Biology subjects

Yvert, G.

Publications and source records attributed to Yvert, G..

8 recordsLinked to original sources

bioRxiv2025

Characterizing drug activity with sensitive interactomes in human living cells

A number of human diseases results from abnormal protein-protein interactions (PPIs) involving key regulatory proteins. Therefore, an important strategy in therapeutics consists in developing inhibitory molecules that should ideally be specific for the aberrant PPI. In this context, it is critical to evaluate the number of PPIs that could be affected by the candidate molecule and to analyze the inhibitory potential before and after the formation of the PPI. Surprisingly, these two molecular aspects are rarely considered, due to a lack of appropriate methodological approaches. In this study, we present a novel methodology that captures drug-sensitive PPIs by considering drug-induced cellular functions in live cell conditions. As a proof-of-concept, we identified interactions of the human core signaling protein ERK1 that are specifically affected by two different inhibitory molecules. In addition, we used a complementary set of innovative tools that allowed visualizing the inhibitory effect on ERK1/cofactor protein complexes after their assembly in living cells. Overall, our work establishes a unique methodological approach for deciphering drug activity for potentially any target bait protein of interest.

molecular biology↗

bioRxiv2025

Trans-acting mutations reveal non-nuclear modulators of both intrinsic and extrinsic gene expression noise in a eukaryote.

Gene expression regulation is a stochastic process that can be modified by mutations not only in a deterministic manner but also in a probabilistic way, for example by changing the extent of cell-to-cell variability in gene expression (also called "gene expression noise"). Although systematic studies have successfully analyzed the properties of cis-acting mutations that modulate expression noise at their own locus, less is known about the type of genetic changes that alter expression noise in trans (at loci distant from the mutation). Here, we applied genetic mapping on yeast strains (Saccharomyces cerevisiae) generated by random mutagenesis and identified three mutations (in chs1, yme2 and msh1 genes) that changed the expression noise of a reporter gene in the nuclear genome regulated by the yeast TDH3 promoter. These mutations affected either extrinsic noise (variability due to cell-specific factors), intrinsic noise (inherent variability within each cell) or both, and their effects were found to not be specific to the TDH3 promoter. Surprisingly, all three mutations targeted proteins located outside of the nucleus: Yme2 and Msh1 being involved in the maintenance of mitochondrial genome integrity, and Chs1 being necessary for the repair of cell-wall defects in freshly-born daughter cells. Our results reveal that mitochondrial state can modulate the extent of intrinsic expression noise of eukaryotic nuclear genes.

genetics↗

bioRxiv2024

Kinetic properties of optogenetic DNA editing by LiCre-loxP

Advances in optogenetics now allow to specifically modify the DNA of live cells with light. However, using these technologies successfully requires to know their properties in terms of sensitivity, efficiency, kinetics and mechanism. We previously developed an optogenetic tool made of a single chimeric protein called LiCre that enables the induction of specific changes in the genome with blue light via DNA recombination between loxP sites [1]. Here, we used in vitro and in vivo experiments combined with kinetic modeling to provide a deeper characterization of the photo-activated LiCre-loxP recombination reaction. We find that LiCre binds DNA with high affinity in absence of light stimulus and that this binding is cooperative although not as much as for the Cre recombinase from which LiCre was derived. In yeast, addition of riboflavin to the culture medium had no effect on LiCres efficiency, even when cells over-expressed riboflavin kinase, suggesting that abundance of the flavin mono-nucleotide co-factor is not limiting for the reaction. However, LiCres efficiency in yeast gradually increased when raising temperature from 20{degrees}C to 37{degrees}C. The recombination kinetics observed in live cells are best explained by a model where photo-activation of two or more DNA-bound LiCre units (happening in seconds) can produce (in several minutes) a functional recombination synapse. This model was able to capture the effect of a point mutation altering LiCres light cycle. This deeper understanding of the LiCre-loxP system provides additional knowledge for designing experiments where specific genetic changes are induced in live cells with light. AUTHOR SUMMARYUsing a technology called optogenetics, scientists are now able to change the DNA sequence of live cells by illuminating them with light. In theory, they can trigger in genetically-engineered organisms a mutation of interest in specific cells at a specific time. This practice, however, is not common because optogenetics relies on light-controlled enzymes that are recent and not well characterized. We previously developed one of these enzymes, called LiCre, which recognizes a specific piece of DNA. Following illumination with blue light, LiCre can switch on or off whatever gene is close by. Here, we combined experiments and computational modeling to better understand how, and how fast, LiCre works. We find that, although it is inactive in the dark, LiCre does not need photo-activation to bind to its DNA partner. We estimated the speed at which LiCre gets deactivated in the dark and the speed at which active LiCre molecules modify DNA. We also showed the effect of temperature and illumination dynamics on LiCres efficiency. These results will help design strategies where LiCre can be used to conduct genetic studies at high spatio-temporal resolution, or implement it in industrial and biomedical applications.

bioengineering↗

bioRxiv2020

A monogenic and fast-responding Light-Inducible Cre recombinase as a novel optogenetic switch

