Biology subjects

Saito, Y.

Publications and source records attributed to Saito, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

RIF1 is a multifunctional protein that regulates DNA replication and repair. RIF1-deficient cells are hypersensitive to DNA replication stress. Of the two alternatively spliced RIF1 isoforms, called RIF1-Short and RIF1-Long, the RIF1-Long isoform is more capable than RIF1-Short in supporting cell recovery from replication stress. Examining replication stress resistance mechanisms specific to RIF1-Long, we find that prolonged replication stress unexpectedly induces interaction of RIF1-Long with BRCA1. Mechanistically, a phosphorylated SPKF motif unique to the RIF1-Long isoform binds the tandem BRCT domain of BRCA1. BRCA1-RIF1-Long interaction is strongly down-regulated through dephosphorylation by RIF1-associated Protein Phosphatase 1. BRCA1-RIF1-Long interaction requires ATR signaling, and occurs predominantly during S phase. Loss of RIF1-Long impairs the formation of RAD51 foci, and reduces the efficiency of homology-mediated repair at broken replication forks. In summary, our investigation establishes RIF1-Long as a new functional binding partner of the BRCA1-BRCT domain, crucial to protect cells from extended DNA replication stress by enabling RAD51-dependent repair of broken replication forks.

molecular biology↗

bioRxiv2025

Establishment of a second-generation transgenic marmoset model of polyglutamine disease recapitulating neurological symptoms and pathology

Neurodegenerative diseases, including polyglutamine diseases, remain a major clinical challenge, partly because of limited animal models that recapitulate human disease. Here, we describe a second-generation transgenic marmoset model of spinocerebellar ataxia 3 (SCA3), a polyglutamine disease, which stably expresses expanded CAG repeats in ATXN3. All five offspring of the founder marmoset harbored the transgene with reduced transgene integration sites and without repeat instability or genetic mosaicism, offering improved construct validity. Three of the five marmosets developed progressive motor impairments that segregated into two distinct phenotypes: early onset with rapid progression and late onset with mild progression, accompanied by corresponding patterns in body weight gain and grip strength. Pathological analysis revealed cerebellar Purkinje cell loss, spinal cord neurodegeneration, and widespread intranuclear inclusions. The severity of motor phenotypes correlated with transgene expression levels in disease-relevant brain regions, including the cerebellum, spinal cord, and striatum. By overcoming the common translational limitations of rodent systems, our second-generation model offers a powerful platform for investigating disease mechanisms and testing potential therapeutic interventions. Our results advance the utility of transgenic marmosets as clinically relevant models of neurodegenerative diseases. Summary StatementSecond-generation transgenic marmoset models of spinocerebellar ataxia 3 replicated the progressive motor deficits and neuropathology of the founder marmoset, providing a powerful platform for studying disease mechanisms and developing therapies.

neuroscience↗

bioRxiv2025

Longitudinal analysis of antibody titers after primary and booster mRNA COVID-19 vaccination can identify individuals at risk for breakthrough infection

A key issue in the post-COVID-19 era is the ongoing administration of COVID-19 vaccines. Repeated vaccination is essential for preparing against currently circulating and newly emerging SARS-CoV-2 variants while enabling people to continue with daily life. Optimizing vaccination strategies is crucial to efficiently manage medical resources and establish an effective vaccination framework. Therefore, it is important to quantitatively understand vaccine-induced immunity dynamics and to be able to identify poor responders with lower sustained antibody titers as potential priorities for revaccination. We investigated longitudinal antibody titer data in a cohort of 2,526 people in Fukushima, Japan, from April 2021 to November 2022 for whom basic demographic and health information was available. Using mathematical modeling and machine learning, we stratified the time-course patterns of antibody titers after 2 primary doses and 1 booster dose of mRNA COVID-19 vaccines. We identified 3 notable populations, which we refer to as the durable, the vulnerable, and the rapid-decliner populations, approximately half of which remained in the same population after the booster dose. Notably, the rapid-decliner population experienced earlier infections than the others. Furthermore, when comparing IgG(S) titers, IgA(S) titers, and T-spot counts between participants who experienced breakthrough infections after booster vaccination and those who did not, we found that IgA(S) titers were significantly lower in breakthrough infected participants during the early stage after booster vaccination. Our computational approach is adaptable to various types of vaccinations. This flexibility can inform policy decisions on vaccine distribution to enhance immunity both in future pandemics and in the post-COVID-19 era.

