESR. These results elaborate the central axis of cellular stress response signaling.

Amyloid fibril formation in the presence of water structure-affecting solutes.

The impact of the differently hydrated non-electrolytes (protein structure destabilizers) on the fibrillation of hen egg white lysozyme (HEWL) was investigated. Two isomeric urea derivatives i.e. butylurea (BU) and N,N,N',N'-tetramethylurea (TMU) were chosen as a tested compounds. The obtained results show that butylurea exerts greater impact on HEWL and its fibrillation than tetramethylurea. Both substances decrease the time of induction of the fibrillation (lag time) but only BU increases the efficiency of amyloidogenesis. For the systems with equivalent reduction of the HEWL stability (250mM BU and 500mM TMU) the not-equivalent increase of the protein fibrillation was recorded (higher for BU). This fact suggests that specific interactions with protein, possibly water mediated, are responsible for the action of the tested substances.

Transcriptional landscape and clinical utility of enhancer RNAs for eRNA-targeted therapy in cancer.

Enhancer RNA (eRNA) is a type of noncoding RNA transcribed from the enhancer. Although critical roles of eRNA in gene transcription control have been increasingly realized, the systemic landscape and potential function of eRNAs in cancer remains largely unexplored. Here, we report the integration of multi-omics and pharmacogenomics data across large-scale patient samples and cancer cell lines. We observe a cancer-/lineage-specificity of eRNAs, which may be largely driven by tissue-specific TFs. eRNAs are involved in multiple cancer signaling pathways through putatively regulating their target genes, including clinically actionable genes and immune checkpoints. They may also affect drug response by within-pathway or cross-pathway means. We characterize the oncogenic potential and therapeutic liability of one eRNA, NET1e, supporting the clinical feasibility of eRNA-targeted therapy. We identify a panel of clinically relevant eRNAs and developed a user-friendly data portal. Our study reveals the transcriptional landscape and clinical utility of eRNAs in cancer.

Monitoring of lysozyme thermal denaturation by volumetric measurements and nanoDSF technique in the presence of N-butylurea.

The results of thermal studies of denaturation of hen egg white lysozyme (HEWL) in water and an aqueous solution of N-butylurea (BU) are presented. High-precision densimetric measurements were used to characterize and analyze the changes of the specific volume, v, during temperature elevation. The temperature of the midpoint of protein denaturation was also determined by nanoDSF technique (differential scanning fluorimetry). The densities of lysozyme solutions were measured at temperatures ranging from 298.15 to 353.15 K with an interval of 5 K at atmospheric pressure (0.1 MPa). The concentration of the protein covered the range from 2 to 20 mg per 1 ml of the solution. The optimal range of the concentration for the densimetric measurements was roughly estimated. In the transition region, the structural changes of the protein are accompanied by the biggest increase of ν values with temperature. Our measurements show that this effect can be monitored from volumetric data without precise determination of protein concentration. The results prove that a two-state model of denaturation could be used for data interpretation. Contrary to common misconception, the volumetric measurements suggest that the denatured protein does not necessarily need to be in a fully extended state. In this way, the 'protein volume paradox' could be explained. The surface area of the protein remains unchanged and thus the increase of the specific volume of the protein is relatively small. Additionally, the self-stabilizing effect of the protein in BU solution was reported. For the HEWL in pure water, this phenomenon was not observed.

Influence of the ionic strength on the amyloid fibrillogenesis of hen egg white lysozyme.

The study investigates the role of the electrostatic interactions in the fibrillation of the hen egg white lysozyme (HEWL). In order to achieve this aim the influence of the cations Na+, Mg2+ and Al3+ on the amyloid fibril formation and amorphous aggregation was tested. The amyloids are formed in the solution without added salt but the Thioflavin T fluorescence gives the false-negative result. In these conditions, the HEWL fibrils are long and curvy. If the ionic strength of the solution is sufficiently high, the formed amyloids are shorter and fragmented. Our study shows that the addition of the aluminium salt promotes protein fibrillation. The amorphous aggregation dominates in the high concentration of electrolyte. The in vitro amyloid fibril formation seems to be regulated by universal mechanisms. The theories implemented in the polymer science or for colloidal solutions give the qualitative description of the aggregation phenomena. However, the specific interactions and the additional effects (e.g. fibril fragmentation) modulate the amyloidogenesis.

Hsf1 Phosphorylation Generates Cell-to-Cell Variation in Hsp90 Levels and Promotes Phenotypic Plasticity.

