Biology subjects

Bertrand, E.

Publications and source records attributed to Bertrand, E..

At least 19 recordsLinked to original sources

bioRxiv2026

A large-scale analysis of the R2TP chaperone network reveals its contribution to the assembly of INO80, SRCAP and TIP60

HSP90/R2TP is an essential quaternary chaperone composed of RPAP3, PIH1D1 and the RUVBL1/RUVBL2 AAA+ ATPases. These enzymes are also part of the INO80, SRCAP and TIP60 complexes, but the relationship between these chromatin remodelers and R2TP remains unclear. Here, we performed systematic analyses of the R2TP-specific subunits RPAP3 and PIH1D1. We validated 115 interaction partners and found that many were sensitive to HSP90 or R2TP inhibition. In yeast, epistatic screens revealed functional interactions with Ino80, Swr1 (SRCAP) and NuA4 (TIP60). Consistently, human RPAP3 physically interacted with subunits of INO80, SRCAP and TIP60 and was required for the formation of these complexes. More specifically, RPAP3 enabled the co-translational association of RUVBL1/RUVBL2 with the motor subunit of these chromatin remodelers. In vitro, the client-binding domain of RUVBL1/RUVBL2 modulated their interaction with RPAP3, suggesting that client subunits displace RPAP3 from nascent complexes. Thus, R2TP is an early chaperone of TIP60, SRCAP and INO80, which leaves RUVBL1/RUVBL2 as resident scaffolding subunits.

molecular biology↗

bioRxiv2026

The R2T(P) complex orchestrates the SHQ1 driven early steps of box H/ACA snoRNP maturation

The chaperones RuvBL1 and RuvBL2 are members of the AAA+ ATPase family and participate in diverse cellular processes, including DNA repair, transcriptional regulation, and assembly of macromolecular complexes such as snoRNPs. The biogenesis of box H/ACA snoRNPs additionally requires the assembly factor SHQ1. These protein-RNA complexes are essential for ribosome biogenesis and telomerase stability and are linked to diseases such as dyskeratosis congenita and cancer. Despite detailed knowledge of mature complexes, their assembly mechanisms and how they can be modulated remain unclear. We characterize a trimeric interaction between SHQ1 and RuvBL1:RuvBL2, providing insight into early maturation of the protein-only precursor of box H/ACA snoRNPs. SHQ1 binds the flexible domain II of RuvBL1:RuvBL2, corresponding to the dodecamerization interface, suggesting disruption of this interface and promotion of hexamer formation. We further purified a complex containing RuvBL1:RuvBL2, SHQ1, and DKC1, the catalytic component of H/ACA snoRNPs and a client of SHQ1. This demonstrates that SHQ1 and DKC1 can simultaneously associate with RuvBL1:RuvBL2, potentially facilitating DKC1 release and subsequent snoRNA binding. Additionally, we identified a direct interaction between SHQ1 and RPAP3, a co-chaperone of RuvBL1:RuvBL2 (within the R2TP). Hence, RPAP3 may be responsible for recruiting SHQ1:DKC1 to hexameric RuvBL1:RuvBL2 and/or assist in the AAA+ mediated dissociation of the dimer. Since SHQ1 shares a domain with PIH1D1, an integral member of the R2TP complex, our findings suggest that early H/ACA snoRNP maturation may involve the R2T instead of the previously proposed R2TP complex.

biophysics↗

bioRxiv2025

Cell heterogeneity contributes to the variable response of HIV-1 to latency reversing agents

