Biology subjects

Huet, S.

Publications and source records attributed to Huet, S..

14 recordsLinked to original sources

bioRxiv2026

Relapse-founding cancer persister cells in follicular lymphoma

Follicular lymphoma (FL) is an incurable, prototypical relapse-remitting cancer, implying the existence of therapy-persistent cells that survive frontline treatment and seed disease recurrence1-3. However, these persister cells remain difficult to study directly in patients because immediate post-treatment sampling is ethically and practically challenging. Using a genetically defined mouse model that allows sampling of persistent cells immediately after frontline R-CHOP therapy, we prospectively isolate and functionally define relapse-founding cancer persister cells (CPC). The CPC is an IgM memory-like B-cell with high germinal center re-entry capacity. This state represents a discrete component of a heterogeneous residual pool indicating that residual disease is polytypic and that relapse potential may depend on which cells persist rather than on residual tumour burden alone. By integrating mouse CPC with human FL datasets, we show that an analogous transcriptional programme is detectable at diagnosis and is enriched in patients with inferior clinical outcome across independent cohorts4,5. These findings support the concept that relapse risk is linked to a conserved, genotype-agnostic CPC programme present before therapy. To explore therapeutic vulnerabilities, we developed a scalable in-vitro platform that models the CPC-like state and used it to identify sensitivity to histone deacetylase inhibition. Romidepsin and panobinostat killed CPC-like cells in-vitro, and decreased therapy-persistent cells after R-CHOP treatment in-vivo and in patient-derived organoids. Together, these data define a tractable CPC state in FL, with a validated clinical readout and an immediately testable therapeutic entry point, opening CPC-directed strategies for durable FL control.

cancer biology↗

bioRxiv2025

A coordinated haptic mechanism ensures efficient DNA sampling by the 8-oxoguanine glycosylase OGG1.

7,8-Dihydro-8-oxoguanine (8-oxoG) is the most frequent base modification occurring upon oxidative stress. This highly mutagenic lesion is specifically recognized and excised by the DNA glycosylase OGG1 when paired with cytosine, initiating the base excision repair pathway. Since 8-oxoG neither significantly impacts the structure of the double helix nor blocks transcription and replication processes, its detection requires a careful inspection of each base pair by OGG1. By monitoring this lesion search process both in vitro and in living cells, we demonstrate that it involves a tight coordination between several conserved amino acids encircling the DNA helix. More specifically, we show that the N149-151 motif, on the target strand, as well as residues R154 and R204, on the opposite strand, both regulate OGG1 engagement on the DNA to ensure fast Y203-mediated base unstacking, a prerequisite for efficient 8-oxoG detection. These findings highlight the early mechanisms that enable OGG1 to maintain rapid sampling kinetics while preserving high specificity for 8-oxoG in the context of the complex architecture displayed by the DNA within the cell nucleus.

genetics↗

bioRxiv2025

Detecting aberrant splicing events in short-read RNA-seq with SAMI, an UMI-aware Nextflow pipeline

SummaryDespite RNA-sequencing solutions have existed for 15 years, successfully replacing micro-arrays for gene expression profiling, their potential to analyse splicing is still to be achieved in clinical settings. Powerful tools were developed to quantify known iso-forms, to compare sample groups or to identify outliers in large datasets, but most fail to address the most common situation in routine diagnostics: identifying non-recurring events in low-dimension data. To fill this gap, we developed SAMI (Splicing Analysis with Molecular Indexes), an UMI-aware pipeline focusing on splicing events which differ from provided annotation. The ability of SAMI and concurrent software to detect intragenic splicing aberrations and gene fusions was assessed, both on real data from a commercial control sample and simulated data generated with ASimulatoR. Availability and ImplementationNextflow pipeline and Singularity container recipe freely available under GPL 3 licence at https://github.com/HCL-HUBL/SAMI Contactsylvain.mareschal@chu-lyon.fr

bioinformatics↗

bioRxiv2024

Single-nucleosome imaging reveals principles of transient multiscale chromatin unfolding triggered by histone ADP-ribosylation at DNA lesions