ABSTRACTOptogenetics enables genome manipulations with high spatiotemporal resolution, opening exciting possibilities for fundamental and applied biological research. Here, we report the development of LiCre, a novel light-inducible Cre recombinase. LiCre is made of a single flavin-containing protein comprising the asLOV2 photoreceptor domain of Avena sativa fused to a Cre variant carrying destabilizing mutations in its N-terminal and C-terminal domains. LiCre can be activated within minutes of illumination with blue light, without the need of additional chemicals. When compared to existing photoactivatable Cre recombinases based on two split units, LiCre displayed faster and stronger activation by light as well as a lower residual activity in the dark. LiCre was efficient both in yeast, where it allowed us to control the production of β-carotene with light, and in human cells. Given its simplicity and performances, LiCre is particularly suited for fundamental and biomedical research, as well as for controlling industrial bioprocesses.Competing Interest StatementThe authors declare the following competing interest: A patent application covering LiCre and its potential applications has been filed. Patent applicant: CNRS; inventors: Helene Duplus-Bottin, Martin Spichty and Gael Yvert.View Full Text

synthetic biology↗

bioRxiv2019

Cell-to-cell expression dispersion of B-cell surface proteins displays genetic variation among humans

Variability in gene expression across a population of homogeneous cells is known to influence various biological processes. In model organisms, natural genetic variants were found that modify expression dispersion (variability at a fixed mean) but whether such effects exist in humans has not been fully demonstrated. Here, we analyzed single-cell expression of four proteins (CD23, CD55, CD63 and CD86) across cell lines derived from individuals of the Yoruba population. Using data from over 30 million cells, we found substantial inter-individual variation of dispersion. We demonstrate, via de novo cell line generation and subcloning experiments, that this variation exceeds the variation associated with cellular immortalization. By association mapping, we linked the expression dispersion of CD63 to the rs971 SNP. Our results show that human DNA variants can have inherently-probabilistic effects on gene expression. Such subtle genetic effects may participate to phenotypic variation and disease predisposition.

genetics↗

bioRxiv2019

The C. elegans SET-2 histone methyltransferase maintains germline fate by preventing progressive transcriptomic deregulation across generations

Chromatin factors contribute to germline maintenance by preserving a germline-appropriate transcriptional program. In the absence of the conserved histone H3 Lys4 (H3K4) methyltransferase SET-2, C. elegans germ cells progressively lose their identity over generations, leading to sterility. How this transgenerational loss of fertility results from the absence of SET-2 is unknown. Here we performed expression profiling across generations on germlines from mutant animals lacking SET-2 activity. We found that gene deregulation occurred in 2 steps: a priming step in early generations progressing to loss of fertility in later generations. By performing Within-Class Analysis (WCA), a derivative of Principal Component Analysis, we identified transcriptional signatures associated with SET-2 inactivation, both at the priming step and later on during loss of fertility. Further analysis showed that repression of germline genes, derepression of somatic programs, and X-chromosome desilencing through interference with PRC2-dependent repression, are priming events driving loss of germline identity in the absence of SET-2. Decreasing expression of identified priming genes, including the C/EBP homologue cebp-1 and TGF-{beta} pathway components, was sufficient to delay the onset of sterility, suggesting that they individually contribute to the loss of germ cell fate. Altogether, our findings illustrate how the loss of a chromatin regulator at one generation can progressively deregulate multiple transcriptional and signaling programs, ultimately leading to loss of appropriate cell fate.

genetics↗

bioRxiv2017

Assigning Function To Natural Allelic Variation Via Dynamic Modeling Of Gene Network Induction

More and more natural DNA variants are being linked to physiological traits. Yet, understanding what differences they make on molecular regulations remains challenging. Important properties of gene regulatory networks can be captured by computational models. If model parameters can be personalized according to the genotype, their variation may then reveal how DNA variants operate in the network. Here, we combined experiments and computations to visualize natural alleles of the yeast GAL3 gene in a space of model parameters describing the galactose response network. Alleles altering the activation of Gal3p by galactose were discriminated from those affecting its activity (production/degradation or efficiency of the activated protein). The approach allowed us to correctly predict that a non-synonymous SNP would change the binding affinity of Gal3p with the Gal80p transcriptional repressor. Our results illustrate how personalizing gene regulatory models can be used for the mechanistic interpretation of genetic variants.

systems biology↗

bioRxiv2017

Genomics of cellular proliferation under periodic stress

Living systems control cell growth dynamically by processing information from their environment. Although responses to one environmental change have been intensively studied, little is known about how cells react to fluctuating conditions. Here we address this question at the genomic scale by measuring the relative proliferation rate (fitness) of 3,568 yeast gene deletion mutants in out-of-equilibrium conditions: periodic oscillations between two salinity conditions. Fitness and its genetic variance largely depended on the stress period. Surprisingly, dozens of mutants displayed pronounced hyperproliferation at short periods, identifying unexpected controllers of growth under fast dynamics. We validated the implication of the high-affinity cAMP phosphodiesterase and of a regulator of protein translocation to mitochondria in this control. The results illustrate how natural selection acts on mutations in a fluctuating environment, highlighting unsuspected genetic vulnerabilities to periodic stress in molecular processes that are conserved across all eukaryotes.

systems biology↗