Abstract
Development of high throughput single-cell sequencing technologies has made it cost-effective to profile thousands of cells from diverse samples containing multiple cell types. To study how ...these different cell types work together, here we develop NATMI (Network Analysis Toolkit for Multicellular Interactions). NATMI uses connectomeDB2020 (a database of 2293 manually curated ligand-receptor pairs with literature support) to predict and visualise cell-to-cell communication networks from single-cell (or bulk) expression data. Using multiple published single-cell datasets we demonstrate how NATMI can be used to identify (i) the cell-type pairs that are communicating the most (or most specifically) within a network, (ii) the most active (or specific) ligand-receptor pairs active within a network, (iii) putative highly-communicating cellular communities and (iv) differences in intercellular communication when profiling given cell types under different conditions. Furthermore, analysis of the Tabula Muris (organism-wide) atlas confirms our previous prediction that autocrine signalling is a major feature of cell-to-cell communication networks, while also revealing that hundreds of ligands and their cognate receptors are co-expressed in individual cells suggesting a substantial potential for self-signalling.
Upon the first publication of the fifth iteration of the Functional Annotation of Mammalian Genomes collaborative project, FANTOM5, we gathered a series of primary data and database systems into the ...FANTOM web resource (http://fantom.gsc.riken.jp) to facilitate researchers to explore transcriptional regulation and cellular states. In the course of the collaboration, primary data and analysis results have been expanded, and functionalities of the database systems enhanced. We believe that our data and web systems are invaluable resources, and we think the scientific community will benefit for this recent update to deepen their understanding of mammalian cellular organization. We introduce the contents of FANTOM5 here, report recent updates in the web resource and provide future perspectives.
Abstract
The Functional ANnoTation Of the Mammalian genome (FANTOM) Consortium has continued to provide extensive resources in the pursuit of understanding the transcriptome, and transcriptional ...regulation, of mammalian genomes for the last 20 years. To share these resources with the research community, the FANTOM web-interfaces and databases are being regularly updated, enhanced and expanded with new data types. In recent years, the FANTOM Consortium's efforts have been mainly focused on creating new non-coding RNA datasets and resources. The existing FANTOM5 human and mouse miRNA atlas was supplemented with rat, dog, and chicken datasets. The sixth (latest) edition of the FANTOM project was launched to assess the function of human long non-coding RNAs (lncRNAs). From its creation until 2020, FANTOM6 has contributed to the research community a large dataset generated from the knock-down of 285 lncRNAs in human dermal fibroblasts; this is followed with extensive expression profiling and cellular phenotyping. Other updates to the FANTOM resource includes the reprocessing of the miRNA and promoter atlases of human, mouse and chicken with the latest reference genome assemblies. To facilitate the use and accessibility of all above resources we further enhanced FANTOM data viewers and web interfaces. The updated FANTOM web resource is publicly available at https://fantom.gsc.riken.jp/.
Cell-to-cell communication across multiple cell types and tissues strictly governs proper functioning of metazoans and extensively relies on interactions between secreted ligands and cell-surface ...receptors. Herein, we present the first large-scale map of cell-to-cell communication between 144 human primary cell types. We reveal that most cells express tens to hundreds of ligands and receptors to create a highly connected signalling network through multiple ligand-receptor paths. We also observe extensive autocrine signalling with approximately two-thirds of partners possibly interacting on the same cell type. We find that plasma membrane and secreted proteins have the highest cell-type specificity, they are evolutionarily younger than intracellular proteins, and that most receptors had evolved before their ligands. We provide an online tool to interactively query and visualize our networks and demonstrate how this tool can reveal novel cell-to-cell interactions with the prediction that mast cells signal to monoblastic lineages via the CSF1-CSF1R interacting pair.
