Centre for Genomic Regulation

In growing cell populations such as tumours, mutations can serve as markers that allow tracking the past evolution from current samples. The genomic analyses of bulk samples and samples from multiple regions have shed light on the evolutionary forces acting on tumours. However, little is known empirically on the spatio-temporal dynamics of tumour evolution. Here, we leverage published data from resected hepatocellular carcinomas, each with several hundred samples taken in two and three dimensions. Using spatial metrics of evolution, we find that tumour cells grow predominantly uniformly within the tumour volume instead of at the surface. We determine how mutations and cells are dispersed throughout the tumour and how cell death contributes to the overall tumour growth. Our methods shed light on the early evolution of tumours in vivo and can be applied to high-resolution data in the emerging field of spatial biology.

Understanding the origin of eukaryotic cells is one of the most difficult problems in all of biology. A key challenge relevant to the question of eukaryogenesis is reconstructing the gene repertoire of the last eukaryotic common ancestor (LECA). As data sets grow, sketching an accurate genomics-informed picture of early eukaryotic cellular complexity requires provision of analytical resources and a commitment to data sharing. Here, we summarise progress towards understanding the biology of LECA and outline a community approach to inferring its wider gene repertoire. Once assembled, a robust LECA gene set will be a useful tool for evaluating alternative hypotheses about the origin of eukaryotes and understanding the evolution of traits in all descendant lineages, with relevance in diverse fields such as cell biology, microbial ecology, biotechnology, agriculture, and medicine. In this Consensus View, we put forth the status quo and an agreed path forward to reconstruct LECA’s gene content.

We present the proteomic profiling of 79 bladder cancers, including treatment‐naïve non‐muscle‐invasive bladder cancer (NMIBC, n = 17), muscle‐invasive bladder cancer (MIBC, n = 51), and neoadjuvant‐treated MIBC ( n = 11). Proteins were extracted from formalin‐fixed, paraffin‐embedded samples and analyzed using data‐independent acquisition, yielding >8,000 quantified proteins. MIBC, compared to NMIBC, shows an extracellular matrix (ECM) and immune response signature as well as alteration of the metabolic proteome together with concomitant depletion of proteins involved in cell–cell adhesion and lipid metabolism. Neoadjuvant treatment did not consistently impact the proteome of the residual tumor mass. NMIBC presents two proteomic subgroups that correlate with histological grade and feature signatures of cell adhesion or lipid/DNA metabolism. Treatment‐naïve MIBC presents three proteomic subgroups with resemblance to the basal‐squamous, stroma‐rich, or luminal subtypes and signatures of metabolism, immune functionality, or ECM. The metabolic subgroup presents an immune‐depleted microenvironment, whereas the ECM and immune subgroups are enriched for markers of M2‐like tumor‐associated macrophages and dendritic cells. Markers for natural killer cells are exclusive for the ECM subgroup, and markers for cytotoxic T cells are a hallmark of the immune subgroup. Endogenous proteolysis is increased in MIBC alongside upregulation of matrix metalloproteases, including MMP‐14. Genomic panel sequencing yielded the prototypical profile of prevalent FGRF3 alterations in NMIBC and TP53 alterations in MIBC. Tumor–stroma interactions of MIBC were investigated by proteomic analysis of patient‐derived xenografts, highlighting specific tumor and stroma contributions to the matrisome and tumor‐induced stromal proteome phenotypes. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The eukaryotic cytoskeleton is formed in part by microtubules, which are relatively rigid filaments with inherent structural polarity. One consequence of this polarity is that the two ends of a microtubule have different properties with important consequences for their cellular roles. These differences are often challenging to probe within the crowded environment of the cell. Fluorescence microscopy–based in vitro assays with purified proteins and stabilized microtubules have been used to characterize polarity-dependent and end-specific behaviors. These assays require ways to visualize the polarity of the microtubules, which has previously been achieved either by the addition of fluorescently tagged motor proteins with known directionality or by fluorescently polarity marking the microtubules themselves. However, classical polarity-marking protocols require a particular chemically modified tubulin and generate microtubules with chemically different plus and minus segments. These chemical differences in the segments may affect the behavior of interacting proteins of interest in an undesirable manner. We present here a new protocol that uses a previously characterized, reversibly binding microtubule plus-end capping protein, a designed ankyrin repeat protein (DARPin), to efficiently produce polarity-marked microtubules with different fluorescently labeled, but otherwise biochemically identical, plus- and minus-end segments. Key features • Produces polarity-marked microtubules with biochemically identical segments • Allows analysis of end-specific and polarity-dependent activities of purified microtubule-associated proteins • Requires purified microtubule plus-end capping DARPin (D1)2 • Concentrations optimized for porcine brain tubulin

