Biology subjects

Inoue, F.

Publications and source records attributed to Inoue, F..

At least 19 recordsLinked to original sources

bioRxiv2026

Immune context unmasks regulatory effects of Neanderthal and Denisovan introgression

Neanderthal and Denisovan introgression have left a pervasive footprint in the human genome, yet its regulatory consequences remain poorly understood. Here we use a massively parallel reporter assay to characterize the cis-regulatory activity of 4,161 high-frequency introgressed variants across respiratory (A549), hepatic (HepG2), and hematopoietic (K562) cells exposed to immune and infectious stimuli. We find that ~18% of variants show differential activity between archaic and modern alleles, including 94 whose effects are revealed or modulated by stimulation, often in a cell type-specific manner. We identify loci, including STAT2, IL23A, and RNF41, where clusters of introgressed alleles exert coordinated regulatory effects consistent with adaptive programs. Finally, we dissect the mechanisms underlying the association between Neanderthal introgression and COVID-19 severity and show that the risk allele rs17713054-A, which displays the strongest effect in our assay, increases activity of a TNF--responsive enhancer in lung epithelial cells, directly upregulating SLC6A20.Together, these findings reveal widespread context-dependent regulatory effects of archaic introgression, with broad evolutionary and biomedical implications.

evolutionary biology↗

bioRxiv2026

A DNA foundation model predicts osteoporosis risk genes without proximity bias

Targets supported by human genetic associations are more than twice as likely to progress from clinical development to approval. Genome-wide association studies are the largest source of genetic evidence for disease risk but linking non-coding variants to effector genes remains a significant barrier to identifying causal targets. Current gene-mapping approaches suffer from proximity bias, largely ignoring distal genes. Here we introduce Rosalind, a DNA foundation model fine-tuned on human genetic variation from GTEx, that directly predicts variant-gene regulatory relationships from sequence without relying on nearest-gene heuristics. We demonstrate Rosalinds accuracy through extensive benchmarking, apply it to multiple complex traits to establish broad utility, and provide experimental validation in osteoporosis using a translational osteoblast assay. We demonstrate that genes distal to osteoporosis risk variants were significantly more likely to alter a bone formation phenotype than nearest genes. Together, these results highlight deep learning-based regulatory models as a general and scalable framework for translating novel genetic insights to drug discovery.

genetics↗

bioRxiv2025

Simultaneous epigenomic profiling and regulatory activity measurement using e2MPRA

Cis-regulatory elements (CREs) have a major effect on phenotypes including disease. They are identified in a genome-wide manner by analyzing the binding of transcription factors (TFs), various co-factors and histone modifications in DNA using assays such as ChIP-seq, Cut&Tag and ATAC-seq. However, these assays are descriptive and require high-throughput technologies, such as massively parallel reporter assays (MPRAs), to test the functional activity and variant effect on these sequences. Currently, technologies that can simultaneously analyze both the regulatory function of a specific sequence and the TFs, cofactors and epigenomic modifications that determine it do not exist. Here, we developed enrichment followed by epigenomic profiling MPRA (e2MPRA), a novel technology that utilizes lentivirus-based MPRA to enrich for the integration of specific CREs into the genome followed by Cut&Tag or ATAC-seq targeted specifically for these sequences. This method allows to simultaneously analyze in a high-throughput manner regulatory activity, protein binding and epigenetic modification of thousands of candidate CREs and their variants. We demonstrate that e2MPRA can be used to dissect the epigenetic functions of TF motifs arranged in synthetic enhancers, as well as to analyze the effect of enhancer sequence variants on epigenetic modifications. In summary, this technology will increase our understanding of the regulatory code, its effect on the epigenome and how its alteration can lead to a variety of phenotypes including human disease.

genomics↗

bioRxiv2025

Capture-C MPRA: A high-throughput method to simultaneously characterize promoter interactions and regulatory activity

Cis regulatory elements (CREs) interact with their target promoters over long genomic distances and can be identified using chromatin conformation capture (3C) assays. Their regulatory activity can be functionally characterized in a high-throughput manner using massively parallel reporter assays (MPRAs) that generally test an enhancer alongside a minimal promoter. Here, we developed a novel technology called Capture-C MPRA (ccMPRA) that combines both technologies and can simultaneously obtain chromatin interactions and measure CRE activity alongside their target promoters. We utilized ccMPRA to analyze the regulatory activity of 650 promoters interacting with 42,719 sequences. As C-based techniques also capture isolated promoters, we were able to obtain promoter baseline activity, enabling the identification of both enhancers and silencers. Analysis of CREs interacting with more than one promoter showed significant activity differences depending on the promoter. In summary, ccMPRA can simultaneously characterize chromatin interactions and regulatory activity, allowing to further dissect regulatory grammar.

