Search

In eukaryotes, capped RNAs include long transcripts such as messenger RNAs and long noncoding RNAs, as well as shorter transcripts such as spliceosomal RNAs, small nucleolar RNAs, and enhancer RNAs. Long capped transcripts can be profiled using cap analysis gene expression (CAGE) sequencing and other methods. Here, we describe a sequencing library preparation protocol for short capped RNAs, apply it to a differentiation time course of the human cell line THP-1, and systematically compare the landscape of short capped RNAs to that of long capped RNAs. Transcription initiation peaks associated with genes in the sense direction have a strong preference to produce either long or short capped RNAs, with one out of six peaks detected in the short capped RNA libraries only. Gene-associated short capped RNAs have highly specific 3′ ends, typically overlapping splice sites. Enhancers also preferentially generate either short or long capped RNAs, with 10% of enhancers observed in the short capped RNA libraries only. Enhancers producing either short or long capped RNAs show enrichment for GWAS-associated disease SNPs. We conclude that deep sequencing of short capped RNAs reveals new families of noncoding RNAs and elucidates the diversity of transcripts generated at known and novel promoters and enhancers.

Within the scope of the FANTOM6 consortium, we perform a large-scale knockdown of 200 long non-coding RNAs (lncRNAs) in human induced pluripotent stem cells (iPSCs) and systematically characterize their roles in self-renewal and pluripotency. We find 36 lncRNAs (18%) exhibiting cell growth inhibition. From the knockdown of 123 lncRNAs with transcriptome profiling, 36 lncRNAs (29.3%) show molecular phenotypes. Integrating the molecular phenotypes with chromatin-interaction assays further reveals cis- and trans-interacting partners as potential primary targets. Additionally, cell-type enrichment analysis identifies lncRNAs associated with pluripotency, while the knockdown of LINC02595, CATG00000090305.1, and RP11-148B6.2 modulates colony formation of iPSCs. We compare our results with previously published fibroblasts phenotyping data and find that 2.9% of the lncRNAs exhibit a consistent cell growth phenotype, whereas we observe 58.3% agreement in molecular phenotypes. This highlights that molecular phenotyping is more comprehensive in revealing affected pathways.

Distinguishing how genetics impact cellular processes can improve our understanding of variable risk for diseases. Although single-cell omics have provided molecular characterization of cell types and states on diverse tissue samples, their genetic ancestry and effects on cellular molecular traits are largely understudied. Here, we developed Monopogen, a computational tool enabling researchers to detect single nucleotide variants (SNVs) from a variety of single cell transcriptomic and epigenomic sequencing data. It leverages linkage disequilibrium from external reference panels to identify germline SNVs from sparse sequencing data and uses Monovar to identify novel SNVs at cluster (or cell type) levels. Monopogen can identify 100K~3M germline SNVs from various single cell sequencing platforms (scRNA-seq, snRNA-seq, snATAC-seq etc), with genotyping accuracy higher than 95%, when compared against matched whole genome sequencing data. We applied Monopogen on human retina, normal breast and Asian immune diversity atlases, showing that that derived genotypes enable accurate global and local ancestry inference and identification of admixed samples from ancestrally diverse donors. In addition, we applied Monopogen on ~4M cells from 65 human heart left ventricle single cell samples and identified novel variants associated with cardiomyocyte metabolic levels and epigenomic programs. In summary, Monopogen provides a novel computational framework that brings together population genetics and single cell omics to uncover genetic determinants of cellular quantitative traits.