immunology↗

bioRxiv2025

Eukaryotes' closest relatives are internally simple syntrophic archaea

Eukaryotes are theorized to have originated from an archaeal phylum Promethearchaeota (formerly Asgard archaea)1,2. The first cultured representatives revealed valuable insight3,4 but are distantly related to the first eukaryotic common ancestor (FECA), leaving many unknowns regarding this archaeons biology. Here, we report isolation of two strains belonging to the order proposed as FECAs closest relative, Hodarchaeales,5 as members of a pure co- and tri-culture with methanogenic partners. Both are obligately anaerobic, syntrophic, peptide-degrading, and mesophilic archaea that have simple internal cell structure and produce protrusions and vesicles, like Promethearchaeales3,4. All strains demonstrate behavior focused on cell construction rather than division, unlike typical prokaryotes6. Strain HC1 possesses genes associated with aerobic lifestyles but lacks complete pathways for aerobic respiration and co-cultures cannot grow under (micro)aerobic conditions, suggesting the genes support oxygen detoxification rather than respiration. Reflecting this, HC1 can survive and grow under microaerobic conditions only when aerobic organisms are present. Phylogenetic analyses indicate FECA may have possessed these genes and thus some aerotolerance. Physiological, genomic, and phylogenetic observations indicate FECA was a simple-celled anaerobic syntrophic peptide-degrading archaeon with a non-growth-centric lifestyle and potential adaptations towards an oxygenated planet--the archaea-eukaryote transition was steep in both cell structure and aerobiosis.

microbiology↗

bioRxiv2024

Fitness translocation: improving variant effect prediction with biologically-grounded data augmentation

Data scarcity limits the characterization of protein fitness landscapes and the development of accurate variant effect prediction models. To address this challenge, we introduce fitness translocation, a data augmentation strategy that generates synthetic variants for a target protein by leveraging variant fitness data previously measured in homologous proteins. Using embeddings from protein language models, the method computes the difference between each homolog variant and its wild type and applies these offsets to the target wild-type embedding to create synthetic variants in embedding space. We illustrate the utility of fitness translocation in the context of variant effect prediction on three protein families: IGPS, GFP, and SARS-CoV-2 spike proteins, across different models and training data sizes. Fitness translocation consistently improves predictive performance, particularly under limited training data, and is effective even when augmenting with remote homologs sharing as little as 35% sequence identity. These results illustrate how biologically grounded data augmentation can expand and diversify protein fitness landscapes, supporting more data-efficient protein engineering. The code is available at https://github.com/adrienmialland/ProtFitTrans.

bioinformatics↗

bioRxiv2024

Key taxa of the gut microbiome associated with the relationship between environmental sensitivity and inflammation-related biomarkers

BackgroundIndividual differences in environmental sensitivity are linked to stress-related psychiatric symptoms. In previous research, we found that high environmental sensitivity can be a risk factor for increased inflammation and gut permeability, particularly when gut microbiome diversity is low. However, the specific gut bacterial taxa involved in this interaction remain unclear. This study aimed to identify the key gut microbiome taxa associated with this relationship. MethodsEnvironmental sensitivity, gut microbiome composition, gut permeability (lipopolysaccharide-binding protein, LBP) and inflammation (C-reactive protein, CRP) biomarkers were evaluated in 88 participants. ResultsThe interaction between environmental sensitivity and the relative abundance of the family Marinifilaceae (genus Butyricimonas) was a predictor of CRP levels. Similarly, the interaction between environmental sensitivity and relative abundance of the family Barnesiellaceae (genus Coprobacter), the family Akkermansiace (genus Akkermansia), the genus Family XIII AD3011 group, the genus GCA-900066225, or the genus Ruminiclostridium 1 predicted LBP levels. Individuals with high environmental sensitivity exhibited elevated CRP or LBP levels when the relative abundance of these taxa was low. Conversely, highly sensitive individuals had lower CRP or LBP levels when the relative abundance of these taxa was high. ConclusionThis study suggests that specific taxa serve as one of the protective factors against inflammation and gut permeability in individuals with high environmental sensitivity.

