Biology subjects

Torres, M.

Publications and source records attributed to Torres, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Single-cell characterization of the human immune landscape reveals dysfunction in monocyte-mediated cellular interactions in symptomatic malaria

Plasmodium falciparum infection results in a wide range of phenotypes including chronic asymptomatic and symptomatic individuals. The malaria outcome depends, at least in part, on host immune responses mediated by specific cell subsets. Here, we investigated the immune landscape of chronic asymptomatic and symptomatic children using single-cell RNA sequencing. Unexpectedly, we uncovered biologically relevant immune cell complexes, which were further confirmed by imaging flow cytometry. Symptomatic infection was associated with a decrease of cell-cell interactions, particularly between non-classical monocytes and T cells. Non-classical monocytes from symptomatic children exhibited profiles with increased inflammation characterized by enhanced S100A8-S100A9 expression together with decreased expression of interaction-associated genes such as CD86 or ITGAL. Moreover, we demonstrated that inflammatory and cell interaction programs could not be activated simultaneously within the same cell. In addition, the concomitant expression of inflammatory genes and members of the LILR gene family was predominantly observed in monocytes from symptomatic individuals. These findings raise the possibility that enhanced S100A8/S100A9-LILRB1 signaling may contribute to the altered functional state of monocytes in symptomatic malaria and, potentially, may impair their interactions with T cells. Together, our results suggest that immune cell complexes may therefore reveal an additional layer of immune regulation associated with the development of clinical malaria.

genomics↗

bioRxiv2026

Pilifers provide rapid mechanosensory feedback during floral tracking in hawkmoths

Hawkmoths expertly hover over flowers during feeding, accurately tracking floral movements even as flowers gently sway in the wind. They use visual and mechanosensory feedback to adjust their position, while their long, flexible mouthpart, the proboscis, stays inserted inside the nectar spur. Although much is known about how hawkmoths detect and process visual motion, far less is understood about how hawkmoths detect mechanical motion cues. Pilifers are hair bundles at the proboscis-head joint, well-positioned to provide feedback about floral motion. Here, we tested if pilifers could provide mechanosensory feedback essential for successful floral tracking. We computed bending at the proboscis base during floral tracking in freely flying hawkmoths and found that floral movements induce small yet significantly correlated lateral bending. Recordings from pilifer afferents showed that they respond to lateral motions of amplitudes similar to those seen in natural floral tracking behaviors. They respond to different directions and speeds of proboscis motion with an average latency of 1.9 ms. Neural tracing revealed extensive sensory arborizations in the sub-esophageal ganglion of the brain and ventral nerve cord, indicating a feedback pathway to the neck and flight motor system that enables rapid coordination of appendages and body for successful feeding.

neuroscience↗

bioRxiv2026

Cytogenomic signatures of hybridisation in the genus Carpobrotus reveal biased parental dominance

O_LIHybridisation is frequently associated with plant invasions; however, its consequences for genome organisation and chromosome evolution remain poorly understood in invasive species. We investigated the extent of hybridisation in the invasive Carpobrotus edulis--acinaciformis hybrid complex and determined the cytogenomic contribution of parental species in hybrid accessions. C_LIO_LIWe combined whole-genome sequencing, population genomic analyses, genome size estimation, repeatome characterisation, chromosome counting and fluorescence in situ hybridisation to compare parental species and hybrid accessions from South Africa and the Mediterranean Basin. C_LIO_LIPopulation genomic analyses revealed widespread hybridisation and introgression, with most invasive accessions showing admixed ancestries. Pattersons D-statistic supported asymmetric allele sharing towards C. edulis. Hybrid accessions displayed genome sizes indistinguishable from C. edulis, whereas C. acinaciformis possessed significantly larger genomes. Repeatome analyses identified marked differences in repetitive DNA composition, particularly in satellite DNA abundance and chromosomal distribution. A newly identified satellite repeat (CarpoSat) showed contrasting chromosomal patterns between parental species, whereas hybrids resembled C. edulis satellite pattern. C_LIO_LIOur results demonstrate that Carpobrotus hybrid accessions are a swarm of later-generation hybrids and backcrosses showing a strong bias towards C. edulis, indicating asymmetric introgression. These findings highlight the value of integrating cytogenetic and genomic approaches to understand genome evolution in invasive hybrid complexes. C_LI

plant biology↗

bioRxiv2026

Highly contiguous reference genome assembly of the endangered Orces blue whiptail Holcosus orcesi