Nuclear paraspeckle assembly transcript 1 (NEAT1) is a long non-coding RNA associated with the promotion of several solid cancers. As part of the FANTOM6 project, we aimed to define its role in acute myeloid leukemia (AML) and myelopoiesis, since it remains largely uninvestigated. We show that NEAT1 expression increases during myelopoiesis, especially in monocytes. NEAT1 expression is elevated in AML compared to normal bone marrow (NBM), especially in AML with inv(16)and t(8;21). In addition, NEAT1 expression correlates positively with ASXL1, KRAS and NRAS mutations, but negatively with TP53 mutant AML. Higher NEAT1 expression is associated with better overall survival in AML, independent of other known risk factors. Knockdown of NEAT1 in AML cells induces monocytic differentiation and upregulated gene expression of genes involved in glucose metabolism. By investigating genome-wide RNA and DNA interactions using RADICL-sequencing, we show that NEAT1 binds to the RUNX2 locus that is associated with differentiation, metabolism, and glucose uptake. The results suggest that the lncRNA NEAT1 has a role in myelopoiesis, AML and is implicated in glycolysis.

Recent efforts on the characterization of long non-coding RNAs (lncRNAs) revealed their functional roles in modulating diverse cellular processes. These include pluripotency maintenance, lineage commitment, carcinogenesis, and pathogenesis of various diseases. By interacting with DNA, RNA and protein, lncRNAs mediate multifaceted mechanisms to regulate transcription, RNA processing, RNA interference and translation. Of more than 173000 discovered lncRNAs, the majority remain functionally unknown. The cell type-specific expression and localization of the lncRNA also suggest potential distinct functions of lncRNAs across different cell types. This highlights the niche of identifying functional lncRNAs in different biological processes and diseases through high-throughput (HTP) screening. This review summarizes the current work performed and perspectives on HTP screening of functional lncRNAs where different technologies, platforms, cellular responses and the downstream analyses are discussed. We hope to provide a better picture in applying different technologies to facilitate functional annotation of lncRNA efficiently.

Infection of the lungs with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via the angiotensin I converting enzyme 2 (ACE2) receptor induces a type of systemic inflammation known as a cytokine storm. However, the precise mechanisms involved in severe coronavirus disease 2019 (COVID-19) pneumonia are unknown. Here, we show that interleukin-10 (IL-10) changed normal alveolar macrophages into ACE2-expressing M2c-type macrophages that functioned as spreading vectors for SARS-CoV-2 infection. The depletion of alveolar macrophages and blockade of IL-10 attenuated SARS-CoV-2 pathogenicity. Furthermore, genome-wide association and quantitative trait locus analyses identified novel mRNA transcripts in human patients, COVID-19 infectivity enhancing dual receptor (CiDRE), which has unique synergistic effects within the IL-10-ACE2 system in M2c-type macrophages. Our results demonstrate that alveolar macrophages stimulated by IL-10 are key players in severe COVID-19. Collectively, CiDRE expression levels are potential risk factors that predict COVID-19 severity, and CiDRE inhibitors might be useful as COVID-19 therapies.Graphical abstract

Alzheimer's disease (AD), a common chronic neurodegenerative disease, has become a major public health concern. Despite years of research, therapeutics for AD are limited. Overexpression of secretory glutaminyl cyclase (sQC) in AD brain leads to the formation of a highly neurotoxic pyroglutamate variant of amyloid beta, pGlu-Aβ, which acts as a potential seed for the aggregation of full length Aβ. Preventing the formation of pGlu-Aβ through inhibition of sQC has become an attractive disease-modifying therapy in AD. In this current study, through a pharmacophore assisted high throughput virtual screening, we report a novel sQC inhibitor (Cpd-41) with a piperidine-4-carboxamide moiety (IC50 = 34 μM). Systematic molecular docking, MD simulations and X-ray crystallographic analysis provided atomistic details of the binding of Cpd-41 in the active site of sQC. The unique mode of binding and moderate toxicity of Cpd-41 make this molecule an attractive candidate for designing high affinity sQC inhibitors.

