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.

ZENBU-Reports: a graphical web-portal builder for interactive visualization and dissemination of genome-scale data.

In the genomic era, data dissemination and visualization is an integral part of scientific publications and research projects involving international consortia producing massive genome-wide data sets, intra-organizational collaborations, or individual labs. However, creating custom supporting websites is oftentimes impractical due to the required programming effort, web server infrastructure, and data storage facilities, as well as the long-term maintenance burden. ZENBU-Reports (https://fantom.gsc.riken.jp/zenbu/reports) is a web application to create interactive scientific web portals by using graphical interfaces while providing storage and secured collaborative sharing for data uploaded by users. ZENBU-Reports provides the scientific visualization elements commonly used in supplementary websites, publications and presentations, presenting a complete solution for the interactive display and dissemination of data and analysis results during the full lifespan of a scientific project both during the active research phase and after publication of the results.

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.

Use of Acupuncture for Adult Health Conditions, 2013 to 2021: A Systematic Review.

Importance: Acupuncture is a popular treatment that has been advocated for dozens of adult health conditions and has a vast evidence base. Objective: To map the systematic reviews, conclusions, and certainty or quality of evidence for outcomes of acupuncture as a treatment for adult health conditions. Evidence Review: Computerized search of PubMed and 4 other databases from 2013 to 2021. Systematic reviews of acupuncture (whole body, auricular, or electroacupuncture) for adult health conditions that formally rated the certainty, quality, or strength of evidence for conclusions. Studies of acupressure, fire acupuncture, laser acupuncture, or traditional Chinese medicine without mention of acupuncture were excluded. Health condition, number of included studies, type of acupuncture, type of comparison group, conclusions, and certainty or quality of evidence. Reviews with at least 1 conclusion rated as high-certainty evidence, reviews with at least 1 conclusion rated as moderate-certainty evidence, and reviews with all conclusions rated as low- or very low-certainty evidence; full list of all conclusions and certainty of evidence. Findings: A total of 434 systematic reviews of acupuncture for adult health conditions were found; of these, 127 reviews used a formal method to rate certainty or quality of evidence of their conclusions, and 82 reviews were mapped, covering 56 health conditions. Across these, there were 4 conclusions that were rated as high-certainty evidence, and 31 conclusions that were rated as moderate-certainty evidence. All remaining conclusions (>60) were rated as low- or very low-certainty evidence. Approximately 10% of conclusions rated as high or moderate-certainty were that acupuncture was no better than the comparator treatment, and approximately 75% of high- or moderate-certainty evidence conclusions were about acupuncture compared with a sham or no treatment. Conclusions and Relevance: Despite a vast number of randomized trials, systematic reviews of acupuncture for adult health conditions have rated only a minority of conclusions as high- or moderate-certainty evidence, and most of these were about comparisons with sham treatment or had conclusions of no benefit of acupuncture. Conclusions with moderate or high-certainty evidence that acupuncture is superior to other active therapies were rare.

Recombination of repeat elements generates somatic complexity in human genomes.

Non-allelic recombination between homologous repetitive elements contributes to evolution and human genetic disorders. Here, we combine short- and long-DNA read sequencing of repeat elements with a new bioinformatics pipeline to show that somatic recombination of Alu and L1 elements is widespread in the human genome. Our analysis uncovers tissue-specific non-allelic homologous recombination hallmarks; moreover, we find that centromeres and cancer-associated genes are enriched for retroelements that may act as recombination hotspots. We compare recombination profiles in human-induced pluripotent stem cells and differentiated neurons and find that the neuron-specific recombination of repeat elements accompanies chromatin changes during cell-fate determination. Finally, we report that somatic recombination profiles are altered in Parkinson's and Alzheimer's disease, suggesting a link between retroelement recombination and genomic instability in neurodegeneration. This work highlights a significant contribution of the somatic recombination of repeat elements to genomic diversity in health and disease.

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.

Combined inhibition of XIAP and BCL2 drives maximal therapeutic efficacy in genetically diverse aggressive acute myeloid leukemia.

Aggressive therapy-resistant and refractory acute myeloid leukemia (AML) has an extremely poor outcome. By analyzing a large number of genetically complex and diverse, primary high-risk poor-outcome human AML samples, we identified specific pathways of therapeutic vulnerability. Through drug screens followed by extensive in vivo validation and genomic analyses, we found inhibition of cytosolic and mitochondrial anti-apoptotic proteins XIAP, BCL2 and MCL1, and a key regulator of mitosis, AURKB, as a vulnerability hub based on patient-specific genetic aberrations and transcriptional signatures. Combinatorial therapeutic inhibition of XIAP with an additional patient-specific vulnerability eliminated established AML in vivo in patient-derived xenografts (PDXs) bearing diverse genetic aberrations, with no signs of recurrence during off-treatment follow-up. By integrating genomic profiling and drug-sensitivity testing, this work provides a platform for a precision-medicine approach for treating aggressive AML with high unmet need.

Comparative transcriptomics of primary cells in vertebrates.

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.

Functional annotation of human long noncoding RNAs via molecular phenotyping.

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.

