Annotation of nuclear lncRNAs based on chromatin interactions.

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. Some specific nuclear lncRNAs have been shown to be important regulatory components acting locally. As RNA-chromatin interaction and Hi-C chromatin conformation data showed that chromatin interactions of nuclear lncRNAs are determined by the local chromatin 3D conformation, we used Hi-C data to identify potential target genes of lncRNAs. RNA-protein interaction data suggested that nuclear lncRNAs act as scaffolds to recruit regulatory proteins to target promoters and enhancers. Nuclear lncRNAs may therefore play a role in directing regulatory factors to locations spatially close to the lncRNA gene. We provide the analysis results through an interactive visualization web portal at https://fantom.gsc.riken.jp/zenbu/reports/#F6_3D_lncRNA.

ROR2 expression predicts human induced pluripotent stem cell differentiation into neural stem/progenitor cells and GABAergic neurons.

Despite the development of various in vitro differentiation protocols for the efficient derivation of specific cell types, human induced pluripotent stem cell (hiPSC) lines have varing ability to differentiate into specific lineages. Therefore, surrogate markers for accurately predicting the differentiation propensity of hiPSC lines may facilitate cell-based therapeutic product development and manufacture. We attempted to identify marker genes that could predict the differentiation propensity of hiPSCs into neural stem/progenitor cells (NS/PCs). Using Spearman's rank correlation coefficients, we investigated genes in the undifferentiated state, the expression levels of which were significantly correlated with the neuronal differentiation propensity of several hiPSC lines. Among genes significantly correlated with NS/PC differentiation (P < 0.01), we identified ROR2 as a novel predictive marker. ROR2 expression in hiPSCs was negatively correlated with NS/PC differentiation tendency, regardless of the differentiation method, whereas its knockdown enhanced differentiation. ROR2 regulates NS/PC differentiation, suggesting that ROR2 is functionally essential for NS/PC differentiation. Selecting cell lines with relatively low ROR2 expression facilitated identification of hiPSCs that can differentiate into NS/PCs. Cells with ROR2 knockdown showed increased efficiency of differentiation into forebrain GABAergic neurons compared to controls. These findings suggest that ROR2 is a surrogate marker for selecting hiPSC lines appropriate for NS/PC and GABAergic neuronal differentiations.

Use of clinical variables for preoperative prediction of lymph node metastasis in endometrial cancer.

OBJECTIVE: Endometrial cancer is the most common gynaecological cancer, and most patients are identified during early disease stages. Noninvasive evaluation of lymph node metastasis likely will improve the quality of clinical treatment, for example, by omitting unnecessary lymphadenectomy. METHODS: The study population comprised 611 patients with endometrial cancer who underwent lymphadenectomy at four types of institutions, comprising seven hospitals in total. We systematically assessed the association of 18 preoperative clinical variables with postoperative lymph node metastasis. We then constructed statistical models for preoperative lymph node metastasis prediction and assessed their performance with a previously proposed system, in which the score was determined by counting the number of high-risk variables among the four predefined ones. RESULTS: Of the preoperative 18 variables evaluated, 10 were significantly associated with postoperative lymph node metastasis. A logistic regression model achieved an area under the curve of 0.85 in predicting lymph node metastasis; this value is significantly higher than that from the previous system (area under the curve, 0.74). When we set the false-negative rate to ~1%, the new predictive model increased the rate of true negatives to 21%, compared with 6.8% from the previous one. We also provide a spreadsheet-based tool for further evaluation of its ability to predict lymph node metastasis in endometrial cancer. CONCLUSIONS: Our new lymph node metastasis prediction method, which was based solely on preoperative clinical variables, performed significantly better than the previous method. Although additional evaluation is necessary for its clinical use, our noninvasive system may help improve the clinical treatment of endometrial cancer, complementing minimally invasive sentinel lymph node biopsy.

Single-Cell RNA-Seq Reveals LRRC75A-Expressing Cell Population Involved in VEGF Secretion of Multipotent Mesenchymal Stromal/Stem Cells Under Ischemia.