The human brain is a complex, three-dimensional structure. To better recapitulate brain complexity, recent efforts have focused on the development of human-specific midbrain organoids. Human iPSC-derived midbrain organoids consist of differentiated and functional neurons, which contain active synapses, as well as astrocytes and oligodendrocytes. However, the absence of microglia, with their ability to remodel neuronal networks and phagocytose apoptotic cells and debris, represents a major disadvantage for the current midbrain organoid systems. Additionally, neuroinflammation-related disease modeling is not possible in the absence of microglia. So far, no studies about the effects of human iPSC-derived microglia on midbrain organoid neural cells have been published. Here we describe an approach to derive microglia from human iPSCs and integrate them into iPSC-derived midbrain organoids. Using single nuclear RNA Sequencing, we provide a detailed characterization of microglia in midbrain organoids as well as the influence of their presence on the other cells of the organoids. Furthermore, we describe the effects that microglia have on cell death and oxidative stress-related gene expression. Finally, we show that microglia in midbrain organoids affect synaptic remodeling and increase neuronal excitability. Altogether, we show a more suitable system to further investigate brain development, as well as neurodegenerative diseases and neuroinflammation., Glia, 70 (7), ISSN:0894-1491, ISSN:1098-1136

Human stem cell-derived organoids have great potential for modelling physiological and pathological processes. They recapitulate in vitro the organization and function of a respective organ or part of an organ. Human midbrain organoids (hMOs) have been described to contain midbrain-specific dopaminergic neurons that release the neurotransmitter dopamine. However, the human midbrain contains also additional neuronal cell types, which are functionally interacting with each other. Here, we analysed hMOs at high-resolution by means of single-cell RNA sequencing (scRNA-seq), imaging and electrophysiology to unravel cell heterogeneity. Our findings demonstrate that hMOs show essential neuronal functional properties as spontaneous electrophysiological activity of different neuronal subtypes, including dopaminergic, GABAergic, glutamatergic and serotonergic neurons. Recapitulating these in vivo features makes hMOs an excellent tool for in vitro disease phenotyping and drug discovery. Electronic supplementary material The online version of this article (10.1007/s00441-020-03249-y) contains supplementary material, which is available to authorized users.

Single-cell transcriptome analysis reveals heterogeneous cell types in complex tissues and leads to unexpected biological findings when compared to bulk populations. However most of the methods focus on the 3'-end of polyadenylated transcripts using droplet-based technology. To achieve complete transcriptome, we describe single-cell 5'-end transcriptome protocol with random primed-cDNA harvesting on the Fluidigm C1™ platform which can isolate and process up to 96 cells from a single run with custom library preparation. The method enables detection of Transcription Start Site (TSS) at the single-cell resolution yielding a more comprehensive overview of gene regulatory elements governing in the EpiSC-like cell (EpiLC) including non-polyadenylated RNA and enhancer RNA activities.

Background The lymphatic and the blood vasculature are closely related systems that collaborate to ensure the organism’s physiological function. Despite their common developmental origin, they present distinct functional fates in adulthood that rely on robust lineage-specific regulatory programs. The recent technological boost in sequencing approaches unveiled long noncoding RNAs (lncRNAs) as prominent regulatory players of various gene expression levels in a cell-type-specific manner. Results To investigate the potential roles of lncRNAs in vascular biology, we performed antisense oligonucleotide (ASO) knockdowns of lncRNA candidates specifically expressed either in human lymphatic or blood vascular endothelial cells (LECs or BECs) followed by Cap Analysis of Gene Expression (CAGE-Seq). Here, we describe the quality control steps adopted in our analysis pipeline before determining the knockdown effects of three ASOs per lncRNA target on the LEC or BEC transcriptomes. In this regard, we especially observed that the choice of negative control ASOs can dramatically impact the conclusions drawn from the analysis depending on the cellular background. Conclusion In conclusion, the comparison of negative control ASO effects on the targeted cell type transcriptomes highlights the essential need to select a proper control set of multiple negative control ASO based on the investigated cell types., BMC Genomic Data, 22 (1)

