Biology subjects

Gaspa-Toneu, L.

Publications and source records attributed to Gaspa-Toneu, L..

4 recordsLinked to original sources

bioRxiv2025

A statistical model for quantitative analysis of single-molecule footprinting data

The binding of sequence-specific TFs (TF) to genomic DNA is fundamental to gene regulation. Emerging single-molecule footprinting (SMF) technologies such as the NOMe-seq and Fiber-seq assays offer unique opportunities for acquiring quantitative information about binding states of TFs and nucleosomes at single-DNA-molecule resolution. Contrasting bulk epigenomic profiling methodologies, SMF enables better molecular characterization of inherently stochastic processes of protein-DNA interactions. Despite the many advantages that SMF technologies bring for studying mechanisms of gene regulation, rigorous statistical models for the analysis of datasets generated using these technologies are still missing. Here, we introduce a novel statistical framework designed for inference of footprint lengths and predictions of footprint positions for unbiased quantitative analysis and interpretation of SMF datasets. We carried out comprehensive computational simulations of SMF experiments and identified experimental parameters that are critical to footprint detection. Finally, we demonstrate the power of this statistical approach for the analysis of genome-wide and amplicon-based NOMe-seq datasets generated for mouse embryonic stem cells.

bioinformatics↗

bioRxiv2025

Modeling host-microbe interactions in immunocompetent engineered human gut tissues

The intestinal mucosal barrier contains microbial organisms within the lumen while preserving the ability to absorb nutrients. Dietary, microbial, and other exposures shaped human barrier evolution and continue to impact disease susceptibility. Here, we established engineered barrier models of the human small intestine and colon composed of a multilineage epithelium, mucus layer, accessible microbial compartment and autologous tissue-resident immune cells. The epithelium has crypt- and villus-like topological domains, with stem cells differentiating into absorptive and secretory lineages with region-specific identities. Secreted mucins accumulate apically, forming a dense mucus layer separating the epithelium from colonizing commensal and pathogenic bacteria. Intestinal memory T cells integrate into and interact with the epithelium. We use the engineered intestinal tissues to identify an epithelial gene regulatory network underlying response to Salmonella Typhimurium infection, and uncover epithelial-immune-pathogen crosstalk coordinating cytokine release and epithelial damage. Overall, this work allows for the modular integration of epithelial, microbial, and immune compartments providing a versatile system for studying human intestinal physiology and pathologies.

bioengineering↗

bioRxiv2025

High-throughput histopathology for complex in vitro models

Human complex in vitro models (CIVMs) have demonstrated remarkable potential to study tissue development, physiology and disease at high-throughput. To effectively employ these miniaturized systems in translational preclinical research, their in-depth benchmarking is pivotal. Histology has been the core of tissue characterization for centuries and the foundation of spatial phenotyping. However, standard histology workflows are inherently low-throughput and centered on large tissue pieces. This does not match the high sample volumes and small sample sizes in CIVM research. Here, we introduce a holistic histo-workflow, utilizing 3D-printed histomolds that facilitate co-planar embedding of CIVMs at high-throughput, resulting in up to 48 samples in one section. We developed a variety of model-specific histomold designs that enable spatially controlled histological sectioning and downstream analyses. We describe these workflows, including mold generation, highplex staining and image analysis, and exemplify their application to histological analyses of various CIVMs. Altogether, the histomolds introduced here afford opportunities for CIVM processing and analysis, while significantly reducing labor and reagent resources, thereby democratizing high-throughput CIVM in histopathology.

pathology↗

bioRxiv2024

DNA methylation modulates nucleosome retention in sperm and H3K4 methylation deposition in early mouse embryos.

DNA methylation (DNAme) serves a stable gene regulatory function in somatic cells (1). In the germ line and during early embryogenesis, however, DNAme undergoes global erasure and re-establishment to support germ cell and embryonic development (2). While de novo DNAme acquisition during male germ cell development is essential for setting genomic DNA methylation imprints, other intergenerational roles for paternal DNAme in defining embryonic chromatin after fertilization are unknown. To approach this question, we reduced levels of DNAme in developing male germ cells through conditional gene deletion of the de novo DNA methyltransferases DNMT3A and DNMT3B in undifferentiated spermatogonia. We observed that DNMT3A serves a DNAme maintenance function in undifferentiated spermatogonia while DNMT3B catalyzes de novo DNAme during spermatogonial differentiation. Mutant male germ cells nevertheless completed their differentiation to sperm. Failing de novo DNAme in Dnmt3a/Dnmt3b double deficient spermatogonia is associated with increased nucleosome occupancy in mature sperm, preferentially at sites with higher CpG content, supporting the model that DNAme modulates nucleosome retention in sperm (3). To assess the impact of altered sperm chromatin in the formation of embryonic chromatin, we measured H3K4me3 occupancy at paternal and maternal alleles in 2-cell embryos using a newly developed transposon-based tagging assay for modified chromatin. Our data show that reduced DNAme in sperm renders paternal alleles permissive for H3K4me3 establishment in early embryos, independently of possible paternal inheritance of sperm born H3K4me3. Together, this study provides first evidence that paternally inherited DNAme directs chromatin formation during early embryonic development.

genetics↗