Biology subjects

Roule, T.

Publications and source records attributed to Roule, T..

10 recordsLinked to original sources

bioRxiv2026

GNOMES: an integrated framework for genome-wide normalization and differential binding analysis of CUT&RUN and ChIP-seq data

BackgroundDespite their use, quantitative comparison of epigenomic datasets such as ChIP-seq and CUT&RUN remains challenging, particularly due to difficulties in signal normalization across samples and conditions. Normalization solely based on sequencing depth is often insufficient due to the high variability in signal-to-noise ratios across samples, even from a same experiment. While exogeneous spike-in normalization can address some issues, robust spike-in controls are not always available, and may introduce additional experimental burden and computational complexity. Furthermore, normalization and differential binding analysis are typically performed using separate bioinformatics tools. Indeed, most differential analysis frameworks operate on raw count matrices, preventing users from visually inspecting normalized signal tracks and evaluating how normalization influences the results. To overcome these challenges, we developed GNOMES (Genome-wide NOrmalization of Mapped Epigenomic Signals), a framework that integrates signal normalization, quality control, and differential binding analysis within a unified workflow. ResultsGNOMES is a user-friendly tool able to process ChIP-seq and CUT&RUN datasets from aligned reads, and generate normalized coverage profiles and differential binding results. The tool implements a robust genome-wide normalization strategy based on percentile scaling of signal local maxima, enabling stable normalization between biological replicates and conditions. GNOMES supports both single- and paired- end sequencing, does not required a negative control (input or IGG), and can be applied to both broad (histone marks) or narrow (transcription factor) enrichment patterns. The workflow includes normalization, optional consensus peak identification, and differential binding analysis. For each step, GNOMES generates extensive quality-control metrics and visual outputs, including normalized bigWig tracks, median signal tracks, BED files of regions with significant changes, and diagnostic plots such as heatmaps and PCA. GNOMES is highly configurable and integrates established tools such as MACS2 for candidate peak regions identification for differential binding analysis, as well as DESeq2 and edgeR for statistical testing. Finally, GNOMES is organism-agnostic and can be applied to epigenomic datasets from any model system. ConclusionsGNOMES provides an integrated and highly customizable environment for normalization and differential binding analysis of epigenomic sequencing data. By integrating signal normalization, with downstream differential statistical method for differential binding analysis, and comprehensive quality control, GNOMES simplifies the analysis of ChIP-seq and CUT&RUN datasets, for the identification of chromatin changes.

bioinformatics↗

bioRxiv2025

YAP levels regulate anteroposterior elongation of hESC-derived gastruloids

Human embryonic stem cells (hESCs) can self-organize into anteroposterior patterned three-dimensional structures, characterized by polarized expression of CDX2 and GATA6, known as human 3D gastruloids (h3D-gastruloids). This patterning emerges through hESC-intrinsic mechanisms that remain poorly understood. Here, we demonstrate that the formation and elongation of h3D-gastruloids is modulated by the activity levels of the Hippo pathway effector YAP. Using complementary chemical and genetic perturbations, we show that elevated nuclear YAP activity inhibits gastruloid elongation, whereas YAP inhibition enhances axial elongation relative to controls. Single-cell RNA sequencing (scRNA-seq) analyses reveal that YAP activation disrupts the establishment of distinct anterior GATA6 and posterior CDX2 poles. Conversely, YAP inhibition promotes clearer segregation of these domains, facilitating symmetry breaking and elongation. Finally, we developed a high-throughput platform for h3D-gastruloid generation, enabling the production of over 500 gastruloids per well. Together, our findings uncover a role for YAP in orchestrating the three-dimensional organization of human gastruloids.

developmental biology↗

bioRxiv2025

YAP1 and QSER1 are Key Modulators of Embryonic Signaling Pathways in the Mammalian Epiblast

YAP1 signaling is essential for development but its specific roles in early embryogenesis remain poorly understood. To shed light on this, we analyzed YAP1s role in regulating the pluripotency of the mammalian epiblast, using scRNAseq approaches. Conditional deletion of Yap1 in the mouse epiblast (Sox2-Cre) altered the expression of signaling genes, including Nodal, Wnt3, and Fgf8. Accordingly, Yap1 loss led to enhanced differentiation of the epiblast toward primitive streak lineages, as evidenced by the upregulation of T/Brachyury and Eomes genes. Furthermore, a proximity labeling assay in human pluripotent stem cells, followed by biochemical assays and molecular modeling predictions, revealed that YAP1 cooperates with QSER1 protein to regulate lineage genes. Our analysis shows that YAP1:TEAD4 enhancers recruit QSER1 to prevent RNA Polymerase II recruitment. Accordingly, QSER1 depletion, similar to YAP1, increases NODAL gene expression and leads to hyperactive NODAL signaling in human 2D-gastruloids. Overall, our findings define a role of YAP1 in the epiblast in vivo and uncovered an interplay with QSER1 controlling the activity of developmental signaling pathways in pluripotent cells.