Transcriptional noise contributes to gene expression variability, but its origin and impact in HIV-1 latency remain incompletely characterized. Here, we combine a dual-copy MS2-tagging system with novel mathematical analysis to investigate the variability of HIV-1 transcription in live cells. In the basal state, the transcriptional activity of proviruses located in the same cell was uncorrelated, indicating that expression variability primarily comes from the intrinsic stochasticity of promoter dynamics. Upon stimulation with diverse latency-reversing agents, viral transcription became more correlated within cells than across them, revealing a shift from promoter-driven to cell state-driven variability. Analysis of the molecular drug targets confirmed variable effects across cells. Our findings indicates that cellular heterogeneity shapes the response to latency reversing agents and demonstrate how quantitative tools can dissect noise sources. This work offers mechanistic insights into HIV-1 latency and informs strategies to target the latent viral reservoir. TeaserLive transcription imaging reveals how cell heterogeneity contributes to the variability of HIV-1 activation.

molecular biology↗

bioRxiv2025

Single-Molecule DNA Footprinting and Transcription Imaging Reveal the Molecular Mechanisms of Promoter Dynamics

Live cell RNA imaging revealed that transcription levels are encoded by the intrinsic dynamics of promoters. However, capturing both kinetic and molecular aspects of promoter fluctuations has been challenging. Here, we resolve this key issue by combining Single Molecule DNA footprinting (SMF) with live transcription imaging. Using HIV-1 as a model, SMF reveals that the promoter functions in two modes depending on the viral transactivator Tat. Without Tat, a nucleosome occupies the core promoter and prevents assembly of the pre-initiation complex. With Tat, this nucleosome is absent while TBP and initiating polymerases are frequently detected. Combining live imaging with SMF provides a mechanistic model of promoter dynamics, which estimates the rates of deposition and removal of promoter nucleosomes (0.7 h-1), TBP binding (0.04 min-1) and polymerase loading (seconds). The data further reveal a kinetic proofreading mechanism of initiating polymerases, which enables Tat to indirectly control promoter nucleosomes by promoting elongation.

molecular biology↗

bioRxiv2025

A Deep Learning approach for time-consistent cell cycle phase prediction from microscopy data

The cell cycle consists of four phases and impacts most cellular processes. In imaging assays, the cycle phase can be identified using dedicated cell-cycle markers. However, such markers occupy fluorescent channels that may be needed for other reporters. Here, we propose to address this limitation by inferring the phase from a widely used fluorescent reporter: SiR-DNA. Our method is based on a variational auto-encoder, enhanced with two auxiliary tasks: predicting the intensity of phase-specific markers and enforcing the latent space temporal consistency. Our model is freely available, along with a new dataset comprising over 600,000 annotated HeLa Kyoto nuclear images.

bioinformatics↗

bioRxiv2025

Biophysical Modeling Uncovers Transcription Factor and Nucleosome Binding on Single DNA Molecules

Gene regulation in eukaryotes emerges from a dynamic interplay between transcription factors (TFs), nucleosomes, and RNA Polymerase II (Pol II), whose competitive and cooperative binding shapes DNA accessibility and transcriptional output. Single-molecule footprinting (SMF) and long-read chromatin accessibility assays such as Fiber-seq now capture these interactions at nucleotide resolution on individual DNA molecules. However, existing computational tools remain insufficient to decode complex binding events from sparse methylation data. Here, we introduce HiddenFoot, a probabilistic modeling framework based on statistical mechanics that quantitatively infers TF, nucleosome, and Pol II occupancy profiles on single DNA molecules by systematically evaluating all thermodynamically plausible binding configurations. Applying HiddenFoot to SMF and Fiber-seq data from mouse, Drosophila, and human cells, we recovered known TF footprints, precisely resolved Pol II pausing, and identified extensive heterogeneity in nucleosome positioning driven by TF binding. HiddenFoot further distinguishes direct TF-TF cooperativity from nucleosome-mediated co-dependency by estimating pairwise interaction energies and comparing to null models under equilibrium. By integrating biophysical modeling with high-resolution single-molecule data, HiddenFoot offers a general, interpretable framework for dissecting regulatory logic in native chromatin with base-pair precision. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/653852v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@46f3b5org.highwire.dtl.DTLVardef@2a2cbdorg.highwire.dtl.DTLVardef@df42f2org.highwire.dtl.DTLVardef@1a4205d_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

