Biology subjects

Kasukawa, T.

Publications and source records attributed to Kasukawa, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Repeat element-anchored enhancer-promoter interactions (REEPIs) shape the regulatory landscape of hormone-resistant breast cancer

Repeat elements (REs) comprise over half of the human genome and are increasingly recognized as regulatory components, yet their role in therapy resistance remains unclear. Here, we integrate CAGE and RADICL-seq to map regulatory elements and RNA-chromatin interactions in estrogen receptor-positive breast cancer cells (MCF7) and their long-term estrogen-deprived (LTED) derivatives. We identify repeat element-anchored enhancer-promoter interactions (REEPIs), defined by the presence of annotated REs at both enhancer and promoter sites. In LTED cells, REEPIs are enriched for ERV1 and ERVL elements and involve specific repeat pairings associated with transcription factor combinations such as SP1-SMAD3 and SP1-NFYC. At the gene level, these REEPIs are preferentially linked to genes involved in chromatin remodeling, DNA repair, and lineage plasticity. Our findings reveal a repeat-guided regulatory architecture associated with transcriptional adaptation in endocrine resistance and identify REEPIs as a previously underexplored component of regulatory reprogramming in hormone-resistant breast cancer.

cancer biology↗

bioRxiv2026

Co-regulatory changes in Homo sapiens prefrontal cortex shape RNA-chromatin interactions

Noncoding RNAs (ncRNAs) regulate gene expression through RNA-chromatin interactions, yet their role in human brain evolution remains unclear. To map these interactions, we integrated FANTOM6 neurogenic data with a comprehensive atlas of regulatory regions active during early cortical development. This integration effort linked ncRNAs and transcription factors (TFs) to their genomic targets, revealing functional interactions captured by RNA-DNA contact mapping, yet largely missed by chromatin conformation data. Overlaying these interactions with nearly fixed variants in Homo sapiens relative to extinct hominins uncovered widespread rewiring of TF binding, affecting genes implicated in progenitor proliferation and neuronal differentiation. Gene regulatory network reconstruction identified TEAD2 and ONECUT2 as key regulators of intermediate progenitors and migrating neurons. Functional perturbation followed by Cut&Tag and ATAC-seq linked the two TFs to disease- and evolutionarily- relevant pathways. Together, these results show how ncRNA-TF cooperation and selective cis-regulatory rewiring contributed to sapiens-specific features of cortical development.

molecular biology↗

bioRxiv2026

Alternative promoters used during myeloid differentiation and upon activation change the gene products available for innate immune programs.

Macrophages are innate immune cells present in most tissues of the body, whose molecular programs are determined by their ontogeny and environment. From the earliest stages of embryonic development, macrophages are recruited into developing tissues where they support organogenesis with trophic factors such as WNT, VEGF and PDGF. While macrophage subsets have been described in different tissues at single cell resolution, little is known about transcript isoforms and proteoforms that underpin their differentiation and function. Here we assessed enhancer, promoter, transcript and proteomic variation as pluripotent stem cells differentiate to macrophages, identifying over 200 previously uncharacterised genes and over 20,000 new mRNA isoforms, updating our current understanding of the human genome, its regulation and potential output. Newly discovered myeloid-expressed transcripts and proteins were enriched for motifs associated with secreted proteins, and these included previously uncharacterised isoforms of growth factors, in which we predict N-terminal changes impact on their location and function. Activation of primary adult monocytes and monocyte-derived macrophages was also characterised by the expression of diverse transcript isoforms, largely arising from alternate transcription initiation sites and predicted to impact on the acute response to bacterial or fungal stimuli. Understanding the full spectrum of gene products expressed by these cells further extends our understanding of the phenotypic plasticity and trophic potential of macrophages in human development and may lead to the discovery of new clinical targets for tissue engineering or immune-related studies. Graphical AbstractIn this manuscript, Berrocal-Rubio and colleagues examined the differentiation of human macrophages using an iPSC model of tissue macrophage biology. Combining long-read sequencing technology with promoter profiling identified over 17, 700 genes implicated in pluripotency-myeloid specification. 7% of transcripts profiled from previously characterised genes were predicted to encode new proteins, and a further 3% of transcripts were derived from genes newly discovered in this project. They confirmed that a high proportion of these alternate transcripts were detectable in primary monocytes but also discovered that activation of primary monocytes led to further alternate promoter usage, with the potential to further diversify the innate immune responses to a broad set of pathogens. The newly described macrophage genes and transcripts encoded proteins enriched for motifs associated with secreted peptides. These data suggest that alternate transcription of macrophage genes leads to new effectors of innate immune function, that include a substantially expanded number of growth factors or secreted proteins. Created in BioRender. Berrocal, M. (2025) https://BioRender.com/d0n47le O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/703142v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@454cf0org.highwire.dtl.DTLVardef@1be28e8org.highwire.dtl.DTLVardef@16fb2e8org.highwire.dtl.DTLVardef@4aca30_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Extending cis-regulatory networks using chromatin-RNA interactions

