Search

SARS-CoV-2 infection can manifest as a wide range of respiratory and systemic symptoms well after the acute phase of infection in over 50% of patients. Key questions remain on the long-term effects of infection on tissue pathology in recovered COVID-19 patients. To address these questions we performed multiplexed imaging of post-mortem lung tissue from 12 individuals who died post-acute COVID-19 (PC) and compare them to lung tissue from patients who died during the acute phase of COVID-19, or patients who died with idiopathic pulmonary fibrosis (IPF), and otherwise healthy lung tissue. We find evidence of viral presence in the lung up to 359 days after the acute phase of disease, including in patients with negative nasopharyngeal swab tests. The lung of PC patients are characterized by the accumulation of senescent alveolar type 2 cells, fibrosis with hypervascularization of peribronchial areas and alveolar septa, as the most pronounced pathophysiological features. At the cellular level, lung disease of PC patients, while distinct, shares pathological features with the chronic pulmonary disease of IPF. which may help rationalize interventions for PC patients. Altogether, this study provides an important foundation for the understanding of the long-term effects of SARS-CoV-2 pulmonary infection at the microanatomical, cellular, and molecular level.

Lung-infiltrating macrophages create a marked inflammatory milieu in a subset of patients with COVID-19 by producing a cytokine storm, which correlates with increased lethality. However, these macrophages are largely not infected by SARS-CoV-2, so the mechanism underlying their activation in the lung is unclear. Type I interferons (IFN-I) contribute to protecting the host against SARS-CoV-2 but may also have some deleterious effect, and the source of IFN-I in the lungs of infected patients is not well defined. Plasmacytoid dendritic cells (pDCs), a key cell type involved in antiviral responses, can produce IFN-I in response to SARS-CoV-2. We observed the infiltration of pDCs in the lungs of SARS-CoV-2–infected patients, which correlated with strong IFN-I signaling in lung macrophages. In patients with severe COVID-19, lung macrophages expressed a robust inflammatory signature, which correlated with persistent IFN-I signaling at the single-cell level. Hence, we observed the uncoupling in the kinetics of the infiltration of pDCs in the lungs and the associated IFN-I signature, with the cytokine storm in macrophages. We observed that pDCs were the dominant IFN-α–producing cells in response to the virus in the blood, whereas macrophages produced IFN-α only when in physical contact with infected epithelial cells. We also showed that IFN-α produced by pDCs, after the sensing of SARS-CoV-2 by TLR7, mediated changes in macrophages at both transcriptional and epigenetic levels, which favored their hyperactivation by environmental stimuli. Together, these data indicate that the priming of macrophages can result from the response by pDCs to SARS-CoV-2, leading to macrophage activation in patients with severe COVID-19.

Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection1,2, but the host response at the lung tissue level is poorly understood. Here we performed single-nucleus RNA sequencing of about 116,000 nuclei from the lungs of nineteen individuals who died of COVID-19 and underwent rapid autopsy and seven control individuals. Integrated analyses identified substantial alterations in cellular composition, transcriptional cell states, and cell-to-cell interactions, thereby providing insight into the biology of lethal COVID-19. The lungs from individuals with COVID-19 were highly inflamed, with dense infiltration of aberrantly activated monocyte-derived macrophages and alveolar macrophages, but had impaired T cell responses. Monocyte/macrophage-derived interleukin-1β and epithelial cell-derived interleukin-6 were unique features of SARS-CoV-2 infection compared to other viral and bacterial causes of pneumonia. Alveolar type 2 cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition into alveolar type 1 cells, resulting in impaired lung regeneration. Furthermore, we identified expansion of recently described CTHRC1+ pathological fibroblasts3 contributing to rapidly ensuing pulmonary fibrosis in COVID-19. Inference of protein activity and ligand–receptor interactions identified putative drug targets to disrupt deleterious circuits. This atlas enables the dissection of lethal COVID-19, may inform our understanding of long-term complications of COVID-19 survivors, and provides an important resource for therapeutic development. Lung samples collected soon after death from COVID-19 are used to provide a single-cell atlas of SARS-CoV-2 infection and the ensuing molecular changes.

Multiplexed imaging and spatial transcriptomics enable highly resolved spatial characterization of cellular phenotypes, but still largely depend on laborious manual annotation to understand higher-order patterns of tissue organization. As a result, higher-order patterns of tissue organization are poorly understood and not systematically connected to disease pathology or clinical outcomes. To address this gap, we developed UTAG, a novel method to identify and quantify microanatomical tissue structures in multiplexed images without human intervention. Our method combines information on cellular phenotypes with the physical proximity of cells to accurately identify organ-specific microanatomical domains in healthy and diseased tissue. We apply our method to various types of images across physiological and disease states to show that it can consistently detect higher level architectures in human organs, quantify structural differences between healthy and diseased tissue, and reveal tissue organization patterns with relevance to clinical outcomes in cancer patients.