Human multipotent mesenchymal stromal/stem cells (MSCs) have been utilized in cell therapy for various diseases and their clinical applications are expected to increase in the future. However, the variation in MSC-based product quality due to the MSC heterogeneity has resulted in significant constraints in the clinical utility of MSCs. Therefore, we hypothesized that it might be important to identify and ensure/enrich suitable cell subpopulations for therapies using MSC-based products. In this study, we aimed to identify functional cell subpopulations to predict the efficacy of angiogenic therapy using bone marrow-derived MSCs (BM-MSCs). To assess its angiogenic potency, we observed various levels of vascular endothelial growth factor (VEGF) secretion among 11 donor-derived BM-MSC lines under in vitro ischemic culture conditions. Next, by clarifying the heterogeneity of BM-MSCs using single-cell RNA-sequencing analysis, we identified a functional cell subpopulation that contributed to the overall VEGF production in BM-MSC lines under ischemic conditions. We also found that leucine-rich repeat-containing 75A (LRRC75A) was more highly expressed in this cell subpopulation than in the others. Importantly, knockdown of LRRC75A using small interfering RNA resulted in significant inhibition of VEGF secretion in ischemic BM-MSCs, indicating that LRRC75A regulates VEGF secretion under ischemic conditions. Therefore, LRRC75A may be a useful biomarker to identify cell subpopulations that contribute to the angiogenic effects of BM-MSCs. Our work provides evidence that a strategy based on single-cell transcriptome profiles is effective for identifying functional cell subpopulations in heterogeneous MSC-based products.

CALML5 is a novel diagnostic marker for differentiating thymic squamous cell carcinoma from type B3 thymoma.

BACKGROUND: Thymic squamous cell carcinoma and type B3 thymoma are primary neoplasms of the anterior mediastinum that are sometimes difficult to differentiate from one another histologically. However, only a few immunohistochemical markers are available for the differential diagnosis. The purpose of this study was to discover a novel marker for differentiating between thymic squamous cell carcinoma and type B3 thymoma. METHODS: We used histological samples of thymic carcinomas (n = 26) and type B3 thymomas (n = 38) which were resected between 1986 and 2017. To search for candidates of differential markers, gene expression levels were evaluated in samples using promoter analysis by cap analysis of gene expression (CAGE) sequencing. RESULTS: Promoter level expression of CALML5 genes was significantly higher in thymic carcinomas than in type B3 thymomas. We further validated the results of the CAGE analysis in all 26 thymic carcinomas and 38 type B3 thymomas by immunohistochemistry (IHC). CALML5 was strongly expressed in the cytoplasm in 19 of 26 cases with thymic carcinoma, whereas positivity at the protein level was shown in two of 38 type B3 thymomas. Thus, the sensitivity (73.1%) and specificity (94.7%) of CALML5 as markers for immunohistochemical diagnosis of thymic carcinoma were extremely high. CONCLUSION: We identified CALML5 as a potential marker for differentiating thymic squamous cell carcinoma from type B3 thymoma. It is assumed that future clinical use of CALML5 may improve the diagnostic accuracy of differentiating between these two diseases.

Antisense-oligonucleotide-mediated perturbation of long non-coding RNA reveals functional features in stem cells and across cell types.

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.

Deep sequencing of short capped RNAs reveals novel families of noncoding RNAs.

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.

High quality genome assembly of the anhydrobiotic midge provides insights on a single chromosome-based emergence of extreme desiccation tolerance.

Non-biting midges (Chironomidae) are known to inhabit a wide range of environments, and certain species can tolerate extreme conditions, where the rest of insects cannot survive. In particular, the sleeping chironomid Polypedilum vanderplanki is known for the remarkable ability of its larvae to withstand almost complete desiccation by entering a state called anhydrobiosis. Chromosome numbers in chironomids are higher than in other dipterans and this extra genomic resource might facilitate rapid adaptation to novel environments. We used improved sequencing strategies to assemble a chromosome-level genome sequence for P. vanderplanki for deep comparative analysis of genomic location of genes associated with desiccation tolerance. Using whole genome-based cross-species and intra-species analysis, we provide evidence for the unique functional specialization of Chromosome 4 through extensive acquisition of novel genes. In contrast to other insect genomes, in the sleeping chironomid a uniquely high degree of subfunctionalization in paralogous anhydrobiosis genes occurs in this chromosome, as well as pseudogenization in a highly duplicated gene family. Our findings suggest that the Chromosome 4 in Polypedilum is a site of high genetic turnover, allowing it to act as a 'sandbox' for evolutionary experiments, thus facilitating the rapid adaptation of midges to harsh environments.