Cis-regulatory networks are essential for determining gene transcriptional states, yet the mechanisms that mediate cellular signaling and enhancer-promoter communications are not yet well understood. Here, we integrate high-resolution enhancer-promoter interactions obtained using targeted chromosome conformation capture and RNA-DNA contacts from RADICL-seq to uncover the role of RNA in modulating enhancer-promoter interactions. Both datasets were generated from human induced pluripotent cells at different stages across neural differentiation. As expected, most enhancer-promoter interactions were dynamic with only 38% shared across all cell states and 68% of promoter interacting regions overlapped at least one annotated neural enhancer. Integration with RADICL-seq data revealed a 9.3-fold enrichment for cis-interacting DNA regions to associate with at least one RNA. Among 18,346 cis-interacting promoters, only 1,170 (6.3%) lacked any RNA association, whereas 3,702 (28%) showed stable RNA association and 13,579 (74%) displayed dynamic RNA association across differentiation. Promoters associated with RNA engaged with higher number of enhancers and exhibited higher expression levels, while enhancers with RNA associations showed higher levels of chromatin activation marks. Promoters with dynamic RNA association were also more likely to be differentially expressed and enriched for relevant biological processes, phenotypes and diseases. Importantly, RNA-DNA association dynamics correlated strongly with DNA-DNA interaction dynamics; gain of RNA association on enhancers was typically accompanied by enhancer-promoter interaction gain, whereas concurrent RNA association gain on both promoter and enhancers frequently led to interaction loss. The correlation of RNA association on cis-interaction dynamics across differentiation were also captured in the community structure of the DNA-RNA network. Together, our results reveal extensive coupling between RNA-DNA and DNA-DNA networks supporting a coordinating role of RNA in gene regulation.

genomics↗

bioRxiv2025

A genome-wide, machine learning-guided exploration of the cis-regulatory code involved in neuronal differentiation

Gene expression is controlled by proximal and distal cis-regulatory elements (CREs), containing DNA motifs bound by various transcription factors (TFs). Other sequence features, such as specific k-mers or low complexity regions, have also been implicated [1-3]. However, in a dynamic biological process such as cell differentiation, we lack an understanding of how the transcriptional activity of CREs progressively change and what sequence features underlie these transitions, which may reflect common and/or coordinated regulatory processes. Here, we use single-cell ATAC-seq and RNA-seq to follow, at a genome scale, CREs along differentiation of induced pluripotent stem cells into cortical neurons and develop a method to automatically identify the diversity of CRE profiles and their underlying sequence features. We propose a machine-learning guided clustering algorithm, STOIC (Statistical learning TO Inform Clustering), that jointly learns an unsupervised clustering of the CREs in the space of the activity profiles and a supervised predictor associated with each cluster in the DNA-sequence space. STOIC is specifically designed to provide readily interpretable results. We show that the method identifies CRE profiles associated with highly predictive sequence features and outperforms methods solely concerned with co-activity clustering on this task. Orthogonal data collected in the same settings link the inferred CRE clusters to specific enhancer or promoter signatures. Furthermore, we show that the DNA features unveiled by STOIC reflect biologically relevant regulators and offer a valuable basis to dissect elements of the cisregulatory grammar. Finally, we demonstrate the general applicability of STOIC by analyzing five bulk CAGE datasets of human cells responding to various treatments.

