Mining alternative splicing patterns in scRNA-seq data using scASfind.

Single-cell RNA-seq (scRNA-seq) is widely used for transcriptome profiling, but most analyses focus on gene-level events, with less attention devoted to alternative splicing. Here, we present scASfind, a novel computational method to allow for quantitative analysis of cell type-specific splicing events using full-length scRNA-seq data. ScASfind utilizes an efficient data structure to store the percent spliced-in value for each splicing event. This makes it possible to exhaustively search for patterns among all differential splicing events, allowing us to identify marker events, mutually exclusive events, and events involving large blocks of exons that are specific to one or more cell types.

Demuxafy: improvement in droplet assignment by integrating multiple single-cell demultiplexing and doublet detection methods.

Recent innovations in single-cell RNA-sequencing (scRNA-seq) provide the technology to investigate biological questions at cellular resolution. Pooling cells from multiple individuals has become a common strategy, and droplets can subsequently be assigned to a specific individual by leveraging their inherent genetic differences. An implicit challenge with scRNA-seq is the occurrence of doublets-droplets containing two or more cells. We develop Demuxafy, a framework to enhance donor assignment and doublet removal through the consensus intersection of multiple demultiplexing and doublet detecting methods. Demuxafy significantly improves droplet assignment by separating singlets from doublets and classifying the correct individual.

Integrative analysis of transcriptomic and epigenomic data reveals distinct patterns for developmental and housekeeping gene regulation.

BACKGROUND: Regulation of transcription is central to the emergence of new cell types during development, and it often involves activation of genes via proximal and distal regulatory regions. The activity of regulatory elements is determined by transcription factors (TFs) and epigenetic marks, but despite extensive mapping of such patterns, the extraction of regulatory principles remains challenging. RESULTS: Here we study differentially and similarly expressed genes along with their associated epigenomic profiles, chromatin accessibility and DNA methylation, during lineage specification at gastrulation in mice. Comparison of the three lineages allows us to identify genomic and epigenomic features that distinguish the two classes of genes. We show that differentially expressed genes are primarily regulated by distal elements, while similarly expressed genes are controlled by proximal housekeeping regulatory programs. Differentially expressed genes are relatively isolated within topologically associated domains, while similarly expressed genes tend to be located in gene clusters. Transcription of differentially expressed genes is associated with differentially open chromatin at distal elements including enhancers, while that of similarly expressed genes is associated with ubiquitously accessible chromatin at promoters. CONCLUSION: Based on these associations of (linearly) distal genes' transcription start sites (TSSs) and putative enhancers for developmental genes, our findings allow us to link putative enhancers to their target promoters and to infer lineage-specific repertoires of putative driver transcription factors, within which we define subgroups of pioneers and co-operators.

Yolk sac cell atlas reveals multiorgan functions during human early development.

The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.

Amelioration of non-alcoholic fatty liver disease by targeting adhesion G protein-coupled receptor F1 (Adgrf1).

Background: Recent research has shown that the adhesion G protein-coupled receptor F1 (Adgrf1; also known as GPR110; PGR19; KPG_012; hGPCR36) is an oncogene. The evidence is mainly based on high expression of Adgrf1 in numerous cancer types, and knockdown Adgrf1 can reduce the cell migration, invasion, and proliferation. Adgrf1 is, however, mostly expressed in the liver of healthy individuals. The function of Adgrf1 in liver has not been revealed. Interestingly, expression level of hepatic Adgrf1 is dramatically decreased in obese subjects. Here, the research examined whether Adgrf1 has a role in liver metabolism. Methods: We used recombinant adeno-associated virus-mediated gene delivery system, and antisense oligonucleotide was used to manipulate the hepatic Adgrf1 expression level in diet-induced obese mice to investigate the role of Adgrf1 in hepatic steatosis. The clinical relevance was examined using transcriptome profiling and archived biopsy specimens of liver tissues from non-alcoholic fatty liver disease (NAFLD) patients with different degree of fatty liver. Results: The expression of Adgrf1 in the liver was directly correlated to fat content in the livers of both obese mice and NAFLD patients. Stearoyl-coA desaturase 1 (Scd1), a crucial enzyme in hepatic de novo lipogenesis, was identified as a downstream target of Adgrf1 by RNA-sequencing analysis. Treatment with the liver-specific Scd1 inhibitor MK8245 and specific shRNAs against Scd1 in primary hepatocytes improved the hepatic steatosis of Adgrf1-overexpressing mice and lipid profile of hepatocytes, respectively. Conclusions: These results indicate Adgrf1 regulates hepatic lipid metabolism through controlling the expression of Scd1. Downregulation of Adgrf1 expression can potentially serve as a protective mechanism to stop the overaccumulation of fat in the liver in obese subjects. Overall, the above findings not only reveal a new mechanism regulating the progression of NAFLD, but also proposed a novel therapeutic approach to combat NAFLD by targeting Adgrf1. Funding: This work was supported by the National Natural Science Foundation of China (81870586), Area of Excellence (AoE/M-707/18), and General Research Fund (15101520) to CMW, and the National Natural Science Foundation of China (82270941, 81974117) to SJ.

