Biology subjects

Costa, I. G.

Publications and source records attributed to Costa, I. G..

15 recordsLinked to original sources

bioRxiv2026

PDE3A-SLFN12 Molecular Glues Target Multiple KIT D816V Cell Types in Preclinical Models of Mast Cell Malignancies

A drug discovery approach was used to specifically target malignant cells with KIT D816V mutation, which is the predominant disease-causing mutation in clonal mast cell malignancies. To this end, KIT D816V cells derived from induced pluripotent stem cells (iPS cells) of KIT D816V patients were employed to screen a library of FDA approved and experimental drugs for specific killing of KIT D816V cells. We discovered the novel compound LDC 3416, which targets multiple malignant KIT D816V cell types, including hematopoietic stem/progenitor cells and mast cells. Importantly, by exploring the LDC 3416 targeting profile, we identified the phosphodiesterase 3A-Schlafen 12 (PDE3A-SLFN12) molecular glue pathway as a novel approach for specific targeting of malignant KIT D816V cells. We found that the KIT D816V mutant protein leads to increased expression of PDE3A and SLFN12 and thus confers a selective molecular vulnerability to PDE3A-SLFN12 molecular glues. Primary malignant mast cells of KIT D816V patients with indolent and advanced systemic mastocytosis also exhibit increased expression of PDE3A and SLFN12. We extended our study to include additional PDE3A-SLFN12 molecular glues and demonstrate their synergistic action with KIT D816V selective tyrosine kinase inhibitors (TKIs) in killing KIT D816V cells. Furthermore, the PDE3A-SLFN12 molecular glues also target KIT D816V megakaryocytes, a cell type that has been underestimated in malignant mast cell pathophysiology and molecular targeting. The identified molecular glues, along with their synergy with TKIs and their simultaneous targeting of multiple KIT D816V cell types, open novel treatment options for KIT D816V mast cell malignancies and other KIT D816V associated diseases.

cancer biology↗

bioRxiv2026

IntraTalker - Modelling Intracellular Signalling in1 Cellular Crosstalk

Single-cell sequencing has advanced the study of cell-cell communication, yet most methods focus on intercellular ligand-receptor interactions while neglecting downstream intracellular signalling cascades and the possibility that downstream target genes themselves encode ligands, thereby propagating communication across multiple cells. We present IntraTalker+CrossTalkeR that combines intracellular (IntraTalker) and intercellular (CrossTalkeR) signalling from multimodal single-cell data. IntraTalker infers cell-type-specific transcription factor activities and constructs receptomes that link receptors to downstream target genes, which are then integrated with ligand-receptor predictions in CrossTalkeR. To prioritize signalling receptors, the framework performs in silico receptor perturbation. In a murine bone marrow dataset, this recovered the known function of the Il1r1 receptor in driving myeloid progenitor cell-state changes. In a human myocardial infarction dataset, it predicted a novel role for IGF1R signalling in driving the differentiation of fibroblasts towards progenitor fibroblast states.

bioinformatics↗

bioRxiv2025

Developmental factors drive the compartmentalised and discontinuous maturation of the small intestinal epithelium during the early postnatal period

The temporal, spatial and cellular diversity of the small intestinal epithelium during the postnatal period, a critical time window that accompanies the transition from placental energy supply to enteral feeding, the establishment of the enteric microbiota and postnatal immune maturation, has not been systematically investigated. Here, we used laser capture microdissection and bulk RNA-Seq, proteomics, and single cell RNA-Seq to analyse the total, organ site, crypt- and villus-specific intestinal epithelium of specific pathogen-free, germ-free and Salmonella-infected mice during the postnatal period. We identified key temporal and organ-site specific expression patterns that revealed a weak effect of the microbiota but strong influence of developmental regulators during early life. We also determined age-dependent signalling pathway and transcription factor activity and characterised age- and cell type-specific developmental trajectories revealing a distinct compartmentalised maturation process along the proximal-to-distal length and crypt-villus axis and a discontinuous appearance of goblet/Paneth cell and absorptive enterocyte transcriptional profiles. Finally, we described the cell type-specific response to neonatal enteric infection. Taken together, our findings identify the epithelium as an integral element in the maturation of postnatal mucosal tissues and in the establishment of host-microbe homeostasis.

immunology↗

bioRxiv2024

SIngle cell level Genotyping Using scRna Data (SIGURD)

