Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for ...transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function.
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•Ψ-seq for high resolution, transcriptome-wide profiling of pseudouridine•Many distinct sites in mRNA; dynamically regulated in heat shock•Sites depend on conserved cognate pseudouridine synthases in yeast and human•Reduced rRNA and TERC pseudouridine in dyskeratosis congenita patients
Transcriptome-wide pseudouridine mapping reveals extensive, dynamic pseudouridylation of mRNA and noncoding RNA in yeast and human.
Neuroimmune interactions have emerged as critical modulators of allergic inflammation, and type 2 innate lymphoid cells (ILC2s) are an important cell type for mediating these interactions. Here, we ...show that ILC2s expressed both the neuropeptide calcitonin gene-related peptide (CGRP) and its receptor. CGRP potently inhibited alarmin-driven type 2 cytokine production and proliferation by lung ILC2s both in vitro and in vivo. CGRP induced marked changes in ILC2 expression programs in vivo and in vitro, attenuating alarmin-driven proliferative and effector responses. A distinct subset of ILCs scored highly for a CGRP-specific gene signature after in vivo alarmin stimulation, suggesting CGRP regulated this response. Finally, we observed increased ILC2 proliferation and type 2 cytokine production as well as exaggerated responses to alarmins in mice lacking the CGRP receptor. Together, these data indicate that endogenous CGRP is a critical negative regulator of ILC2 responses in vivo.
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•ILC2s express the neuropeptide CGRP and its receptor•CGRP inhibits type 2 cytokine production and proliferation of activated ILC2s•Treatment with CGRP negatively regulates IL-33-induced airway inflammation•CGRP receptor-deficient mice show increased ILC2 responses to alarmins
Cross-talk between neurons and ILC2s regulates tissue inflammation. Here, Wallrapp et al. show that the neuropeptide CGRP negatively regulates ILC2 responses to alarmins and inhibits airway inflammation in vivo. Mice lacking the CGRP receptor have exacerbated responses to alarmins, indicating this is an important pathway that controls type 2 inflammation.
Finding the components of cellular circuits and determining their functions systematically remains a major challenge in mammalian cells. Here, we introduced genome-wide pooled CRISPR-Cas9 libraries ...into dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the Tlr4 pathway. We found many of the known regulators of Tlr4 signaling, as well as dozens of previously unknown candidates that we validated. By measuring protein markers and mRNA profiles in DCs that are deficient in known or candidate genes, we classified the genes into three functional modules with distinct effects on the canonical responses to LPS and highlighted functions for the PAF complex and oligosaccharyltransferase (OST) complex. Our findings uncover new facets of innate immune circuits in primary cells and provide a genetic approach for dissection of mammalian cell circuits.
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•Cytokine stain readout for genome-wide CRISPR screen in primary immune cells•Individual validation of dozens of known and novel regulators of the LPS response•Regulators cluster into three modules by knockout effect on expression•General multi-pronged approach to CRISPR screens
A protein marker-based, genome-wide CRISPR screen has been developed in primary immune cells to identify genes that control the induction of tumor necrosis factor. Many of the known regulators, as well as dozens of previously unknown candidates, have been identified, individually validated, and classified into three functional modules.
Aneuploidy, an abnormal number of chromosomes, is a widespread phenomenon found in unicellulars such as yeast, as well as in plants and in mammalians, especially in cancer. Aneuploidy is a ...genome-scale aberration that imposes a severe burden on the cell, yet under stressful conditions specific aneuploidies confer a selective advantage. This dual nature of aneuploidy raises the question of whether it can serve as a stable and sustainable evolutionary adaptation. To clarify this, we conducted a set of laboratory evolution experiments in yeast and followed the long-term dynamics of aneuploidy under diverse conditions. Here we show that chromosomal duplications are first acquired as a crude solution to stress, yet only as transient solutions that are eliminated and replaced by more efficient solutions obtained at the individual gene level. These transient dynamics of aneuploidy were repeatedly observed in our laboratory evolution experiments; chromosomal duplications gained under stress were eliminated not only when the stress was relieved, but even if it persisted. Furthermore, when stress was applied gradually rather than abruptly, alternative solutions appear to have emerged, but not aneuploidy. Our findings indicate that chromosomal duplication is a first evolutionary line of defense, that retains survivability under strong and abrupt selective pressures, yet it merely serves as a “quick fix,” whereas more refined and sustainable solutions take over. Thus, in the perspective of genome evolution trajectory, aneuploidy is a useful yet short-lived intermediate that facilitates further adaptation.