bioinformatics↗

bioRxiv2025

Cell type-specific intronic RNAs shape genome architecture during neuronal lineage specification

Cell differentiation towards neurons is accompanied by widespread changes in three-dimensional (3D) genome organization and gene expression. Chromatin-associated RNAs have been proposed to be important regulators of such changes; however, the type, abundance, and role of these RNAs during neuronal differentiation remain largely unexplored. Here, we integrate multi-omic data generated in the frame of the Functional ANnoTation Of the Mammalian genome (FANTOM6) to chart 3D genome, RNA-DNA contactome, and transcriptome changes occurring during in vitro differentiation of human induced pluripotent stem cells to neural stem cells and neurons. We reveal a previously unreported phenomenon, in which intronic RNAs engage in long-distance contacts with DNA loci distributed all over the genome. These trans-contacting intronic RNAs (TIRs) are produced from exceptionally long (mean length: 750 kilobases, kb) protein-coding genes that carry ultra-long introns and are selectively expressed in neurons. We show that TIRs do not undergo rapid co-transcriptional degradation but rather accumulate in the nucleus of neuronal cells, forming large dot clouds around their source loci and spreading across the nucleus, as visualized by single-molecule RNA fluorescence in situ hybridization. TIRs engage in contacts with a set of genomic regions (TIR-contacted regions or TIRCs) that carry much shorter (mean length: [~]30 kb) neuronally expressed genes forming high-connectivity hubs. We also show that the expression of genes within TIRCs contacted by the same set of TIRs is highly co-varied, and that TIR source genes, especially their introns, are enriched in genetic risk loci for neurodevelopmental and neuropsychiatric disorders. Our findings point to a functional role of the persistence of long intronic RNAs in the nucleus of neuronal cells and might contribute to explain why neurons uniquely express many ultra-long genes. We propose a model in which TIRs form pan-nuclear scaffolds--which we propose to name introsomes--that constitute a dynamically self-renewing regulatory layer, where transcription itself continuously regenerates the very scaffold that organizes genome function in neurons, and that might be involved in the pathogenesis of neurodevelopmental and neuropsychiatric disorders.

genomics↗

bioRxiv2025

Genome-wide mapping of the Galleria mellonella larvae transcription start sites during fungal infection and treatment

Using Low Quantity single strand CAGE (LQ-ssCAGE), we mapped the transcription start sites (TSS). We annotated the 5 end of the invertebrate Galleria mellonella, an upcoming and booming experimental model in infectious disease and immunology research. However, the current genome annotation of this model organism lacks annotation of the 5 end and TSS information. G. mellonella larva was infected with the fungal pathogen Madurella mycetomatis to map TSS under healthy and infection conditions. Larvae were first treated with itraconazole or ravuconazole, and then RNA-seq and LQ-ssCAGE libraries were prepared and sequenced 4, 30, and 52 hours following infection. The LQ-ssCAGE data was processed to identify CAGE transcription start site (CTSS), uni-, and bi-directional clusters. LQ-ssCAGE enabled us to precisely identify 39,410 TSS and 249 active enhancers; we assigned genomic features to the resulting TSSs and enhancers. The majority of the TSS peaks are annotated as promoter regions, while the enhancers were annotated as intergenic and genic. Furthermore, we confirmed the quality of TSS calling by promoter shapes and GC bias. Furthermore, we identified a set of super-enhancers and predicted de-novo motifs. The raw and processed data was deposited to NCBI GEO GSE282923. CTSS, TSS peaks, and enhancers coordinated are available through the ZENBU Genome browser. In this study, we reported the first atlas of TSS and active enhancers of G. mellonella.

genomics↗

bioRxiv2024

CFC-seq: identification of full-length capped RNAs unveil enhancer-derived transcription