The mammalian immune system implements a remarkably effective set of mechanisms for fighting pathogens1. Its main components are haematopoietic immune cells, including myeloid cells that control innate immunity, and lymphoid cells that constitute adaptive immunity2. However, immune functions are not unique to haematopoietic cells, and many other cell types display basic mechanisms of pathogen defence3–5. To advance our understanding of immunology outside the haematopoietic system, here we systematically investigate the regulation of immune genes in the three major types of structural cells: epithelium, endothelium and fibroblasts. We characterize these cell types across twelve organs in mice, using cellular phenotyping, transcriptome sequencing, chromatin accessibility profiling and epigenome mapping. This comprehensive dataset revealed complex immune gene activity and regulation in structural cells. The observed patterns were highly organ-specific and seem to modulate the extensive interactions between structural cells and haematopoietic immune cells. Moreover, we identified an epigenetically encoded immune potential in structural cells under tissue homeostasis, which was triggered in response to systemic viral infection. This study highlights the prevalence and organ-specific complexity of immune gene activity in non-haematopoietic structural cells, and it provides a high-resolution, multi-omics atlas of the epigenetic and transcriptional networks that regulate structural cells in the mouse. Structural cells implement a broad range of immune-regulatory functions beyond their roles as barriers and connective tissues, and they utilize an epigenetically encoded potential for immune gene activation in their rapid response to viral infection.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib provides effective treatment for patients with chronic lymphocytic leukemia (CLL), despite extensive heterogeneity in this disease. To define the underlining regulatory dynamics, we analyze high-resolution time courses of ibrutinib treatment in patients with CLL, combining immune-phenotyping, single-cell transcriptome profiling, and chromatin mapping. We identify a consistent regulatory program starting with a sharp decrease of NF-κB binding in CLL cells, which is followed by reduced activity of lineage-defining transcription factors, erosion of CLL cell identity, and acquisition of a quiescence-like gene signature. We observe patient-to-patient variation in the speed of execution of this program, which we exploit to predict patient-specific dynamics in the response to ibrutinib based on the pre-treatment patient samples. In aggregate, our study describes time-dependent cellular, molecular, and regulatory effects for therapeutic inhibition of B cell receptor signaling in CLL, and it establishes a broadly applicable method for epigenome/transcriptome-based treatment monitoring., Ibrutinib, a Bruton tyrosine kinase inhibitor, provides effective treatment for chronic lymphocytic leukemia (CLL). Here, the authors describe time-dependent molecular changes to malignant cells and to the immune system in patients undergoing ibrutinib therapy, with can be used for therapy monitoring.

Summary Regulatory B cells (Bregs) play a critical role in the control of autoimmunity and inflammation. IL-10 production is the hallmark for the identification of Bregs. However, the molecular determinants that regulate the transcription of IL-10 and control the Breg developmental program remain unknown. Here, we demonstrate that aryl hydrocarbon receptor (AhR) regulates the differentiation and function of IL-10-producing CD19+CD21hiCD24hiBregs and limits their differentiation into B cells that contribute to inflammation. Chromatin profiling and transcriptome analyses show that loss of AhR in B cells reduces expression of IL-10 by skewing the differentiation of CD19+CD21hiCD24hiB cells into a pro-inflammatory program, under Breg-inducing conditions. B cell AhR-deficient mice develop exacerbated arthritis, show significant reductions in IL-10-producing Bregs and regulatory T cells, and show an increase in T helper (Th) 1 and Th17 cells compared with B cell AhR-sufficient mice. Thus, we identify AhR as a relevant contributor to the transcriptional regulation of Breg differentiation., Graphical Abstract, Highlights • IL-10+ Bregs are identified by high expression of AhR • B cell AhR deficiency leads to exacerbated arthritis and impaired Breg function • AhR directly binds to and regulates the expression of IL-10 in Bregs • AhR maintains Breg phenotype by suppressing pro-inflammatory gene expression, The transcriptional control of interleukin-10 (IL-10) in regulatory B cells (Bregs) remains undefined. Piper et al. identify the aryl hydrocarbon receptor (AhR) as an important transcription factor involved in Breg differentiation and show a direct role of AhR in the regulation of IL-10 transcription.

Background Organizing and annotating biological sample data is critical in data-intensive bioinformatics. Unfortunately, metadata formats from a data provider are often incompatible with requirements of a processing tool. There is no broadly accepted standard to organize metadata across biological projects and bioinformatics tools, restricting the portability and reusability of both annotated datasets and analysis software. Results To address this, we present the Portable Encapsulated Project (PEP) specification, a formal specification for biological sample metadata structure. The PEP specification accommodates typical features of data-intensive bioinformatics projects with many biological samples. In addition to standardization, the PEP specification provides descriptors and modifiers for project-level and sample-level metadata, which improve portability across both computing environments and data processing tools. PEPs include a schema validator framework, allowing formal definition of required metadata attributes for data analysis broadly. We have implemented packages for reading PEPs in both Python and R to provide a language-agnostic interface for organizing project metadata. Conclusions The PEP specification is an important step toward unifying data annotation and processing tools in data-intensive biological research projects. Links to tools and documentation are available at http://pep.databio.org/.