Being overweight or obese increases the risk of developing numerous medical conditions including non-alcoholic fatty liver disease (NAFLD), where excess fat accumulates in the liver. NAFLD is a major global health issue affecting about 25% of the world's population and, if left untreated, can lead to liver inflammation as well as serious complications such as type 2 diabetes, heart disease, and liver cancer. Currently, there are no medications which specifically treat NFALD. Instead, only medications which help to manage the associated health complications are available. Therefore, a better understanding of NFALD is required to help to develop new strategies for diagnosing and treating the progression of this disease. A family of proteins known as GPCRs have crucial roles in regulating various bodily processes and are therefore commonly targeted for the treatment of disease. By identifying the GPCRs specifically involved in liver fat accumulation, new treatments for NFALD could be identified. Previous studies identified a GPCR known as Adgrf1 that is mainly found in liver cells, but its role remained unclear. To investigate the function of Adgrf1 in the liver, Wu et al. studied obese mice and human patients with NAFLD. The experiments showed that elevated levels of Adgrf1 in human and mouse livers led to increased fat accumulation. On the other hand, livers with lower levels of Adgrf1 exhibited reduced fat levels. A technique called RNA sequencing revealed that Adgrf1 induces expression of enzymes involved in fat synthesis, including a key regulator called Scd1. Treating mice with high levels of liver fat with molecules that inhibit Scd1 decreased the symptoms of Adgrf1-mediated fatty liver disease. These findings suggest therapies that decrease the levels of Adgrf1 may help to stop too much fat accumulating in the liver of human patients who are at risk of developing NAFLD. Further research is needed to confirm the effectiveness and safety of targeting Adgrf1 in humans and to develop suitable candidate drugs for the task.

Production of human entorhinal stellate cell-like cells by forward programming shows an important role of Foxp1 in reprogramming.

Stellate cells are principal neurons in the entorhinal cortex that contribute to spatial processing. They also play a role in the context of Alzheimer's disease as they accumulate Amyloid beta early in the disease. Producing human stellate cells from pluripotent stem cells would allow researchers to study early mechanisms of Alzheimer's disease, however, no protocols currently exist for producing such cells. In order to develop novel stem cell protocols, we characterize at high resolution the development of the porcine medial entorhinal cortex by tracing neuronal and glial subtypes from mid-gestation to the adult brain to identify the transcriptomic profile of progenitor and adult stellate cells. Importantly, we could confirm the robustness of our data by extracting developmental factors from the identified intermediate stellate cell cluster and implemented these factors to generate putative intermediate stellate cells from human induced pluripotent stem cells. Six transcription factors identified from the stellate cell cluster including RUNX1T1, SOX5, FOXP1, MEF2C, TCF4, EYA2 were overexpressed using a forward programming approach to produce neurons expressing a unique combination of RELN, SATB2, LEF1 and BCL11B observed in stellate cells. Further analyses of the individual transcription factors led to the discovery that FOXP1 is critical in the reprogramming process and omission of RUNX1T1 and EYA2 enhances neuron conversion. Our findings contribute not only to the profiling of cell types within the developing and adult brain's medial entorhinal cortex but also provides proof-of-concept for using scRNAseq data to produce entorhinal intermediate stellate cells from human pluripotent stem cells in-vitro.