Long-read sequencing has transformed transcriptome profiling, yet capturing full-length, non-polyadenylated transcripts like enhancer RNAs (eRNAs) remains challenging. Here, we introduce CFC-seq, combining cap-trapping and in vitro poly(A)-tailing to sequence poly(A) and non-poly(A) RNAs with precise transcription start site. Paired with our assembler, SALA, we identified 39,425 novel transcriptional units, including [~]24,000 eRNAs. Our data reveal a distinct genomic code governing eRNA fate dictated by core promoter architecture. CpG-island enhancers show high chromatin connectivity but yield short, exosome-sensitive RNAs. Conversely, TATA-box enhancers systematically co-opt LTR retrotransposons to inherit structural motifs that produce long, stable, and spliced RNAs. Mechanistically, the pioneer factor NF-Y activates these viral elements to license transcription, balanced by TEAD4 activity across a dual-gear regulatory axis. Finally, non-poly(A) eRNAs terminate via exosome-associated processing at structural-depleted cleavage zones. This comprehensive annotation links enhancer sequence architecture to RNA fate, providing a new transformative framework for decoding the functional human genome. HighlightsO_LIExpanded genomic architecture: CFC-seq unmasks a hidden layer of human transcriptome, identifying 39,425 novel transcriptional units with high-confidence TSS support, including [~]24,000 eRNAs. C_LIO_LITSS-first assembler: We introduce SALA, a specialized long-read assembler that prioritizes authentic 5 Cap-trapped ends to accurately reconstruct the TSS-resolved transcript models. C_LIO_LIGenomic code of eRNA fate: CGI enhancers drive short and exosome-sensitive transcripts associated with repressive H3K27me3 mark and high chromatin connectivity. TATA-box enhancers produce cell-type-specific, long, stable, and frequently spliced eRNAs. C_LIO_LIEvolutionary co-option of retrotransposons: A major fraction of TATA-box eRNAs originate from LTR retrotransposons, providing a direct mechanism for integration of viral elements into the human regulatory landscape. C_LIO_LIA dual-gear pioneering axis: The pioneer factor NF-Y activates unprimed LTR-TATA enhancers to license transcription independent of histone acetylation cascades, operating in parallel with TEAD4-mediated activation. C_LIO_LIStructural determinants of eRNA termination: Non-poly(A) eRNA TES features a secondary structure depletion zone that coordinates pol II termination and calibrates exosome-mediated turnover. C_LI

genomics↗

bioRxiv2024

Impacts of Cell Ranger versions on Chromium gene expression data

In droplet-based single cell gene expression data, cell barcode processing by Cell Ranger (CR) is a standard pipeline. But no systematic evaluation of the impact of CR version on single cell gene expression data has been conducted. To comprehensively evaluate the impact of CR version, we considered six molecular quality criteria, quantified gene expression, and performed downstream analysis for 12 single-cell datasets. Each dataset was processed by 15 versions of CR. We demonstrated that different versions of CR yield different numbers of cell barcodes with significant variation in detected UMIs, features, molecular qualities and average gene expression of protein-coding and lncRNA for the same dataset. Our analysis finds distinction between two diverse categories of cell barcodes: common barcodes unmasked by all versions of CR, and specific barcodes only unmasked/masked by some versions. Surprisingly, we observed variation in molecular read-out between common cell barcodes when called by different versions of CR. The specific barcodes yield skewed gene body coverage and form distinct clusters. The choice of CR version affects scores for quality, average gene expression, clustering results, and top cluster marker genes of the dataset.

bioinformatics↗

bioRxiv2023

Abundant capped RNAs are derived from mRNA cleavage at 3'UTR G-Quadruplexes

The 3 untranslated region (3UTR) plays a crucial role in determining mRNA stability, localisation, translation and degradation. Cap analysis gene expression (CAGE), a method for the detection of capped 5 ends of mRNAs, additionally reveals a large number of apparently 5 capped RNAs derived from 3UTRs. Here we provide the first direct evidence that these 3UTR-derived RNAs are indeed capped and often more abundant than the corresponding full-length mRNAs. By using a combination of AGO2 enhanced individual nucleotide resolution UV crosslinking and immunoprecipitation (eiCLIP) and CAGE following siRNA knockdowns, we find that these 3UTR-derived RNAs likely originate from AGO2-mediated cleavage, and most often occur at locations with potential to form RNA-G-quadruplexes and are enriched by RNA-binding protein UPF1. High-resolution imaging and long-read sequencing analysis validates several 3UTR-derived RNAs, demonstrates their abundance and shows that they tend not to co-localise with the parental mRNAs. We also find that production of 3UTR-derived RNA could explain the previously reported role of a 3UTR G-quadruplex in regulating the production of APP protein. Taken together, we provide new insights into the origin and abundance of 3UTR-derived RNAs, show the utility of CAGE-seq for their quantitative detection, and provide a rich dataset for exploring new biology of a poorly understood new class of RNAs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/538568v3_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@3f2ca7org.highwire.dtl.DTLVardef@18ca1b6org.highwire.dtl.DTLVardef@1ccdd8dorg.highwire.dtl.DTLVardef@e58938_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Iron regulatory pathways differentially expressed during Madurella mycetomatis grain development in Galleria mellonella