genomics↗

bioRxiv2024

Comprehensive network modeling approaches unravel dynamic enhancer-promoter interactions across neural differentiation

BackgroundIncreasing evidence suggests that a substantial proportion of disease-associated mutations occur in enhancers, regions of non-coding DNA essential to gene regulation. Understanding the structures and mechanisms of regulatory programs this variation affects can shed light on the apparatuses of human diseases. ResultsWe collected epigenetic and gene expression datasets from seven early time points during neural differentiation. Focusing on this model system, we constructed networks of enhancer-promoter interactions, each at an individual stage of neural induction. These networks served as the base for a rich series of analyses, through which we demonstrated their temporal dynamics and enrichment for various disease-associated variants. We applied the Girvan-Newman clustering algorithm to these networks to reveal biologically relevant substructures of regulation. Additionally, we demonstrated methods to validate predicted enhancer-promoter interactions using transcription factor overexpression and massively parallel reporter assays. ConclusionsOur findings suggest a generalizable framework for exploring gene regulatory programs and their dynamics across developmental processes. This includes a comprehensive approach to studying the effects of disease-associated variation on transcriptional networks. The techniques applied to our networks have been published alongside our findings as a computational tool, E-P-INAnalyzer. Our procedure can be utilized across different cellular contexts and disorders.

genomics↗

bioRxiv2023

Detailed microbiome analysis of sticker-stripped surface materials of acne lesions revealed acne-related Cutibacterium acnes subtypes: A pilot study

Cutibacterium acnes (C. acnes) is known to play a central role in pathogenesis of acne vulgaris. It has been understood that multiple phylotypes of C. acnes exist, with certain types being more prevalent in patient with acne vulgaris and others more common in healthy individuals. In this context, we conducted a preliminary study using self-collected samples via an adhesive sticker (MySkin(R) patch) to analyze the skin microbiome of Japanese women. The study aimed to determine the role of C. acnes and its specific phylotypes in the development of acne vulgaris. Participants in this study were Japanese females aged between their 20s and 40s. Dermatologists evaluate the data from web-based questionnaires and smartphone image submissions to classify subjects into either Acne group (n = 219) or Non-acne group (n = 77). Quality assessment of DNA extracted from the sticker was conducted, followed by amplification of the 16S rRNA region using PCR. Subsequent microbial community analysis was performed using next-generation sequencing techniques. Genetic classification of C. acnes was accomplished through single locus sequence typing. Results indicated a bacterial community composition on the facial skin surface predominantly consisting of C. acnes clusters, with over half of these clusters constituted by C. acnes. Notably, the Acne group exhibited a significantly higher proportion of C. acnes relative to total bacterial presence compared to the Non-acne group. Analysis of C. acnes phylotypes revealed a markedly lower presence of type III (subspecies elongatum) in the Acne group (vs. Non-acne group, p < 0.05). No significant differences were observed in the prevalence of Types IA1, IA2, II, and IB between the two groups. The predominantsequence types (ST) of C. acnes identified were IA2_2_F0 (23.9%), IA1_4_A0 (20.6%), and II_2_K0 (18.6%). Within the Acne group, an increase in IA2_1_F1 and a decrease in III_1_L0 were observed (vs. Non-acne group, p < 0.05). This study underscores the feasibility of using self-collected and mailed-in samples for qPCR and microbiome analysis, maintaining diagnostic quality comparable to in-person assessments. Furthermore, the variation in the expression of C. acnes phylotypes across skin surfaces between acne-afflicted and healthy individuals could suggest that shifts in phylotype expression patterns may be indicative of skin susceptibilities to acne development.