Holcosus orcesi, the Orces Blue Whiptail, is a Critically Endangered lizard endemic to the upper Jubones River basin in southern Ecuador. Restricted to a narrow elevational range within semi-arid Andean shrublands, it represents one of the few montane members of a predominantly lowland lineage. Here we present the first high-quality reference genome for H. orcesi, generated using Oxford Nanopore Technologies long-read sequencing. The assembly spans 1.68 Gb across only 91 contigs, with an N50 of 76.2 Mb and a BUSCO completeness of 96.8%, making it among the most contiguous and complete squamate genomes to date. Structural annotation predicted 25,682 genes, of which 85% showed homology to known proteins and 45% were assigned Gene Ontology terms. Repetitive elements accounted for 46.3% of the genome, with LINEs representing the predominant class. This genome provides a foundational resource for future evolutionary, comparative and conservation-genomic research of H. orcesi and other mountain reptiles, enabling studies of population genomics, local adaptation, and genomic erosion in isolated populations. By expanding the genomic representation of tropical montane reptiles, this work helps address longstanding phylogenetic and geographic gaps in global biodiversity genomics and provides a foundation for evidence-based conservation of H. orcesi and related taxa.

genomics↗

bioRxiv2026

TIM3+ Tumor Associated M2 Macrophages Impair Antitumor T Cell Immunity and Promote Gastric Cancer Progression and Peritoneal Metastasis

Peritoneal metastases (PM) are the leading cause of cancer-related death in gastric cancer (GC) patients with survival typically < 9 months. Here, we demonstrate that TIM3 and its ligands are increased along the GC continuum and associated with poor survival. Integrated omics analyses and functional studies revealed highly enriched TIM3 in CD163+ tumor associated M2 immunosuppressive macrophages significantly promote tumor cell invasion and tumor growth in vivo, while TIM3 depletion in macrophages reduced tumor cell malignant attributes and increased T cell immunity from PBMCs or CD45+ immune cells of malignant ascites in co-culture system. By cytokine and kinase arrays, we discovered that depletion of TIM3 in macrophages reduced the production of notable secretome of cytokines/chemokines from M2 macrophages; and the protumor function of TIM3+ macrophages rely on the p90RSK1/2/CCL20 axis. Finally, we reveal that TIM3 blockage or genetic KO had superior antitumor activity in combination with anti-PD1 immunotherapy and mitomycin C (MMC) chemotherapy. Together, this study uncovers an important role for TIM3 in tumor associated M2 macrophages and underscores the potential of TIM3 blockage in GC patients with PM. Statement of significanceIn this study, we show TIM3 increases along GC continuum, and highly enriched on tumor associated M2 macrophages that fuel tumor growth; and suppress T cell function via p90RSK1/2/CCL20 axis. TIM3 depletion restores T-cell immunity and curbs tumor growth. TIM3 blockade combined with anti-PD1 and mitomycin C provide a novel therapeutic strategy for GC patients with PM.

immunology↗

bioRxiv2025

AXL governs axolotl cardiac regeneration and directs mammalian cardiomyocyte dedifferentiation

Cardiac injury outcomes vary widely across species, from complete regeneration to irreversible scarring. Using single-nucleus multiomics and spatial transcriptomics, we generated a spatially resolved atlas of axolotl heart regeneration following injury, identifying a distinct border-zone cardiomyocyte population with a pro-regenerative transcriptional program. Ligand-receptor analysis of the border zone niche identified enrichment of the receptor tyrosine kinase AXL in injury-responsive cardiomyocytes and its ligand Gas6 in endothelial cells. Functional perturbation demonstrated AXL requirement for border-zone cardiomyocyte activation and axolotl heart regeneration. In murine cardiomyocytes, AXL overexpression induced sarcomere disassembly, metabolic rewiring, and immature gene expression without triggering proliferation. These findings show that AXL signaling induces cardiomyocyte dedifferentiation and uncouples dedifferentiation from cell cycle re-entry, providing mechanistic insight into cellular plasticity during heart regeneration.

cell biology↗

bioRxiv2025

Functional rescue of critical-size bone defect using molecular network analysis of axolotl limb regeneration.