The Transcriptional Network That Controls Growth Arrest and Macrophage Differentiation in the Human Myeloid Leukemia Cell Line THP-1.

The response of the human acute myeloid leukemia cell line THP-1 to phorbol esters has been widely studied to test candidate leukemia therapies and as a model of cell cycle arrest and monocyte-macrophage differentiation. Here we have employed Cap Analysis of Gene Expression (CAGE) to analyze a dense time course of transcriptional regulation in THP-1 cells treated with phorbol myristate acetate (PMA) over 96 h. PMA treatment greatly reduced the numbers of cells entering S phase and also blocked cells exiting G2/M. The PMA-treated cells became adherent and expression of mature macrophage-specific genes increased progressively over the duration of the time course. Within 1-2 h PMA induced known targets of tumor protein p53 (TP53), notably CDKN1A, followed by gradual down-regulation of cell-cycle associated genes. Also within the first 2 h, PMA induced immediate early genes including transcription factor genes encoding proteins implicated in macrophage differentiation (EGR2, JUN, MAFB) and down-regulated genes for transcription factors involved in immature myeloid cell proliferation (MYB, IRF8, GFI1). The dense time course revealed that the response to PMA was not linear and progressive. Rather, network-based clustering of the time course data highlighted a sequential cascade of transient up- and down-regulated expression of genes encoding feedback regulators, as well as transcription factors associated with macrophage differentiation and their inferred target genes. CAGE also identified known and candidate novel enhancers expressed in THP-1 cells and many novel inducible genes that currently lack functional annotation and/or had no previously known function in macrophages. The time course is available on the ZENBU platform allowing comparison to FANTOM4 and FANTOM5 data.

What Is the Optimal Primary Care Panel Size?: A Systematic Review.

Background: Primary care for a panel of patients is a central component of population health, but the optimal panel size is unclear. Purpose: To review evidence about the association of primary care panel size with health care outcomes and provider burnout. Data Sources: English-language searches of multiple databases from inception to October 2019 and Google searches performed in September 2019. Study Selection: English-language studies of any design, including simulation models, that assessed the association between primary care panel size and safety, efficacy, patient-centeredness, timeliness, efficiency, equity, or provider burnout. Data Extraction: Independent, dual-reviewer extraction; group consensus rating of certainty of evidence. Data Synthesis: Sixteen hypothesis-testing studies and 12 simulation modeling studies met inclusion criteria. All but 1 hypothesis-testing study were cross-sectional assessments of association. Three studies each provided low-certainty evidence that increasing panel size was associated with no or modestly adverse effects on patient-centered and effective care. Eight studies provided low-certainty evidence that increasing panel size was associated with variable effects on timely care. No studies assessed the effect of panel size on safety, efficiency, or equity. One study provided very-low-certainty evidence of an association between increased panel size and provider burnout. The 12 simulation studies evaluated 5 models; all used access as the only outcome of care. Five and 2 studies, respectively, provided moderate-certainty evidence that adjusting panel size for case mix and adding clinical conditions to the case mix resulted in better access. Limitation: No studies had concurrent comparison groups, and published and unpublished studies may have been missed. Conclusion: Evidence is insufficient to make evidence-based recommendations about the optimal primary care panel size for achieving beneficial health outcomes. Primary Funding Source: Veterans Affairs Quality Enhancement Research Initiative.

Massage for Pain: An Evidence Map.

Objectives: Massage therapy has been proposed for painful conditions, but it can be difficult to understand the breadth and depth of evidence, as various painful conditions may respond differently to massage. The authors conducted an evidence mapping process and generated an "evidence map" to visually depict the distribution of evidence available for massage and various pain indications to identify gaps in evidence and to inform future research priorities. Design: The authors searched PubMed, Embase, and Cochrane for systematic reviews reporting pain outcomes for massage therapy. The authors assessed the quality of each review using the Assessing the Methodological Quality of Systematic Reviews (AMSTAR) criteria. The authors used a bubble plot to depict the number of included articles, pain indication, effect of massage for pain, and strength of findings for each included systematic review. Results: The authors identified 49 systematic reviews, of which 32 were considered high quality. Types of pain frequently included in systematic reviews were cancer pain, low back pain, and neck pain. High quality reviews concluded that there was low strength of evidence of potential benefits of massage for labor, shoulder, neck, low back, cancer, arthritis, postoperative, delayed onset muscle soreness, and musculoskeletal pain. Reported attributes of massage interventions include style of massage, provider, co-interventions, duration, and comparators, with 14 high-quality reviews reporting all these attributes in their review. Conclusion: Prior reviews have conclusions of low strength of evidence because few primary studies of large samples with rigorous methods had been conducted, leaving evidence gaps about specific massage type for specific pain. Primary studies often do not provide adequate details of massage therapy provided, limiting the extent to which reviews are able to draw conclusions about characteristics such as provider type.

C1 CAGE detects transcription start sites and enhancer activity at single-cell resolution.

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-ß 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.

Update of the FANTOM web resource: expansion to provide additional transcriptome atlases.