microbiology↗

bioRxiv2023

Cryptic endogenous retrovirus subfamilies in the primate lineage

Many endogenous retroviruses (ERVs) in the human genome are primate-specific and have contributed novel cis-regulatory elements and transcripts. However, current approaches for classifying and annotating ERVs and their long terminal repeats (LTRs) have limited resolution and are inaccurate. Here, we developed a new annotation based on phylogenetic analysis and cross-species conservation. Focusing on the evolutionary young MER11A/B/C subfamilies, we revealed the presence of 4 new subfamilies, that better explained the epigenetic heterogeneity observed within the MER11 instances, suggesting a new annotation for 412 (19.8%) of these repeat elements. Furthermore, we functionally validated the regulatory potential of these four new subfamilies using a massively parallel reporter assay (MPRA), which also identified motifs associated with their differential activities. Combining MPRA with new annotations across primates revealed an apes-specific gain of SOX related motifs through a single-nucleotide deletion. Lastly, by applying our approach across 53 simian-enriched LTR subfamilies, we defined a total of 75 new subfamilies and found that 3,807 (30.0%) instances from 26 LTR subfamilies could be categorized into a novel annotation, many of which with a distinct epigenetic profile. Thus, with our refined annotation of simian-enriched LTRs, it will be possible to better understand the evolution in primate genomes and potentially identify new roles for ERVs and their LTRs in the hosts.

genomics↗

bioRxiv2023

Best practices for perturbation MPRA--a computational evaluation framework of sequence design strategies

The advent of the perturbation-based massively parallel reporter assays (MPRAs) technique has enabled delineating of the roles of non-coding regulatory elements in orchestrating gene expression. However, computational efforts remain scant to evaluate and establish guidelines for sequence design strategies for perturbation MPRAs. Here, we propose a framework for evaluating and comparing various perturbation strategies for MPRA experiments. Under this framework, we benchmark three different perturbation approaches from the perspectives of alteration in motif-based profiles, consistency of MPRA outputs, and robustness of models that predict the activities of putative regulatory motifs. Although our analyses show similar while significant results in multiple metrics, the method of randomly shuffling nucleotides outperform the other two methods. Thus, we still recommend designing sequences by randomly shuffling the nucleotides of the perturbed site in perturbation-MPRA. The evaluation framework, together with the benchmarking findings in our work, creates a resource of computational pipelines and illustrates the promise of perturbation-MPRA for predicting non-coding regulatory activities.

bioinformatics↗

bioRxiv2023

Characterization of enhancer activity in early human neurodevelopment using Massively parallel reporter assay (MPRA) and forebrain organoids

AO_SCPLOWBSTRACTC_SCPLOWRegulation of gene expression through enhancers is one of the major processes shaping the structure and function of the human brain during development. High-throughput assays have predicted thousands of enhancers involved in neurodevelopment, and confirming their activity through orthogonal functional assays is crucial. Here, we utilized Massively Parallel Reporter Assays (MPRAs) in stem cells and forebrain organoids to evaluate the activity of [~]7,000 gene-linked enhancers previously identified in human fetal tissues and brain organoids. We used a Gaussian mixture model to evaluate the contribution of background noise in the measured activity signal to confirm the activity of [~]35% of the tested enhancers, with most showing temporal-specific activity, suggesting their evolving role in neurodevelopment. The temporal specificity was further supported by the correlation of activity with gene expression. Our findings provide a valuable gene regulatory resource to the scientific community. AO_SCPLOWUTHORC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWSUMMARYC_SCPLOWEnhancers are non-coding elements that play a crucial role in the regulation of gene expression during brain development. Despite the availability of various techniques available to identify enhancers, their functional activity is relatively less understood, leaving a gap in our understanding of how enhancer behavior might regulate complex transitions of neurodevelopment. To address this, we utilized forebrain organoids, a 3D model system which closely mimics the complex cellular environment of the developing human brain, and employed Massively Parallel Reporter Assay (MPRA) to validate enhancer activity at various stages of forebrain differentiation, from induced pluripotent stem cells (iPSCs) to neuronal progenitors and cortical neurons. Our study provides a comprehensive catalog of over 2,300 enhancers, showcasing their temporal activity profiles during early neuronal development and offering valuable insights into their likely biological functions. This research advances our understanding of enhancer dynamics in brain development and offers new avenues for further investigations in this field.

genomics↗

bioRxiv2023

Massively parallel characterization of transcriptional regulatory elements in three diverse human cell types