developmental biology↗

bioRxiv2024

Single-cell RNA sequencing of mutant whole mouse embryos: from the epiblast to the end of gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, we designed a robust pipeline to perform single-cell and nuclei analysis on mouse embryos from E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to WT perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, we present a methodology designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with FAST genotyping protocol (3 hours) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. We also include guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula. SUMMARYWe establish a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

developmental biology↗

bioRxiv2024

IMPDH2 filaments protect from neurodegeneration in AMPD2 deficiency

Metabolic dysregulation is one of the most common causes of pediatric neurodegenerative disorders. However, how the disruption of ubiquitous and essential metabolic pathways predominantly affect neural tissue remains unclear. Here we use mouse models of AMPD2 deficiency to study cellular and molecular mechanisms that lead to selective neuronal vulnerability to purine metabolism imbalance. We show that AMPD deficiency in mice primarily leads to hippocampal dentate gyrus degeneration despite causing a generalized reduction of brain GTP levels. Remarkably, we found that neurodegeneration resistant regions accumulate micron sized filaments of IMPDH2, the rate limiting enzyme in GTP synthesis. In contrast, IMPDH2 filaments are barely detectable in the hippocampal dentate gyrus, which shows a progressive neuroinflammation and neurodegeneration. Furthermore, using a human AMPD2 deficient neural cell culture model, we show that blocking IMPDH2 polymerization with a dominant negative IMPDH2 variant, impairs AMPD2 deficient neural progenitor growth. Together, our findings suggest that IMPDH2 polymerization prevents detrimental GTP deprivation in neurons with available GTP precursor molecules, providing resistance to neurodegeneration. Our findings open the possibility of exploring the involvement of IMPDH2 assembly as a therapeutic intervention for neurodegeneration.

neuroscience↗

bioRxiv2024

Evolution of cell-type-specific accessible chromatin regions and the cis-regulatory elements that drive lineage-specific innovation

Cis-regulatory elements (CREs) are critical in regulating gene expression, and yet understanding of CRE evolution remains challenging. Here, we constructed a comprehensive single-cell atlas of chromatin accessibility in Oryza sativa, integrating data from 103,911 nuclei representing 126 discrete cell states across nine distinct organs. We used comparative genomics to compare cell-type resolved chromatin accessibility between O. sativa and 57,552 nuclei from four additional grass species (Zea mays, Sorghum bicolor, Panicum miliaceum, and Urochloa fusca). Accessible chromatin regions (ACRs) had different levels of conservation depending on the degree of cell-type specificity. We found a complex relationship between ACRs with conserved noncoding sequences, cell-type specificity, conservation, and tissue-specific switching. Additionally, we found that epidermal ACRs were less conserved compared to other cell types, potentially indicating that more rapid regulatory evolution has occurred in the L1-derived epidermal layer of these species. Finally, we identified and characterized a conserved subset of ACRs that overlapped the repressive histone modification H3K27me3, implicating them as potentially silencer-like CREs maintained by evolution. Collectively, this comparative genomics approach highlights the dynamics of plant cell-type-specific CRE evolution.

plant biology↗

bioRxiv2023

High density SNP array and reanalysis of genome sequencing uncovers CNVs associated with neurodevelopmental disorders in KOLF2.1J iPSCs

The KOLF2.1J iPSC line was recently proposed as a reference iPSC to promote the standardization of research studies in the stem cell field. Due to overall good performance differentiating to neural cell lineages, high gene editing efficiency, and absence of genetic variants associated to neurological disorders KOLF2.1J iPSC line was particularly recommended for neurodegenerative disease modeling. However, our work uncovers that KOLF2.1J hPSCs carry heterozygous small copy number variants (CNVs) that cause DTNBP1, JARID2 and ASTN2 haploinsufficiencies, all of which are associated with neurological disorders. We further determine that these CNVs arose in vitro over the course of KOLF2.1J iPSC generation from a healthy donor-derived KOLF2 iPSC line and affect the expression of DNTBP1, JARID2 and ASTN2 proteins in KOLF2.1J iPSCs and neural progenitors. Therefore, our study suggests that KOLF2.1J iPSCs carry genetic variants that may be deleterious for neural cell lineages. This data is essential for a careful interpretation of neural cell studies derived from KOLF2.1J iPSCs and highlights the need for a catalogue of iPSC lines that includes a comprehensive genome characterization analysis.