Intestinal epithelial cells absorb nutrients, respond to microbes, function as a barrier and help to coordinate immune responses. Here we report profiling of 53,193 individual epithelial cells from ...the small intestine and organoids of mice, which enabled the identification and characterization of previously unknown subtypes of intestinal epithelial cell and their gene signatures. We found unexpected diversity in hormone-secreting enteroendocrine cells and constructed the taxonomy of newly identified subtypes, and distinguished between two subtypes of tuft cell, one of which expresses the epithelial cytokine Tslp and the pan-immune marker CD45, which was not previously associated with non-haematopoietic cells. We also characterized the ways in which cell-intrinsic states and the proportions of different cell types respond to bacterial and helminth infections: Salmonella infection caused an increase in the abundance of Paneth cells and enterocytes, and broad activation of an antimicrobial program; Heligmosomoides polygyrus caused an increase in the abundance of goblet and tuft cells. Our survey highlights previously unidentified markers and programs, associates sensory molecules with cell types, and uncovers principles of gut homeostasis and response to pathogens.
In the small intestine, a niche of accessory cell types supports the generation of mature epithelial cell types from intestinal stem cells (ISCs). It is unclear, however, if and how immune cells in ...the niche affect ISC fate or the balance between self-renewal and differentiation. Here, we use single-cell RNA sequencing (scRNA-seq) to identify MHC class II (MHCII) machinery enrichment in two subsets of Lgr5+ ISCs. We show that MHCII+ Lgr5+ ISCs are non-conventional antigen-presenting cells in co-cultures with CD4+ T helper (Th) cells. Stimulation of intestinal organoids with key Th cytokines affects Lgr5+ ISC renewal and differentiation in opposing ways: pro-inflammatory signals promote differentiation, while regulatory cells and cytokines reduce it. In vivo genetic perturbation of Th cells or MHCII expression on Lgr5+ ISCs impacts epithelial cell differentiation and IEC fate during infection. These interactions between Th cells and Lgr5+ ISCs, thus, orchestrate tissue-wide responses to external signals.
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•Intestinal stem cells (ISCs) are non-conventional antigen-presenting cells by MHCII•Interactions between ISCs and T helper subsets modulate distinct ISC fates•Epithelial MHCII is important for epithelial-cell remodeling following infection•Regulatory T cells and their key cytokine IL-10 support ISC renewal
Intestinal stem cells act as non-conventional antigen presenting cells, and these interactions with T helper cells modulate ISC renewal and differentiation to shape the intestine.
The choroid plexus (ChP) in each brain ventricle produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier. Here, we construct a single-cell and spatial atlas of each ChP in the developing, ...adult, and aged mouse brain. We delineate diverse cell types, subtypes, cell states, and expression programs in epithelial and mesenchymal cells across ages and ventricles. In the developing ChP, we predict a common progenitor pool for epithelial and neuronal cells, validated by lineage tracing. Epithelial and fibroblast cells show regionalized expression by ventricle, starting at embryonic stages and persisting with age, with a dramatic transcriptional shift with maturation, and a smaller shift in each aged cell type. With aging, epithelial cells upregulate host-defense programs, and resident macrophages upregulate interleukin-1β (IL-1β) signaling genes. Our atlas reveals cellular diversity, architecture and signaling across ventricles during development, maturation, and aging of the ChP-brain barrier.
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•A detailed map of mouse choroid plexus (ChP) cells across ventricles and ages•Ventricle- and age-dependent expression programs in epithelial and fibroblast cells•A common progenitor pool for embryonic epithelial and neuronal cells uncovered•Cell-cell interaction networks found cell-type-specific IL-1β signaling in aged ChP
Single-cell and spatial atlas of the developing, adult, and aged mouse brain choroid plexus uncovers its cellular diversity, organization, and signaling to provide insights into the development and function of this essential brain barrier.
Genome-wide association studies (GWAS) have revealed risk alleles for ulcerative colitis (UC). To understand their cell type specificities and pathways of action, we generate an atlas of 366,650 ...cells from the colon mucosa of 18 UC patients and 12 healthy individuals, revealing 51 epithelial, stromal, and immune cell subsets, including BEST4+ enterocytes, microfold-like cells, and IL13RA2+IL11+ inflammatory fibroblasts, which we associate with resistance to anti-TNF treatment. Inflammatory fibroblasts, inflammatory monocytes, microfold-like cells, and T cells that co-express CD8 and IL-17 expand with disease, forming intercellular interaction hubs. Many UC risk genes are cell type specific and co-regulated within relatively few gene modules, suggesting convergence onto limited sets of cell types and pathways. Using this observation, we nominate and infer functions for specific risk genes across GWAS loci. Our work provides a framework for interrogating complex human diseases and mapping risk variants to cell types and pathways.