microbiology↗

bioRxiv2024

Beyond dichotomy: diversity of rat's ultrasonic vocalizations

Rats emit ultrasonic vocalizations (USVs) in diverse contexts, and these USVs are thought to serve many biological functions, such as socio-coordinating and alarming functions. In particular, rat USVs have been established as a good model for studying emotional expressions. Since the discovery of USVs in rats, it has gradually been revealed that two families of juvenile and adult rat USVs with distinct acoustic features are closely linked to different affective states: 50-kHz (high-short) calls are associated with positive affective states, whereas 22-kHz (low-long) calls are associated with negative affective states. Although various subtypes within high-short and low-long calls have been proposed and used, most studies adopt the dichotomous framework of first dividing USVs into high-short or low-long calls. The diversity of rat USVs may have been overlooked due to this high-short / low-long dichotomous framework, as such labeling could discourage reporting of actual measured acoustic properties. Considering that several recent studies claim to have found new USV categories outside this framework, we conducted a systematic survey of descriptions of rat USVs in the literature. We identified 15 articles reporting USVs that are outside the dichotomous framework. Our results support the existence of diverse USVs beyond the dichotomy, highlighting the importance of research on a broader range of vocalizations that might reflect complex affective states beyond the simple distinction between positive and negative.

animal behavior and cognition↗

bioRxiv2024

Estimating and correcting index hopping misassignments in single-cell RNA-seq data

BackgroundIndex hopping causes read assignment errors in data from multiplexed sequencing libraries. This issue has become more prevalent with the widespread use of high-capacity sequencers and highly multiplexed single-cell RNA sequencing (scRNA- seq). ResultsWe conducted deep, plate-based scRNA-seq on a mixed population of mouse skin cells. Analysis of transcriptomes from 1152 cells identified four distinct cell types. To estimate the error rate in sample assignment due to index hopping, we employed differential expression analysis to identify signature genes that were highly and specifically expressed in each cell type. We quantified the proportion of misassigned reads by examining the detection rates of signature genes in other cell types. Remarkably, regardless of gene expression levels, we estimated that 0.65% of reads per gene were assigned to incorrect cell across our data. To computationally compensate for index hopping, we developed a simple correction method wherein, for each gene, 0.65% of the librarys average expression level was subtracted from the expression in each cell. This correction had notable effects on transcriptome analyses, including increased cell-cell clustering distance and alterations in intermediate state assignments of cell differentiation. ConclusionsIndex hopping misassignments are measurable and can impact the experimental interpretation of sequencing results. We devised a straightforward method to estimate and correct for the index hopping rate by quantifying misassigned genes in distinct cell types within an scRNA-seq library. This approach can be applied to any barcoded, multiplexed scRNA-seq library containing cells with distinct expression profiles, allowing for correction of the expression matrix before conducting biological analysis.

genomics↗

bioRxiv2024

Postnatal xanthine metabolism regulates cardiac regeneration in mammals

Postnatal cardiomyocyte cell cycle withdrawal is a critical step wherein the mammalian heart loses regenerative potential after birth. Here, we conducted interspecies multi-omic comparisons between the mouse heart and that of the opossum, which have different postnatal time-windows for cardiomyocyte cell cycle withdrawal. Xanthine metabolism was activated in both postnatal hearts in parallel with cardiomyocyte cell cycle arrest. The pentose phosphate pathway (PPP) which produces NADPH was found to decrease simultaneously. Postnatal myocardial tissues became oxidized accordingly, and administration of antioxidants to neonatal mice altered the PPP and suppressed the postnatal activation of cardiac xanthine metabolism. These results suggest a redox-dependent postnatal switch from purine synthesis to degradation in the heart. Inhibition of xanthine metabolism in the postnatal heart extended postnatal duration of cardiomyocyte proliferation and maintained postnatal heart regeneration potential in mice. These findings highlight a novel role of xanthine metabolism as a metabolic regulator of cardiac regeneration potential.

