Biology subjects

Poulet, A.

Publications and source records attributed to Poulet, A..

8 recordsLinked to original sources

bioRxiv2026

Systemic RNAi in planarians depends on spread of RNPs from active stem cells

RNAi is a powerful cellular defense mechanism against genomic invaders that rely on dsRNA intermediates, such as viruses and mobile elements. Due to its specificity and ease of use, RNAi is also widely used as an experimental and therapeutic strategy to reduce levels of specific RNAs. For many systems however, delivery of the silencing agents into each individual cell is a major challenge. A mechanistic understanding of the processes involved in intercellular spread in systems with effective systemic RNAi thus is key to improve applications, and to increase our understanding of this defense mechanism. Remarkably, outside of C. elegans and plants, our molecular understanding of systemic RNAi remains very limited. We here investigated the highly-effective systemic RNAi of the planarian S. mediterranea, which can be triggered by introduction of dsRNA via food or injection and rapidly spreads through the entire body. Notwithstanding its efficiency, we find no evidence of an RNA amplification mechanism or of transgenerational effects as are found in C. elegans, and rather find that planarian RNAi effects are limited in time. We identify the biogenesis factors involved in the planarian RNAi mechanism, and find that these are independent of the miRNA pathway, enabling the separation of the effects from these small RNA pathways. Surprisingly, we find that planarian systemic RNAi relies on active stem cells. Further, we identify Argonaute-siRNA complexes as the mobile agent that effectuates systemic spread of RNAi throughout the tissue. These findings provide new insights into the mechanisms by which small RNAs spread between cells, and by which organisms can extend protection to all their cells upon encounter of a novel invading element. Additionally, our findings may have important implications for the design of effective applications in other systems.

molecular biology↗

bioRxiv2026

Two tiers of piRNA clusters balance diversification of piRNAs with limitation of off-target effects

PIWI-interacting RNAs (piRNAs) are important factors in the protection of the genome against nucleic acid invaders such as transposons. piRNAs are encoded in the genome in regions known as piRNA clusters, but major questions remain surrounding their regulation. Two such questions are how the piRNA clusters are recognized as piRNA sources, and how the piRNA response can expand from these regions to facilitate the recognition of novel invading sequences without risking the targeting of essential cellular mRNAs. Here, we investigated the piRNA clusters of the planarian S, mediterranea, and found that the clusters differ regarding the PIWI proteins their piRNAs bind to, as well as their chromatin features. We uncovered a subset of piRNA clusters that had unique chromatin features and atypical transcription. piRNAs from these clusters were depleted of genic content, stabilized by 3 methylation and were not dependent on the ping-pong mechanism. We therefore named these clusters Seed clusters. We identified a second set of piRNA clusters that we called Spread clusters, which had features reminiscent of pseudogenes or aberrant genic transcripts. piRNA generation from these clusters relied on ping-pong, and piRNAs largely remained unmethylated. Further, we found that many more regions in the genome generated single ping-pong events, suggesting that ping-pong is used as a means to diversify the collection of piRNA-generating transcripts beyond the Seed clusters. We propose that this two-tiered organization of the piRNA clusters allows the stable targeting of known genomic threats by the piRNAs generated from the Seed clusters, while the flexible generation of additional diverse piRNAs from Spread clusters as well as other aberrant transcripts increases the sequence space probed by the piRNA system. The absence of methylation on these additional piRNAs decreases their lifespan and limits the chances of a run-away response. This two-tiered organization thus solves a core challenge of the piRNA system and may be a widespread feature of piRNA systems across animals.

molecular biology↗

bioRxiv2025

Single-cell SNP-DNA sequencing precisely maps genotoxic events in CRISPR-edited primary cells

