Optimized single-nucleus transcriptional profiling by combinatorial indexing.

Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) is a powerful method for recovering gene expression data from an exponentially scalable number of individual cells or nuclei. However, sci-RNA-seq is a complex protocol that has historically exhibited variable performance on different tissues, as well as lower sensitivity than alternative methods. Here, we report a simplified, optimized version of the sci-RNA-seq protocol with three rounds of split-pool indexing that is faster, more robust and more sensitive and has a higher yield than the original protocol, with reagent costs on the order of 1 cent per cell or less. The total hands-on time from nuclei isolation to final library preparation takes 2-3 d, depending on the number of samples sharing the experiment. The improvements also allow RNA profiling from tissues rich in RNases like older mouse embryos or adult tissues that were problematic for the original method. We showcase the optimized protocol via whole-organism analysis of an E16.5 mouse embryo, profiling ~380,000 nuclei in a single experiment. Finally, we introduce a 'Tiny-Sci' protocol for experiments in which input material is very limited.

The continuum of Drosophila embryonic development at single-cell resolution.

Drosophila melanogaster is a powerful, long-standing model for metazoan development and gene regulation. We profiled chromatin accessibility in almost 1 million and gene expression in half a million nuclei from overlapping windows spanning the entirety of embryogenesis. Leveraging developmental asynchronicity within embryo collections, we applied deep neural networks to infer the age of each nucleus, resulting in continuous, multimodal views of molecular and cellular transitions in absolute time. We identify cell lineages; infer their developmental relationships; and link dynamic changes in enhancer usage, transcription factor (TF) expression, and the accessibility of TFs' cognate motifs. With these data, the dynamics of enhancer usage and gene expression can be explored within and across lineages at the scale of minutes, including for precise transitions like zygotic genome activation.

Bi-fated tendon-to-bone attachment cells are regulated by shared enhancers and KLF transcription factors.

The mechanical challenge of attaching elastic tendons to stiff bones is solved by the formation of a unique transitional tissue. Here, we show that murine tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, under regulation of shared regulatory elements and Krüppel-like factors (KLFs) transcription factors. High-throughput bulk and single-cell RNA sequencing of humeral attachment cells revealed expression of hundreds of chondrogenic and tenogenic genes, which was validated by in situ hybridization and single-molecule ISH. ATAC sequencing showed that attachment cells share accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis revealed enhancer signatures for most of these regions. Transgenic mouse enhancer reporter assays verified the shared activity of some of these enhancers. Finally, integrative chromatin and motif analyses and transcriptomic data implicated KLFs as regulators of attachment cells. Indeed, blocking expression of both Klf2 and Klf4 in developing limb mesenchyme impaired their differentiation.

A human cell atlas of fetal gene expression.

The gene expression program underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of gene expression and chromatin accessibility in fetal tissues. For gene expression, we applied three-level combinatorial indexing to >110 samples representing 15 organs, ultimately profiling ~4 million single cells. We leveraged the literature and other atlases to identify and annotate hundreds of cell types and subtypes, both within and across tissues. Our analyses focused on organ-specific specializations of broadly distributed cell types (such as blood, endothelial, and epithelial), sites of fetal erythropoiesis (which notably included the adrenal gland), and integration with mouse developmental atlases (such as conserved specification of blood cells). These data represent a rich resource for the exploration of in vivo human gene expression in diverse tissues and cell types.

Host-Viral Infection Maps Reveal Signatures of Severe COVID-19 Patients.

Viruses are a constant threat to global health as highlighted by the current COVID-19 pandemic. Currently, lack of data underlying how the human host interacts with viruses, including the SARS-CoV-2 virus, limits effective therapeutic intervention. We introduce Viral-Track, a computational method that globally scans unmapped single-cell RNA sequencing (scRNA-seq) data for the presence of viral RNA, enabling transcriptional cell sorting of infected versus bystander cells. We demonstrate the sensitivity and specificity of Viral-Track to systematically detect viruses from multiple models of infection, including hepatitis B virus, in an unsupervised manner. Applying Viral-Track to bronchoalveloar-lavage samples from severe and mild COVID-19 patients reveals a dramatic impact of the virus on the immune system of severe patients compared to mild cases. Viral-Track detects an unexpected co-infection of the human metapneumovirus, present mainly in monocytes perturbed in type-I interferon (IFN)-signaling. Viral-Track provides a robust technology for dissecting the mechanisms of viral-infection and pathology.

Dissecting cellular crosstalk by sequencing physically interacting cells.