developmental biology↗

bioRxiv2024

Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C

Neurodegenerative diseases are characterised by the abnormal filamentous assembly of specific proteins in the central nervous system1. Human genetic studies established a causal role for protein assembly in neurodegeneration2. However, the underlying molecular mechanisms remain largely unknown, which is limiting progress in developing clinical tools for these diseases. Recent advances in electron cryo-microscopy (cryo-EM) have enabled the structures of the protein filaments to be determined from patient brains1. All diseases studied to date have been characterised by the self-assembly of a single intracellular protein in homomeric amyloid filaments, including that of TAR DNA-binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) Types A and B3,4. Here, we used cryo-EM to determine filament structures from the brains of individuals with FTLD-TDP Type C, one of the most common forms of sporadic FTLD-TDP. Unexpectedly, the structures revealed that a second protein, annexin A11 (ANXA11), co-assembles with TDP-43 in heteromeric amyloid filaments. The ordered filament fold is formed by TDP-43 residues G282/284-N345 and ANXA11 residues L39-L74 from their respective low-complexity domains (LCDs). Regions of TDP-43 and ANXA11 previously implicated in protein-protein interactions form an extensive hydrophobic interface at the centre of the filament fold. Immunoblots of the filaments revealed that the majority of ANXA11 exists as a [~]22 kDa N-terminal fragment (NTF) lacking the annexin core domain. Immunohistochemistry of brain sections confirmed the co-localisation of ANXA11 and TDP-43 in inclusions, redefining the histopathology of FTLD-TDP Type C. This work establishes a central role for ANXA11 in FTLD-TDP Type C. The unprecedented formation of heteromeric amyloid filaments in human brain revises our understanding of amyloid assembly and may be of significance for the pathogenesis of neurodegenerative diseases.

neuroscience↗

bioRxiv2024

PRDX6 dictates ferroptosis sensitivity by directing cellular selenium mobilization

Selenium-dependent glutathione peroxidase 4 (GPX4) is the guardian of ferroptosis and prevents unrestrained (phospho)lipid peroxidation by directly reducing phospholipid hydroperoxides (PLOOH) to their corresponding alcohols. However, it remains unclear whether other phospholipid peroxidases can also contribute to ferroptosis prevention, albeit to a varying degree. Here we show that cells lacking GPX4 still exhibit substantial PLOOH reduction capacity, arguing for the presence of alternative PLOOH peroxidases. By scrutinizing potential candidates, we showed that while overexpression of peroxiredoxin 6 (PRDX6), a thiol-specific antioxidant enzyme with reported PLOOH-reducing activity, failed to prevent ferroptosis, its genetic loss markedly sensitizes cancer cells to ferroptosis. Mechanistically, we uncover that PRDX6 facilitates intracellular selenium handling, which is crucial for selenium incorporation into selenoproteins, including GPX4. Consequently, PRDX6 modulates GPX4 expression, thereby dictating the sensitivity of cells to undergo ferroptosis. Our study highlights PRDX6 as a critical factor in ferroptosis prevention by directing cellular selenium mobilization.

cell biology↗

bioRxiv2024

Selective agonism of GPR34 stimulates microglial uptake and clearance of amyloid β fibrils

Microglia, the primary immune cells of the central nervous system, play a crucial role in maintaining brain homeostasis through phagocytosis of various substrates, including amyloid-{beta} (A{beta}) fibrils, a hallmark of Alzheimer disease (AD) pathology. However, the molecular mechanisms regulating microglial A{beta} uptake remain poorly understood. Here, we identified GPR34, a Gi/o-coupled receptor highly expressed in microglia, as a novel regulator of fibrillar A{beta} phagocytosis. Treatment with a selective GPR34 agonist, M1, specifically enhanced uptake of A{beta} fibrils, but not its monomer or oligomer, in both mouse and human microglia. Mechanistically, M1 reduced intracellular cAMP levels, which inversely correlated with A{beta} uptake activity. Importantly, a single intrahippocampal injection of M1 in an AD mouse model significantly increased microglial A{beta} uptake in vivo. Furthermore, single-nucleus RNA-sequencing analysis of Japanese AD patient samples revealed a significant reduction of GPR34 expression in microglia from AD patients compared to controls. We also observed an age-dependent decline in microglial GPR34 expression in both human and mouse datasets, suggesting a potential contribution of GPR34 downregulation to age-related A{beta} accumulation and AD risk. Collectively, our findings identify GPR34 as a promising target for modulating microglial A{beta} clearance and highlight the therapeutic potential of GPR34 agonists in AD. Significance statementAlzheimer disease (AD) is characterized by amyloid-{beta} (A{beta}) accumulation in the brain. Microglia, the brains immune cells, play a crucial role in the metabolism of A{beta}. We discovered that activating the microglial receptor GPR34 with a selective agonist enhances the phagocytosis of A{beta} fibrils, a key pathogenic form of A{beta}. Importantly, GPR34 expression decreases with aging and AD progression, potentially contributing to impaired A{beta} clearance. Our findings highlight GPR34 as a promising therapeutic target for AD, as boosting its activity could promote A{beta} clearance and slow disease progression. This study provides valuable insights into microglial function in AD and offers a novel strategy for developing disease-modifying therapies.