Genome editing by CRISPR-Cas9 is promising for gene therapy. However, safety concerns remain, particularly regarding the ON-target genotoxicity associated with protocols using nucleases. Monitoring the genotoxicity of edited cells before and after graft is essential, especially to assay potentially deleterious megabase-scale genomic rearrangements induced at the targeted locus. High sensitivity requires single-cell resolution. Here, we developed an integrated approach combining targeted single-cell DNA sequencing focused on single nucleotide polymorphism (scSNP-DNAseq) with complementary micronuclei and LOH cytometry-reporter assays. This multiplexed strategy enables orthogonal readouts to accurately monitor CRISPR-mediated genotoxicity in primary cells. Using this approach, we detected, mapped and characterized various types of induced-losses of heterozygosity (terminal, interstitial, copy-loss and copy-neutral). Our compelling workflow assessed editing-associated chromosomal instability linked to double strand break after editing. Importantly, palbociclib prevented the appearance of such genomic rearrangements in hematopoietic stem/progenitor cells without impairing cell fate or graft capability. Conversely, short-term risk was significantly increased with DNA-PKcs inhibitor AZD7648 (HDR booster) in HSPCs and fibroblasts. Fortunately, targeting HBG1/2 in Chr11p in HSPCs, scSNP-DNA-seq revealed that ON-target genotoxic events were no longer detectable after long-term xenografts, even in AZD7648-treated cells. This work demonstrates that scSNP-DNA-seq should be routinely implemented to monitor chromosomal rearrangements before and after CRISPR-edited cell infusions.

bioengineering↗

bioRxiv2024

H3.1K27M-induced misregulation of the TSK/TONSL-H3.1 pathway causes genomic instability

The oncomutation lysine 27-to-methionine in histone H3 (H3K27M) is frequently identified in tumors of patients with diffuse midline glioma-H3K27 altered (DMG-H3K27a). H3K27M inhibits the deposition of the histone mark H3K27me3, which affects the maintenance of transcriptional programs and cell identity. Cells expressing H3K27M are also characterized by defects in genome integrity, but the mechanisms linking expression of the oncohistone to DNA damage remain mostly unknown. In this study, we demonstrate that expression of H3.1K27M in the model plant Arabidopsis thaliana interferes with post-replicative chromatin maturation mediated by the H3.1K27 methyltransferases ATXR5 and ATXR6. As a result, H3.1 variants on nascent chromatin remain unmethylated at K27 (H3.1K27me0), leading to ectopic activity of TONSOKU (TSK), which induces DNA damage and genomic alterations. Elimination of TSK activity suppresses the genome stability defects associated with H3.1K27M expression, while inactivation of specific DNA repair pathways prevents survival of H3.1K27M-expressing plants. Overall, our results suggest that H3.1K27M disrupts the chromatin-based mechanisms regulating TSK/TONSL activity, which causes genomic instability and may contribute to the etiology of DMG-H3K27a.

genetics↗

bioRxiv2022

Deficiency of Heterogeneous Nuclear Ribonucleoprotein U leads to delayed neurogenesis

Genetic variants affecting Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU) have been identified in several neurodevelopmental disorders (NDDs). HNRNPU is widely expressed in the human brain and shows the highest postnatal expression in the cerebellum. Recent studies have investigated the role of HNRNPU in cerebral cortical development, but the effects of HNRNPU deficiency on cerebellar development remain unknown. Here, we describe the molecular and cellular outcomes of HNRNPU locus deficiency during in vitro neural differentiation of patient-derived and isogenic neuroepithelial stem cells with a hindbrain profile. We demonstrate that HNRNPU deficiency leads to chromatin remodeling of A/B compartments, and transcriptional rewiring, partly by impacting exon inclusion during mRNA processing. Genomic regions affected by the chromatin restructuring and host genes of exon usage differences show a strong enrichment for genes implicated in epilepsies, intellectual disability, and autism. Lastly, we show that at the cellular level. HNRNPU downregulation leads to altered neurogenesis and an increased fraction of neural progenitors in the maturing neuronal population. We conclude that, HNRNPU locus is involved in delayed commitment of neural progenitors to neuronal maturation in cell types with hindbrain profile.