BackgroundMycetoma is a neglected, chronic granulomatous infection of the subcutaneous tissue, most often caused by the fungal pathogen Madurella mycetomatis. Characteristic of the infection is the formation of grains. However, knowledge of the function and formation of the grain is limited. To map the processes leading to M. mycetomatis grain formation, we used a Galleria mellonella larvae infection model and time-course transcriptomic profiling. MethodsGalleria mellonella larvae were infected with M. mycetomatis genome strain mm55. At 4h, 24h, 72h and 168h post-inoculation, RNA was extracted from larval content. Two types of sequencing libraries were prepared for time-course transcriptomic profiling and analysis. FindingsIn the infected G. mellonella, 88.0% of the RNA sequence reads mapped to G. mellonella, while only 0.01% mapped to M. mycetomatis. Differential Gene Expression analysis revealed that 3,498 G. mellonella and 136 M. mycetomatis genes were differentially expressed during infection. Most of the enriched GO terms of both host and pathogen are linked to energy pathways, nucleobase metabolic process as well as cation and iron transport. Genes related to iron transport were highly expressed by both G. mellonella (transferrin and ferritin) and M. mycetomatis (SidA, SidD and SidI). A protein-protein interaction network analysis of D. melanogaster homologous genes in M. mycetomatis revealed the expression of the entire siderophore biosynthesis pathway throughout infection. InterpretationThe identification of the importance of iron acquisition during grain formation can be exploited as a potential novel diagnostic and therapeutic strategy for mycetoma. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSMycetoma is a chronic, neglected tropical infectious disease, characterised by a large subcutaneous mass and the formation of black grains in the affected tissue. Treatment for mycetoma is disappointing as in 25-50% of the patients recurrences are noted and up to 15% of patients will have to undergo amputation. The main reason behind this poor treatment response is the formation of protective structures by the pathogen upon entering the human body. These structures are called grains and provide a strong barrier for antifungal agents. Although grains are the hallmark of mycetoma, it is currently not known how these grains are formed. To improve the current therapy, it is important to gain insights in grain formation. Added value of this studyWe unravel the processes leading to grain formation and development in an invertebrate model of Madurella mycetomatis grain. We were able to build a model of grain formation and demonstrated that iron sequestering plays an important role in this process. Our findings were an important milestone in understanding the pathogenesis of mycetoma which has been a mystery for decades. Implications of all the available evidenceThe findings, will provide leads for future drug development of mycetoma treatment and therefore, improve patients live and end the need for amputations.