The salamander limb has served as a canonical model for successful regeneration that has yielded numerous molecular insights on its basic mechanism. Harnessing such information to induce regeneration in a non-regenerative setting has been a long-sought goal. The amputated salamander limb efficiently regenerates all of its missing bones, but paradoxically, a large bone gap without amputation - commonly called a Critical Size Defect (CSD) - is not regenerated, similarly to other vertebrates1. This non-regenerating injury provides a human-relevant setting to understand how to rescue lack-of-regeneration. Satoh and colleagues demonstrated in axolotl that transplantation of blastema cells from an amputated limb into a CSD yields cartilage bridging of the CSD2. This work provided a roadmap for rescuing the CSD. Here we asked, what are the crucial molecular differences in cells populating a regenerating blastema versus the CSD and can they be used to elicit CSD bridging? Previous genetic fate mapping and single cell transcriptomics showed that limb regeneration occurs via migration and dedifferentiation of fibroblastic, soft connective tissue (CT) cells that form a multipotent, skeletal stem cell 3, 4. Here using single cell transcriptomics and genetic fate mapping we found that the CSD is populated by CT cells that undergo a divergent molecular transition compared to the blastema. Using gene regulatory network (GRN) modeling, and lipid nanoparticle (LNP) delivery of mRNA we could express a single molecular factor, Wnt3a, to induce cartilage bridging of the CSD. Our results demonstrate the power of using molecular information from successful regeneration to rescue a non-regenerating injury.

cell biology↗

bioRxiv2025

Mitophagy upregulates WNT5A/Ca2+ signalling to accelerate fibroblast migration and wound healing

In the event of dysregulated wound healing, hard-to-heal chronic wounds form and can place a significant burden on healthcare systems, yet gaps in knowledge surrounding the cellular and molecular processes involved have resulted in a lack of effective treatments. Here, we show that ubiquitin-independent mitophagy is upregulated in the early- and mid-wound healing stages. Additionally, enhancing mitophagy through Urolithin A treatment improved wound healing, in particular by accelerating fibroblast migration. RNAseq analysis demonstrated an upregulation of non-canonical WNT5A signalling in Urolithin A-treated fibroblasts, which was underpinned by elevated cytosolic Ca2+ buffering and CREB phosphorylation, ultimately leading to enhanced actin polymerisation and fibroblast migration. This study is thus the first to elucidate a role for mitophagy specifically in fibroblasts during wound healing; to demonstrate an important role for mitophagy in Ca2+-mediated WNT5A signalling cascades; and indicate the potential therapeutic benefits of treating non-healing wounds with mitophagy inducers such as Urolithin A.

developmental biology↗

bioRxiv2025

Mitophagy promotes metabolic reprogramming to enhance keratinocyte migration via ANGPTL4 during wound healing

Mitochondrial function and quality control is emerging as a key regulator of keratinocyte migration in both wounding and non-wound healing contexts, yet the cellular mechanisms that support this process are incompletely understood. In this study, using single-cell RNA sequencing data of human wounded tissue we identified a distinct population of migrating keratinocytes marked by the high expression of the mitophagy regulator BNIP3 during the proliferative stage of wound healing. Pharmacological induction of mitophagy with Urolithin A accelerated keratinocyte migration in vitro as well as keratinocyte function and regeneration in aged zebrafish, whilst RNA sequencing of primary human keratinocytes revealed the transcriptional upregulation of ANGPTL4 in Urolithin-A treated cells. Mechanistically, Urolithin A increased metabolic switching to a more glycolytic phenotype, leading to AKTGSK3 pathway activity and FOSL1-mediated ANGPTL4 transcription, ultimately promoting keratinocyte migration through enhanced laminin-332 production. Overall, our findings uncover a novel role for mitophagy in promoting keratinocyte migration during wound repair, and demonstrate that pharmacologically enhancing mitophagy promotes regenerative epithelial responses by enhancing FOSL1-mediated ANGPTL4 signalling through the modulation of metabolic switching. These insights significantly expand the understanding of the role of mitophagy on keratinocyte function during wound healing, linking mitophagy to metabolic adaption in keratinocytes, and provide a mechanistic basis for targeting mitophagy or downstream genes in wound healing therapies.