Clonal populations of cells exhibit cell-to-cell variation in the transcription of individual genes. In addition to this noise in gene expression, heterogeneity in the proteome and the proteostasis network expands the phenotypic diversity of a population. Heat shock factor 1 (Hsf1) regulates chaperone gene expression, thereby coupling transcriptional noise to proteostasis. Here we show that cell-to-cell variation in Hsf1 activity is an important determinant of phenotypic plasticity. Budding yeast cells with high Hsf1 activity were enriched for the ability to acquire resistance to an antifungal drug, and this enrichment depended on Hsp90, a known phenotypic capacitor and canonical Hsf1 target. We show that Hsf1 phosphorylation promotes cell-to-cell variation, and this variation, rather than absolute Hsf1 activity, promotes antifungal resistance. We propose that Hsf1 phosphorylation enables differential tuning of the proteostasis network in individual cells, allowing populations to access a range of phenotypic states.

Hsf1 and Hsp70 constitute a two-component feedback loop that regulates the yeast heat shock response.

Models for regulation of the eukaryotic heat shock response typically invoke a negative feedback loop consisting of the transcriptional activator Hsf1 and a molecular chaperone. Previously we identified Hsp70 as the chaperone responsible for Hsf1 repression and constructed a mathematical model that recapitulated the yeast heat shock response (Zheng et al., 2016). The model was based on two assumptions: dissociation of Hsp70 activates Hsf1, and transcriptional induction of Hsp70 deactivates Hsf1. Here we validate these assumptions. First, we severed the feedback loop by uncoupling Hsp70 expression from Hsf1 regulation. As predicted by the model, Hsf1 was unable to efficiently deactivate in the absence of Hsp70 transcriptional induction. Next, we mapped a discrete Hsp70 binding site on Hsf1 to a C-terminal segment known as conserved element 2 (CE2). In vitro, CE2 binds to Hsp70 with low affinity (9 µM), in agreement with model requirements. In cells, removal of CE2 resulted in increased basal Hsf1 activity and delayed deactivation during heat shock, while tandem repeats of CE2 sped up Hsf1 deactivation. Finally, we uncovered a role for the N-terminal domain of Hsf1 in negatively regulating DNA binding. These results reveal the quantitative control mechanisms underlying the heat shock response.

Unique agreement of experimental and computational infrared spectroscopy: a case study of lithium bromide solvation in an important electrochemical solvent.

Infrared (IR) spectroscopy is a widely used and invaluable tool in the studies of solvation phenomena in electrolyte solutions. Using state-of-the-art chemometric analysis of a spectral series measured in a concentration-dependent manner, the spectrum of the solute-affected solvent can be extracted, providing a detailed view of the structural and energetic states of the solvent molecules influenced by the solute. Concurrently, ab initio molecular dynamics (AIMD) simulations provide the solvation shell picture at an atomistic detail level and allow for a consistent decomposition of the theoretical IR spectrum in terms of distance-dependent contributions of the solvent molecules. Here, we show for the first time how the chemometric techniques designed with the analysis of experimental data in mind can be harnessed to extract corresponding information from the computed IR spectra for mutual benefit, but without any mutual input. The wide applicability of this two-track approach is demonstrated using lithium bromide solvation in γ-butyrolactone (GBL) as a showcase. GBL is a cyclic ester with extensive applications as a solvent in electrochemistry and we are particularly motivated by its usefulness in the rechargeable cell industry which justifies further studies of lithium cation solvation in GBL. The combination of experiment and simulations firmly asserts the strong solvent structuring character of Li+ and a comparatively weak influence exerted on the solvent by Br-.

Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response.

Protein AMPylation is a conserved posttranslational modification with emerging roles in endoplasmic reticulum homeostasis. However, the range of substrates and cell biological consequences of AMPylation remain poorly defined. We expressed human and Caenorhabditis elegans AMPylation enzymes-huntingtin yeast-interacting protein E (HYPE) and filamentation-induced by cyclic AMP (FIC)-1, respectively-in Saccharomyces cerevisiae, a eukaryote that lacks endogenous protein AMPylation. Expression of HYPE and FIC-1 in yeast induced a strong cytoplasmic Hsf1-mediated heat shock response, accompanied by attenuation of protein translation, massive protein aggregation, growth arrest, and lethality. Overexpression of Ssa2, a cytosolic heat shock protein (Hsp)70, was sufficient to partially rescue growth. In human cell lines, overexpression of active HYPE similarly induced protein aggregation and the HSF1-dependent heat shock response. Excessive AMPylation also abolished HSP70-dependent influenza virus replication. Our findings suggest a mode of Hsp70 inactivation by AMPylation and point toward a role for protein AMPylation in the regulation of cellular protein homeostasis beyond the endoplasmic reticulum.