The molecular characteristics of metastatic upper tract urothelial carcinoma (UTUC) are unknown. The genomic and transcriptomic differences between primary and metastatic UTUC is not well described either. We combined whole-exome sequencing, RNA-sequencing, and Imaging Mass Cytometry™ (IMC™) of 44 tumor samples from 28 patients with high-grade primary and metastatic UTUC. IMC enables spatially resolved single-cell analyses to examine the evolution of cancer cell, immune cell, and stromal cell markers using mass cytometry with lanthanide metal-conjugated antibodies. We discovered that actionable genomic alterations are frequently discordant between primary and metastatic UTUC tumors in the same patient. In contrast, molecular subtype membership and immune depletion signature were stable across primary and matched metastatic UTUC. Molecular and immune subtypes were consistent between bulk RNA-sequencing and mass cytometry of protein markers from 340,798 single-cells. Molecular subtyping at the single cell level was highly conserved between primary and metastatic UTUC tumors within the same patient.

SUMMARYInflammation is essential to the disruption of tissue homeostasis, and, in the pancreas, can destabilize the identity of terminally differentiated acinar cells. Herein we employ lineage-traced mouse models to delineate the chromatin dynamics that accompany the cycle of metaplasia and regeneration following pancreatitis, and unveil the presence of an epigenetic memory of inflammation in the pancreatic acinar cell compartment. We observe that despite histologic resolution of pancreatitis, acinar cells fail to return to their molecular baseline after several months, representing an incomplete cell fate decision. In vivo, this epigenetic memory controls lineage plasticity, with diminished metaplasia in response to a second inflammatory insult but increased tumorigenesis with an oncogenic Kras mutation. We demonstrate that both persistent chromatin and transcriptional changes constituting memory are recalled with oncogenic stress. Together, our findings define the dynamics and recall of an epigenetic memory of inflammation that impacts cell fate decisions in a context-dependent manner.

The molecular mechanisms underlying the clinical manifestations of COVID-19 and what distinguishes them from common seasonal influenza virus and other lung injury states such as Acute Respiratory Distress Syndrome, remains poorly understood. To address these challenges, we combine transcriptional profiling of 646 clinical nasopharyngeal swabs and 39 patient autopsy tissues to define body-wide transcriptome changes in response to COVID-19. We then match this data with spatial protein and expression profiling across 357 tissue sections from 16 representative patient lung samples and identify tissue compartment-specific damage wrought by SARS-CoV-2 infection, evident as a function of varying viral loads during the clinical course of infection and tissue type specific expression states. Overall, our findings reveal a systemic disruption of canonical cellular and transcriptional pathways across all tissues, which can inform subsequent studies to combat the mortality of COVID-19 and to better understand the molecular dynamics of lethal SARS-CoV-2 and other respiratory infections., Graphical Abstract, Park et al. report system-wide transcriptome damage and tissue identity loss wrought by SARS-CoV-2, influenza, and bacterial infection across multiple organs (heart, liver, lung, kidney, and lymph nodes) and provide spatio-temporal landscape of COVID-19 in the lung.

Aberrations in genes coding for subunits of the BRG1/BRM associated factor (BAF) chromatin remodeling complexes are highly abundant in human cancers. Currently, it is not understood how these mostly loss-of-function mutations contribute to cancer development and how they can be targeted therapeutically. The cancer-type-specific occurrence patterns of certain subunit mutations suggest subunit-specific effects on BAF complex function, possibly by the formation of aberrant residual complexes. Here, we systematically characterize the effects of individual subunit loss on complex composition, chromatin accessibility and gene expression in a panel of knockout cell lines deficient for 22 BAF subunits. We observe strong, specific and sometimes discordant alterations dependent on the targeted subunit and show that these explain intracomplex codependencies, including the synthetic lethal interactions SMARCA4–ARID2, SMARCA4–ACTB and SMARCC1–SMARCC2. These data provide insights into the role of different BAF subcomplexes in genome-wide chromatin organization and suggest approaches to therapeutically target BAF-mutant cancers. The authors generate cell lines deficient for 22 BAF subunits, studying effects on complex composition, chromatin accessibility and gene expression. They identify synthetic lethal interactions between SMARCA4–ARID2, SMARCA4–ACTB and SMARCC1–SMARCC2.

The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.

Supplemental Digital Content is available in the text., Background: Cardiovascular risk in diabetes remains elevated despite glucose-lowering therapies. We hypothesized that hyperglycemia induces trained immunity in macrophages, promoting persistent proatherogenic characteristics. Methods: Bone marrow–derived macrophages from control mice and mice with diabetes were grown in physiological glucose (5 mmol/L) and subjected to RNA sequencing (n=6), assay for transposase accessible chromatin sequencing (n=6), and chromatin immunoprecipitation sequencing (n=6) for determination of hyperglycemia-induced trained immunity. Bone marrow transplantation from mice with (n=9) or without (n=6) diabetes into (normoglycemic) Ldlr −/− mice was used to assess its functional significance in vivo. Evidence of hyperglycemia-induced trained immunity was sought in human peripheral blood mononuclear cells from patients with diabetes (n=8) compared with control subjects (n=16) and in human atherosclerotic plaque macrophages excised by laser capture microdissection. Results: In macrophages, high extracellular glucose promoted proinflammatory gene expression and proatherogenic functional characteristics through glycolysis-dependent mechanisms. Bone marrow–derived macrophages from diabetic mice retained these characteristics, even when cultured in physiological glucose, indicating hyperglycemia-induced trained immunity. Bone marrow transplantation from diabetic mice into (normoglycemic) Ldlr −/− mice increased aortic root atherosclerosis, confirming a disease-relevant and persistent form of trained innate immunity. Integrated assay for transposase accessible chromatin, chromatin immunoprecipitation, and RNA sequencing analyses of hematopoietic stem cells and bone marrow–derived macrophages revealed a proinflammatory priming effect in diabetes. The pattern of open chromatin implicated transcription factor Runt-related transcription factor 1 (Runx1). Similarly, transcriptomes of atherosclerotic plaque macrophages and peripheral leukocytes in patients with type 2 diabetes were enriched for Runx1 targets, consistent with a potential role in human disease. Pharmacological inhibition of Runx1 in vitro inhibited the trained phenotype. Conclusions: Hyperglycemia-induced trained immunity may explain why targeting elevated glucose is ineffective in reducing macrovascular risk in diabetes and suggests new targets for disease prevention and therapy.