developmental biology↗

bioRxiv2025

Unraveling Fitness Landscapes in Plant-Associated Bacteroidota

Members of the bacterial phylum Bacteroidota inhabit a wide range of ecosystems. Although significant progress has been made over the past decade in understanding the metabolic functions of clinically relevant Bacteroidota, far less is known about the environmental species. Despite being one of the dominant phyla linked to crops, there is limited information on the mechanisms that enable Bacteroidota to thrive in plant and soil environments. A key obstacle in understanding gene fitness in Bacteroidota is the lack of genome-wide functional studies, largely due to their inherent resistance to antibiotics, which complicates genetic manipulation. In this study, we used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) to measure gene fitness in a plant-associated Bacteroidota, Mucilaginibacter yixingensis YX-36. Our data sheds light on pathways involved in rhizosphere colonization, gliding motility, stress tolerance, and carbon metabolism. Notably, we found that phylum-specific genes such as Polysaccharide Utilization Loci (PULs) and Carbohydrate-Active enzymes (CAZymes) are needed for fitness in the plant niche. Overall, this work advances our understanding of gene functions in environmental Bacteroidota species and provides a foundation for future research on their roles in plant-microbe interactions.

microbiology↗

bioRxiv2025

MIF-induced CD74+ microglia/macrophages are clinically relevant disease-associated subpopulations in brain metastasis and other CNS disorders

The upregulation of CD74, a chaperone involved in MHC-II antigen processing 1,2, has been broadly reported in virtually all brain disorders analyzed by single-cell RNA sequencing 3-6. However, its expression is usually interpreted as indicative of antigen presentation. In parallel, CD74 expression has also been described in cancer cells across multiple tumor types, but interestingly in glioma its expression has been mainly identified in the microenvironment. However, the functional contribution of CD74 to disease progression in the brain, and specifically in secondary brain tumors, has not been directly addressed. Here we described that, in contrast to what it has been assumed, the presence of CD74+ microglia/macrophages, which is induced by increased levels of interferon gamma in the brain affected by metastases, does not relate to its canonical pathway. Instead, CD74s alternative function as cytokine receptor is pivotal. Rewired by increasing levels of its ligand MIF, produced by proliferating cancer cells, the CD74 receptor, upon binding to this ligand, translocates to the nucleus activating a NF-{kappa}B-dependent program promoting metastasis progression. A brain metastasis-associated CD74 signature involves a more aggressive progression of the local disease in patients, while it has no clinical correlation with the matched primary tumor. Furthermore, we identified the CD74+ myeloid population in additional brain disorders including Alzheimers disease and multiple sclerosis, which shared a pan-disease non-canonical signature with clinical relevance. The brain-penetrant drug ibudilast, which prevents the binding of MIF to CD74, decreases brain metastases in experimental models in vivo and in patient-derived organotypic cultures ex vivo in a primary tumor-agnostic manner. Our findings suggest that MIF/CD74-induced reprogramming of myeloid cells in brain disorders is a novel vulnerability that could be exploited therapeutically against brain metastases, and possibly other brain disorders, guided by a non-invasive molecular strategy.

cancer biology↗

bioRxiv2025

Quantitative computerized analysis demonstrates strongly compartmentalized tissue deformation patterns underlying mammalian heart tube formation

The quantitative analysis of tissue deformation at cellular resolution remains an important challenge in mammalian organogenesis. Here, we developed a new computational workflow to extract regional and temporal patterns of tissue deformation and applied it to a collection of live microscopy datasets from mouse cardiogenesis. We devised a method to track tissue deformation directly from time-lapse raw images and experimentally validated the method by comparison with actual cell tracks. We then used a machine-learning approach to temporally and spatially align different specimens and reconstruct a single statistical model of tissue motion, deducing maps of strain, anisotropy, and tissue growth. We also implemented a virtual fate mapping tool that allows tracking any initial position in the cardiac primordium onto the linear heart tube. Our study reveals predominant local cellular coherence during the deformation of the cardiac tissue, whereas strong compartmentalization of tissue deformation patterns transforms the bilateral cardiac primordium into a 3D longitudinal heart tube. At the future outer curvature of the primitive tube, the ventricular chamber forms by expansion of the tissue in a hemi-barrel shape with two harnessing belts; one that constrains tissue expansion at the arterial pole and one that constrains the expansion at the venous pole. Our study provides a new approach to understanding heart morphogenesis and proposes a new model of primitive heart tube formation.

developmental biology↗

bioRxiv2025

A method for analysing tissue motion and deformation during mammalian organogenesis