Timely access to DNA lesions is crucial for genome integrity. This process requires profound remodeling of densely packed chromatin to establish a repair-competent architecture. However, limited resolution has made it impossible to fully understand these remodeling events. Here, combining microirradiation with live-cell multiscale imaging, we report that DNA damage-induced changes in genome packing rely on the conformational behaviour of the chromatin fiber. Immediately after damage, a transient increase in nucleosome mobility switches chromatin from a densely-packed state to a looser conformation, making it accessible to repair. While histone poly-ADP-ribosylation is required to trigger this switch, mono-ADP-ribosylation is sufficient to maintain the open-chromatin state. The removal of these histone marks by the ARH3 hydrolase then leads to chromatin recondensation. Together, our multiscale study of chromatin dynamics establishes a global model: distinct waves of histone ADP-ribosylation control nucleosome mobility, triggering a transient breathing of chromatin, crucial for initiating the DNA damage response.

cell biology↗

bioRxiv2024

DNA damage-induced PARP/ALC1 activation leads to Epithelial-to-Mesenchymal transition stimulating homologous recombination.

Epithelial-to-mesenchymal transition (EMT) allows cancer cells to metastasize while acquiring resistance to apoptosis and to chemotherapeutic agents with significant implications in patients prognosis and survival. Despite its clinical relevance, the mechanisms initiating EMT during cancer progression remain poorly understood. We demonstrate that DNA damage triggers EMT by activating PARP and the PARP-dependent chromatin remodeler ALC1 (CHD1L). We show that this activation directly facilitates the access to chromatin of EMT transcriptional factors (TFs) which then initiate cell reprogramming. We also show that EMT-TFs bind to the RAD51 promoter to stimulate its expression and to promote DNA repair by recombination. Importantly, a clinically relevant PARP inhibitor totally reversed or prevented EMT in response to DNA damage while resensitizing tumor cells to other genotoxic agents. Overall, our observations shed light on the intricate relationship between EMT, DNA damage response and PARP inhibitors, providing valuable insights for future therapeutic strategies in cancer treatment.

cancer biology↗

bioRxiv2023

The loss of DNA polymerase epsilon accessory subunits POLE3-POLE4 leads to BRCA1-independent PARP inhibitor sensitivity

The clinical success of PARP1/2 inhibitors prompts the expansion of their applicability beyond homologous recombination deficiency. Here, we demonstrate that the loss of the accessory subunits of DNA polymerase epsilon, POLE3 and POLE4, sensitizes cells to PARP inhibitors. We show that the sensitivity of POLE4 knockouts is not due to a compromised response to DNA damage or homologous recombination deficiency. Instead, POLE4 deletion generates replication stress with the accumulation of single-stranded DNA gaps upon PARP inhibitor treatment. In POLE4 knockouts, replication stress leads to elevated DNA-PK signaling revealing a role of POLE4 in regulating DNA-PK activation. Moreover, POLE4 knockouts show synergistic sensitivity to the co-inhibition of ATR and PARP. Finally, POLE4 loss enhances the sensitivity of BRCA1-deficient cells to PARP inhibitors and counteracts acquired resistance consecutive to restoration of homologous recombination. Altogether, our findings establish POLE4 as a promising target to improve PARP inhibitor driven therapies and hamper acquired PARP inhibitor resistance.

cell biology↗

bioRxiv2023

Functional precision oncology for follicular lymphoma with patient-derived xenograft in avian embryos

BackgroundFollicular lymphoma (FL) is an incurable B-cell malignancy that constitutes a quarter of all lymphomas. Although RCHOP immuno-chemotherapy induces high rates of complete remission, almost all FL patients experience multiple relapses post-treatment. The limited understanding of treatment response heterogeneity is due to the absence of in vitro or in vivo experimental models, primarily because tumor cells heavily rely on their microenvironment to survive. In this study, we present an innovative xenograft model of primary FL cells in avian embryos, circumventing these limitations. MethodsWe developed the FL-AVI-PDX model by transplanting 20 biopsy FL samples, including good (n=11) and poor clinical responders (POD24, n=9), into chicken embryos. Each set of embryos was treated with RCHOP or vehicle intravenously. We evaluated the effect of immuno-chemotherapy on tumor volume by light sheet microscopy and on tumor biology by transcriptomic analysis at the single-cell level. ResultsWe successfully engrafted all samples in avian embryos. We found that RCHOP treatment in ovo led to tumor volume reduction, which predicted progression-free survival in multivariate analysis, demonstrating the models capacity to capture clinical heterogeneity at the patient level. The FL-AVI-PDX model also provided a unique opportunity to analyze the transcriptomic impact of RCHOP on FL cells using single-cell RNA sequencing. We identified a signature of 21 genes upregulated after RCHOP exposure, displaying significant intra-tumoral heterogeneity. As a proof of concept, we validated the functional involvement of BAX, a gene from the RCHOP-induced signature, as a critical effector of immuno- chemotherapy in vitro and in avian embryos. ConclusionsThe FL-AVI-PDX model is a platform for functional precision oncology in primary FL cells that captures both inter- and intra-patient heterogeneity of clinical response to a complex therapeutic regimen. It offers a unique opportunity to better understand FL biology, opening perspectives for the development of new drugs.