Profiling of cis-regulatory elements (CREs, mostly promoters and enhancers) in single cells allows the interrogation of the cell-type and cell-state-specific contexts of gene regulation and genetic predisposition to diseases. Here we demonstrate single-cell RNA-5′end-sequencing (sc-end5-seq) methods can detect transcribed CREs (tCREs), enabling simultaneous quantification of gene expression and enhancer activities in a single assay at no extra cost. We showed enhancer RNAs can be detected using sc-end5-seq methods with either random or oligo(dT) priming. To analyze tCREs in single cells, we developed SCAFE (Single Cell Analysis of Five-prime Ends) to identify genuine tCREs and analyze their activities (https://github.com/chung-lab/scafe). As compared to accessible CRE (aCRE, based on chromatin accessibility), tCREs are more accurate in predicting CRE interactions by co-activity, more sensitive in detecting shifts in alternative promoter usage and more enriched in diseases heritability. Our results highlight additional dimensions within sc-end5-seq data which can be used for interrogating gene regulation and disease heritability.

The human genome is pervasively transcribed and produces a wide variety of long non-coding RNAs (lncRNAs), constituting the majority of transcripts across human cell types. Studying lncRNAs is challenging due to their low expression level, cell type-specific occurrence, poor sequence conservation between orthologs, and lack of information about RNA domains. LncRNAs direct the regulatory factors in the locations that are in cis to their transcription sites. We designed a model to predict if an lncRNA acts in cis based on its features and trained it using RNA-chromatin interaction data. The trained model is cell type-independent and does not require RNA-chromatin data. Combining RNA-chromatin and Hi-C data, we showed that lncRNA-chromatin binding sites are determined by chromosome conformation. For each lncRNA, the spatially proximal genes were identified as their potential targets by combining Hi-C and Cap Analysis Gene Expression (CAGE) data in 18 human cell types. RNA-protein and RNA-chromatin interaction data suggested that lncRNAs act as scaffolds to recruit regulatory proteins to target promoters and enhancers. We provide the data through an interactive visualization web portal at https://fantom.gsc.riken.jp/zenbu/reports/#F6_3D_lncRNA.

Human brain organoids provide us the means to investigate human brain development and neurological diseases, and single-cell RNA-sequencing (scRNA-seq) technologies allow us to identify homologous cell types and the molecular heterogeneity between individual cells. Previously established human brain organoids of brainstem (hBSOs) and midbrain (hMBOs) were analyzed by scRNA-seq, but the difference in cellular composition between these organoids remains unclear. Here, we integrated and compared the single-cell transcriptome of hBSOs and hMBOs. Our analysis demonstrated that the hBSOs and hMBOs contain some unique cell types, including inflammatory and mesenchymal cells. Further comparison of the hBSOs and hMBOs with publicly available scRNA-seq dataset of human fetal midbrain (hMB) showed high similarity in their neuronal components. These results provide new insights into human brain organoid technologies.

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-to-date lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

Long noncoding RNAs (lncRNAs) have emerged as key coordinators of biological and cellular processes. Characterizing lncRNA expression across cells and tissues is key to understanding their role in determining phenotypes, including human diseases. We present here FC-R2, a comprehensive expression atlas across a broadly defined human transcriptome, inclusive of over 109,000 coding and noncoding genes, as described in the FANTOM CAGE-Associated Transcriptome (FANTOM-CAT) study. This atlas greatly extends the gene annotation used in the originalrecount2resource. We demonstrate the utility of the FC-R2 atlas by reproducing key findings from published large studies and by generating new results across normal and diseased human samples. In particular, we (a) identify tissue-specific transcription profiles for distinct classes of coding and noncoding genes, (b) perform differential expression analysis across thirteen cancer types, identifying novel noncoding genes potentially involved in tumor pathogenesis and progression, and (c) confirm the prognostic value for several enhancer lncRNAs expression in cancer. Our resource is instrumental for the systematic molecular characterization of lncRNA by the FANTOM6 Consortium. In conclusion, comprised of over 70,000 samples, the FC-R2 atlas will empower other researchers to investigate functions and biological roles of both known coding genes and novel lncRNAs.