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•51 cell subsets in colon mucosa of 18 ulcerative colitis and 12 healthy individuals•M-like cells, inflammatory monocytes and fibroblasts, and CD8+IL-17+ T cells expand in disease•Oncostatin M circuit in inflammatory monocytes and fibroblasts may affect drug response•Co-expression of genes within cells allows inference of causal genes across risk loci
Single-cell analyses of colon biopsy specimens from patients with ulcerative colitis delineate how expression patterns and shifting cell populations may shape disease and drug resistance, and provide a framework for linking GWAS risk loci with specific cell types and functional pathways.
Reversing the dysfunctional T cell state that arises in cancer and chronic viral infections is the focus of therapeutic interventions; however, current therapies are effective in only some patients ...and some tumor types. To gain a deeper molecular understanding of the dysfunctional T cell state, we analyzed population and single-cell RNA profiles of CD8+ tumor-infiltrating lymphocytes (TILs) and used genetic perturbations to identify a distinct gene module for T cell dysfunction that can be uncoupled from T cell activation. This distinct dysfunction module is downstream of intracellular metallothioneins that regulate zinc metabolism and can be identified at single-cell resolution. We further identify Gata-3, a zinc-finger transcription factor in the dysfunctional module, as a regulator of dysfunction, and we use CRISPR-Cas9 genome editing to show that it drives a dysfunctional phenotype in CD8+ TILs. Our results open novel avenues for targeting dysfunctional T cell states while leaving activation programs intact.
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•Distinct gene modules for T cell dysfunction and activation can be uncoupled•Single-cell profiling of CD8 TILs shows that these modules are exclusive•Metallothioneins, zinc regulators, promote T cell dysfunction•CRISPR-Cas9 targeting shows Gata-3, a zinc-finger TF, promotes dysfunction
Single-cell profiling of tumor-infiltrating lymphocytes identifies critical regulators of T cell dysfunction in cancer, opening new avenues for targeting dysfunctional T cell states while leaving activation programs intact.
In tumors, a subset of CD8+ T cells expressing the transcription factor TCF-1 drives the response to immune checkpoint blockade. We examined the mechanisms that maintain these cells in an ...autochthonous model of lung adenocarcinoma. Longitudinal sampling and single-cell sequencing of tumor-antigen specific TCF-1+ CD8+ T cells revealed that while intratumoral TCF-1+ CD8+ T cells acquired dysfunctional features and decreased in number as tumors progressed, TCF-1+ CD8+ T cell frequency in the tumor draining LN (dLN) remained stable. Two discrete intratumoral TCF-1+ CD8+ T cell subsets developed over time—a proliferative SlamF6+ subset and a non-cycling SlamF6− subset. Blocking dLN egress decreased the frequency of intratumoral SlamF6+ TCF-1+ CD8+ T cells. Conventional type I dendritic cell (cDC1) in dLN decreased in number with tumor progression, and Flt3L+anti-CD40 treatment recovered SlamF6+ T cell frequencies and decreased tumor burden. Thus, cDC1s in tumor dLN maintain a reservoir of TCF-1+ CD8+ T cells and their decrease contributes to failed anti-tumor immunity.
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•Longitudinal scRNA-seq of tumor-specific TCF-1+ CD8+ T cells in KP lung adenocarcinoma•Identified a proliferative Slamf6+ TCF-1+ T cell subset and a non-cycling SlamF6− subset•The lymph node contains a recruitable reservoir of functional TCF-1+ CD8+ T cells•Flt3L+CD40 boosts cDC1, increases TCF-1+CD8+ T cell frequencies, decreases tumor burden
In tumors, TCF-1+ CD8+ T cells drive the response to immune checkpoint blockade. Schenkel, Herbst et al. reveal that, in lung adenocarcinoma, a reservoir of TCF-1+ CD8+ T cells is maintained in tumor draining lymph nodes by conventional type I dendritic cell (cDC1). Decrease of these cDC1 as the tumor progresses contributes to failed anti-tumor immunity.
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