genetics↗

bioRxiv2020

ChronoRoot: High-throughput phenotyping by deep segmentation networks reveals novel temporal parameters of plant root system architecture

BackgroundDeep learning methods have outperformed previous techniques in most computer vision tasks, including image-based plant phenotyping. However, massive data collection of root traits and the development of associated artificial intelligence approaches have been hampered by the inaccessibility of the rhizosphere. Here we present ChronoRoot, a system which combines 3D printed open-hardware with deep segmentation networks for high temporal resolution phenotyping of plant roots in agarized medium. ResultsWe developed a novel deep learning based root extraction method which leverages the latest advances in convolutional neural networks for image segmentation, and incorporates temporal consistency into the root system architecture reconstruction process. Automatic extraction of phenotypic parameters from sequences of images allowed a comprehensive characterization of the root system growth dynamics. Furthermore, novel time-associated parameters emerged from the analysis of spectral features derived from temporal signals. ConclusionsAltogether, our work shows that the combination of machine intelligence methods and a 3D-printed device expands the possibilities of root high-throughput phenotyping for genetics and natural variation studies as well as the screening of clock-related mutants, revealing novel root traits.

plant biology↗

bioRxiv2020

The lncRNA MARS modulates the epigenetic reprogramming of the marneral cluster in response to ABA

Clustered organization of biosynthetic non-homologous genes is emerging as a characteristic feature of plant genomes. The co-regulation of clustered genes seems to largely depend on epigenetic reprogramming and three-dimensional chromatin conformation. Here we identified the long noncoding RNA (lncRNA) MARneral Silencing (MARS), localized inside the Arabidopsis marneral cluster, which controls the local epigenetic activation of its surrounding region in response to ABA. MARS modulates the POLYCOMB REPRESSIVE COMPLEX 1 (PRC1) component LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1) binding throughout the cluster in a dose-dependent manner, determining H3K27me3 deposition and chromatin condensation. In response to ABA, MARS decoys LHP1 away from the cluster and promotes the formation of a chromatin loop bringing together the MARNERAL SYNTHASE 1 (MRN1) locus and a distal ABA-responsive enhancer. The enrichment of co-regulated lncRNAs in clustered metabolic genes in Arabidopsis suggests that the acquisition of novel noncoding transcriptional units may constitute an additional regulatory layer driving the evolution of biosynthetic pathways.

genetics↗

bioRxiv2019

Two ecotype-related long non-coding RNAs in the environmental control of root growth

BackgroundRoot architecture varies widely between species and even between ecotypes of the same species despite the strong conservation of the protein-coding portion of their genomes. In contrast, non-coding RNAs evolved rapidly between ecotypes and may control their differential responses to the environment as several long non-coding RNAs (lncRNAs) can quantitatively regulate gene expression.\n\nResultsRoots from Columbia (Col) and Landsberg erecta (Ler) ecotypes respond differently to phosphate starvation. We compared complete transcriptomes (mRNAs, lncRNAs and small RNAs) of root tips from these two ecotypes during early phosphate starvation. We identified thousands of new lncRNAs categorized as intergenic or antisense RNAs that were largely conserved at DNA level in these ecotypes. In contrast to coding genes, many lncRNAs were specifically transcribed in one ecotype and/or differentially expressed between ecotypes independently of the phosphate condition. These ecotype-related lncRNAs were characterized by analyzing their sequence variability among plants and their link with siRNAs. Our analysis identified 675 lncRNAs differentially expressed between the two ecotypes including specific antisense RNAs targeting key regulators of root growth responses. Mis-regulation of several intergenic lncRNAs showed that at least two ecotype-related lncRNAs regulate primary root growth in Col.\n\nConclusionsThe in depth exploration of the non-coding transcriptome of two ecotypes identified thousands of new lncRNAs showing specific expression in root apexes. De-regulation of two ecotype-related lncRNAs revealed a new pathway involved in the regulation of primary root growth. The non-coding genome may reveal novel mechanisms involved in ecotype adaptation of roots to different soil environments.

plant biology↗