Recent studies have revealed the importance of long noncoding RNAs (lncRNAs) as tissue-specific regulators of gene expression. There is ample evidence that distinct types of vasculature undergo tight transcriptional control to preserve their structure, identity, and functions. We determined, for the first time, the global lineage-specific lncRNAome of human dermal blood and lymphatic endothelial cells (BECs and LECs), combining RNA-Seq and CAGE-Seq. A subsequent genome-wide antisense oligonucleotide-knockdown screen of a robust set of BEC- and LEC-specific lncRNAs identified LETR1 as a critical gatekeeper of the global LEC transcriptome. Deep RNA-DNA, RNA-protein, and phenotype rescue analyses revealed that LETR1 acts as a nuclear trans-acting lncRNA modulating, via key epigenetic factors, the expression of essential target genes, including KLF4 and SEMA3C, governing the growth and migratory ability of LECs. Together, our study provides new evidence supporting the intriguing concept that every cell type expresses precise lncRNA signatures to control lineage-specific regulatory programs.

Thrombospondin-2 (TSP2) is an anti-angiogenic matricellular protein that inhibits tumor growth and angiogenesis. Tumor-associated blood vascular endothelial cells (BECs) were isolated from human invasive bladder cancers and from matched normal bladder tissue by immuno-laser capture microdissection. Exon expression profiling analyses revealed a particularly high expression of a short TSP2 transcript containing only the last 9 (3′) exons of the full-length TSP2 transcript. Using 5′ and 3′ RACE (rapid amplification of cDNA ends) and Sanger sequencing, we confirmed the existence of the shorter transcript of TSP2 (sTSP2) and determined its sequence which completely lacked the anti-angiogenic thrombospondin type 1 repeats domain. The largest open reading frame predicted within the transcript comprises 209 amino acids and matches almost completely the C-terminal lectin domain of full-length TSP2. We produced recombinant sTSP2 and found that unlike the full-length TSP2, sTSP2 did not inhibit vascular endothelial growth factor-A-induced proliferation of cultured human BECs, but in contrast when combined with TSP2 blocked the inhibitory effects of TSP2 on BEC proliferation. In vivo studies with stably transfected A431 squamous cell carcinoma cells revealed that full-length TSP2, but not sTSP2, inhibited tumor growth and angiogenesis. This study reveals that the transcriptional program of tumor stromal cells can change to transcribe a new version of an endogenous angiogenesis inhibitor that has lost its anti-angiogenic activity., Oncogene, 37 (19), ISSN:0950-9232, ISSN:1476-5594

The Human Cell Atlas is a large, international consortium that aims to identify and describe every cell type in the human body. The comprehensive cellular maps that arise from this ambitious effort have the potential to transform many aspects of fundamental biology and clinical practice. Here, we discuss the technical approaches that could be used today to generate such a resource and also the technical challenges that will be encountered.

Published single-cell datasets are rich resources for investigators who want to address questions not originally asked by the creators of the datasets. The single-cell datasets might be obtained by different protocols and diverse analysis strategies. The main challenge in utilizing such single-cell data is how we can make the various large-scale datasets to be comparable and reusable in a different context. To challenge this issue, we developed the single-cell centric database ‘SCPortalen’ (http://single-cell.clst.riken.jp/). The current version of the database covers human and mouse single-cell transcriptomics datasets that are publicly available from the INSDC sites. The original metadata was manually curated and single-cell samples were annotated with standard ontology terms. Following that, common quality assessment procedures were conducted to check the quality of the raw sequence. Furthermore, primary data processing of the raw data followed by advanced analyses and interpretation have been performed from scratch using our pipeline. In addition to the transcriptomics data, SCPortalen provides access to single-cell image files whenever available. The target users of SCPortalen are all researchers interested in specific cell types or population heterogeneity. Through the web interface of SCPortalen users are easily able to search, explore and download the single-cell datasets of their interests.