neuroscience↗

bioRxiv2024

Fetal Kidney Transplantation for In Utero Fetuses

Potter sequence, characterized by bilateral renal agenesis, oligohydramnios, and consequent pulmonary hypoplasia, presents a significant challenge in the management of affected neonates. Due to their prematurity and associated abdominal complications, these infants often fail to reach a stage where dialysis can be safely initiated and sustained, leading to an exceedingly high mortality rate. Therefore, there is hopeful anticipation that interventions serving as a bridge to achieve a state where dialysis can be safely performed will markedly improve life expectancy. We have developed a unique approach of "transplantation of fetal kidneys from a different species during the fetal period" as a bridge therapy until stable dialysis therapy can be implemented. This is a new concept of fetal therapy, targeting the fetus in utero and utilizing fetal kidneys of an appropriate size for transplantation. In this study, we first validated the approach using allogeneic transplantation. Fetal kidneys with bladders from GFP-expressing rats (gestational age 14.0-16.5 days) were transplanted subcutaneously into allogeneic rat fetuses in utero (gestational age 18.0-18.5 days) using a special needle transuterinally, and live pups were successfully obtained. The transplanted fetal kidneys with bladders were confirmed to have urine production capability. By periodic aspiration of the subcutaneous urinary cyst after birth, urine produced by the transplanted fetal kidney was successfully drained outside the body for an extended period (up to 150 days). Biochemical tests confirmed the solute removal capacity of the transplanted fetal kidney. Furthermore, despite allogeneic transplantation, long-term urine production was sustained without the use of immunosuppressants, confirming that organ transplantation into fetuses is associated with lower rejection compared to adult transplantation. Next, xenotransplantation was performed. When GFP-expressing mouse fetal kidneys (gestational age 13.0-13.5 days) were transplanted into rat fetuses in utero, maturation of renal tissue structures was confirmed even in the interspecies setting.

bioengineering↗

bioRxiv2024

Human iPSC-derived Committed Cardiac Progenitors Generate Cardiac Tissue Grafts in a Swine Ischemic Cardiomyopathy Model without Triggering Ventricular Arrhythmias

BackgroundIntramyocardial injection of human pluripotent stem cell-derived cardiomyocytes following a myocardial infarction (MI) improves cardiac function in large animal models, but associated ventricular arrhythmias are major safety concern. We hypothesized that transendocardial injection of human induced pluripotent stem cell (hiPSC)-derived committed cardiac progenitor cells (CCPs), combined with cardiac fibroblast-derived extracellular matrix (cECM) to enhance cell retention, will generate cardiac tissue grafts improving contractility without triggering ventricular arrhythmias. MethodshiPSCs were differentiated using bioreactors and small molecules to produce committed cardiac progenitor cells (CCPs). MI was created using a coronary artery balloon occlusion and reperfusion model in Yucatan mini pigs. Four weeks later, epicardial needle injections of CCPs+cECM were performed in a small initial feasibility cohort (n=6), and then transendocardial injections of CCPs+cECM (n=14), CCPs alone (n=14), cECM alone (n=4) or vehicle control (n=13) into the peri-infarct region in a randomized cohort. Arrhythmias were evaluated using implanted event recorders. Magnetic resonance imaging (MRI) and invasive pressure-volume assessment were used to evaluate left ventricular anatomic and functional performance. Detailed histology was performed to detect and characterize human grafts. ResultsA scalable biomanufacturing protocol was developed generating CCPs which can efficiently differentiate into cardiomyocytes or endothelial cells in vitro. Intramyocardial delivery of CCPs to post-MI porcine hearts resulted in engraftment and differentiation of CCPs to form ventricular cardiomyocyte rich grafts. There was no significant difference in cardiac MRI-based measured cardiac volumes or function between control, CCP and CCP+cECM groups; however, pressure-volume analysis showed an improvement in dobutamine-stimulated functional reserve in CCP and CCP+cECM groups. Delivery of CCPs did not result in tumors or ventricular arrhythmias. ConclusionsTransendocardial delivery of CCPs with or without cECM into post-MI porcine hearts resulted in comparable human cardiomyocyte grafts which did not improve resting LV function but did improve stress-induced contractile reserve without triggering ventricular arrhythmias.