EGFR-Mediated Mechanotransduction in Aortic Valve Cells: A Key Pathway in Response to Wall Shear Stress

AimBlood flow-induced mechanical forces, particularly wall shear stress (WSS), play a fundamental role in aortic valve remodeling and maturation. Dysregulation of these processes contributes to age-related valve diseases, such as aortic stenosis and regurgitation. While epidermal growth factor receptor (EGFR) signaling has been implicated in valve development, its role in mechanotransduction remains unclear. This study aims to investigate how EGFR regulates WSS-induced signaling in valvular cells and explore its interaction with the mechanosensitive ion channel PIEZO1. Methods and ResultsTo investigate the role of EGFR in valvular cell mechanotransduction, we used conditional Egfrflox allele to selectively delete Egfr in valvular cells. Histological analysis revealed increased valve leaflet thickness and hyperproliferation of mesenchymal cells when Egfr was deleted both endothelial (Tie2-Cre lineage) and mesenchymal (Sm22-Cre lineage) cells. This was accompanied by a reduction in maturation-related genes (Egr1, Nos3, Tgf-{beta}) and extracellular matrix (ECM) components. We previously demonstrated that Egr1 expression is regulated by WSS in valvular endothelial cells, prompting further exploration of Egr1s role in valvular cells. In vitro, Egr1 overexpression and shRNA-mediated knockdown confirmed its role in regulating Nos3, Col1a1, and Tgf-{beta}, key mediators of valve remodeling. Using a pulsatile WSS-mimicking device, we found that WSS induces Erk1/2 phosphorylation and Egr1 expression in valvular cells, both of which were abolished by EGFR inhibition. However, direct EGFR activation via EGF failed to replicate WSS-induced Egr1 expression, suggesting the involvement of additional mechanosensitive pathways. Pharmacological studies further revealed that PIEZO1 inhibition impaired WSS-induced Egr1 expression, while PIEZO1 activation (via YODA) mimicked WSS effects on Erk1/2 phosphorylation and Egr1 expression. These findings suggest a functional interaction between EGFR and PIEZO1 in mechanotransduction, linking mechanical forces to key molecular pathways in valve remodeling. ConclusionOur findings establish EGFR as a critical mediator of WSS-induced mechanotransduction in valve remodeling, working in synergy with PIEZO1 to regulate flow-sensitive transcription factors such as Egr1. This study provides new insights into the molecular mechanisms governing valve maturation and highlights potential therapeutic targets for age-related valve pathologies linked to abnormal WSS responses. Translational PerspectiveOur study highlights the pivotal role of EGFR and the mechanosensitive ion channel PIEZO1 in aortic valve cell responses to wall shear stress (WSS), offering new insights into valve remodeling. The findings suggest that dysregulated EGFR signaling contributes to valve thickening and stenosis, key factors in age-related valvular heart disease. By identifying EGFR-PIEZO1 as a critical mechanotransduction pathway, our work provides a potential therapeutic target for early intervention in aortic valve disease. Modulating EGFR or PIEZO1 activity could help mitigate pathological valve remodeling, presenting novel strategies for treating or preventing degenerative valve disorders.

developmental biology↗

bioRxiv2025

Co-translational determination of quaternary structures in chaperone factories

The HSP90/R2TP quaternary chaperone assembles key cellular machines, including the three nuclear RNA polymerases and many non-coding RNPs. Here, we characterized the RNA associated to R2TP and found that it binds many partners co-translationally. Its co-translational interactome further reveals many novel potential clients and identifies clients bound only co-translationally, only post-translationally, or both. For pairs of subunits assembling together and bound co-translationally by R2TP, only a marginal proportion of their mRNAs is co-localized and co-translated. Instead, the HSP90 and R2TP chaperones induce the formation of condensates accumulating client mRNAs and thus favoring co-translational interactions between chaperones and clients. The R2TP then cycles between co- and post-translational steps and this is regulated by ATP: it binds co-translationally in absence of ATP and becomes released from post-translational assembly intermediates by ATP hydrolysis. Assembly of protein complexes is thus initiated early by chaperones and this mechanism, dubbed co-translational chaperone channeling (cha-cha), substitutes for the rarity of co-localized/co-translated mRNAs.