Sequencing of cell-free DNA in the blood of cancer patients (liquid biopsy) provides attractive opportunities for early diagnosis, assessment of treatment response, and minimally invasive disease monitoring. To unlock liquid biopsy analysis for pediatric tumors with few genetic aberrations, we introduce an integrated genetic/epigenetic analysis method and demonstrate its utility on 241 deep whole-genome sequencing profiles of 95 patients with Ewing sarcoma and 31 patients with other pediatric sarcomas. Our method achieves sensitive detection and classification of circulating tumor DNA in peripheral blood independent of any genetic alterations. Moreover, we benchmark different metrics for cell-free DNA fragmentation analysis, and we introduce the LIQUORICE algorithm for detecting circulating tumor DNA based on cancer-specific chromatin signatures. Finally, we combine several fragmentation-based metrics into an integrated machine learning classifier for liquid biopsy analysis that exploits widespread epigenetic deregulation and is tailored to cancers with low mutation rates. Clinical associations highlight the potential value of cfDNA fragmentation patterns as prognostic biomarkers in Ewing sarcoma. In summary, our study provides a comprehensive analysis of circulating tumor DNA beyond recurrent genetic aberrations, and it renders the benefits of liquid biopsy more readily accessible for childhood cancers., Liquid biopsies enable minimally invasive applications for diagnosis and treatment monitoring. Here the authors analyse fragmentation patterns of circulating tumour DNA on multiple levels and develop a bioinformatic tool, LIQUORICE, to accurately detect and classify paediatric cancers with low mutational burden.

Cancer-associated, loss-of-function mutations in genes encoding subunits of the BRG1/BRM-associated factor (BAF) chromatin-remodeling complexes1–8 often cause drastic chromatin accessibility changes, especially in important regulatory regions9–19. However, it remains unknown how these changes are established over time (for example, immediate consequences or long-term adaptations), and whether they are causative for intracomplex synthetic lethalities, abrogating the formation or activity of BAF complexes9,20–24. In the present study, we use the dTAG system to induce acute degradation of BAF subunits and show that chromatin alterations are established faster than the duration of one cell cycle. Using a pharmacological inhibitor and a chemical degrader of the BAF complex ATPase subunits25,26, we show that maintaining genome accessibility requires constant ATP-dependent remodeling. Completely abolishing BAF complex function by acute degradation of a synthetic lethal subunit in a paralog-deficient background results in an almost complete loss of chromatin accessibility at BAF-controlled sites, especially also at superenhancers, providing a mechanism for intracomplex synthetic lethalities. Acute degradation of the BAF subunit SMARCA4 shows rapid chromatin accessibility changes, thus suggesting that maintenance of genome accessibility requires constant ATP-dependent remodeling.

Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection

SummaryRecent studies have provided insights into the pathology and immune response to coronavirus disease 2019 (COVID-19)1–8. However thorough interrogation of the interplay between infected cells and the immune system at sites of infection is lacking. We use high parameter imaging mass cytometry9targeting the expression of 36 proteins, to investigate at single cell resolution, the cellular composition and spatial architecture of human acute lung injury including SARS-CoV-2. This spatially resolved, single-cell data unravels the disordered structure of the infected and injured lung alongside the distribution of extensive immune infiltration. Neutrophil and macrophage infiltration are hallmarks of bacterial pneumonia and COVID-19, respectively. We provide evidence that SARS-CoV-2 infects predominantly alveolar epithelial cells and induces a localized hyper-inflammatory cell state associated with lung damage. By leveraging the temporal range of COVID-19 severe fatal disease in relation to the time of symptom onset, we observe increased macrophage extravasation, mesenchymal cells, and fibroblasts abundance concomitant with increased proximity between these cell types as the disease progresses, possibly as an attempt to repair the damaged lung tissue. This spatially resolved single-cell data allowed us to develop a biologically interpretable landscape of lung pathology from a structural, immunological and clinical standpoint. This spatial single-cell landscape enabled the pathophysiological characterization of the human lung from its macroscopic presentation to the single-cell, providing an important basis for the understanding of COVID-19, and lung pathology in general.