microbiology↗

bioRxiv2022

Systematic functional annotation workflow for insects

Next generation sequencing has revolutionized entomological study, rendering it possible to analyze the genomes and transcriptomes of non-model insects. However, use of this technology is often limited to obtaining nucleotide sequences of target or related genes, with many of the acquired sequences remaining unused because other available sequences are not sufficiently annotated. To address this issue, we have developed a functional annotation workflow for transcriptome-sequenced insects to determine transcript descriptions, which represents a significant improvement over the previous method (functional annotation pipeline for insects). The developed workflow attempts to annotate not only the protein sequences obtained from transcriptome analysis but also the ncRNA sequences obtained simultaneously. In addition, the workflow integrates the expression level information obtained from transcriptome sequencing for application as functional annotation information. Using the workflow, functional annotation was performed on the sequences obtained from transcriptome sequencing of stick insect (Entoria okinawaensis) and silkworm (Bombyx mori), yielding richer functional annotation information than that obtained in our previous study. The improved workflow allows more comprehensive exploitation of transcriptome data and is applicable to other insects because the workflow has been openly developed on GitHub. Simple SummaryThe function of all genes encoded in the genome should be studied for genome editing. The genome editing technology can speeds up insect research for functional analysis of genes. Our knowledge about the functional information of genes is still incomplete currently while genome sequencing of an organism can be completed. The functional information has been annotated based solely on the information that has been obtained from the result of previous biological research. However, this information will be important in determining the target genes for genome editing. In particular, it is very important that this information is in machine-readable form because computer programs mainly parse this information for the understanding of biological systems. In this paper, we describe a workflow-based method for annotating gene functions in insects that make use of transcribed sequence information as well as reference genome and protein sequence databases. Using the developed workflow, we annotated functional information of Japanese stick insect and silkworm, including gene expression as well as sequence analysis. The functional annotation information obtained by the workflow will greatly expand the possibilities of entomological research using genome editing.

bioinformatics↗

bioRxiv2022

The high-throughput perturbation of long non-coding RNA reveals functional features in stem cells and across cell-types

Within the scope of FANTOM6 consortium, we perform a large-scale knockdown of 200 long non-coding RNAs (lncRNAs) in human induced pluripotent stem (iPS) cells, 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 iPS cells. We compare our results with previously published fibroblasts phenotyping data and find that 2.9% of the lncRNAs exhibit consistent cell growth phenotype, whereas we observe 58.3% agreement in molecular phenotypes. This highlights molecular phenotyping is more comprehensive in revealing affected pathways.

genomics↗

bioRxiv2021

Functional annotation of human long noncoding RNAs using chromatin conformation data

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. Studying lncRNAs is challenging due to their low expression level, cell type-specific occurrence, poor sequence conservation between orthologs, and lack of information about RNA domains. LncRNAs direct the regulatory factors in the locations that are in cis to their transcription sites. We designed a model to predict if an lncRNA acts in cis based on its features and trained it using RNA-chromatin interaction data. The trained model is cell type-independent and does not require RNA-chromatin data. Combining RNA-chromatin and Hi-C data, we showed that lncRNA-chromatin binding sites are determined by chromosome conformation. For each lncRNA, the spatially proximal genes were identified as their potential targets by combining Hi-C and Cap Analysis Gene Expression (CAGE) data in 18 human cell types. RNA-protein and RNA-chromatin interaction data suggested that lncRNAs act as scaffolds to recruit regulatory proteins to target promoters and enhancers. We provide the data through an interactive visualization web portal at https://fantom.gsc.riken.jp/zenbu/reports/#F6_3D_lncRNA.

genomics↗

bioRxiv2020

Single-cell transcriptomics, scRNA-Seq and C1 CAGE discovered distinct phases of pluripotency during naïve-to-primed conversion in mice

BackgroundTwo types of mammalian pluripotent stem cells (PSC), i.e. naive and primed possess distinct cellular characteristics. It is largely unknown how these differences are generated during naive-to-primed transition process. We have established a robust in vitro transition system using a Wnt inhibitor for the first time and analyzed dynamic changes in cellular status via single-cell RNA-sequencing and C1 CAGE analyses. ResultsAnalysis of known marker genes suggested that the cell transition process progresses as expected. However, cluster analyses revealed a sudden increase in expression profile diversities three and four days after induction of the transition. These expression diversities can be reconciled by the presence of two subpopulations with distinct transcription profiles emerging at these time points. One of the subpopulations appears transiently, and surprisingly these cells showed a global downregulation of gene expression. Moreover, initiation of random X chromosome inactivation (XCI) coincides with the appearance of these transient cells. The other subpopulation can be maintained as a stem cell line and possesses expression profiles more similar to those of primed epiblast stem cells (EpiSC) than embryonic stem cells (ESC). However, there are important differences in gene expression related to epithelial-mesenchymal transition (EMT), suggesting that this subpopulation may represent a novel pluripotent state that has an intermediate cellular phenotype between ESC and EpiSC. ConclusionsThese findings should contribute to our understanding of the establishment and maintenance of distinct differentiation statuses of mammalian PSCs and provide new insights into the pluripotency spectrum in general.