cell biology↗

bioRxiv2025

R2TP-like Quaternary Chaperones: a comprehensive overview to understand the dynamic R2SP complex

The human R2SP complex belongs to the R2TP-like quaternary chaperone family and consists of RUVBL1, RUVBL2, SPAG1 and PIH1D2. R2SP is crucial for the correct assembly of motile cilia (SPAG1 null mutations cause Primary Ciliary Dyskinesia) and the organization of the synaptic zone. RUVBL1/2 ATPases are the powerhouse of this molecular machinery, while SPAG1 and PIH1D2 would be adaptors that interact with specific clients to promote their quaternary assembly. Despite these functional data, little is known about the structure of R2SP and the precise mode of action of these R2TP-like complexes. We have combined biochemical and structural approaches (NMR, structural mass spectrometry and cryo-EM) to investigate the 3D organization of the human R2SP complex, its mode of assembly and ATPase activity. Our study reveals a three-dimensional structure similar to that of the canonical R2TP complex, but also highlights differences in the mode of action of its RUVBL1/2 ATPase core as well as the binding of its adaptors SPAG1 and PIH1D2.

biophysics↗

bioRxiv2024

Universal dynamics of cohesin-mediated loop extrusion

Most animal genomes are partitioned into Topologically Associating Domains (TADs), created by cohesin-mediated loop extrusion and defined by convergently oriented CTCF sites. The dynamics of loop extrusion and its regulation remains poorly characterized in vivo. Here, we tracked TAD anchors in living human cells to visualize and quantify cohesin-dependent loop extrusion across multiple endogenous genomic regions. We show that TADs are dynamic structures whose anchors are brought in proximity about once per hour and for 6-19 min ([~]16% of the time). TADs are continuously subjected to extrusion by multiple cohesin complexes, extruding loops at [~]0.1 kb/s. Remarkably, despite strong differences of Hi-C patterns between the chromatin regions, their dynamics is consistent with the same density, residence time and speed of cohesin. Our results suggest that TAD dynamics is governed primarily by CTCF site location and affinity, which allows genome-wide predictive models of cohesin-dependent interactions.

genetics↗

bioRxiv2024

Cell cycle-dependent mRNA localization in P-bodies

Understanding the dynamics of RNA targeting to membraneless organelles is essential to disentangle their functions. Here, we investigate how P-bodies (PBs) evolve during cell cycle progression. PB purification across the cell cycle uncovers widespread changes in their RNA content, which are partly uncoupled from cell cycle-dependent changes in RNA expression. Single molecule FISH shows various mRNA localization patterns in PBs peaking in G1, S, or G2, with examples illustrating the timely capture of mRNAs in PBs when their encoded protein becomes dispensable. Yet, rather than directly reflecting absence of translation, cyclic mRNA localization in PBs can be controlled by RBPs, such as HuR in G2, and by RNA features. Indeed, while PB mRNAs are AU-rich at all cell cycle phases, they are specifically longer in G1, possibly related to post-mitotic PB reassembly. Altogether, our study supports a model where PBs are more than a default location for excess untranslated mRNAs.

cell biology↗

bioRxiv2024

Co-translational sorting enables a single mRNA to generate distinct polysomes with different localizations and protein fates