pathology↗

bioRxiv2023

Role of Immature Choroid Plexus in the Pathology of Autism Spectrum Disorder

During gestation, the choroid plexus (ChP) produces protein-rich cerebrospinal fluid and matures prior to brain development. It is assumed that ChP dysfunction has a profound effect on developmental neuropsychiatric disorders, such as Autism Spectrum Disorder (ASD). However, the mechanisms linking immature ChP to the onset of ASD remain unclear. In this study, we found that ChP-specific CAMDI-knockout mice developed an immature ChP, alongside decreased multiciliogenesis and expression of differentiation marker genes following disruption of the cerebrospinal fluid barrier. These mice exhibited ASD-like behaviors, including impaired socialization with delayed critical period. Additionally, administration of Metformin, an FDA-approved drug, before the critical period achieved ChP maturation and restored social behaviors. Furthermore, ASD model mice and ASD patient-derived organoids developed immature ChP. These results indicate towards involvement of immature ChP in the pathogenesis of ASD and suggest the targeting of functional maturation of ChP as a therapeutic strategy for ASD.

neuroscience↗

bioRxiv2023

Can Colpoda travel across oceans? Salinity tolerance of resting cysts may enable global dispersal of the species.

Protist species are distributed worldwide. The processes that enabled this global distribution are unclear. One possible means is through the oceanic dispersal of freshwater protists, although this has not been investigated in detail to date. Here, the ability of resting cysts to tolerate saline conditions is examined as a possible mechanism that enables the oceanic dispersal of protists. Resting cysts of Colpoda cucullus, a freshwater soil protist, can tolerate saline conditions of at least 3.5% NaCl for more than one week. A transcriptome analysis showed that the relative levels of expression of genes associated with membrane function are increased in resting cysts, indicating that salinity tolerance is associated with reconstruction of the cell membrane. Additionally, the outer layer of the cyst wall, a shell-like ectocyst, includes chitin. This may function as a form of "biological armor" that protects the cell from physical stress during oceanic dispersal. Teaser: Fresh water protists may be dispersed across oceans by formation of resting cysts that can tolerate saline conditions.

zoology↗

bioRxiv2023

Endocytic vesicles act as vehicles for glucose uptake in response to growth factor stimulation

Glycolysis is a fundamental cellular process, yet its regulatory mechanisms remain incompletely understood. Here, we show that a subset of glucose transporter 1 (GLUT1/SLC2A1) co-endocytoses with platelet-derived growth factor (PDGF) receptor (PDGFR) upon PDGF-stimulation. Furthermore, multiple glycolytic enzymes localize to these endocytosed PDGFR/GLUT1-containing vesicles adjacent to mitochondria. Contrary to current models, which emphasize the importance of glucose transporters on the cell surface, we find that PDGF-stimulated glucose uptake depends on receptor/transporter endocytosis. Our results suggest that growth factors generate glucose-loaded endocytic vesicles that deliver glucose to the glycolytic machinery in proximity to mitochondria, and argue for a new layer of regulation for glycolytic control governed by cellular membrane dynamics.

cell biology↗

bioRxiv2023

Unidirectional expression of enhancers with cell type-dependent direction of transcription

Enhancers are genomic regulatory elements that can affect expression of genes over megabases of genomic distances. As previous evidence suggested that transcription initiation at enhancers occurs in a bidirectional pattern, balanced expression of divergently transcribed capped RNAs has been used as a hallmark for enhancer detection from transcriptome data. By analyzing deep Cap Analysis Gene Expression (CAGE) data from FANTOM5, FANTOM6, and other studies, we show that enhancers are usually unidirectionally transcribed in a given cell type. However, since the preferred direction of transcription of an enhancer may switch between cell types, enhancers may appear to be bidirectionally expressed when using CAGE data aggregated over many cell types. By analyzing expression directionality of enhancers across cell types, we classified enhancers into bidirectionally expressed, unidirectionally expressed with a consistent direction of transcription, and unidirectionally expressed with a switching direction, which was the largest category. We conclude that requiring bidirectional expression during enhancer prediction from transcriptome data may lead to false negatives. Also, the preference of a given cell type for a specific direction of transcription at an enhancer suggests that enhancer RNAs are not transcriptional noise, but may be functional.

genomics↗