The choice of negative control antisense oligonucleotides dramatically impacts downstream analysis depending on the cellular background.

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.

Low Quantity Single Strand CAGE (LQ-ssCAGE) Maps Regulatory Enhancers and Promoters.

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.

Discovery of widespread transcription initiation at microsatellites predictable by sequence-based deep neural network.

Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism.

Nanopore sequencing reveals TACC2 locus complexity and diversity of isoforms transcribed from an intronic promoter.

Gene expression is controlled at the transcriptional and post-transcriptional levels. The TACC2 gene was known to be associated with tumors but the control of its expression is unclear. We have reported that activity of the intronic promoter p10 of TACC2 in primary lesion of endometrial cancer is indicative of lymph node metastasis among a low-risk patient group. Here, we analyze the intronic promoter derived isoforms in JHUEM-1 endometrial cancer cells, and primary tissues of endometrial cancers and normal endometrium. Full-length cDNA amplicons are produced by long-range PCR and subjected to nanopore sequencing followed by computational error correction. We identify 16 stable, 4 variable, and 9 rare exons including 3 novel exons validated independently. All variable and rare exons reside N-terminally of the TACC domain and contribute to isoform variety. We found 240 isoforms as high-confidence, supported by more than 20 reads. The large number of isoforms produced from one minor promoter indicates the post-transcriptional complexity coupled with transcription at the TACC2 locus in cancer and normal cells.

Cancer-associated fibroblast migration in non-small cell lung cancers is modulated by increased integrin α11 expression.

Cancer-associated fibroblasts (CAFs) regulate cancer progression through the modulation of extracellular matrix (ECM) and cancer cell adhesion. While undergoing a series of phenotypic changes, CAFs control cancer-stroma interactions through integrin receptor signaling. Here, we isolated CAFs from patients with non-small-cell lung cancer (NSCLC) and examined their gene expression profiles. We identified collagen type XI α1 (COL11A1), integrin α11 (ITGA11), and the ITGA11 major ligand collagen type I α1 (COL1A1) among the 390 genes that were significantly enriched in NSCLC-associated CAFs. Increased ITGA11 expression in cancer stroma was correlated with a poor clinical outcome in patients with NSCLC. Increased expression of fibronectin and collagen type I induced ITGA11 expression in CAFs. The cellular migration of CAFs toward collagen type I and fibronectin was promoted via ERK1/2 signaling, independently of the fibronectin receptor integrin α5ß1. Additionally, ERK1/2 signaling induced ITGA11 and COL11A1 expression in cancer stroma. We, therefore, propose that targeting ITGA11 and COL11A1 expressing CAFs to block cancer-stroma interactions may serve as a novel, promising anti-tumor strategy.

Ex vivo conditioning of peripheral blood mononuclear cells of diabetic patients promotes vasculogenic wound healing.

The quality and quantity of endothelial progenitor cells (EPCs) are impaired in patients with diabetes mellitus patients, leading to reduced tissue repair during autologous EPC therapy. This study aimed to address the limitations of the previously described serum-free Quantity and Quality Control Culture System (QQc) using CD34+ cells by investigating the therapeutic potential of a novel mononuclear cell (MNC)-QQ. MNCs were isolated from 50 mL of peripheral blood of patients with diabetes mellitus and healthy volunteers (n = 13 each) and subjected to QQc for 7 days in serum-free expansion media with VEGF, Flt-3 ligand, TPO, IL-6, and SCF. The vascular regeneration capability of MNC-QQ cells pre- or post-QQc was evaluated with an EPC colony-forming assay, FACS, EPC culture, tube formation assay, and quantitative real time PCR. For in vivo assessment, 1 × 104 pre- and post-MNC-QQc cells from diabetic donors were injected into a murine wound-healing model using Balb/c nude mice. The percentage of wound closure and angio-vasculogenesis was then assessed. This study revealed vasculogenic, anti-inflammatory, and wound-healing effects of MNC-QQ therapy in both in vitro and in vivo models. This system addresses the low efficiency and efficacy of the current naïve MNC therapy for wound-healing in diabetic patients. As this technique requires a simple blood draw, isolation, and peripheral blood MNC suspension culture for only a week, it can be used as a simple and effective outpatient-based vascular and regenerative therapy for patients with diabetes mellitus.