cancer biology↗

bioRxiv2022

Identification of key residues of the DNA glycosylase OGG1 controlling efficient DNA scanning and recruitment to oxidized bases in living cells

The DNA-glycosylase OGG1 oversees the detection and clearance of the 7,8-dihydro-8-oxoguanine (8-oxoG), which is the most frequent form of oxidized base in the genome. This lesion is deeply buried within the double-helix and its detection requires careful inspection of the bases by OGG1 via a mechanism that remains only partially understood. By analyzing OGG1 dynamics in the nucleus of living human cells, we demonstrate that the glycosylase constantly scans the DNA by rapidly alternating between diffusion within the nucleoplasm and short transits on the DNA. This scanning process, that we find to be tightly regulated by the conserved residue G245, is crucial for the rapid recruitment of OGG1 at oxidative lesions induced by laser micro-irradiation. Furthermore, we show that residues Y203, N149 and N150, while being all involved in early stages of 8-oxoG probing by OGG1 based on previous structural data, differentially regulate the scanning of the DNA and recruitment to oxidative lesions.

cell biology↗

bioRxiv2021

HPF1-dependent histone ADP-ribosylation triggers chromatin relaxation to promote the recruitment of repair factors at sites of DNA damage

PARP1 activity is regulated by its cofactor HPF1. The binding of HPF1 on PARP1 controls the grafting of ADP-ribose moieties on serine residues of proteins nearby the DNA lesions, mainly PARP1 and histones. However, the impact of HPF1 on DNA repair regulated by PARP1 remains unclear. Here, we show that HPF1 controls both the number and the length of the ADP-ribose chains generated by PARP1 at DNA lesions. We demonstrate that HPF1-dependent histone ADP-ribosylation, rather than auto-modification of PARP1, triggers the rapid unfolding of the chromatin structure at the DNA damage sites and promotes the recruitment of the repair factors CHD4 and CHD7. Together with the observation that HPF1 contributes to efficient repair both by homologous recombination and non-homologous end joining, our findings highlight the key roles played by this PARP1 cofactor at early stages of the DNA damage response.

cell biology↗

bioRxiv2021

A stochastic process modelling of maize phyllochron enables to characterize environmental and genetic effects.

The times between appearance of successive leaves or phyllochron characterize the vegetative development of annual plants. Hypothesis testing models, which enables to compare phyllochron between genetic groups or conditions, are usually based on regression of thermal time on the number of leaves, most of the time assuming a constant leaf appearance rate. However these models are both statistically biased and inappropriate in terms of modelling. We propose a stochastic process model in which the emergence of new leaves is considered as successive time-to-events, which provides a flexible and more accurate modelling as well as unbiased testing procedures. The model was applied on an original maize dataset collected in fields for three years on plants originating from two divergent selection experiments for flowering time conducted in two maize inbred lines. We showed that the main differences in phyllochron were not observed between selection populations (Early or Late), but rather between ancestral lines, years of experimentation, and leaf ranks. Our results highlight a strong departure from the assumption of a constant leaf appearance rate in one year that could be related to climate variations, even if the impact of each climatic variables individually was not clearly elucidated.

plant biology↗

bioRxiv2020

RhoA- and Ran-induced antagonistic forces underlie symmetry breaking and spindle rotation in mouse oocytes.