The human genome contains millions of candidate cis-regulatory elements (CREs) with cell-type-specific activities that shape both health and myriad disease states. However, we lack a functional understanding of the sequence features that control the activity and cell-type-specific features of these CREs. Here, we used lentivirus-based massively parallel reporter assays (lentiMPRAs) to test the regulatory activity of over 680,000 sequences, representing a nearly comprehensive set of all annotated CREs among three cell types (HepG2, K562, and WTC11), finding 41.7% to be functional. By testing sequences in both orientations, we find promoters to have significant strand orientation effects. We also observe that their 200 nucleotide cores function as non-cell-type-specific on switches providing similar expression levels to their associated gene. In contrast, enhancers have weaker orientation effects, but increased tissue-specific characteristics. Utilizing our lentiMPRA data, we develop sequence-based models to predict CRE function with high accuracy and delineate regulatory motifs. Testing an additional lentiMPRA library encompassing 60,000 CREs in all three cell types, we further identified factors that determine cell-type specificity. Collectively, our work provides an exhaustive catalog of functional CREs in three widely used cell lines, and showcases how large-scale functional measurements can be used to dissect regulatory grammar.

genomics↗

bioRxiv2023

Massively parallel characterization of psychiatric disorder-associated and cell-type-specific regulatory elements in the developing human cortex

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and disease. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 sequences, including differentially accessible cell-type specific regions in the developing cortex and single-nucleotide variants associated with psychiatric disorders. In primary cells, we identified 46,802 active enhancer sequences and 164 disorder-associated variants that significantly alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning, we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development. One Sentence SummaryWe identify 46,802 enhancers and 164 psychiatric disorder variants with regulatory effects in the developing cortex and organoids.

genomics↗

bioRxiv2022

Three-dimensional genome re-wiring in loci with Human Accelerated Regions

Human Accelerated Regions (HARs) are conserved genomic loci that evolved at an accelerated rate in the human lineage and may underlie human-specific traits. We generated HARs and chimpanzee accelerated regions with the largest alignment of mammalian genomes to date. To facilitate exploration of accelerated evolution in other lineages, we implemented an open-source Nextflow pipeline that runs on any computing platform. Combining deep-learning with chromatin capture experiments in human and chimpanzee neural progenitor cells, we discovered a significant enrichment of HARs in topologically associating domains (TADs) containing human-specific genomic variants that change three-dimensional (3D) genome organization. Differential gene expression between humans and chimpanzees at these loci in multiple cell types suggests rewiring of regulatory interactions between HARs and neurodevelopmental genes. Thus, comparative genomics together with models of 3D genome folding revealed enhancer hijacking as an explanation for the rapid evolution of HARs. One-Sentence SummaryHuman-specific changes to 3D genome organization may have contributed to rapid evolution of mammalian-conserved loci in the human genome.

evolutionary biology↗

bioRxiv2021

Massively parallel reporter perturbation assay uncovers temporal regulatory architecture during neural differentiation

Gene regulatory elements play a key role in orchestrating gene expression during cellular differentiation, but what determines their function over time remains largely unknown. Here, we performed perturbation-based massively parallel reporter assays at seven early time points of neural differentiation to systematically characterize how regulatory elements and motifs within them guide cellular differentiation. By perturbing over 2,000 putative DNA binding motifs in active regulatory regions, we delineated four categories of functional elements, and observed that activity direction is mostly determined by the sequence itself, while the magnitude of effect depends on the cellular environment. We also find that fine-tuning transcription rates is often achieved by a combined activity of adjacent activating and repressing elements. Our work provides a blueprint for the sequence components needed to induce different transcriptional patterns in general and specifically during neural differentiation.

genomics↗

bioRxiv2020

The cis-regulatory effects of modern human-specific variants

The Neanderthal and Denisovan genomes enabled the discovery of sequences that differ between modern and archaic humans, the majority of which are noncoding. However, our understanding of the regulatory consequences of these differences remains limited, in part due to the decay of regulatory marks in ancient samples. Here, we used a massively parallel reporter assay in embryonic stem cells, neural progenitor cells and bone osteoblasts to investigate the regulatory effects of the 14,042 single-nucleotide modern human-specific variants. Overall, 1,791 (13%) of sequences containing these variants showed active regulatory activity, and 407 (23%) of these drove differential expression between human groups. Differentially active sequences were associated with divergent transcription factor binding motifs, and with genes enriched for vocal tract and brain anatomy and function. This work provides insight into the regulatory function of variants that emerged along the modern human lineage and the recent evolution of human gene expression.