{beta}-catenin is a multi-functional protein playing essential roles in tissue homeostasis and cancer. It bridges E-cadherin to the cytoskeleton and also activates transcription in response to Wnt. Plasma membrane {beta}-catenin is stable whereas without Wnt, cytoplasmic {beta}-catenin is degraded by the destruction complex, composed of APC and Axin. Here, we show that APC and Axin associate with many mRNAs and that this occurs via the nascent protein chains. Notably, APC and Axin bind {beta}-catenin mRNAs present as either single polysome or polysome condensates, and co-translational interactions constitute the major fraction of their binding to the {beta}-catenin protein. Remarkably, E-cadherin also binds {beta}-catenin co-translationally, and {beta}-catenin mRNAs localize either with APC in the cytosol or E-cadherin at the plasma membrane. Thus, co-translational interactions sort {beta}-catenin mRNAs into distinct polysome populations that spatially segregate in cells and synthesize proteins with different functions. Co-translational polysome sorting provides a mechanism to regulate the fate of multi-functional proteins.

molecular biology↗

bioRxiv2023

Boosting the toolbox for live imaging of translation

Live imaging of translation based on tag recognition by a single chain antibody is a powerful technique to assess translation regulation in living cells. However, especially in a multicellular organism, this approach is challenging and requires optimization in terms of expression level and detection sensitivity of the system. Here, we improved existing fluorescent tools and developed new ones to image and quantify nascent translation in the living Drosophila embryo and in mammalian cells. We tested and characterized five different Green Fluorescent Protein variants fused to the single chain fragment variable (scFv) and uncover photobleaching, aggregation and intensity disparities. Using different strengths of germline and somatic drivers, we determined that the availability of the scFv is critical in order to detect translation throughout development. We introduced a new translation imaging method based on a nanobody/tag system named ALFA-array, allowing the sensitive and simultaneous detection of the translation of several distinct mRNA species. Finally, we developed a largely improved RNA imaging system based on an MCP-tdStaygold fusion.

developmental biology↗

bioRxiv2023

G-quadruplexes are promoter elements controlling nucleosome exclusion and RNA polymerase II pausing

Despite their central role in transcription, it has been difficult to define universal sequences associated to eukaryotic promoters. Within chromatin context, recruitment of the transcriptional machinery requires opening of the promoter but how DNA elements could contribute to this process has remained elusive. Here, we show that G-quadruplex (G4) secondary structures are highly enriched mammalian core promoter elements. G4s are located at the deepest point of nucleosome exclusion at promoters and correlate with maximum promoter activity. We found that experimental G4s exclude nucleosomes both in vivo and in vitro and display a strong positioning potential. At model promoters, impairing G4s affected both transcriptional activity and chromatin opening. G4 destabilization also resulted in an inactive promoter state and affected transition to effective RNA production in live imaging experiments. Finally, G4 stabilization resulted in global reduction of proximal promoter pausing. Altogether, our data introduce G4s as bona fide promoter elements allowing nucleosome exclusion and facilitating pause release by the RNA Polymerase II.

genomics↗

bioRxiv2022

Chromatin organization drives the exploration strategy of nuclear factors

Nuclear Factors (NFs) rapidly scan the genome for their targets, but the role of nuclear organization in such search is uncharted. Here we analyzed how multiple NFs explore chromatin, by combining live-cell single-molecule tracking with multifocal structured illumination of DNA density. We find that NFs displaying higher bound fractions sample DNA dense regions more exhaustively. Focusing on the tumor-suppressor p53, we demonstrated that this NF search for its targets by alternating between rapid diffusion in the interchromatin compartment and compact sampling of chromatin dense regions. Efficient p53 targeting requires balanced IDR/chromatin interactions: adding an exogenous IDR potentiates p53-mediated target gene activation, but excessive IDR/IDR interactions lead to p53 condensates, derailing its search and downregulating transcription. Our findings highlight the role of NF IDRs on their search and showcase a powerful method to generate traffic maps of the eukaryotic nucleus and dissect how nuclear organization guides NFs action.