Recent studies have provided insights into the pathology of and immune response to COVID-191–8. However, a thorough investigation of the interplay between infected cells and the immune system at sites of infection has been lacking. Here we use high-parameter imaging mass cytometry9 that targets the expression of 36 proteins to investigate the cellular composition and spatial architecture of acute lung injury in humans (including injuries derived from SARS-CoV-2 infection) at single-cell resolution. These spatially resolved single-cell data unravel the disordered structure of the infected and injured lung, alongside the distribution of extensive immune infiltration. Neutrophil and macrophage infiltration are hallmarks of bacterial pneumonia and COVID-19, respectively. We provide evidence that SARS-CoV-2 infects predominantly alveolar epithelial cells and induces a localized hyperinflammatory cell state that is associated with lung damage. We leverage the temporal range of fatal outcomes of COVID-19 in relation to the onset of symptoms, which reveals increased macrophage extravasation and increased numbers of mesenchymal cells and fibroblasts concomitant with increased proximity between these cell types as the disease progresses—possibly as a result of attempts to repair the damaged lung tissue. Our data enable us to develop a biologically interpretable landscape of lung pathology from a structural, immunological and clinical standpoint. We use this landscape to characterize the pathophysiology of the human lung from its macroscopic presentation to the single-cell level, which provides an important basis for understanding COVID-19 and lung pathology in general. Imaging mass cytometry of the human lung reveals the cellular composition and spatial architecture during COVID-19 and other acute injuries, enabling the characterization of lung pathophysiology from structural, immunological and clinical perspectives.

Organizing and annotating biological sample data is critical in data-intensive bioinformatics. Unfortunately, metadata formats from a data provider are often incompatible with requirements of a processing tool. There is no broadly accepted standard to organize metadata across biological projects and bioinformatics tools, restricting the portability and reusability of both annotated datasets and analysis software. To address this, we present Portable Encapsulated Projects (PEP), a formal specification for biological sample metadata structure. The PEP specification accommodates typical features of data-intensive bioinformatics projects with many samples, whether from individual experiments, organisms, or single cells. In addition to standardization, the PEP specification provides descriptors and modifiers for different organizational layers of a project, which improve portability among computing environments and facilitate use of different processing tools. PEP includes a schema validator framework, allowing formal definition of required metadata attributes for any type of biomedical data analysis. We have implemented packages for reading PEPs in both Python and R to provide a language-agnostic interface for organizing project metadata. PEP therefore presents an important step toward unifying data annotation and processing tools in data-intensive biological research projects.

With a rising incidence of COVID-19-associated morbidity and mortality worldwide, it is critical to elucidate the innate and adaptive immune responses that drive disease severity. We performed longitudinal immune profiling of peripheral blood mononuclear cells from 45 patients and healthy donors. We observed a dynamic immune landscape of innate and adaptive immune cells in disease progression and absolute changes of lymphocyte and myeloid cells in severe versus mild cases or healthy controls. Intubation and death were coupled with selected natural killer cell KIR receptor usage and IgM+ B cells and associated with profound CD4 and CD8 T cell exhaustion. Pseudo-temporal reconstruction of the hierarchy of disease progression revealed dynamic time changes in the global population recapitulating individual patients and the development of an eight-marker classifier of disease severity. Estimating the effect of clinical progression on the immune response and early assessment of disease progression risks may allow implementation of tailored therapies.

NOTCH signaling represents a promising therapeutic target in chronic lymphocytic leukemia (CLL). We compared the anti-neoplastic effects of the nuclear NOTCH2 inhibitor gliotoxin and the pan-NOTCH &gamma, secretase inhibitor RO4929097 in primary CLL cells with special emphasis on the individual roles of the different NOTCH receptors. Gliotoxin rapidly induced apoptosis in all CLL cases tested, whereas RO4929097 exerted a variable and delayed effect on CLL cell viability. Gliotoxin-induced apoptosis was associated with inhibition of the NOTCH2/FCER2 (CD23) axis together with concomitant upregulation of the NOTCH3/NR4A1 axis. In contrast, RO4929097 downregulated the NOTCH3/NR4A1 axis and counteracted the spontaneous and gliotoxin-induced apoptosis. On the cell surface, NOTCH3 and CD23 expression were mutually exclusive, suggesting that downregulation of NOTCH2 signaling is a prerequisite for NOTCH3 expression in CLL cells. ATAC-seq confirmed that gliotoxin targeted the canonical NOTCH signaling, as indicated by the loss of chromatin accessibility at the potential NOTCH/CSL site containing the gene regulatory elements. This was accompanied by a gain in accessibility at the NR4A1, NF&kappa, B, and ATF3 motifs close to the genes involved in B-cell activation, differentiation, and apoptosis. In summary, these data show that gliotoxin recovers a non-canonical tumor-suppressing NOTCH3 activity, indicating that nuclear NOTCH2 inhibitors might be beneficial compared to pan-NOTCH inhibitors in the treatment of CLL.

Cell atlas projects and single-cell CRISPR screens hit the limits of current technology, as they require cost-effective profiling for millions of individual cells. To satisfy these enormous throughput requirements, we developed “single-cell combinatorial fluidic indexing” (scifi) and applied it to single-cell RNA sequencing. The resulting scifi-RNA-seq assay combines one-step combinatorial pre-indexing of single-cell transcriptomes with subsequent single-cell RNA-seq using widely available droplet microfluidics. Pre-indexing allows us to load multiple cells per droplet, which increases the throughput of droplet-based single-cell RNA-seq up to 15-fold, and it provides a straightforward way of multiplexing hundreds of samples in a single scifi-RNA-seq experiment. Compared to multi-round combinatorial indexing, scifi-RNA-seq provides an easier, faster, and more efficient workflow, thereby enabling massive-scale scRNA-seq experiments for a broad range of applications ranging from population genomics to drug screens with scRNA-seq readout. We benchmarked scifi-RNA-seq on various human and mouse cell lines, and we demonstrated its feasibility for human primary material by profiling TCR activation in T cells.