Crosstalk between neighboring cells underlies many biological processes, including cell signaling, proliferation and differentiation. Current single-cell genomic technologies profile each cell separately after tissue dissociation, losing information on cell-cell interactions. In the present study, we present an approach for sequencing physically interacting cells (PIC-seq), which combines cell sorting of physically interacting cells (PICs) with single-cell RNA-sequencing. Using computational modeling, PIC-seq systematically maps in situ cellular interactions and characterizes their molecular crosstalk. We apply PIC-seq to interrogate diverse interactions including immune-epithelial PICs in neonatal murine lungs. Focusing on interactions between T cells and dendritic cells (DCs) in vitro and in vivo, we map T cell-DC interaction preferences, and discover regulatory T cells as a major T cell subtype interacting with DCs in mouse draining lymph nodes. Analysis of T cell-DC pairs reveals an interaction-specific program between pathogen-presenting migratory DCs and T cells. PIC-seq provides a direct and broadly applicable technology to characterize intercellular interaction-specific pathways at high resolution.

Single-Cell Analysis of Diverse Pathogen Responses Defines a Molecular Roadmap for Generating Antigen-Specific Immunity.

The immune system generates pathogen-tailored responses. The precise innate immune cell types and pathways that direct robust adaptive immune responses have not been fully characterized. By using fluorescent pathogens combined with massively parallel single-cell RNA-seq, we comprehensively characterized the initial 48 h of the innate immune response to diverse pathogens. We found that across all pathogens tested, most of the lymph node cell types and states showed little pathogen specificity. In contrast, the rare antigen-positive cells displayed pathogen-specific transcriptional programs as early as 24 h after immunization. In addition, mycobacteria activated a specific NK-driven IFNγ response. Depletion of NK cells and IFNγ showed that IFNγ initiated a monocyte-specific signaling cascade, leading to the production of major chemokines and cytokines that promote Th1 development. Our systems immunology approach sheds light on early events in innate immune responses and may help further development of safe and efficient vaccines.

The bone marrow is patrolled by NK cells that are primed and expand in response to systemic viral activation.

The bone marrow hosts NK cells whose distribution, motility and response to systemic immune challenge are poorly understood. At steady state, two-photon microscopy of the bone marrow in Ncr1gfp/+ mice captured motile NK cells interacting with dendritic cells. NK cells expressed markers and effector molecules of mature cells. Following poly (I:C) injection, RNA-Seq of NK cells revealed three phases of transcription featuring immune response genes followed by posttranscriptional processes and proliferation. Functionally, poly (I:C) promoted upregulation of granzyme B, enhanced cytotoxicity in vitro and in vivo, and, in the same individual cells, triggered proliferation. Two-photon imaging revealed that the proportion of sinusoidal NK cells decreased, while at the same time parenchymal NK cells accelerated, swelled and divided within the bone marrow. MVA viremia induced similar responses. Our findings demonstrate that the bone marrow is patrolled by mature NK cells that rapidly proliferate in response to systemic viral challenge while maintaining their effector functions.

CD74 is a novel transcription regulator.

CD74 is a cell-surface receptor for the cytokine macrophage migration inhibitory factor. Macrophage migration inhibitory factor binding to CD74 induces its intramembrane cleavage and the release of its cytosolic intracellular domain (CD74-ICD), which regulates cell survival. In the present study, we characterized the transcriptional activity of CD74-ICD in chronic lymphocytic B cells. We show that following CD74 activation, CD74-ICD interacts with the transcription factors RUNX (Runt related transcription factor) and NF-κB and binds to proximal and distal regulatory sites enriched for genes involved in apoptosis, immune response, and cell migration. This process leads to regulation of expression of these genes. Our results suggest that identifying targets of CD74 will help in understanding of essential pathways regulating B-cell survival in health and disease.

M-sec regulates polarized secretion of inflammatory endothelial chemokines and facilitates CCL2-mediated lymphocyte transendothelial migration.