biophysics↗

bioRxiv2021

FISH-quant v2: a scalable and modular analysis tool for smFISH image analysis

Regulation of RNA abundance and localization is a key step in gene expression control. Single-molecule RNA fluorescence in-situ hybridization (smFISH) is a widely used single-cell-single-molecule imaging technique enabling a quantitative understanding of gene expression and its regulatory mechanisms. Recent progress in experimental techniques provides larger data-sets, requiring adequate tools for data analysis and exploration. Here, we present FISH-quant v2, a highly modular analysis tool accessible both for non-experts and experts, which we validated and applied on large-scale smFISH image datasets. Our package allows the user to detect isolated and clustered mRNA spots, segment nuclei and cells, quantify RNA localization patterns and visualize these results at the single-cell level.

bioinformatics↗

bioRxiv2021

RNA labelling in live plants reveals single cell transcriptional dynamics: application to phosphate signaling

Plants are sessile organisms constantly adapting to ambient fluctuations through spatial and temporal transcriptional responses. Here, we implemented the latest generation RNA imaging system and combined it with microfluidics to visualize transcriptional regulation in living Arabidopsis plants. This enabled quantitative measurements of the transcriptional activity of single loci in single cells, real time and changing environmental conditions. Using phosphate responsive genes as model, we found that active genes displayed high transcription initiation rates ([~]3s) and frequently clustered together in endoreplicated cells. We observed gene bursting and large allelic differences in single cells, revealing that at steady-state, intrinsic noise dominated extrinsic variations. Moreover, we established that transcriptional repression triggered in roots by phosphate, a crucial macronutrient limiting plant development, occurred with unexpected fast kinetics ([~]minutes) and striking heterogeneity between neighboring cells. Access to single cell RNA polymerase II dynamics within live plants will benefit future studies of signaling processes.

plant biology↗

bioRxiv2021

Long non-coding RNA Neat1 is a key translational regulator in hypoxia.

Internal ribosome entry sites (IRESs) drive translation initiation during stress. In response to hypoxia, (lymph)angiogenic factors responsible for tissue revascularization in ischemic diseases are induced by the IRES-dependent mechanism. Here we searched for IRES trans-acting factors (ITAFs) active in early hypoxia in mouse cardiomyocytes. Using knock-down and proteomics approaches, we show a link between a stressed-induced nuclear body, the paraspeckle, and IRES-dependent translation. Furthermore, smiFISH experiments demonstrate the recruitment of IRES-containing mRNA into paraspeckle during hypoxia. Our data reveal that the long non-coding RNA Neat1, an essential paraspeckle component, is a key translational regulator, active on IRESs of (lymph)angiogenic and cardioprotective factor mRNAs. In addition, paraspeckle proteins p54nrb and PSPC1 as well as nucleolin and Rps2, two p54nrb-interacting proteins identified by mass spectrometry, are ITAFs for IRES subgroups. Paraspeckle thus appears as a platform to recruit IRES-containing mRNAs and possibly host IRESome assembly. Polysome PCR array shows that Neat1 isoforms regulate IRES-dependent translation and, more widely, translation of mRNAs involved in stress response. HighlightsO_LIParaspeckle formation correlates with activation of translation via internal ribosome entry sites (IRES) in mouse hypoxic cardiomyocytes as well as in tumoral cells. C_LIO_LIThe long non-coding RNA Neat1, an essential paraspeckle component, is a key translational regulator of (lymph)angiogenic and cardioprotective factor expression in this process. C_LIO_LIIRES-containing mRNA is recruited into paraspeckles during hypoxia. C_LIO_LIParaspeckle proteins p54nrb and PSPC1 as well as two p54nrb-interacting proteins, nucleolin and RPS2, contribute to this process. C_LIO_LIParaspeckle appears as a platform for IRESome formation in the nucleus. C_LIO_LIThe Neat1 isoforms widely regulate the translation of mRNAs containing IRESs and of genes involved in the stress response. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/430272v2_ufig1.gif" ALT="Figure 1"> View larger version (98K): org.highwire.dtl.DTLVardef@1741648org.highwire.dtl.DTLVardef@62e741org.highwire.dtl.DTLVardef@13c86a8org.highwire.dtl.DTLVardef@acae63_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