Cell atlas projects and high-throughput perturbation screens require single-cell sequencing at a scale that is challenging with current technology. To enable cost-effective single-cell sequencing for millions of individual cells, we developed ‘single-cell combinatorial fluidic indexing’ (scifi). The scifi-RNA-seq assay combines one-step combinatorial preindexing of entire transcriptomes inside permeabilized cells with subsequent single-cell RNA-seq using microfluidics. Preindexing allows us to load several cells per droplet and computationally demultiplex their individual expression profiles. Thereby, scifi-RNA-seq massively increases the throughput of droplet-based single-cell RNA-seq, and provides a straightforward way of multiplexing thousands of samples in a single experiment. Compared with multiround combinatorial indexing, scifi-RNA-seq provides an easy and efficient workflow. Compared to cell hashing methods, which flag and discard droplets containing more than one cell, scifi-RNA-seq resolves and retains individual transcriptomes from overloaded droplets. We benchmarked scifi-RNA-seq on various human and mouse cell lines, validated it for primary human T cells and applied it in a highly multiplexed CRISPR screen with single-cell transcriptome readout of T cell receptor activation. Combining whole-transcriptome preindexing with standard droplet microfluidics, scifi-RNA-seq enables single-cell RNA-seq with massive throughput and built-in sample multiplexing.

The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here we map neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S(+) neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S(+) inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of hypothalamic organization and function.

Rationale: Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to define their biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood. Objective: To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells. Methods and Results: LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO. Conclusions: Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.

Chronic lymphocytic leukemia (CLL) is a genetically, epigenetically, and clinically heterogeneous disease. Despite this heterogeneity, the Bruton tyrosine kinase (BTK) inhibitor ibrutinib provides effective treatment for the vast majority of CLL patients. To define the underlining regulatory program, we analyzed high-resolution time courses of ibrutinib treatment in closely monitored patients, combining cellular phenotyping (flow cytometry), single-cell transcriptome profiling (scRNA-seq), and chromatin mapping (ATAC-seq). We identified a consistent regulatory program shared across all patients, which was further validated by an independent CLL cohort. In CLL cells, this program starts with a sharp decrease of NF-κB binding, followed by reduced regulatory activity of lineage-defining transcription factors (including PAX5 and IRF4) and erosion of CLL cell identity, finally leading to the acquisition of a quiescence-like gene signature which was shared across several immune cell types. Nevertheless, we observed patient-to-patient variation in the speed of its execution, which we exploited to predict patient-specific dynamics in the response to ibrutinib based on pre-treatment samples. In aggregate, our study describes the cellular, molecular, and regulatory effects of therapeutic B cell receptor inhibition in CLL at high temporal resolution, and it establishes a broadly applicable method for epigenome/transcriptome-based treatment monitoring.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib has substantially improved therapeutic options for chronic lymphocytic leukemia (CLL). Although ibrutinib is not curative, it has a profound effect on CLL cells and may create new pharmacologically exploitable vulnerabilities. To identify such vulnerabilities, we developed a systematic approach that combines epigenome profiling (charting the gene-regulatory basis of cell state) with single-cell chemosensitivity profiling (quantifying cell-type-specific drug response) and bioinformatic data integration. By applying our method to a cohort of matched patient samples collected before and during ibrutinib therapy, we identified characteristic ibrutinib-induced changes that provide a starting point for the rational design of ibrutinib combination therapies. Specifically, we observed and validated preferential sensitivity to proteasome, PLK1, and mTOR inhibitors during ibrutinib treatment. More generally, our study establishes a broadly applicable method for investigating treatment-specific vulnerabilities by integrating the complementary perspectives of epigenetic cell states and phenotypic drug responses in primary patient samples.

The histone acetyl-reader BRD4 is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1. We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression, and pharmacologic inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin-associated suggests a direct role for nuclear metabolism in the control of gene expression.

Summary The differentiation of IL-10-producing regulatory B cells (Bregs) in response to gut-microbiota-derived signals supports the maintenance of tolerance. However, whether microbiota-derived metabolites can modulate Breg suppressive function remains unknown. Here, we demonstrate that rheumatoid arthritis (RA) patients and arthritic mice have a reduction in microbial-derived short-chain fatty acids (SCFAs) compared to healthy controls and that in mice, supplementation with the SCFA butyrate reduces arthritis severity. Butyrate supplementation suppresses arthritis in a Breg-dependent manner by increasing the level of the serotonin-derived metabolite 5-Hydroxyindole-3-acetic acid (5-HIAA), which activates the aryl-hydrocarbon receptor (AhR), a newly discovered transcriptional marker for Breg function. Thus, butyrate supplementation via AhR activation controls a molecular program that supports Breg function while inhibiting germinal center (GC) B cell and plasmablast differentiation. Our study demonstrates that butyrate supplementation may serve as a viable therapy for the amelioration of systemic autoimmune disorders., Graphical Abstract, Highlights • Stool butyrate levels are reduced in patients with RA compared to healthy controls • Supplementation with butyrate suppresses arthritis severity in a mouse model • Suppression of arthritis by butyrate supplementation depends upon AhR+Bregs • Butyrate increases serotonin-derived 5-HIAA, which directly activates AhR+Bregs, The environmental signals that influence Breg function are not yet fully defined. Here, Rosser et al. demonstrate that the short-chain fatty acid butyrate supports Breg function by increasing the level of the serotonin-derived metabolite 5-Hydroxyindole-3-acetic acid (5-HIAA). 5-HIAA, in turn, activates the aryl-hydrocarbon receptor, a newly discovered transcriptional regulator for Bregs.