Activation of endothelial cells by IL-1ß triggers the expression of multiple inflammatory cytokines and leukocyte-attracting chemokines. The machineries involved in the secretion of these inducible proteins are poorly understood. With the use of genome-wide transcriptional analysis of inflamed human dermal microvascular endothelial cells, we identified several IL-1ß-induced candidate regulators of these machineries and chose to focus our study on TNF-α-induced protein 2 (myeloid-secretory). The silencing of myeloid-secretory did not affect the ability of inflamed endothelial cells to support the adhesion and crawling of effector T lymphocytes. However, the ability of these lymphocytes to complete transendothelial migration across myeloid-secretory-silenced human dermal microvascular endothelial cells was inhibited significantly. These observed effects on lymphocyte transendothelial migration were recovered completely when exogenous promigratory chemokine CXCL12 was overlaid on the endothelial barrier. A polarized secretion assay suggested that the silencing of endothelial myeloid-secretory impairs T effector transendothelial migration by reducing the preferential secretion of endothelial-produced CCL2, a key transendothelial migration-promoting chemokine for these lymphocytes, into the basolateral endothelial compartment. Myeloid-secretory silencing also impaired the preferential secretion of other endothelial-produced inflammatory chemokines, as well as cytokines, such as IL-6 and GM-CSF, into the basolateral endothelial compartment. This is the first evidence of a novel inflammation-inducible machinery that regulates polarized secretion of endothelial CCL2 and other inflammatory chemokines and cytokines into basolateral endothelial compartments and facilitates the ability of endothelial CCL2 to promote T cell transendothelial migration.

Sumoylation coordinates the repression of inflammatory and anti-viral gene-expression programs during innate sensing.

Innate sensing of pathogens initiates inflammatory cytokine responses that need to be tightly controlled. We found here that after engagement of Toll-like receptors (TLRs) in myeloid cells, deficient sumoylation caused increased secretion of transcription factor NF-κB-dependent inflammatory cytokines and a massive type I interferon signature. In mice, diminished sumoylation conferred susceptibility to endotoxin shock and resistance to viral infection. Overproduction of several NF-κB-dependent inflammatory cytokines required expression of the type I interferon receptor, which identified type I interferon as a central sumoylation-controlled hub for inflammation. Mechanistically, the small ubiquitin-like modifier SUMO operated from a distal enhancer of the gene encoding interferon-ß (Ifnb1) to silence both basal and stimulus-induced activity of the Ifnb1 promoter. Therefore, sumoylation restrained inflammation by silencing Ifnb1 expression and by strictly suppressing an unanticipated priming by type I interferons of the TLR-induced production of inflammatory cytokines.

Distinct Murine Mucosal Langerhans Cell Subsets Develop from Pre-dendritic Cells and Monocytes.

Langerhans cells (LCs) populate the mucosal epithelium, a major entry portal for pathogens, yet their ontogeny remains unclear. We found that, in contrast to skin LCs originating from self-renewing radioresistant embryonic precursors, oral mucosal LCs derive from circulating radiosensitive precursors. Mucosal LCs can be segregated into CD103(+)CD11b(lo) (CD103(+)) and CD11b(+)CD103(-) (CD11b(+)) subsets. We further demonstrated that similar to non-lymphoid dendritic cells (DCs), CD103(+) LCs originate from pre-DCs, whereas CD11b(+) LCs differentiate from both pre-DCs and monocytic precursors. Despite this ontogenetic discrepancy between skin and mucosal LCs, the transcriptomic signature and immunological function of oral LCs highly resemble those of skin LCs but not DCs. These findings, along with the epithelial position, morphology, and expression of the LC-associated phenotype strongly suggest that oral mucosal LCs are genuine LCs. Collectively, in a tissue-dependent manner, murine LCs differentiate from at least three distinct precursors (embryonic, pre-DC, and monocytic) in steady state.

Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment.

Macrophages are critical for innate immune defense and also control organ homeostasis in a tissue-specific manner. They provide a fitting model to study the impact of ontogeny and microenvironment on chromatin state and whether chromatin modifications contribute to macrophage identity. Here, we profile the dynamics of four histone modifications across seven tissue-resident macrophage populations. We identify 12,743 macrophage-specific enhancers and establish that tissue-resident macrophages have distinct enhancer landscapes beyond what can be explained by developmental origin. Combining our enhancer catalog with gene expression profiles and open chromatin regions, we show that a combination of tissue- and lineage-specific transcription factors form the regulatory networks controlling chromatin specification in tissue-resident macrophages. The environment is capable of shaping the chromatin landscape of transplanted bone marrow precursors, and even differentiated macrophages can be reprogrammed when transferred into a new microenvironment. These results provide a comprehensive view of macrophage regulatory landscape and highlight the importance of the microenvironment, along with pioneer factors in orchestrating identity and plasticity.

Chronic exposure to TGFß1 regulates myeloid cell inflammatory response in an IRF7-dependent manner.