Mammalian oocyte meiotic divisions are highly asymmetric and produce a large haploid gamete and two small polar bodies. This relies on the ability of the cell to break symmetry and position its spindle close to the cortex before the anaphase occurs. In metaphase II arrested mouse oocytes, the spindle is actively maintained close and parallel to the cortex, until the fertilization triggers the sister chromatids segregation and the rotation of the spindle. The latter must indeed reorient perpendicular to the cortex to enable the cytokinesis ring closure at the base of the polar body. However, the mechanisms underlying symmetry breaking and spindle rotation have remained elusive. In this study, we show that the spindle rotation results from two antagonistic forces. First, an inward contraction of the cytokinesis furrow dependent on RhoA signaling and second, an outward attraction exerted on both lots of chromatids by a RanGTP dependent polarization of the actomyosin cortex. By combining live segmentation and tracking with numerical modelling, we demonstrate that this configuration becomes unstable as the ingression progresses. This leads to spontaneous symmetry breaking, which implies that neither the rotation direction nor the lot of chromatids that eventually gets discarded are biologically predetermined.

developmental biology↗

bioRxiv2020

Performances of targeted RNA-sequencing for the analysis of fusion transcripts, gene mutation and expression in haematological malignancies.

RNA sequencing holds great promise to improve the diagnostic of haematological malignancies, because this technique enables to detect fusion transcripts, to look for somatic mutations in oncogenes, and to capture transcriptomic signatures nosological entities. However, the analytical performances of targeted RNA sequencing have not been extensively described in diagnostic samples. Using a targeted panel of 1385 cancer-related genes in a series of 100 diagnosis samples and 8 controls, we detected all the already known fusion transcripts, and also discovered unknown and/or unsuspected fusion transcripts in 12 samples. Regarding the analysis of transcriptomic profiles, we show that targeted RNA sequencing is performant to discriminate acute lymphoblastic leukemia entities driven by different oncogenic translocations. Additionally, we show that 86% of the mutations identified at the DNA level are also detectable at the mRNA level, except for nonsense mutations which are subjected to mRNA decay. We conclude that targeted RNA sequencing might improve the diagnosis of haematological malignancies. Standardization of the preanalytical steps and further refinements of the panel design and of the bioinformatical pipelines will be an important step towards its use in standard diagnostic procedures.

genomics↗

bioRxiv2019

The RNA export factor Mex67 functions as a mobile nucleoporin

The RNA export factor Mex67 is essential for the transport of mRNA through the nuclear pore complex (NPC) in yeast, but the molecular mechanism of this export process remains poorly understood. Here, we use quantitative fluorescence micro-scopy techniques in live budding yeast cells to investigate how Mex67 facilitates mRNA export. We show that Mex67 exhibits little interaction with mRNA in the nucleus and localizes to the NPC independently of mRNA, occupying a set of binding sites offered by FG repeats in the NPC. The ATPase Dbp5, which is thought to remove Mex67 from transcripts, does not affect the interaction of Mex67 with the NPC. Strikingly, we find that the essential function of Mex67 is spatially restricted to the NPC since a fusion of Mex67 to the nucleoporin Nup116 rescues a deletion of MEX67. Thus, Mex67 functions as a mobile nuclear pore component, which receives mRNA export substrates in the central channel of the NPC to facilitate their translocation to the cytoplasm.

cell biology↗

bioRxiv2017

Correlative live and super-resolution imaging reveals the dynamic structure of replication domains

Chromosome organization in higher eukaryotes controls gene expression, DNA replication, and DNA repair. Genome mapping has revealed the functional units of chromatin at the sub-megabase scale as self-interacting regions called topologically associating domains (TADs) and showed they correspond to replication domains (RDs). A quantitative structural and dynamic description of RD behavior in the nucleus is however missing, as visualization of dynamic subdiffraction-sized RDs remains challenging. Using fluorescence labeling of RDs combined with correlative live and super-resolution microscopy in situ, we determined biophysical parameters to characterize the internal organization, spacing and mechanical coupling of RDs. We found that RDs are typically 150 nm in size and contain four co-replicating regions spaced 60 nm apart. Spatially neighboring RDs are spaced 300 nm apart and connected by highly flexible linker regions that couple their motion only below 550 nm. Our pipeline allows a robust quantitative characterization of chromosome structure in situ, and provides important biophysical parameters to understand general principles of chromatin organization.

cell biology↗