Understanding tissue morphogenesis is an important goal in developmental biology and tissue engineering. Accurately describing tissue deformation processes and how cell rearrangements contribute to these is a challenging task. Live analysis of morphogenesis in 3D is frequently used to obtain source data that allow to extract such features from developing organs. However, several limitations are encountered when applying these methodologies to mammalian embryos. The mouse embryo is the most frequently used model, but most studies use a very limited number of specimens and present only individual acquisitions due to constraints imposed by embryo culture and imaging. Here we used data from live analysis of embryonic heart development to develop methodologies that allow to 1) deduce tissue deformation and cell movements from image intensity flows, 2) stage and register data from several specimens/acquisitions to a dynamic consensus model of heart development, 3) statistically calculate and map to the model tissue growth and anisotropy and 4) generate a digital model of heart development that allows in-silico pseudo-cell fate mapping. Our methodology will greatly facilitate the understanding of morphogenetic processes underlying mammalian organogenesis.

developmental biology↗

bioRxiv2025

Neuronal SEL1L-HRD1 ERAD regulates one-carbon metabolism and is essential for motor function and survival

Hypomorphic variants in the SEL1L-HRD1 ER-associated degradation (ERAD) complex have been linked to severe neurological syndromes in children, including neurodevelopmental delay, intellectual disability, motor dysfunction, and early death. Despite this association, its physiological importance and underlying mechanisms in neurons remain poorly understood. Here, we show that neuronal SEL1L-HRD1 ERAD is essential for maintaining one-carbon metabolism, motor function, and overall viability. Neuron-specific deletion of Sel1L in mice (Sel1LSynCre) resulted in growth retardation, severe motor impairments, and early mortality by 9 weeks of age--mirroring core clinical features observed in affected patients--despite preserved neuronal numbers and only modest ER stress. Multi-omics analyses, including single-nucleus RNA sequencing and metabolomics, revealed significant dysregulation of one-carbon metabolism in ERAD-deficient brains. This included activation of the serine, folate, and methionine pathways, accompanied by elevated levels of S-adenosylmethionine and related metabolites, likely resulted from induction of the integrated stress response (ISR). Together, these findings uncover a previously unappreciated role for neuronal SEL1L-HRD1 ERAD in coordinating ER protein quality control with metabolic adaptation, providing new insight into the molecular basis of ERAD-related neurodevelopmental disease. SummaryUsing a neuron-specific Sel1L knockout mouse model, we demonstrate that Sel1L deficiency activates integrated stress responses, rewires one-carbon metabolism, and impairs motor function and survival.

cell biology↗

bioRxiv2025

Synergistic Control of Mitochondrial Dynamics and Function by ERAD and Autophagy in Brown Adipocytes

Mitochondrial quality control is essential for maintaining cellular energy homeostasis, particularly in brown adipocytes where dynamic mitochondrial remodeling supports thermogenesis. Although the SEL1L-HRD1 endoplasmic reticulum (ER)-associated degradation (ERAD) pathway and autophagy are two major proteostatic systems, how these pathways intersect to regulate mitochondrial integrity in metabolically active tissues remains poorly understood. Here, using adipocyte-specific genetic mouse models combined with high-resolution 2D and 3D ultrastructural imaging technologies, we reveal an unexpected synergy between SEL1L-HRD1 ERAD and autophagy in maintaining mitochondrial structure and function in brown adipocytes. Loss of ERAD alone triggers compensatory autophagy, whereas combined deletion of both pathways (double knockout, DKO) results in severe mitochondrial abnormalities, including the accumulation of hyperfused megamitochondria penetrated by ER tubules, even under basal room temperature conditions. These phenotypes are absent in mice lacking either pathway individually or in SEL1L-IRE1 DKO, highlighting the pathway-specific coordination between ERAD and autophagy. Mechanistically, dual loss of ERAD and autophagy induces ER expansion, excessive ER-mitochondria contact, upregulation of mitochondria-associated membrane (MAM) tethering proteins, impaired calcium transfer, and defective mitochondrial turnover. As a result, DKO adipocytes accumulate dysfunctional mitochondria, exhibit respiratory deficits, and fail to sustain thermogenesis. Collectively, our study uncovers a cooperative and previously unrecognized mechanism of mitochondrial surveillance, emphasizing the critical role of ERAD-autophagy crosstalk in preserving mitochondrial integrity and thermogenic capacity in brown fat. One-sentence summaryOur study uncovers a previously unrecognized synergy between SEL1L-HRD1 ERAD and autophagy that is essential for preserving mitochondrial integrity and thermogenic capacity in brown adipocytes, revealing new opportunities for targeting mitochondrial dysfunction in metabolic disease.