Summary Specialized regulatory T (Treg) cells accumulate and perform homeostatic and regenerative functions in nonlymphoid tissues. Whether common precursors for nonlymphoid-tissue Treg cells exist and how they differentiate remain elusive. Using transcription factor nuclear factor, interleukin 3 regulated (Nfil3) reporter mice and single-cell RNA-sequencing (scRNA-seq), we identified two precursor stages of interleukin 33 (IL-33) receptor ST2-expressing nonlymphoid tissue Treg cells, which resided in the spleen and lymph nodes. Global chromatin profiling of nonlymphoid tissue Treg cells and the two precursor stages revealed a stepwise acquisition of chromatin accessibility and reprogramming toward the nonlymphoid-tissue Treg cell phenotype. Mechanistically, we identified and validated the transcription factor Batf as the driver of the molecular tissue program in the precursors. Understanding this tissue development program will help to harness regenerative properties of tissue Treg cells for therapy., Graphical Abstract, Highlights • Two precursor stages of ST2+ nonlymphoid-tissue Tregs are evident in the spleen and LNs • Precursor stages are defined by differential expression of Nfil3, PD1, and Klrg1 • Chromatin accessibility and scRNA-seq suggests a stepwise precursor reprogramming • Batf drives the molecular tissue program in the precursors, Whether a common precursor exists for nonlymphoid-tissue Treg cells is unclear. Delacher et al. identify two precursor stages for tissue-resident ST2+ Treg cells. These precursors undergo a stepwise reprogramming in the lymphoid organs toward the nonlymphoid-tissue Treg cell phenotype. Chromatin accessibility profiling identified Batf as a key driver of the tissue program in the progenitor cells.

Metastatic melanoma is hallmarked by its ability to switch oncogenic MITF expression. Here we tested the impact of STAT3 on melanoma onset and progression in association with MITF expression levels. We established a mouse melanoma model for deleting Stat3 specifically in melanocytes with specific expression of human hyperactive NRASQ61K in an Ink4a deficient background. Mice with tissue specific Stat3 deletion showed an early onset of disease, but displayed significantly diminished lung metastases. Whole genome expression profiling also revealed a reduced invasion phenotype, which was functionally confirmed in 3D melanoma model systems. Notably, loss or knockdown of STAT3 in mouse or human cells resulted in up-regulation of MITF and induction of cell proliferation. Mechanistically we show that STAT3 induced CEBPa/b expression was sufficient to suppress MITF transcription. Epigenetic analysis by ATAC-seq confirmed that STAT3 enabled CEBPa/b binding to the MITF enhancer region thereby silencing it. We conclude that STAT3 is a metastasis driver in melanoma able to antagonize the MITF oncogene via direct induction of CEBP family member transcription facilitating RAS-RAF-driven melanoma metastasis.List of AbbreviationsATAC-seq, Assay for Transposase-Accessible Chromatin using sequencing; CEBP, CAAT Box Enhancer Binding Protein; CRE, Cre recombinase; EGF, Epidermal Growth Factor; GEO, Gene Expression Omnibus; GSEA, Gene Set Enrichment Analysis; HSC70, Heat Shock 70 kDa protein; IHC, Immunohistochemistry; IL-6, Interleukin-6; JAK, Janus Kinase; MITF, Microphthalmia-Associated Transcription Factor; NSG, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice; OSM, Oncostatin M; PDGF, Platelet-Derived Growth Factor; pS, phosphoserine; pY, phosphotyrosine; RAS, Rat Sarcoma; RAF, Rapidly Accelerated Fibrosarcoma; RTK, Receptor Tyrosine Kinase; RT-PCR, Reverse Transcription Polymerase Chain Reaction; S100b, Calcium Binding Protein S100 beta; shRNA, short hairpin RNA; SOX10, Sex Determining Region Y-10; STAT, Signal Transducer and Activator of Transcription; TCGA, The Cancer Genome Atlas; TMA, Tissue Micro Array