Hepatic PRMT1 ameliorates diet-induced hepatic steatosis via induction of PGC1α.

Rationale: Over-nutrition will lead to overexpression of PRMT1 but protein hypomethylation is observed in the liver of obese subjects. The dynamic alteration of the expression and methyltransferase activity of PRMT1 in the progression of fatty liver diseases remains elusive. Methods: We used recombinant adeno-associated virus-mediated gene delivery system to manipulate the hepatic PRMT1 expression level in diet-induced obese mice to investigate the role of PRMT1 in hepatic steatosis. We further utilized a cohort of obese humans with biopsy-proven nonalcoholic fatty liver disease to support our observations in mouse model. Results: We demonstrated that knockdown of PRMT1 promoted steatosis development in liver of high-fat diet (HFD) fed mice. Over-expression of wild-type PRMT1, but not methyltransferase-defective mutant PRMT1G80R, could alleviate diet-induced hepatic steatosis. The observation is conserved in the specimens of obese humans with biopsy-proven nonalcoholic fatty liver disease. Mechanistically, methyltransferase activity of PRMT1 was required to induce PGC-1α mRNA expression via recruitment of HNF-4α to the promoter of PGC-1α, and hence attenuated HFD-induced hepatic steatosis by enhancing PGC-1α-mediated fatty acid oxidation. Conclusions: Our results identify that activation of the PRMT1/HNF-4α/PGC-1α signaling is a potential therapeutic strategy for combating non-alcoholic fatty liver disease of obese subjects.

A yeast-optimized single-cell transcriptomics platform elucidates how mycophenolic acid and guanine alter global mRNA levels.

Stochastic gene expression leads to inherent variability in expression outcomes even in isogenic single-celled organisms grown in the same environment. The Drop-Seq technology facilitates transcriptomic studies of individual mammalian cells, and it has had transformative effects on the characterization of cell identity and function based on single-cell transcript counts. However, application of this technology to organisms with different cell size and morphology characteristics has been challenging. Here we present yeastDrop-Seq, a yeast-optimized platform for quantifying the number of distinct mRNA molecules in a cell-specific manner in individual yeast cells. Using yeastDrop-Seq, we measured the transcriptomic impact of the lifespan-extending compound mycophenolic acid and its epistatic agent guanine. Each treatment condition had a distinct transcriptomic footprint on isogenic yeast cells as indicated by distinct clustering with clear separations among the different groups. The yeastDrop-Seq platform facilitates transcriptomic profiling of yeast cells for basic science and biotechnology applications.

Fast searches of large collections of single-cell data using scfind.

Single-cell technologies have made it possible to profile millions of cells, but for these resources to be useful they must be easy to query and access. To facilitate interactive and intuitive access to single-cell data we have developed scfind, a single-cell analysis tool that facilitates fast search of biologically or clinically relevant marker genes in cell atlases. Using transcriptome data from six mouse cell atlases, we show how scfind can be used to evaluate marker genes, perform in silico gating, and identify both cell-type-specific and housekeeping genes. Moreover, we have developed a subquery optimization routine to ensure that long and complex queries return meaningful results. To make scfind more user friendly, we use indices of PubMed abstracts and techniques from natural language processing to allow for arbitrary queries. Finally, we show how scfind can be used for multi-omics analyses by combining single-cell ATAC-seq data with transcriptome data.

The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity.