cell biology↗

bioRxiv2025

Integrating structural homology with deep learning to achieve highly accurate protein-protein interface prediction for the human interactome

A significant portion of disease-causing mutations occur at protein-protein interfaces however, the number of structurally resolved multi-protein complexes is extremely small. Here we present a computational pipeline, PIONEER2, that integrates 3D structural similarity with geometric deep learning to accurately predict protein binding partner-specific interfacial residues. We compare the performance of PIONEER2 to that of AlphaFold3 and found, using a test set of PDB structures, that their performance is quite similar. However, about 20% of AlphaFold3 predictions for protein-protein complexes in the PDB have AlphaFold3 ranking scores below 0.5, which indicates an uncertain model. For these structures, PIONEER2 outperforms AlphaFold3 at discriminating interfacial from non-interfacial residues. Further, about half of the AlphaFold3 ranking scores on high confidence protein-protein interactions (PPIs) not associated with a PDB structure are below 0.5 indicating that PIONEER2 offers superior interface prediction for a large number of PPIs for which structures are not available. We created a comprehensive 3D structurally informed interactome encompassing all 352,124 experimentally detected binary human PPIs in the current literature and made PIONEER2 interface predictions for each. We experimentally validated these predictions by generating 1,866 mutations and testing their disruptive impact on 5,010 mutation-interaction pairs. PIONEER2-predicted interfaces are found to be comparable to PDB structures in their ability to predict disruptive mutations while AlphaFold3 performance is reduced. Similarly, PIONEER2-predicted interfaces outperform AlphaFold3 in accounting for the depletion of non-deleterious common population variants and the enrichment of disease-related mutations on protein surfaces. Overall, our results suggest that PIONEER2-predicted interfaces provide a valuable tool for studying disease etiology, advancing personalized medicine and for fundamental research. We further implemented PIONEER2 as a user-friendly web server (https://pioneer2.yulab.org) platform for users to explore our 3D interactome models and conduct genome-wide functional genomics studies.

bioinformatics↗

bioRxiv2025

Forecasting oncogene amplification and tumour suppressor deletion

Oncogene amplification and tumour suppressor deletion can drive tumour initiation, progression and treatment resistance. Detection at diagnosis often signals poor prognosis, but it can also enable opportunities for treatment with highly effective targeted therapies. Predicting the likelihood that a patient will acquire these driver alterations in the future using a genomic test represents an opportunity to realise the benefits of interventions earlier, potentially with preventative intent. Here, we present a forecasting framework that takes as input a DNA copy number profile and predicts whether the tumour will acquire an oncogene amplification or tumour suppressor deletion in the future. This framework leverages mutation rate estimates from the input tumour, alongside gene-specific selection coefficients derived from a large cohort of 7,880 tumours. We demonstrate feasibility using 7,042 single-time-point samples and longitudinally collected tumour pairs from 44 prostate and 100 lung cancers, identifying tumours that went on to acquire amplifications at a later time point with an average AUC of 0.87. We show potential clinical utility by forecasting poor prognosis in low-grade gliomas via CDK4/PDGFRA amplification or CDKN2A deletion, and osimertinib resistance in lung cancers via MET amplification. This study serves as a proof-of-concept for a new class of biomarker, wherein selective pressures and mutation-generating processes can be harnessed to anticipate future genomic alterations.

genomics↗

bioRxiv2025

Exonic enhancers are a widespread class of dual-function regulatory elements

Exonic enhancers (EEs) occupy an under-appreciated niche in gene regulation. By integrating transcription factor binding, chromatin accessibility, and high-throughput enhancer-reporter assays, we demonstrate that many protein-coding exons possess enhancer activity across species. These EEs exhibit characteristic epigenomic signatures, form long-range interactions with gene promoters, and can be altered by both nonsynonymous and synonymous variants. CRISPR-mediated inactivation demonstrated the involvement of EEs in the cis-regulation of host and distal gene expression. Through large-scale cancer genome analyses, we reveal that EE mutations correlate with dysregulated target-gene expression and clinical outcomes, highlighting their potential relevance in disease. Evolutionary comparisons show that EEs exhibit both strong sequence constraint and lineage-specific plasticity, suggesting that they serve ancient regulatory functions while also contributing to species divergence. Our findings redefine the landscape of functional elements by establishing EEs as a component of gene regulation, while revealing how coding regions can simultaneously fulfil both protein-coding and cis-regulatory roles.

genomics↗