Summary Enhancers are genetic elements that regulate spatiotemporal gene expression. Enhancer function requires transcription factor (TF) binding and correlates with histone modifications. However, the extent to which TF binding and histone modifications functionally define active enhancers remains unclear. Here, we combine chromatin immunoprecipitation with a massively parallel reporter assay (ChIP-STARR-seq) to identify functional enhancers in human embryonic stem cells (ESCs) genome-wide in a quantitative unbiased manner. Although active enhancers associate with TFs, only a minority of regions marked by NANOG, OCT4, H3K27ac, and H3K4me1 function as enhancers, with activity markedly changing under naive versus primed culture conditions. We identify an enhancer set associated with functions extending to non-ESC-specific processes. Moreover, although transposable elements associate with putative enhancers, only some exhibit activity. Similarly, within super-enhancers, large tracts are non-functional, with activity restricted to small sub-domains. This catalog of validated enhancers provides a valuable resource for further functional dissection of the regulatory genome., Graphical Abstract, Highlights • Massively parallel reporter assay assessed over 350,000 genome regions • ChIP-STARR-seq catalogs functional enhancers in primed and naive hESCs • Identification of transcription factors and transposable elements linked to enhancers • ChIP-STARR-seq dissects super-enhancers into small functional units, Barakat et al. use a combination of chromatin immunoprecipitation and a massively parallel reporter assay to identify functional enhancers in primed and naive human embryonic stem cells. This genome-wide catalog of validated enhancers provides a valuable resource for the further dissection of the regulatory genome.

Diet-related health issues such as nonalcoholic fatty liver disease and cardiovascular disorders are known to have a major inflammatory component. However, the exact pathways linking diet-induced changes (e.g., hyperlipidemia) and the ensuing inflammation have remained elusive so far. We identified biological processes related to innate immunity and oxidative stress as prime response pathways in livers of low-density lipoprotein receptor-deficient mice on a Western-type diet using RNA sequencing and in silico functional analyses of transcriptome data. The observed changes were independent of the presence of microbiota and thus indicative of a role for sterile triggers. We further show that malondialdehyde (MDA) epitopes, products of lipid peroxidation and markers for enhanced oxidative stress, are detectable in hepatic inflammation predominantly on dying cells and stimulate cytokine secretion as well as leukocyte recruitment in vitro and in vivo. MDA-induced cytokine secretion in vitro was dependent on the presence of the scavenger receptors CD36 and MSR1. Moreover, in vivo neutralization of endogenously generated MDA epitopes by intravenous injection of a specific MDA antibody results in decreased hepatic inflammation in low-density lipoprotein receptor-deficient mice on a Western-type diet. Conclusion Accumulation of MDA epitopes plays a major role during diet-induced hepatic inflammation and can be ameliorated by administration of an anti-MDA antibody. (Hepatology 2017;65:1181-1195).

CRISPR-based genetic screens are accelerating biological discovery, but current methods have inherent limitations. Widely used pooled screens are restricted to simple readouts including cell proliferation and sortable marker proteins. Arrayed screens allow for comprehensive molecular readouts such as transcriptome profiling, but at much lower throughput. Here we combine pooled CRISPR screening with single-cell RNA sequencing into a broadly applicable workflow, directly linking guide RNA expression to transcriptome responses in thousands of individual cells. Our method for CRISPR droplet sequencing (CROP-seq) enables pooled CRISPR screens with single-cell transcriptome resolution, which will facilitate high-throughput functional dissection of complex regulatory mechanisms and heterogeneous cell populations.

ChIP-seq combines chromatin immunoprecipitation with sequencing in order tomeasure genome-wide distributions of chromatin proteins. Despite recent efforts tooptimize and improve ChIP-seq, current protocols are still quite tedious, time-consuming, and costly. The development of a hyperactive Tn5 transposase thatenables simultaneous DNA fragmentation and adapter tagging (“tagmentation”)presents an opportunity for faster and cheaper library preparation (Ref. 1). Here, wedemonstrate tagmentation of immunoprecipitated chromatin in a robust one-stepreaction performed directly on bead-bound chromatin (Schmidl et al. 2015 NatureMethods, Ref. 2). This method – which we call ChIPmentation – provides a fast,cheap, low-input ChIP-seq workflow and yields excellent results for both histonemarks and transcription factors. Using ChIPmentation we observed footprints thatresemble those in ATAC-seq data. Moreover, histone ChIPmentation data shows astriking periodicity indicative of nucleosome stability, and an ordered signal aroundnucleosome dyads that might be used for accurate nucleosome positioning.

Despite considerable differences in morphology and complexity of body plans among animals, a great part of the gene set is shared among Bilateria and their basally branching sister group, the Cnidaria. This suggests that the common ancestor of eumetazoans already had a highly complex gene repertoire. At present it is therefore unclear how morphological diversification is encoded in the genome. Here we address the possibility that differences in gene regulation could contribute to the large morphological divergence between cnidarians and bilaterians. To this end, we generated the first genome-wide map of gene regulatory elements in a nonbilaterian animal, the sea anemone Nematostella vectensis. Using chromatin immunoprecipitation followed by deep sequencing of five chromatin modifications and a transcriptional cofactor, we identified over 5000 enhancers in the Nematostella genome and could validate 75% of the tested enhancers in vivo. We found that in Nematostella, but not in yeast, enhancers are characterized by the same combination of histone modifications as in bilaterians, and these enhancers preferentially target developmental regulatory genes. Surprisingly, the distribution and abundance of gene regulatory elements relative to these genes are shared between Nematostella and bilaterian model organisms. Our results suggest that complex gene regulation originated at least 600 million yr ago, predating the common ancestor of eumetazoans.