genomics↗

bioRxiv2020

Inducing Human Retinal Pigment Epithelium-like Cells from Somatic Tissue

Regenerative medicine relies on basic research to find safe and useful outcomes that are only practical when cost-effective. The human eyeball requires the retinal pigment epithelium (RPE) for support and maintenance that interfaces the neural retina and the choroid at large. Nearly 200 million people suffer from age-related macular degeneration (AMD), a blinding multifactor genetic disease among other retinal pathologies related to RPE degradation. Recently, autologous pluripotent stem cell-derived RPE cells were prohibitively expensive due to time, therefore we developed a new simplified cell reprogramming system. We stably induced RPE-like cells (iRPE) from human fibroblasts by conditional overexpression of broad plasticity and lineage-specific pioneering transcription factors. iRPE cells showed features of modern RPE benchmarks and significant in-vivo integration in transplanted chimeric hosts. Herein, we detail the iRPE system with comprehensive modern single-cell RNA (scRNA) sequencing profiling to interpret and characterize its best cells. We anticipate that our system may enable robust retinal cell induction for regenerative medicine research and affordable autologous human RPE tissue for cell therapy.

bioengineering↗

bioRxiv2020

Automatic identification of small molecules that promote cell conversion and reprogramming

Controlling cell fate has great potential for regenerative medicine, drug discovery, and basic research. Although numerous transcription factors have been discovered that are able to promote cell reprogramming and trans-differentiation, methods based on their up-regulation tend to show low efficiency. The identification of small molecules that can facilitate conversion between cell types can ameliorate this problem working through safe, rapid, and reversible mechanisms. Here we present DECCODE, an unbiased computational method for the identification of such molecules solely based on transcriptional data. DECCODE matches the largest available collection of drug-induced profiles (the LINCS database) for drug treatments against the largest publicly available dataset of primary cell transcriptional profiles (FANTOM5), to identify drugs that either alone or in combination enhance cell reprogramming and cell conversion. Extensive in silico and in vitro validation of DECCODE in the context of human induced pluripotent stem cells (hIPSCs) generation shows that the method is able to prioritize drugs enhancing cell reprogramming. We also generated predictions for cell conversion with single drugs and drug combinations for 145 different cell types and made them available for further studies.

bioinformatics↗

bioRxiv2020

Assessment of mapping strategies for determining the 5'-end of mRNAs and long-noncoding RNAs with short read sequences

BackgroundGenome mapping is an essential step in data processing for transcriptome analysis, and many previous studies have evaluated various methods and strategies for mapping RNA-seq data. Cap Analysis of Gene Expression (CAGE) is a sequencing-based protocol particularly designed to capture the 5{square}-ends of transcripts for quantitatively measuring the expression levels of transcription start sites genome-wide. Because CAGE analysis can also predict the activities of promoters and enhancers, this protocol has been an essential tool in studies of transcriptional regulation. Typically, the same mapping software is used to align both RNA-seq data and CAGE reads to a reference genome, but which mapping software and options are most appropriate for mapping the 5{square}-end sequence reads obtained through CAGE has not previously been evaluated systematically. ResultsHere we assessed various strategies for aligning CAGE reads, particularly [~]50-bp sequences, with the human genome by using the HISAT2, LAST, and STAR programs both with and without a reference transcriptome. One of the major inconsistencies among the tested strategies involves alignments to pseudogenes and parent genes: some of the strategies prioritized alignments with pseudogenes even when the read could be aligned with coding genes with fewer mismatches. Another inconsistency concerned the detection of exon-exon junctions. These preferences depended on the program applied and whether a reference transcriptome was included. Overall, the choice of strategy yielded different mapping results for approximately 2% of all promoters. ConclusionsAlthough the various alignment strategies produced very similar results overall, we noted several important and measurable differences. In particular, using the reference transcriptome in STAR yielded alignments with the fewest mismatches. In addition, the inconsistencies among the strategies were especially noticeable regarding alignments to pseudogenes and novel splice junctions. Our results indicate that the choice of alignment strategy is important because it might affect the biological interpretation of the data.

bioinformatics↗