The human body consists of 37 trillion single cells represented by over 50 organs that are stitched together to make us who we are, yet we still have very little understanding about the basic units of our body: what cell types and states make up our organs both compositionally and spatially. Previous efforts to profile a wide range of human cell types have been attempted by the FANTOM and GTEx consortia. Now, with the advancement in genomic technologies, profiling the human body at single-cell resolution is possible and will generate an unprecedented wealth of data that will accelerate basic and clinical research with tangible applications to future medicine. To date, several major organs have been profiled, but the challenges lie in ways to integrate single-cell genomics data in a meaningful way. In recent years, several consortia have begun to introduce harmonization and equity in data collection and analysis. Herein, we introduce existing and nascent single-cell genomics consortia, and present benefits to necessitate single-cell genomic consortia in a regional environment to achieve the universal human cell reference dataset., Genomics: Collaborative efforts to map the human body—single cell at a time Profiling the human body at the single cell resolution will reveal a map to enable researchers to compare cell types that are found in healthy and disease tissues at a much finer resolution. Large international projects such as FANTOM (Functional ANnoTation Of the Mammalian genome) and GTEx (Genotype-Tissue Expression) have profiled gene expression in hundreds of human cell types. However, the data were obtained from ‘bulk’ samples. The possibility of sequencing large numbers of single cells at a reasonable cost offers the opportunity to create a much more detailed human cell reference map. Jay Shin, at the RIKEN Institute, Yokohama, Japan, and colleagues review rapidly growing single cell genomics consortia and highlight the advantages of initiating national collaborations to harmonize procedures and to obtain a more accurate representation of regional ethnic diversity.

SummaryThe brainstem controls heartbeat, blood pressure and respiration, which are life-sustaining functions, therefore, disorders of the brainstem can be lethal. Brain organoids derived from human pluripotent stem cells recapitulate the course of human brain development and are expected to be useful for medical research on central nervous system disorders. However, existing organoid models have limitations, hampering the elucidation of diseases affecting specific components of the brain. Here, we developed a method to generate human brainstem organoids (hBSOs), containing neural crest stem cells as well as midbrain/hindbrain progenitors, noradrenergic and cholinergic neurons, and dopaminergic neurons, demonstrated by specific electrophysiological signatures. Single-cell RNA sequence analysis, together with proteomics and electrophysiology, revealed that the cellular population in these organoids was similar to that of the human brainstem and neural crest, which raises the possibility of making use of hBSOs in grafting for transplantation, efficient drug screenings and modeling the neural crest diseases.

Long non-coding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes and yet, their functions remain largely unknown. We systematically knockdown 285 lncRNAs expression in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNA exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest to-date lncRNA knockdown dataset with molecular phenotyping (over 1,000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

Summary Classical transection studies suggest that, in addition to the hypothalamus, the brainstem is essential for non-rapid eye movement (NREM) sleep. The circuits underlying this function, however, have remained largely unknown. We identified a circuit distributed in the midbrain, pons, and medulla that promotes NREM sleep in mice. We focused on the sublaterodorsal tegmentum, an area implicated in dual regulation of REM and NREM sleep. Transcriptomic and genetic analyses revealed that neurons positive for the neuropeptide neurotensin promote NREM sleep. Further analyses identified downstream NREM sleep-promoting neurons in the dorsal deep mesencephalic nucleus, the lateral part of the periaqueductal gray, and the medial vestibular nucleus that were also neurotensinergic. Infusion of neurotensin into the fourth ventricle induced NREM sleep-like cortical activity, whereas mice deficient for neurotensin exhibited increased REM sleep, implicating the involvement of the neuropeptide itself. These findings identify a widely distributed NREM sleep-regulating circuit in the brainstem with a common molecular property.