Medicinal and industrial properties of phytochemicals (e.g. glycyrrhizin) from the root of Glycyrrhiza uralensis (licorice plant) made it an attractive, multimillion-dollar trade item. Bioengineering ...is one of the solutions to overcome such high market demand and to protect plants from extinction. Unfortunately, limited genomic information on medicinal plants restricts their research and thus biosynthetic mechanisms of many important phytochemicals are still poorly understood. In this work we utilized the de novo (no reference genome sequence available) assembly of Illumina RNA-Seq data to study the transcriptome of the licorice plant. Our analysis is based on sequencing results of libraries constructed from samples belonging to different tissues (root and leaf) and collected in different seasons and from two distinct strains (low and high glycyrrhizin producers). We provide functional annotations and the expression profile of 43,882 assembled unigenes, which are suitable for various further studies. Here, we searched for G. uralensis-specific enzymes involved in isoflavonoid biosynthesis as well as elucidated putative cytochrome P450 enzymes and putative vacuolar saponin transporters involved in glycyrrhizin production in the licorice root. To disseminate the data and the analysis results, we constructed a publicly available G. uralensis database. This work will contribute to a better understanding of the biosynthetic pathways of secondary metabolites in licorice plants, and possibly in other medicinal plants, and will provide an important resource to further advance transcriptomic studies in legumes.
The Cap Analysis of Gene Expression (CAGE) is a powerful method to identify Transcription Start Sites (TSSs) of capped RNAs while simultaneously measuring transcripts expression level. CAGE allows ...mapping at single nucleotide resolution at all active promoters and enhancers. Large CAGE datasets have been produced over the years from individual laboratories and consortia, including the Encyclopedia of DNA Elements (ENCODE) and Functional Annotation of the Mammalian Genome (FANTOM) consortia. These datasets constitute open resource for TSS annotations and gene expression analysis. Here, we provide an experimental protocol for the most recent CAGE method called Low Quantity (LQ) single strand (ss) CAGE "LQ-ssCAGE", which enables cost-effective profiling of low quantity RNA samples. LQ-ssCAGE is especially useful for samples derived from cells cultured in small volumes, cellular compartments such as nuclear RNAs or for samples from developmental stages. We demonstrate the reproducibility and effectiveness of the method by constructing 240 LQ-ssCAGE libraries from 50 ng of THP-1 cell extracted RNAs and discover lowly expressed novel enhancer and promoter-derived lncRNAs.
Background Children with problematic severe asthma have poor disease control despite high doses of inhaled corticosteroids and additional therapy, leading to personal suffering, early deterioration ...of lung function, and significant consumption of health care resources. If no exacerbating factors, such as smoking or allergies, are found after extensive investigation, these children are given a diagnosis of therapy-resistant (or therapy-refractory) asthma (SA). Objective We sought to deepen our understanding of childhood SA by analyzing gene expression and modeling the underlying regulatory transcription factor networks in peripheral blood leukocytes. Methods Gene expression was analyzed by using Cap Analysis of Gene Expression in children with SA (n = 13), children with controlled persistent asthma (n = 15), and age-matched healthy control subjects (n = 9). Cap Analysis of Gene Expression sequencing detects the transcription start sites of known and novel mRNAs and noncoding RNAs. Results Sample groups could be separated by hierarchical clustering on 1305 differentially expressed transcription start sites, including 816 known genes and several novel transcripts. Ten of 13 tested novel transcripts were validated by means of RT-PCR and Sanger sequencing. Expression of RAR-related orphan receptor A (RORA) , which has been linked to asthma in genome-wide association studies, was significantly upregulated in patients with SA. Gene network modeling revealed decreased glucocorticoid receptor signaling and increased activity of the mitogen-activated protein kinase and Jun kinase cascades in patients with SA. Conclusion Circulating leukocytes from children with controlled asthma and those with SA have distinct gene expression profiles, demonstrating the possible development of specific molecular biomarkers and supporting the need for novel therapeutic approaches.