CREB3L1 overexpression as a potential diagnostic marker of Philadelphia chromosome-negative myeloproliferative neoplasms.

Discrimination of Philadelphia-negative myeloproliferative neoplasms (Ph-MPNs) from reactive hypercytosis and myelofibrosis requires a constellation of testing including driver mutation analysis and bone marrow biopsies. We searched for a biomarker that can more easily distinguish Ph-MPNs from reactive hypercytosis and myelofibrosis by using RNA-seq analysis utilizing platelet-rich plasma (PRP)-derived RNAs from patients with essential thrombocythemia (ET) and reactive thrombocytosis, and CREB3L1 was found to have an extremely high impact in discriminating the two disorders. To validate and further explore the result, expression levels of CREB3L1 in PRP were quantified by reverse-transcription quantitative PCR and compared among patients with ET, other Ph-MPNs, chronic myeloid leukemia (CML), and reactive hypercytosis and myelofibrosis. A CREB3L1 expression cutoff value determined based on PRP of 18 healthy volunteers accurately discriminated 150 driver mutation-positive Ph-MPNs from other entities (71 reactive hypercytosis and myelofibrosis, 6 CML, and 18 healthy volunteers) and showed both sensitivity and specificity of 1.0000. Importantly, CREB3L1 expression levels were significantly higher in ET compared with reactive thrombocytosis (P < .0001), and polycythemia vera compared with reactive erythrocytosis (P < .0001). Pathology-affirmed triple-negative ET (TN-ET) patients were divided into a high- and low-CREB3L1-expression group, and some patients in the low-expression group achieved a spontaneous remission during the clinical course. In conclusion, CREB3L1 analysis has the potential to single-handedly discriminate driver mutation-positive Ph-MPNs from reactive hypercytosis and myelofibrosis, and also may identify a subgroup within TN-ET showing distinct clinical features including spontaneous remission.

Scanning single-molecule counting system for Eprobe with highly simple and effective approach.

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

Deep phenotyping of myalgic encephalomyelitis/chronic fatigue syndrome in Japanese population.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex and debilitating disease with no molecular diagnostics and no treatment options. To identify potential markers of this illness, we profiled 48 patients and 52 controls for standard laboratory tests, plasma metabolomics, blood immuno-phenotyping and transcriptomics, and fecal microbiome analysis. Here, we identified a set of 26 potential molecular markers that distinguished ME/CFS patients from healthy controls. Monocyte number, microbiome abundance, and lipoprotein profiles appeared to be the most informative markers. When we correlated these molecular changes to sleep and cognitive measurements of fatigue, we found that lipoprotein and microbiome profiles most closely correlated with sleep disruption while a different set of markers correlated with a cognitive parameter. Sleep, lipoprotein, and microbiome changes occur early during the course of illness suggesting that these markers can be examined in a larger cohort for potential biomarker application. Our study points to a cluster of sleep-related molecular changes as a prominent feature of ME/CFS in our Japanese cohort.

Development of a Tet-On Inducible Expression System for the Anhydrobiotic Cell Line, Pv11.

The Pv11 cell line established from an African chironomid, Polypedilum vanderplanki, is the only cell line tolerant to complete desiccation. In Pv11 cells, a constitutive expression system for Pv11 cells was previously exploited and several reporter genes were successfully expressed. Here we report the identification of an effective minimal promoter for Pv11 cells and its application to the Tet-On inducible expression system. First, using a luciferase reporter assay, we showed that a 202 bp deletion fragment derived from the constitutively active 121-promoter functions in Pv11 cells as an appropriate minimal promoter with the Tet-On inducible expression system. The AcGFP1 (Aequorea coerulescens green fluorescent protein) was also successfully expressed in Pv11 cells using the inducible system. In addition to these reporter genes, the avian myeloblastosis virus reverse transcriptase α subunit (AMV RTα), which is one of the most widely commercially available RNA-dependent DNA polymerases, was successfully expressed through the inducible expression system and its catalytic activity was verified. These results demonstrate the establishment of an inducible expression system in cells that can be preserved in the dry state and highlight a possible application to the production of large and complex proteins.