Long non-coding RNAs (lncRNAs) have emerged as key coordinators of biological and cellular processes. Characterizing lncRNA expression across cells and tissues is key to understanding their role in determining phenotypes including human diseases. We present here FC-R2, a comprehensive expression atlas across a broadly-defined human transcriptome, inclusive of over 109,000 coding and non-coding genes, as described in the FANTOM CAGE-Associated Transcriptome (FANTOM-CAT) study. This atlas greatly extends the gene annotation used in the original recount2 resource. We demonstrate the utility of the FC-R2 atlas by reproducing key findings from published large studies and by generating new results across normal and diseased human samples. In particular, we (a) identify tissue specific transcription profiles for distinct classes of coding and non-coding genes, (b) perform differential expression analyses across thirteen cancer types, providing new insights linking promoter and enhancer lncRNAs expression to tumor pathogenesis, and (c) confirm the prognostic value of several enhancers in cancer. Comprised of over 70,000 samples, the FC-R2 atlas will empower other researchers to investigate functions and biological roles of both known coding genes and novel lncRNAs. Most importantly, access to the FC-R2 atlas is available from https://jhubiostatistics.shinyapps.io/recount/, the recount Bioconductor package, and http://marchionnilab.org/fcr2.html.

Significance Exceptionally long-lived people such as supercentenarians tend to spend their entire lives in good health, implying that their immune system remains active to protect against infections and tumors. However, their immunological condition has been largely unexplored. We profiled thousands of circulating immune cells from supercentenarians at single-cell resolution and identified CD4 T cells that have cytotoxic features. This characteristic is very unique to supercentenarians, because generally CD4 T cells have helper, but not cytotoxic, functions under physiological conditions. We further profiled their T cell receptors and revealed that the cytotoxic CD4 T cells were accumulated through clonal expansion. The conversion of helper CD4 T cells to a cytotoxic variety might be an adaptation to the late stage of aging., Supercentenarians, people who have reached 110 y of age, are a great model of healthy aging. Their characteristics of delayed onset of age-related diseases and compression of morbidity imply that their immune system remains functional. Here we performed single-cell transcriptome analysis of 61,202 peripheral blood mononuclear cells (PBMCs), derived from 7 supercentenarians and 5 younger controls. We identified a marked increase of cytotoxic CD4 T cells (CD4 cytotoxic T lymphocytes [CTLs]) as a signature of supercentenarians. Furthermore, single-cell T cell receptor sequencing of 2 supercentenarians revealed that CD4 CTLs had accumulated through massive clonal expansion, with the most frequent clonotypes accounting for 15 to 35% of the entire CD4 T cell population. The CD4 CTLs exhibited substantial heterogeneity in their degree of cytotoxicity as well as a nearly identical transcriptome to that of CD8 CTLs. This indicates that CD4 CTLs utilize the transcriptional program of the CD8 lineage while retaining CD4 expression. Indeed, CD4 CTLs extracted from supercentenarians produced IFN-γ and TNF-α upon ex vivo stimulation. Our study reveals that supercentenarians have unique characteristics in their circulating lymphocytes, which may represent an essential adaptation to achieve exceptional longevity by sustaining immune responses to infections and diseases.

SummaryThe conversion of cell fates is controlled by hierarchical gene regulatory networks (GRNs) that induce remarkable alterations in cellular and transcriptome states. The identification of key regulators within these networks from myriad of candidate genes, however, poses a major research challenge. Here we present Convert-seq, combining single-cell RNA sequencing (scRNA-seq) and pooled ectopic gene expression with a new strategy to discriminate sequencing reads derived from exogenous and endogenous transcripts. We demonstrate Convert-seq by associating hundreds of single cells during reprogramming of human fibroblasts to induced neurons (iN) with exogenous and endogenous transcriptional signatures. Convert-seq identified GRNs modulating the emergence of developmental trajectories and predicted combinatorial activation of exogenous transcription factors controlling iN subtype specification. Functional validation of iN subtypes generated by novel combinations of exogenous transcription factors establish Convert-seq as a broadly applicable workflow to rapidly identify key transcription factors and GRNs orchestrating the reprogramming of virtually any cell type.