MotivationBy accounting for variants within measured transcripts, it is possible to evaluate the status of somatic variants using single-cell RNA-sequencing (scRNA-seq) and to characterize their clonality. However, the sparsity (very few reads per transcript) or bias in protocols (favoring 3 ends of the transcripts) makes the chance of capturing somatic variants very unlikely. This can be overcome by targeted sequencing or the use of mitochondrial variants as natural barcodes for clone identification. Currently, available computational tools focus on genotyping, but do not provide functionality for combined analysis of somatic and mitochondrial variants and functional analysis such as characterization of gene expression changes in detected clones. ResultsHere, we propose SIGURD, which is an R-based pipeline for the clonal analysis of single-cell RNA-seq data. This allows the quantification of clones by leveraging both somatic and mitochondrial variants. SIGURD also allows for functional analysis after clonal detection: association of clones with cell populations, detection of differentially expressed genes across clones and association of somatic and mitochondrial variants. Here, we demonstrate the power of SIGURD by analyzing single-cell data of colony-forming cells derived from patients with myeloproliferative neoplasms. AvailabilityCode and tutorial of SIGURD are available at GitHub https://github.com/CostaLab/sigurd. Contactivan.costa@rwth-aachen.de, skoschmieder@ukaachen.de Supplementary InformationSupplementary data are available online.

bioinformatics↗

bioRxiv2024

Weighted 2D-kernel density estimations provide a new probabilistic measure for epigenetic age

BackgroundEpigenetic aging signatures can provide insights into the human aging process. Within the last decade many alternative epigenetic clocks have been described, which are typically based on linear regression analysis of DNA methylation at multiple CG dinucleotides (CpGs). However, this approach assumes that the epigenetic modifications follow either a continuous linear or logarithmic trajectory. In this study, we explored an alternative non-parametric approach using 2D-kernel density estimation (KDE) to determine epigenetic age. ResultsWe used Illumina BeadChip profiles of blood samples of various studies, exemplarily selected the 27 CpGs with highest linear correlation with chronological age (R2 > 0.7), and computed KDEs for each of them. The probability profiles for individual KDEs were further integrated by a genetic algorithm to assign an optimal weight to each CpG. Our weighted 2D-kernel density estimation model (WKDE) facilitated age-predictions with similar correlation and precision (R2 = 0.81, median absolute error = 4 years) as other commonly used clocks. Furthermore, our approach provided a variation score, which reflects the inherent variation of age-related epigenetic changes at different CpG sites within a given sample. An increase of the variation score by one unit reduced the mortality risk by 9.2% (95% CI (0.8387, 0.9872), P <0.0160) in the Lothian Birth Cohort 1921 after adjusting for chronological age and sex. ConclusionsWe describe a new method using weighted 2D-kernel density estimation (WKDE) for accurate epigenetic age-predictions and to calculate variation scores, which provide an additional variable to estimate biological age.

genomics↗

bioRxiv2024

Cancer-associated fibroblasts reveal aberrant DNA methylation across different types of cancer

Cancer-associated fibroblasts (CAFs) are critical components of the tumor microenvironment. Several studies demonstrated molecular differences between CAFs and normal tissue-associated fibroblasts (NAFs). In this study, we isolated CAFs and NAFs from liver tumors and analyzed their DNA methylation profiles. A subset of the CAFs exhibited aberrant DNA methylation, which was also reflected on gene expression level. The DNA methylation at liver-CAF-specific CG dinucleotides (CpGs) was associated with survival in liver cancer data. An integrative analysis with public datasets of CAF versus NAF in different cancer types, including lung, prostate, esophagus, and gastric cancer, revealed overlapping epigenetic aberrations. CpGs with common aberrations in DNA methylation included cg09809672 (EDARADD), cg07134930 (HDAC4), and cg05935904 (intergenic). Aberrant DNA methylation at these sites was associated with prognosis in several cancer types. Thus, activation of CAFs by the tumor environment is associated with characteristic epigenetic modifications that could be used as biomarkers for disease stratification.

cancer biology↗

bioRxiv2022

Neonatal Peyer's patch cDC activation as a pacemaker of postnatal immune maturation

Marked differences exist between the mucosal immune system of the neonate and adult host. The pronounced influence of the enteric microbiota in adults suggests a causal relationship between postnatal colonization and immune maturation. However, using metagenomic, metaproteomic, and functional immunological analyses we demonstrate an early presence of bacteria and immunogenic microbial antigens preceding immune maturation in the small intestine, the primary inductive site of intestinal immunity. Instead, transcriptomic, flow cytometric and histological analysis indicated neonatal Peyers patch (PP) mononuclear phagocytes (MNP) as rate limiting factor of postnatal immune maturation. Despite the early presence of MNPs, conventional dendritic cells (cDC) of type 1, 2a and 2b exhibited significant age-dependent differences in tissue distribution and cellular composition. Single cell transcriptional profiling and functional assays revealed decreased antimicrobial and antigen processing/presentation capacity, an overall retarded cell maturation and reduced antigen uptake. In cDC2a this resulted in a reduced proportion of CCR7+ migratory cells and a consequent defect in CD4 T cell priming. Interestingly, transcriptional profiling of neonatal DC subsets identified reduced expression of type I interferon (IFN)-stimulated genes (ISG). Type I IFN induction by oral administration of the TLR7 agonist R848 accelerated MNP maturation and enhanced cognate antigen CD4 T cell priming. However, humoral responses to oral vaccination in the presence of R848 were significantly reduced. Together, our results identify PP MNP maturation as pacemaker of postnatal mucosal immune priming, indicate the biological role of delayed maturation and demonstrate that targeted interventional strategies allow manipulation of mucosal responses in early life.