neuroscience↗

bioRxiv2021

NODeJ: an ImageJ plugin for 3D segmentation of nuclear objects

BackgroundThe three-dimensional nuclear arrangement of chromatin impacts many cellular processes operating at the DNA level in animal and plant systems. Chromatin organization is a dynamic process that can be affected by biotic and abiotic stresses. Three-dimensional imaging technology allows to follow these dynamic changes, but only a few semi-automated processing methods currently exist for quantitative analysis of the 3D chromatin organization. ResultsWe present an automated method, Nuclear Object DetectionJ (NODeJ), developed as an imageJ plugin. This program segments and analyzes high intensity domains in nuclei from 3D images. NODeJ performs a Laplacian convolution on the mask of a nucleus to enhance the contrast of intra-nuclear objects and allows their detection. We reanalyzed public datasets and determined that NODeJ is able to accurately identify heterochromatin domains from a diverse set of Arabidopsis thaliana nuclei stained with DAPI or Hoechst. NODeJ is also able to detect signals in nuclei from DNA FISH experiments, allowing for the analysis of specific targets of interest. Conclusion and availabilityNODeJ allows for efficient automated analysis of subnuclear structures by avoiding the semi-automated steps, resulting in reduced processing time and analytical bias. NODeJ is written in Java and provided as an ImageJ plugin with a command line option to perform more high-throughput analyses. NODeJ can be downloaded from https://gitlab.com/axpoulet/image2danalysis/-/releases with source code, documentation and further information avaliable at https://gitlab.com/axpoulet/image2danalysis. The images used in this study are publicly available at https://www.brookes.ac.uk/indepth/images/ and https://doi.org/10.15454/1HSOIE.

bioinformatics↗

bioRxiv2020

H3.1K27me1 maintains transcriptional silencing and genome stability by preventing GCN5-mediated histone acetylation

In plants, genome stability is maintained during DNA replication by the H3.1K27 methyltransferases ATXR5 and ATXR6, which catalyze the deposition of K27me1 on replicationdependent H3.1 variants. Loss of H3.1K27me1 in atxr5 atxr6 double mutants leads to heterochromatin defects, including transcriptional de-repression and genomic instability, but the molecular mechanisms involved remain largely unknown. In this study, we identified the conserved histone acetyltransferase GCN5 as a mediator of transcriptional de-repression and genomic instability in the absence of H3.1K27me1. GCN5 is part of a SAGA-like complex in plants that requires ADA2b and CHR6 to mediate the heterochromatic defects of atxr5 atxr6 mutants. Our results show that Arabidopsis GCN5 acetylates multiple lysine residues on H3.1 variants in vitro, but that H3.1K27 and H3.1K36 play key roles in inducing genomic instability in the absence of H3.1K27me1. Overall, this work reveals a key molecular role for H3.1K27me1 in maintaining genome stability by restricting histone acetylation in plants.

plant biology↗

bioRxiv2018

RT States: systematic annotation of the human genome using cell type-specific replication timing programs

The replication timing (RT) program has been linked to many key biological processes including cell fate commitment, 3D chromatin organization and transcription regulation. Significant technology progress now allows to characterize the RT program in the entire human genome in a high-throughput and high-resolution fashion. These experiments suggest that RT changes dynamically during development in coordination with gene activity. Since RT is such a fundamental biological process, we believe that an effective quantitative profile of the local RT program from a diverse set of cell types in various developmental stages and lineages can provide crucial biological insights for a genomic locus. In the present study, we explored recurrent and spatially coherent combinatorial profiles from 42 RT programs collected from multiple lineages at diverse differentiation states. We found that a Hidden Markov Model with 15 hidden states provide a good model to describe these genome-wide RT profiling data. Each of the hidden state represents a unique combination of RT profiles across different cell types which we refer to as \"RT states\". To understand the biological properties of these RT states, we inspected their relationship with chromatin states, gene expression, functional annotation and 3D chromosomal organization. We found that the newly defined RT states possess interesting genome-wide functional properties that add complementary information to the existing annotation of the human genome.\n\nAUTHOR SUMMARYThe replication timing (RT) program is an important cellular mechanism and has been linked to many key biological processes including cell fate commitment, 3D chromatin organization and transcription regulation. Significant technology progress now allows us to characterize the RT program in the entire human genome. Results from these experiments suggest that RT changes dynamically across different developmental stages. Since RT is such a fundamental biological process, we believe that the local RT program from a diverse set of cell types in various developmental stages can provide crucial biological insights for a genomic locus. In the present study, we explored combinatorial profiles from 42 RT programs collected from multiple lineages at diverse differentiation states. We developed a statistical model consist of 15 \"RT states\" to describe these genome-wide RT profiling data. To understand the biological properties of these RT states, we inspected the relationship between RT states and other types of functional annotations of the genome. We found that the newly defined RT states possess interesting genome-wide functional properties that add complementary information to the existing annotation of the human genome.

bioinformatics↗