Single-cell transcriptomic profiling is a powerful tool to explore cellular heterogeneity. However, most of these methods focus on the 3′-end of polyadenylated transcripts and provide only a partial view of the transcriptome. We introduce C1 CAGE, a method for the detection of transcript 5′-ends with an original sample multiplexing strategy in the C1TM microfluidic system. We first quantifiy the performance of C1 CAGE and find it as accurate and sensitive as other methods in the C1 system. We then use it to profile promoter and enhancer activities in the cellular response to TGF-β of lung cancer cells and discover subpopulations of cells differing in their response. We also describe enhancer RNA dynamics revealing transcriptional bursts in subsets of cells with transcripts arising from either strand in a mutually exclusive manner, validated using single molecule fluorescence in situ hybridization., Single-cell transcriptomic profiling allows the exploration of cellular heterogeneity but commonly focuses on the 3′-end of the transcript. Here the authors introduce C1 CAGE, which detects the 5′ transcript end in a multiplexed microfluidic system.

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

Transdifferentiation, the process of converting from one cell type to another without going through a pluripotent state, has great promise for regenerative medicine. The identification of key transcription factors for reprogramming is currently limited by the cost of exhaustive experimental testing of plausible sets of factors, an approach that is inefficient and unscalable. Here we present a predictive system (Mogrify) that combines gene expression data with regulatory network information to predict the reprogramming factors necessary to induce cell conversion. We have applied Mogrify to 173 human cell types and 134 tissues, defining an atlas of cellular reprogramming. Mogrify correctly predicts the transcription factors used in known transdifferentiations. Furthermore, we validated two new transdifferentiations predicted by Mogrify. We provide a practical and efficient mechanism for systematically implementing novel cell conversions, facilitating the generalization of reprogramming of human cells. Predictions are made available to help rapidly further the field of cell conversion.

Basic leucine zipper transcription factor Batf2 is poorly described, whereas Batf and Batf3 have been shown to play essential roles in dendritic cell, T cell, and B cell development and regulation. Batf2 was drastically induced in IFN-γ–activated classical macrophages (M1) compared with unstimulated or IL-4–activated alternative macrophages (M2). Batf2 knockdown experiments from IFN-γ–activated macrophages and subsequent expression profiling demonstrated important roles for regulation of immune responses, inducing inflammatory and host-protective genes Tnf, Ccl5, and Nos2. Mycobacterium tuberculosis (Beijing strain HN878)–infected macrophages further induced Batf2 and augmented host-protective Batf2-dependent genes, particularly in M1, whose mechanism was suggested to be mediated through both TLR2 and TLR4 by LPS and heat-killed HN878 (HKTB) stimulation experiments. Irf1 binding motif was enriched in the promoters of Batf2-regulated genes. Coimmunoprecipitation study demonstrated Batf2 association with Irf1. Furthermore, Irf1 knockdown showed downregulation of IFN-γ– or LPS/HKTB-activated host-protective genes Tnf, Ccl5, Il12b, and Nos2. Conclusively, Batf2 is an activation marker gene for M1 involved in gene regulation of IFN-γ–activated classical macrophages, as well as LPS/HKTB-induced macrophage stimulation, possibly by Batf2/Irf1 gene induction. Taken together, these results underline the role of Batf2/Irf1 in inducing inflammatory responses in M. tuberculosis infection.

Vascular endothelial growth factors (VEGFs) and their receptors play crucial roles in the formation of blood and lymphatic vessels during embryogenesis, and also under pathologic conditions in the adult. Despite intensive efforts over the last decades to elucidate the precise functions of VEGFs, transcriptional responses to VEGF receptor stimulation are still not fully characterized. To investigate the specific transcriptional effects of VEGFR-2 and VEGFR-3 activation, we performed a correlation analysis of previously published CAGE sequencing and microarray data of human lymphatic endothelial cells (LECs) stimulated with distinct VEGFs acting through either VEGFR-2 or VEGFR-3. We identified that specific activation of VEGFR-3 by VEGF-C156S results in the downregulation of many genes involved in immune regulation and inflammation, suggesting that VEGFR-3 stimulation has direct anti-inflammatory effects. Comparing CAGE and microarray data sets, we furthermore identified a small number of genes that showed a receptor-dependent response in LECs, demonstrating that these receptors, despite activating very similar signaling pathways, fulfill overlapping but not identical functions within the same cell type (LECs)., Scientific Data, 4, ISSN:2052-4463