immunology↗

bioRxiv2022

Induction of senescence upon loss of the Ash2l core subunit of H3K4 methyltransferase complexes

Gene expression is controlled in part by post-translational modifications of core histones. Methylation of lysine 4 of histone H3 (H3K4), associated with open chromatin and gene transcription, is catalyzed by type 2 lysine methyltransferase complexes that require WDR5, RBBP5, ASH2L and DPY30 as core subunits. Ash2l is essential during embryogenesis and for maintaining adult tissues. To expand on the mechanistic understanding of Ash2l, we generated mouse embryo fibroblasts (MEFs) with conditional Ash2l alleles. Upon loss of Ash2l, methylation of H3K4 and gene expression were downregulated, which correlated with inhibition of proliferation and cell cycle progression. Moreover, we observed induction of senescence concomitant with a set of downregulated signature genes but independent of SASP. Many of the signature genes are FoxM1 responsive. Indeed, exogenous FOXM1 was sufficient to delay senescence. Thus, although the loss of Ash2l in MEFs has broad and complex consequences, a distinct set of downregulated genes promotes senescence.

cell biology↗

bioRxiv2021

HIF1α-AS1 is a DNA:DNA:RNA triplex-forming lncRNA interacting with the HUSH complex

DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1-AS1 was retrieved as a top hit. Endogenous HIF1-AS1 reduced the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1-AS1 was down-regulated in pulmonary hypertension and loss-of-function approaches not only resulted in gene de-repression but also enhanced angiogenic capacity. As exemplified here with HIF1-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control.

molecular biology↗

bioRxiv2021

CALR frameshift mutations in MPN patient-derived iPS cells accelerate maturation of megakaryocytes

Calreticulin (CALR) mutations are driver mutations in myeloproliferative neoplasms (MPNs), leading to activation of the thrombopoietin receptor, and causing abnormal megakaryopoiesis. Here, we generated patient-derived CALRins5- or CALRdel52-positive induced pluripotent stem (iPS) cells to establish a MPN disease model for molecular and mechanistic studies. We demonstrated myeloperoxidase deficiency in CD15+ granulocytic cells derived from homozygous CALR-mutant iPS cells, rescued by repairing the mutation using CRISPR/Cas9. iPS cell-derived megakaryocytes showed characteristics of primary megakaryocytes such as formation of demarcation membrane system and cytoplasmic pro-platelets protrusions. Importantly, CALR mutations led to enhanced megakaryopoiesis and accelerated megakaryocytic development in a thrombopoietin-independent manner. Mechanistically, our study identified differentially regulated pathways in mutated vs. unmutated megakaryocytes, such as hypoxia signaling, which represents a potential target for therapeutic intervention. Altogether, we demonstrate key aspects of mutated CALR-driven pathogenesis, dependent on its zygosity and found known and novel therapeutic targets, making our model a valuable tool for clinical drug screening in MPNs.

cancer biology↗

bioRxiv2021

Comparison of Resources and Methods to infer Cell-Cell Communication from Single-cell RNA Data

The growing availability of single-cell data has sparked an increased interest in the inference of cell-cell communication from this data. Many tools have been developed for this purpose. Each of them consists of a resource of intercellular interactions prior knowledge and a method to predict potential cell-cell communication events. Yet the impact of the choice of resource and method on the resulting predictions is largely unknown. To shed light on this, we created a framework, available at https://github.com/saezlab/ligrec_decoupler, to facilitate a comparative assessment of methods for inferring cell-cell communication from single cell transcriptomics data and then compared 15 resources and 6 methods. We found few unique interactions and a varying degree of overlap among the resources, and observed uneven coverage in terms of pathways and biological categories. We analysed a colorectal cancer single cell RNA-Seq dataset using all possible combinations of methods and resources. We found major differences among the highest ranked intercellular interactions inferred by each method even when using the same resources. The varying predictions lead to fundamentally different biological interpretations, highlighting the need to benchmark resources and methods. FindingsO_LIBuilt a framework to systematically combine 15 resources and 6 methods to estimate cell-cell communication from single-cell RNA data C_LIO_LICell-cell communication resources are often built from the same original databases and very few interactions are unique to a single resource. Yet overlap varies among resources and certain biological terms are unevenly represented C_LIO_LIDifferent methods and resources provided notably different results C_LIO_LIThe observed disagreement among the methods could have a considerable impact on the interpretation of results C_LI

bioinformatics↗

bioRxiv2020

The spatial self-organization within pluripotent stem cell colonies is continued in detaching aggregates