evolutionary biology↗

bioRxiv2019

Genomic and epigenomic mapping of leptin-responsive neuronal populations involved in body weight regulation

Genome wide association studies (GWAS) in obesity have identified a large number of noncoding loci located near genes expressed in the central nervous system. However, due to the difficulties in isolating and characterizing specific neuronal subpopulations, few obesity-associated SNPs have been functionally characterized. Leptin responsive neurons in the hypothalamus are essential in controlling energy homeostasis and body weight. Here, we combine FACS-sorting of leptin-responsive hypothalamic neuron nuclei with genomic and epigenomic approaches (RNA-seq, ChIP-seq, ATAC-seq) to generate a comprehensive map of leptin-response specific regulatory elements, several of which overlap obesity-associated GWAS variants. We demonstrate the usefulness of our leptin-response neuron regulome, by functionally characterizing a novel enhancer near Socs3, a leptin response-associated transcription factor. We envision our data to serve as a useful resource and a blueprint for functionally characterizing obesity-associated SNPs in the hypothalamus.

genomics↗

bioRxiv2019

A systematic evaluation of the design, orientation, and sequence context dependencies of massively parallel reporter assays

Massively parallel reporter assays (MPRAs) functionally screen thousands of sequences for regulatory activity in parallel. Although MPRAs have been applied to address diverse questions in gene regulation, there has been no systematic comparison of how differences in experimental design influence findings. Here, we screen a library of 2,440 sequences, representing candidate liver enhancers and controls, in HepG2 cells for regulatory activity using nine different approaches (including conventional episomal, STARR-seq, and lentiviral MPRA designs). We identify subtle but significant differences in the resulting measurements that correlate with epigenetic and sequence-level features. We also test this library in both orientations with respect to the promoter, validating en masse that enhancer activity is robustly independent of orientation. Finally, we develop and apply a novel method to assemble and functionally test libraries of the same putative enhancers as 192-mers, 354-mers, and 678-mers, and observe surprisingly large differences in functional activity. This work provides a framework for the experimental design of high-throughput reporter assays, suggesting that the extended sequence context of tested elements, and to a lesser degree the precise assay, influence MPRA results.

genomics↗

bioRxiv2018

Saturation mutagenesis of disease-associated regulatory elements

The majority of common variants associated with common diseases, as well as an unknown proportion of causal mutations for rare diseases, fall in noncoding regions of the genome. Although catalogs of noncoding regulatory elements are steadily improving, we have a limited understanding of the functional effects of mutations within them. Here, we performed saturation mutagenesis in conjunction with massively parallel reporter assays on 20 disease-associated gene promoters and enhancers, generating functional measurements for over 30,000 single nucleotide substitution and deletion mutations. We find that the density of putative transcription factor binding sites varies widely between regulatory elements, as does the extent to which evolutionary conservation or various integrative scores predict functional effects. These data provide a powerful resource for interpreting the pathogenicity of clinically observed mutations in these disease-associated regulatory elements, and also comprise a gold-standard dataset for the further development of algorithms that aim to predict the regulatory effects of noncoding mutations.

genetics↗

bioRxiv2018

Massively parallel characterization of regulatory dynamics during neural induction

The molecular components governing neural induction remain largely unknown. Here, we applied a suite of genomic and computational tools to comprehensively identify these components. We performed RNA-seq, ChIP-seq (H3K27ac, H3K27me3) and ATAC-seq on human embryonic stem cells (hESCs) at seven early neural differentiation time points (0-72 hours) and identified thousands of induced genes and regulatory regions. We analyzed the function of ~2,500 selected regions using massively parallel reporter assays at all time points. We found numerous temporal enhancers that correlated with similarly timed epigenetic marks and gene expression. Development of a prioritization method that incorporated all genomic data identified key transcription factors (TFs) involved in neural induction. Individual overexpression of eleven TFs and several combinations in hESCs found novel neural induction regulators. Combined, our results provide a comprehensive map of genes and functional regulatory elements involved in neural induction and identify master regulator TFs that are instrumental for this process.\n\nOne Sentence SummaryUsing numerous genomic assays and computational tools we characterized the dynamic changes that take place during neural induction.

genomics↗