The production of type 1 conventional dendritic cells (cDC1s) requires high expression of the transcription factor IRF8. Three enhancers at the Irf8 3′ region function in a differentiation ...stage-specific manner. However, whether and how these enhancers interact physically and functionally remains unclear. Here, we show that the Irf8 3′ enhancers directly interact with each other and contact the Irf8 gene body during cDC1 differentiation. The +56 kb enhancer, which functions from multipotent progenitor stages, activates the other 3′ enhancers through an IRF8-dependent transcription factor program, that is, in trans. Then, the +32 kb enhancer, which operates in cDC1-committed cells, reversely acts in cis on the other 3′ enhancers to maintain the high expression of Irf8. Indeed, mice with compound heterozygous deletion of the +56 and +32 kb enhancers are unable to generate cDC1s. These results illustrate how multiple enhancers cooperate to induce a lineage-determining transcription factor gene during cell differentiation.
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•Irf8 3′ enhancers interact with each other and contact the gene body in cDC1 differentiation•The +56 and +32 kb enhancers functionally cooperate during cDC1 differentiation•The +56 and +32 kb enhancers act on other 3′ enhancers via trans and cis mechanisms•Cooperation between the +56 and +32 kb enhancers supports cDC1 differentiation in vivo
Irf8 enhancers function in a differentiation stage-specific manner. Yamasaki et al. analyze physical and functional interaction during cDC1 differentiation. Their results illustrate why and how multiple enhancers mutually cooperate using distinct mechanisms to regulate the proper expression of a lineage-determining transcription factor gene.
Gene expression profiles in homologous tissues have been observed to be different between species, which may be due to differences between species in the gene expression program in each cell type, ...but may also reflect differences in cell type composition of each tissue in different species. Here, we compare expression profiles in matching primary cells in human, mouse, rat, dog, and chicken using Cap Analysis Gene Expression (CAGE) and short RNA (sRNA) sequencing data from FANTOM5. While we find that expression profiles of orthologous genes in different species are highly correlated across cell types, in each cell type many genes were differentially expressed between species. Expression of genes with products involved in transcription, RNA processing, and transcriptional regulation was more likely to be conserved, while expression of genes encoding proteins involved in intercellular communication was more likely to have diverged during evolution. Conservation of expression correlated positively with the evolutionary age of genes, suggesting that divergence in expression levels of genes critical for cell function was restricted during evolution. Motif activity analysis showed that both promoters and enhancers are activated by the same transcription factors in different species. An analysis of expression levels of mature miRNAs and of primary miRNAs identified by CAGE revealed that evolutionary old miRNAs are more likely to have conserved expression patterns than young miRNAs. We conclude that key aspects of the regulatory network are conserved, while differential expression of genes involved in cell-to-cell communication may contribute greatly to phenotypic differences between species.
Purpose
Depending on its histological subtype, salivary gland carcinoma (SGC) may have a poor prognosis. Due to the scarcity of preclinical experimental models, its molecular biology has so far ...remained largely unknown, hampering the development of new treatment modalities for patients with these malignancies. The aim of this study was to generate experimental human SGC models of multiple histological subtypes using patient-derived xenograft (PDX) and organoid culture techniques.
Methods
Tumor specimens from surgically resected SGCs were processed for the preparation of PDXs and patient-derived organoids (PDOs). Specimens from SGC PDXs were also processed for PDX-derived organoid (PDXO) generation. In vivo tumorigenicity was assessed using orthotopic transplantation of SGC organoids. The pathological characteristics of each model were compared to those of the original tumors using immunohistochemistry. RNA-seq was used to analyze the genetic traits of our models.
Results
Three series of PDOs, PDXs and PDXOs of salivary duct carcinomas, one series of PDOs, PDXs and PDXOs of mucoepidermoid carcinomas and PDXs of myoepithelial carcinomas were successfully generated. We found that PDXs and orthotopic transplants from PDOs/PDXOs showed similar histological features as the original tumors. Our models also retained their genetic traits, i.e., transcription profiles, genomic variants and fusion genes of the corresponding histological subtypes.
Conclusion
We report the generation of SGC PDOs, PDXs and PDXOs of multiple histological subtypes, recapitulating the histological and genetical characteristics of the original tumors. These experimental SGC models may serve as a useful resource for the development of novel therapeutic strategies and for investigating the molecular mechanisms underlying the development of these malignancies.
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