Tissue microenvironment influences the function of resident and infiltrating myeloid-derived cells. In the central nervous system (CNS), resident microglia and freshly recruited infiltrating monocyte-derived macrophages (mo-MΦ) display distinct activities under pathological conditions, yet little is known about the microenvironment-derived molecular mechanism that regulates these differences. Here, we demonstrate that long exposure to transforming growth factor-ß1 (TGFß1) impaired the ability of myeloid cells to acquire a resolving anti-inflammatory phenotype. Using genome-wide expression analysis and chromatin immunoprecipitation followed by next-generation sequencing, we show that the capacity to undergo pro- to anti-inflammatory (M1-to-M2) phenotype switch is controlled by the transcription factor interferon regulatory factor 7 (IRF7) that is down-regulated by the TGFß1 pathway. RNAi-mediated perturbation of Irf7 inhibited the M1-to-M2 switch, while IFNß1 (an IRF7 pathway activator) restored it. In vivo induction of Irf7 expression in microglia, following spinal cord injury, reduced their pro-inflammatory activity. These results highlight the key role of tissue-specific environmental factors in determining the fate of resident myeloid-derived cells under both physiological and pathological conditions.

Deterministic direct reprogramming of somatic cells to pluripotency.

Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.

High-throughput chromatin immunoprecipitation for genome-wide mapping of in vivo protein-DNA interactions and epigenomic states.

Dynamic protein binding to DNA elements regulates genome function and cell fate. Although methods for mapping in vivo protein-DNA interactions are becoming crucial for every aspect of genomic research, they are laborious and costly, thereby limiting progress. Here we present a protocol for mapping in vivo protein-DNA interactions using a high-throughput chromatin immunoprecipitation (HT-ChIP) approach. By using paramagnetic beads, we streamline the entire ChIP and indexed library construction process: sample transfer and loss is minimized and the need for manually labor-intensive procedures such as washes, gel extraction and DNA precipitation is eliminated. All of this allows for fully automated, cost effective and highly sensitive 96-well ChIP sequencing (ChIP-seq). Sample preparation takes 3 d from cultured cells to pooled libraries. Compared with previous methods, HT-ChIP is more suitable for large-scale in vivo studies, specifically those measuring the dynamics of a large number of different chromatin modifications/transcription factors or multiple perturbations.

A high-throughput chromatin immunoprecipitation approach reveals principles of dynamic gene regulation in mammals.

Understanding the principles governing mammalian gene regulation has been hampered by the difficulty in measuring in vivo binding dynamics of large numbers of transcription factors (TF) to DNA. Here, we develop a high-throughput Chromatin ImmunoPrecipitation (HT-ChIP) method to systematically map protein-DNA interactions. HT-ChIP was applied to define the dynamics of DNA binding by 25 TFs and 4 chromatin marks at 4 time-points following pathogen stimulus of dendritic cells. Analyzing over 180,000 TF-DNA interactions we find that TFs vary substantially in their temporal binding landscapes. This data suggests a model for transcription regulation whereby TF networks are hierarchically organized into cell differentiation factors, factors that bind targets prior to stimulus to prime them for induction, and factors that regulate specific gene programs. Overlaying HT-ChIP data on gene-expression dynamics shows that many TF-DNA interactions are established prior to the stimuli, predominantly at immediate-early genes, and identified specific TF ensembles that coordinately regulate gene-induction.

M(odu)LLating the innate response.

The integration of chromatin modifiers into specific regulatory networks is not fully understood. In this issue of Immunity, Austenaa et al. (2012) demonstrate a specific role for the histone methyltransferase MLL4 (Wbp7) in controlling the expression of critical molecules in the Toll-like receptor pathway.

Release of serine/threonine-phosphorylated adaptors from signaling microclusters down-regulates T cell activation.

Antigen recognition within immunological synapses triggers and sustains T cell activation by nucleating protein microclusters that gather T cell receptors (TCRs), kinases, and adaptors. Dissipation of these microclusters results in signal termination, but how this process is regulated is unclear. In this paper, we reveal that release of the adaptors SLP76 and GADS from signaling microclusters is induced by the serine/threonine protein kinase HPK1 and that phosphorylation of GADS plays a major role in this process. We found that HPK1 was recruited into microclusters and triggered their dissipation by inducing the phosphorylation of a threonine-containing motif of GADS, together with the previously described serine phosphorylation of SLP76. These events induced the cooperative binding of 14-3-3 proteins to SLP76-GADS complexes, leading to their uncoupling from the transmembrane adaptor LAT and consequently reducing microcluster persistence and activation-induced gene transcription. These results demonstrate that serine/threonine phosphorylation of multiple TCR-proximal effectors controls the stability of signaling microclusters, thereby determining the intensity of T cell responses.