Type 2 cytokines are important signals triggering biogenesis of thermogenic beige adipocytes in white adipose tissue (WAT) during cold acclimation. However, how cold activates type 2 immunity in WAT remains obscure. Here we show that cold-induced type 2 immune responses and beiging in subcutaneous WAT (scWAT) are abrogated in mice with adipose-selective ablation of FGF21 or its co-receptor ß-Klotho, whereas such impairments are reversed by replenishment with chemokine CCL11. Mechanistically, FGF21 acts on adipocytes in an autocrine manner to promote the expression and secretion of CCL11 via activation of ERK1/2, which drives recruitment of eosinophils into scWAT, leading to increases in accumulation of M2 macrophages, and proliferation and commitment of adipocyte precursors into beige adipocytes. These FGF21-elicited type 2 immune responses and beiging are blocked by CCL11 neutralization. Thus, the adipose-derived FGF21-CCL11 axis triggers cold-induced beiging and thermogenesis by coupling sympathetic nervous system to activation of type 2 immunity in scWAT.

Adipose-derived lipocalin 14 alleviates hyperglycaemia by suppressing both adipocyte glycerol efflux and hepatic gluconeogenesis in mice.

AIMS/HYPOTHESIS: Growing evidence supports that dysregulation of adipose tissue-derived factors contributes to the pathogenesis of diabetes and its complications. Since our global gene profiling analysis has identified lipocalin-14 (LCN14)-a secretory protein with lipid-binding properties-as a potential adipokine highly expressed in white adipose tissue (WAT), this study aims to explore the metabolic roles of LCN14 in obese mice, and to investigate the functional mechanisms involved. METHODS: Immunoassays and western blotting were performed to determine the circulating level and tissue distribution of LCN14, respectively. Recombinant adeno-associated virus (rAAV)-mediated gene delivery was used to overexpress LCN14 in diet-induced obese (DIO) mice and the effects on glucose and lipid metabolism were examined. RESULTS: LCN14 is expressed predominantly in WAT. Both circulating levels of LCN14 and its expression in adipose tissues are repressed in DIO and genetically inherited diabetic (db/db) mice. Overexpression of LCN14 by rAAV-mediated gene delivery in DIO mice significantly increased insulin sensitivity in major metabolic tissues and ameliorated hyperglycaemia by inhibiting hepatic gluconeogenesis. The reduced hepatic glucose production is attributed to the suppressive effects of LCN14 on the expression of gluconeogenic genes and on glycerol efflux in adipocytes, possibly by reducing the expression of aquaporin-7. CONCLUSIONS/INTERPRETATION: Reduced LCN14 expression is involved in the pathogenesis of obesity-related metabolic dysregulation. LCN14 exerts its beneficial effects on glucose homeostasis and insulin sensitivity via its actions in both adipocytes and hepatocytes.

Coupling and coordination in gene expression processes with pre-mRNA splicing.

A processing is a tightly regulated and highly complex pathway which includes transcription, splicing, editing, transportation, translation and degradation. It has been well-documented that splicing of RNA polymerase II medicated nascent transcripts occurs co-transcriptionally and is functionally coupled to other RNA processing. Recently, increasing experimental evidence indicated that pre-mRNA splicing influences RNA degradation and vice versa. In this review, we summarized the recent findings demonstrating the coupling of these two processes. In addition, we highlighted the importance of splicing in the production of intronic miRNA and circular RNAs, and hence the discovery of the novel mechanisms in the regulation of gene expression.

Adropin is a brain membrane-bound protein regulating physical activity via the NB-3/Notch signaling pathway in mice.

Adropin is a highly conserved polypeptide that has been suggested to act as an endocrine factor that plays important roles in metabolic regulation, insulin sensitivity, and endothelial functions. However, in this study, we provide evidence demonstrating that adropin is a plasma membrane protein expressed abundantly in the brain. Using a yeast two-hybrid screening approach, we identified NB-3/Contactin 6, a brain-specific, non-canonical, membrane-tethered Notch1 ligand, as an interaction partner of adropin. Furthermore, this interaction promotes NB3-induced activation of Notch signaling and the expression of Notch target genes. We also generated and characterized adropin knockout mice to explore the role of adropin in vivo. Adropin knockout mice exhibited decreased locomotor activity and impaired motor coordination coupled with defective synapse formation, a phenotype similar to NB-3 knockout mice. Taken together, our data suggest that adropin is a membrane-bound protein that interacts with the brain-specific Notch1 ligand NB3. It regulates physical activity and motor coordination via the NB-3/Notch signaling pathway and plays an important role in cerebellum development in mice.