Genome-wide premortem DNA methylation patterns can be computationally reconstructed from high-coverage DNA sequences of ancient samples. Because DNA methylation is more conserved across species than across tissues, and ancient DNA is typically extracted from bones and teeth, previous works utilizing ancient DNA methylation maps focused on studying evolutionary changes in the skeletal system. Here we suggest that DNA methylation patterns in one tissue may, under certain conditions, be informative on DNA methylation patterns in other tissues of the same individual. Using the fact that tissue-specific DNA methylation builds up during embryonic development, we identified the conditions that allow for such cross-tissue inference and devised an algorithm that carries it out. We trained the algorithm on methylation data from extant species and reached high precisions of up to 0.92 for validation datasets. We then used the algorithm on archaic humans, and identified more than 1,850 positions for which we were able to observe differential DNA methylation in prefrontal cortex neurons. These positions are linked to hundreds of genes, many of which are involved in neural functions such as structural and developmental processes. Six positions are located in the neuroblastoma breaking point family (NBPF) gene family, which probably played a role in human brain evolution. The algorithm we present here allows for the examination of epigenetic changes in tissues and cell types that are absent from the palaeontological record, and therefore provides new ways to study the evolutionary impacts of epigenetic changes.

GENCODE produces comprehensive reference gene annotation for human and mouse. Entering its twentieth year, the project remains highly active as new technologies and methodologies allow us to catalog the genome at ever-increasing granularity. In particular, long-read transcriptome sequencing enables us to identify large numbers of missing transcripts and to substantially improve existing models, and our long non-coding RNA catalogs have undergone a dramatic expansion and reconfiguration as a result. Meanwhile, we are incorporating data from state-of-the-art proteomics and Ribo-seq experiments to fine-tune our annotation of translated sequences, while further insights into function can be gained from multi-genome alignments that grow richer as more species’ genomes are sequenced. Such methodologies are combined into a fully integrated annotation workflow. However, the increasing complexity of our resources can present usability challenges, and we are resolving these with the creation of filtered genesets such as MANE Select and GENCODE Primary. The next challenge is to propagate annotations throughout multiple human and mouse genomes, as we enter the pangenome era. Our resources are freely available at our web portal www.gencodegenes.org, and via the Ensembl and UCSC genome browsers.

Antigen cross‐presentation is the process whereby small peptides derived from exogenous antigens are attached to MHC‐I molecules triggering CD8+ T lymphocyte activation. The endocytic route of dendritic cells (DCs) is highly specialised for cross‐presentation to initiate cytotoxic immune responses against numerous intracellular pathogens and tumours. In this study, we identify the endosomal protein sorting nexin (SNX) 17 as a key regulator of antigen internalisation and cross‐presentation by DCs. SNX17 expression in DCs guarantees optimal cross‐presentation of soluble, particulate, and Toxoplasma gondii ‐associated antigens. The silencing of SNX17 expression in DCs significantly affected the internalisation of exogenous antigens by fluid‐phase endocytosis, phagocytosis, and more strikingly, T . gondii invasion. We show that SNX17 controls proper integrin recycling, actin cytoskeleton organisation, and phagosomal maturation. Altogether, our findings provide compelling evidence that SNX17 plays a central role in the modulation of the DC endocytic network, which is essential for competent antigen cross‐presentation.