The FANTOM web resource (http://fantom.gsc.riken.jp/) was developed to provide easy access to the data produced by the FANTOM project. It contains the most complete and comprehensive sets of actively transcribed enhancers and promoters in the human and mouse genomes. We determined the transcription activities of these regulatory elements by CAGE (Cap Analysis of Gene Expression) for both steady and dynamic cellular states in all major and some rare cell types, consecutive stages of differentiation and responses to stimuli. We have expanded the resource by employing different assays, such as RNA-seq, short RNA-seq and a paired-end protocol for CAGE (CAGEscan), to provide new angles to study the transcriptome. That yielded additional atlases of long noncoding RNAs, miRNAs and their promoters. We have also expanded the CAGE analysis to cover rat, dog, chicken, and macaque species for a limited number of cell types. The CAGE data obtained from human and mouse were reprocessed to make them available on the latest genome assemblies. Here, we report the recent updates of both data and interfaces in the FANTOM web resource.

Author Correction: Transcription start site profiling of 15 anatomical regions of the Macaca mulatta central nervous system.

The authors regret that Luba M. Pardo was omitted in error from the author list of the original version of this Data Descriptor. This omission has now been corrected in the HTML and PDF versions. The authors also regret that Anemieke Rozemuller was omitted in error from the Acknowledgements of the original version of this Data Descriptor. This omission has now been corrected in the HTML and PDF versions.

Monitoring transcription initiation activities in rat and dog.

The promoter landscape of several non-human model organisms is far from complete. As a part of FANTOM5 data collection, we generated 13 profiles of transcription initiation activities in dog and rat aortic smooth muscle cells, mesenchymal stem cells and hepatocytes by employing CAGE (Cap Analysis of Gene Expression) technology combined with single molecule sequencing. Our analyses show that the CAGE profiles recapitulate known transcription start sites (TSSs) consistently, in addition to uncover novel TSSs. Our dataset can be thus used with high confidence to support gene annotation in dog and rat species. We identified 28,497 and 23,147 CAGE peaks, or promoter regions, for rat and dog respectively, and associated them to known genes. This approach could be seen as a standard method for improvement of existing gene models, as well as discovery of novel genes. Given that the FANTOM5 data collection includes dog and rat matched cell types in human and mouse as well, this data would also be useful for cross-species studies.

Transcription start site profiling of 15 anatomical regions of the Macaca mulatta central nervous system.

Rhesus macaque was the second non-human primate whose genome has been fully sequenced and is one of the most used model organisms to study human biology and disease, thanks to the close evolutionary relationship between the two species. But compared to human, where several previously unknown RNAs have been uncovered, the macaque transcriptome is less studied. Publicly available RNA expression resources for macaque are limited, even for brain, which is highly relevant to study human cognitive abilities. In an effort to complement those resources, FANTOM5 profiled 15 distinct anatomical regions of the aged macaque central nervous system using Cap Analysis of Gene Expression, a high-resolution, annotation-independent technology that allows monitoring of transcription initiation events with high accuracy. We identified 25,869 CAGE peaks, representing bona fide promoters. For each peak we provide detailed annotation, expanding the landscape of 'known' macaque genes, and we show concrete examples on how to use the resulting data. We believe this data represents a useful resource to understand the central nervous system in macaque.

Linking FANTOM5 CAGE peaks to annotations with CAGEscan.

The FANTOM5 expression atlas is a quantitative measurement of the activity of nearly 200,000 promoter regions across nearly 2,000 different human primary cells, tissue types and cell lines. Generation of this atlas was made possible by the use of CAGE, an experimental approach to localise transcription start sites at single-nucleotide resolution by sequencing the 5' ends of capped RNAs after their conversion to cDNAs. While 50% of CAGE-defined promoter regions could be confidently associated to adjacent transcriptional units, nearly 100,000 promoter regions remained gene-orphan. To address this, we used the CAGEscan method, in which random-primed 5'-cDNAs are paired-end sequenced. Pairs starting in the same region are assembled in transcript models called CAGEscan clusters. Here, we present the production and quality control of CAGEscan libraries from 56 FANTOM5 RNA sources, which enhances the FANTOM5 expression atlas by providing experimental evidence associating core promoter regions with their cognate transcripts.

FANTOM5 CAGE profiles of human and mouse reprocessed for GRCh38 and GRCm38 genome assemblies.

The FANTOM5 consortium described the promoter-level expression atlas of human and mouse by using CAGE (Cap Analysis of Gene Expression) with single molecule sequencing. In the original publications, GRCh37/hg19 and NCBI37/mm9 assemblies were used as the reference genomes of human and mouse respectively; later, the Genome Reference Consortium released newer genome assemblies GRCh38/hg38 and GRCm38/mm10. To increase the utility of the atlas in forthcoming researches, we reprocessed the data to make them available on the recent genome assemblies. The data include observed frequencies of transcription starting sites (TSSs) based on the realignment of CAGE reads, and TSS peaks that are converted from those based on the previous reference. Annotations of the peak names were also updated based on the latest public databases. The reprocessed results enable us to examine frequencies of transcription initiations on the recent genome assemblies and to refer promoters with updated information across the genome assemblies consistently.