Colonies of induced pluripotent stem cells (iPSCs) reveal aspects of self-organization even under culture conditions that maintain pluripotency. To investigate the dynamics of this process under spatial confinement, we used either polydimethylsiloxane (PDMS) pillars or micro-contact printing of vitronectin. There was a progressive upregulation of OCT4, E-cadherin, and NANOG within 70 {micro}m from the outer rim of iPSC colonies. Single- cell RNA-sequencing demonstrated that OCT4high subsets have pronounced up-regulation of the TGF-{beta} pathway, particularly of NODAL and its inhibitor LEFTY, at the rim of the colonies. Furthermore, calcium-dependent cell-cell interactions were found to be relevant for the self-organization. Interestingly, after 5 to 7 days, the iPSC colonies detached spontaneously from micro-contact printed substrates to form 3D aggregates. This new method allowed generation of embryoid bodies (EBs) of controlled size, without any enzymatic or mechanical treatment. Within the early 3D aggregates, the radial organization and differential gene expression continued in analogy to the changes observed during self-organization of iPSC colonies. Our results provide further insight into the gradual self-organization within iPSC colonies and at their transition into EBs.

cell biology↗

bioRxiv2019

scOpen: chromatin-accessibility estimation of single-cell ATAC data

A major drawback of single cell ATAC (scATAC) is its sparsity, i.e. open chromatin regions with no reads due to loss of DNA material during the scATAC-seq protocol. We propose scOpen, a computational method for imputing and quantifying the open chromatin status of regulatory regions from sparse scATAC-seq experiments. We show that scOpen improves crucial down-stream analysis steps of scATAC-seq data as clustering, visualisation, cis-regulatory DNA interactions and delineation of regulatory features. We demonstrate the power of scOpen to dissect regulatory changes in the development of fibrosis in the kidney. This identified a novel role of Runx1 and target genes by promoting fibroblast to myofibroblast differentiation driving kidney fibrosis.

bioinformatics↗

bioRxiv2019

Unique and assay specific features of NOMe-, ATAC- and DNase I-seq data

Chromatin accessibility maps are important for the functional interpretation of the genome. Here, we systematically analysed assay specific differences between DNase I-Seq, ATAC-Seq and NOMe-Seq in a side by side experimental and bioinformatic setup. We observe that most prominent nucleosome depleted regions (NDRs, e.g. in promoters) are roboustly called by all three or at least two assays. However we also find a high proportion of assay specific NDRs that are often "called" by only one of the assays. We show evidence that these assay specific NDRs are indeed genuine open chromatin sites and contribute important information for accurate gene expression prediction. While technically ATAC-Seq and DNAse I-Seq provide a high NDR calling rate for relatively low sequencing costs in comparison to NOMe-Seq, NOMe-Seq singles out as it provides a multitude of information: it allows to not only detect NDRs but also endogenous DNA methylation, genome wide segmentation into heterochromatic A/B domains and local phasing of nucleosomes outside of NDRs. In summary our comparison strongly suggest to consider assay specific differences for the experimental desgin and for generalized and comparative functional interpretations.

genomics↗

bioRxiv2017

Topological Demarcation By HMGB2 Is Disrupted Early Upon Senescence Entry Across Cell Types And Induces CTCF Clustering

Ageing-relevant processes, like cellular senescence, are characterized by complex, often stochastic, events giving rise to heterogeneous cell populations. We hypothesized that entry into senescence of different primary human cells can be triggered by one early molecular event affecting the spatial organization of chromosomes. To test this, we combined whole-genome chromosome conformation capture, population and single-cell transcriptomics, super-resolution imaging, and functional analyses applied on proliferating and replicatively-senescent populations from three distinct human cell types. We found a number of genes involved in DNA conformation maintenance being suppressed upon senescence across cell types. Of these, the abundant high mobility group (HMG) B1 and B2 nuclear factors are quantitatively removed from cell nuclei before typical senescence markers appear, and mark a subset of topologically-associating domain (TAD) boundaries. Their loss coincides with obvious reorganization of chromatin interactions via the dramatic spatial clustering of CTCF foci. HMGB2 knock-down recapitulates this senescence-induced CTCF clustering, while also affecting insulation at TAD boundaries. We accordingly propose that HMGB-mediated deregulation of chromosome conformation constitutes a primer for the ensuing senescent program across cell types.

genomics↗