The Complex Portal (www.ebi.ac.uk/complexportal) is a manually curated reference database for molecular complexes. It is a unifying web resource linking aggregated data on composition, topology and the function of macromolecular complexes from 28 species. In addition to significantly extending the number of manually curated complexes, we have massively extended the coverage of the human complexome through the incorporation of high confidence assemblies predicted by machine-learning algorithms trained on large-scale experimental data. The current content of the portal comprising 2150 human complexes has been augmented by 14 964 machine-learning (ML) predicted complexes from hu.MAP3.0. We have refactored the website to enable easy search and filtering of these different classes of protein complexes and have implemented the Complex Navigator, a visualisation tool to facilitate comparison of related complexes in the context of orthology or paralogy. We have embedded the Rhea reaction visualisation tool into the website to enable users to view the catalytic activity of enzyme complexes.

Investigating the binding between proteins and aptamers, such as peptides or RNA molecules, is of crucial importance both for understanding the molecular mechanisms that regulate cellular activities and for therapeutic applications in several pathologies. Here, a new computational procedure, employing mainly docking, clustering analysis, and molecular dynamics simulations, was designed to estimate the binding affinities between a protein and some RNA aptamers, through the investigation of the dynamical behavior of the predicted molecular complex. Using the state‐of‐the‐art software catRAPID, we computationally designed a set of RNA aptamers interacting with the TAR DNA‐binding protein 43 (TDP‐43), a protein associated with several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). We thus devised a computational protocol to predict the RNA–protein molecular complex, so that the structural and dynamical behavior of such a complex can be investigated through extensive molecular dynamics simulation. We hypothesized that the coordinated and synchronized motion of the protein‐binding residues, when in contact with RNA molecule, is a critical requisite in order to have a stable binding. Indeed, we calculated the motion covariance exhibited by the interface residues during molecular dynamics simulation: we tested the results against experimental measurements of binding affinity (in this case, the dissociation constant) for six RNA molecules, resulting in a linear correlation of about 0.9. Our findings suggest that the synchronized movement of interface residues plays a pivotal role in ensuring the stability within RNA–protein complexes, moreover providing insights into the contribution of each interface residue. This promising pipeline could thus contribute to the design of RNA aptamers interacting with proteins.

The non-pathogenic Mycoplasma pneumoniae engineered chassis (Mycochassis) has demonstrated the ability to express therapeutic molecules in vitro and to be effective for treatment of lung infectious diseases in in vivo mouse models. However, the expression of heterologous molecules, whether secreted or exposed on the bacterial membrane has not been optimized to ensure sufficient secretion and/or exposure levels to exert a maximum in vivo biological effect. Here, we have improved the currently used secretion signal from MPN142 protein. We found that mutations at P1’ position of the signal peptide cleavage site do not abrogate secretion but affect it. Increasing hydrophobicity and mutations at the C-terminal of the signal peptide increases secretion. We tested different lipoprotein signal peptides as possible N-terminal protein anchoring motifs on the Mpn cell surface. Unexpectedly we found that these peptides exhibit variable retention and secretion rates of the protein, with some sequences behaving as full secretion motifs. This raises the question of the biological role of the lipobox motif traditionally thought to anchor membrane proteins without a helical transmembrane domain. These results altogether represent a step forward in chassis optimization, offering different sequences for secretion or membrane retention, which could be used to improve Mycochassis as a delivery vector, and broadening its therapeutic possibilities.

Fibroblast growth factor receptor 2 (FGFR2) has been associated with breast cancer. We performed in silico analyses to investigate the FGFR2 mRNA expression and splice variants associated with breast cancer subtypes. Online databases, including cBioPortal and TCGA SpliceSeq, were used to examine the association between the FGFR2 expression and splice variants with breast cancer subtypes. A higher FGFR2 mRNA was significantly associated with luminal, oestrogen receptor (ER)-positive breast cancers, and invasive lobular carcinomas, whereas a lower FGFR2 was associated with human epidermal growth factor receptor 2 (HER2)-positive breast cancer and invasive ductal carcinomas. The epithelial alternatively spliced FGFR2 IIIb isoform was significantly enriched in ER+ breast cancer, while the mesenchymal FGFR2 IIIc isoform was significantly prevalent in HER2+ cancer. Increased levels of FGFR2 and IIIb splice isoforms are associated with less aggressive breast cancer phenotypes, while decreased levels of FGFR2 and increased IIIc splice isoform are associated with more aggressive phenotypes.

Background: The pre-surgical evaluation for drug-resistant epilepsy achieves seizure freedom in only 50–60% of patients. Efforts to identify quantitative intracranial EEG (qEEG) biomarkers of epileptogenicity are needed. This review summarizes and evaluates the design of qEEG studies, discusses barriers to biomarker adoption, and proposes refinements of qEEG study protocols. Methods: We included exploratory and prediction prognostic studies from MEDLINE and Scopus published between 2017 and 2023 that investigated qEEG markers for identifying the epileptogenic network as the surgical target. Cohort parameters, ground truth references, and analytical approaches were extracted. Results: Out of 1789 search results, 128 studies were included. The study designs were highly heterogeneous. Half of the studies included a non-consecutive cohort, with sample sizes ranging from 2 to 166 patients (median of 16). The most common minimum follow-up was one year, and the seizure onset zone was the most common ground truth. Prediction studies were heterogeneous in their analytical approaches, and only 25 studies validated the marker through post-surgical outcome prediction. Outcome prediction performance decreased in larger cohorts. Conversely, longer follow-up periods correlated with higher prediction accuracy, and connectivity-based approaches yielded better predictions. The data and code were available in only 9% of studies. Conclusions: To enhance the validation qEEG markers, we propose standardizing study designs to resemble clinical trials. This includes using a consecutive cohort with long-term follow-up, validating against surgical resection as ground truth, and evaluating markers through post-surgical outcome prediction. These considerations would improve the reliability and clinical adoption of qEEG markers.

Nuclear metabolism and DNA damage response are intertwined processes, but the precise molecular links remain elusive. Here, we explore this crosstalk using triple-negative breast cancer (TNBC) as a model, a subtype often prone to DNA damage accumulation. We show that the de novo purine synthesis enzyme IMPDH2 is enriched on chromatin in TNBC compared to other subtypes. IMPDH2 chromatin localization is DNA damage dependent, and IMPDH2 repression leads to DNA damage accumulation. On chromatin, IMPDH2 interacts with and modulates PARP1 activity by controlling the nuclear availability of NAD⁺ to fine-tune the DNA damage response. However, when IMPDH2 is restricted to the nucleus, it depletes nuclear NAD⁺, leading to PARP1 cleavage and cell death. Our study identifies a non-canonical nuclear role for IMPDH2, acting as a convergence point of nuclear metabolism and DNA damage response.

Subcellular compartmentalization of metabolic enzymes establishes a unique metabolic environment that elicits specific cellular functions. Indeed, the nuclear translocation of certain metabolic enzymes is required for epigenetic regulation and gene expression control. Here, we show that the nuclear localization of the mitochondrial enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) ensures mitosis progression. Nuclear MTHFD2 interacts with proteins involved in mitosis regulation and centromere stability, including the methyltransferases KMT5A and DNMT3B. Loss of MTHFD2 induces severe methylation defects and impedes correct mitosis completion. MTHFD2 deficient cells display chromosome congression and segregation defects and accumulate chromosomal aberrations. Blocking the catalytic nuclear function of MTHFD2 recapitulates the phenotype observed in MTHFD2 deficient cells, whereas restricting MTHFD2 to the nucleus is sufficient to ensure correct mitotic progression. Our discovery uncovers a nuclear role for MTHFD2, supporting the notion that translocation of metabolic enzymes to the nucleus is required to meet precise chromatin needs.

The spliceosome is the complex molecular machinery that sequentially assembles on eukaryotic messenger RNA precursors to remove introns (pre-mRNA splicing), a physiologically regulated process altered in numerous pathologies. We report transcriptome-wide analyses upon systematic knock down of 305 spliceosome components and regulators in human cancer cells and the reconstruction of functional splicing factor networks that govern different classes of alternative splicing decisions. The results disentangle intricate circuits of splicing factor cross-regulation, reveal that the precise architecture of late-assembling U4/U6.U5 tri–small nuclear ribonucleoprotein (snRNP) complexes regulates splice site pairing, and discover an unprecedented division of labor among protein components of U1 snRNP for regulating exon definition and alternative 5′ splice site selection. Thus, we provide a resource to explore physiological and pathological mechanisms of splicing regulation.

During cell division, the microtubule cytoskeleton undergoes dramatic cell cycle-driven reorganizations of its architecture. Coordinated by changes in the phosphorylation patterns of a multitude of microtubule associated proteins, the mitotic spindle first self-assembles to capture the chromosomes and then reorganizes in anaphase as the chromosomes are segregated. A key protein for this reorganization is PRC1 which is differentially phosphorylated by the mitotic kinases CDK1 and PLK1. How the phosphorylation state of PRC1 orchestrates spindle reorganization is not understood mechanistically. Here, we reconstitute in vitro the transition between metaphase and anaphase-like microtubule architectures triggered by the changes in PRC1 phosphorylation. We find that whereas PLK1 regulates its own recruitment by PRC1, CDK1 controls the affinity of PRC1 for antiparallel microtubule binding. Dephosphorylation of CDK1-phosphorylated PRC1 is required and sufficient to trigger the reorganization of a minimal anaphase midzone in the presence of the midzone length controlling kinesin KIF4A. These results demonstrate how phosphorylation-controlled affinity changes regulate the architecture of active microtubule networks, providing new insight into the mechanistic underpinnings of the cell cycle-driven reorganization of the central spindle during mitosis.

The genomic revolution has fueled rapid progress in synthetic and systems biology, opening up new possibilities for using live biotherapeutic products (LBP) to treat, attenuate or prevent human diseases. Among LBP, bacteria-based therapies are particularly promising due to their ability to colonize diverse human tissues, modulate the immune system and secrete or deliver complex biological products. These bacterial LBP include engineered pathogenic species designed to target specific diseases, and microbiota species that promote microbial balance and immune system homeostasis, either through local administration or the gut-body axes. This review focuses on recent advancements in preclinical and clinical trials of bacteria-based LBP, highlighting both on-site and long-reaching strategies.

The genome of Hepatitis B virus (HBV) persists in infected hepatocytes as a nuclear episome (cccDNA) that is responsible for the transcription of viral genes and viral rebound, following antiviral treatment arrest in chronically infected patients. There is currently no clinically approved therapeutic strategy able to efficiently target cccDNA (Lucifora J 2016). The development of alternative strategies aiming at permanently abrogating HBV RNA production requires a thorough understanding of cccDNA transcriptional and post-transcriptional regulation. In a previous study, we discovered that 1C8, a compound that inhibits the phosphorylation of some cellular RNA-binding proteins, could decrease the level of HBV RNAs. Here, we aimed at identifying kinases responsible for this effect. Among the kinases targeted by 1C8, we focused on DYRK1A, a dual-specificity kinase that controls the transcription of cellular genes by phosphorylating transcription factors, histones, chromatin regulators as well as RNA polymerase II. The results of a combination of genetic and chemical approaches using HBV-infected hepatocytes, indicated that DYRK1A positively regulates the production of HBV RNAs. In addition, we found that DYRK1A associates with cccDNA, and stimulates the production of HBV nascent RNAs. Finally, reporter gene assays showed that DYRK1A up-regulates the activity of the HBV enhancer 1/X promoter in a sequence-dependent manner. Altogether, these results indicate that DYRK1A is a proviral factor that may participate in the HBV life cycle by stimulating the production of HBx, a viral factor absolutely required to trigger the complete cccDNA transcriptional program.