Biology subjects

Guillou, E.

Publications and source records attributed to Guillou, E..

5 recordsLinked to original sources

bioRxiv2025

The Mediator complex subunit Med19 extends healthy lifespan in Drosophila by preventing cellular and organismal frailty

Aging involves a progressive decline in physiological functions, often marked by the onset of a "frailty point" just before survival rates decrease rapidly. Here, we investigate how the Mediator subunit Med19 modulates this transition in Drosophila. We find that upregulating Med19 extends the median lifespan by nearly 90% and postpones the onset of accelerated mortality, suggesting that Med19 helps preserve the resilience phase of aging. In contrast, Med19 downregulation sharply reduces both median and maximum lifespan, advancing the frailty threshold. We show that Med19 knockdown increases fly vulnerability to environmental insults such as oxidative and genotoxic challenges whereas Med19 upregulation helps them resist these stresses, underscoring Med19s protective role in maintaining genomic integrity. We link these phenotypes to altered stress-response pathways at the cellular level: Med19-depleted cells show a compensatory upregulation of genes involved in iron-sulfur cluster biogenesis, glutathione metabolism, and DNA damage repair. At the cellular level, Med19 depletion triggers a "loser" phenotype in cell competition assays, activating the JNK pathway and undergoing apoptosis, highlighting a form of "cellular frailty" that parallels organismal frailty. Finally, we found that the Med19 protein level naturally decreases with age and showed that restoring Med19 expression in aged flies increases fitness and delays the onset of frailty even in older, "frail" individuals, underscoring its significance as an aging regulator. Altogether, our findings establish Med19 as a crucial mediator of lifespan and stress resilience, suggesting it acts as a rheostat that modulates the transition from healthy aging to frailty in Drosophila. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=180 HEIGHT=200 SRC="FIGDIR/small/659864v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1a5e398org.highwire.dtl.DTLVardef@14658org.highwire.dtl.DTLVardef@1b14089org.highwire.dtl.DTLVardef@41060_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- At the organismal level, Med19 upregulation extends Drosophila lifespan by 90%, delays the onset of frailty and enhances oxidative and DNA damage stress resistance, while its depletion has the opposite effect. - Med19 depletion alters the expression of stress response genes and induces a loser-cell phenotype in cell competition assays revealing its crucial function in controlling cellular fitness. - Med19 protein levels naturally decline with age, and its restoration in aged flies improves both healthspan and lifespan.

cell biology↗

bioRxiv2023

Circadian PERIOD complex regulates TC-DSB repair through anchoring to the nuclear envelope

Repair of DNA Double-Strand Breaks (DSBs) produced in transcriptionally active chromatin occurs through a poorly characterized pathway called Transcription-Coupled DSB repair (TC-DSBR). Here, using a screening approach scoring multiple outputs in human cells, we identified proteins from the PERIOD complex, a key module ensuring circadian oscillations, as novel TC-DSBR players. We show that the core PER complex protein PER2 is recruited at TC-DSBs and that it contributes to the targeting of TC-DSBs at the nuclear envelope (NE). At the NE, SUN1 and the Nuclear Pore Complex (NPC) act as docking sites for TC-DSBs and TC-DSB anchoring fosters RAD51 assembly. Impaired DSB localization at the NE results in elevated DSB clustering and translocation rate. In agreement, the circadian clock regulates TC-DSB anchoring to the NE, RAD51 assembly, and DSB clustering. Our study shows that DSB localization to the NPC is a conserved molecular pathway that also occurs in human cells and provides a direct link between the circadian rhythm and the response to DSBs occurring in active genes. This opens new therapeutic strategies for chemotherapies based on drugs that are inducing DSBs in active loci such as topoisomerase poisons.

molecular biology↗

bioRxiv2022

Inducible degradation of the Drosophila Mediator subunit Med19 reveals its role in regulating developmental but not constitutively-expressed genes

The multi-subunit Mediator complex plays a critical role in gene expression by bridging enhancer-bound transcription factors and the RNA polymerase II machinery. Although experimental case studies suggest differential roles of Mediator subunits, a comprehensive view of the specific set of genes regulated by individual subunits in a developing tissue is still missing. Here we address this fundamental question by focusing on the Med19 subunit and using the Drosophila wing imaginal disc as a developmental model. By coupling auxin- inducible degradation of endogenous Med19 in vivo with RNA-seq, we got access to the early consequences of Med19 elimination on gene expression. Differential gene expression analysis reveals that Med19 is not globally required for mRNA transcription but specifically regulates positively or negatively less than a quarter of the expressed genes. By crossing our transcriptomic data with those of Drosophila gene expression profile database, we found that Med19-dependent genes are highly enriched with spatially-regulated genes while the expression of most constitutively expressed genes is not affected upon Med19 loss. Whereas globally downregulation does not exceed upregulation, we identified a functional class of genes encoding spatially-regulated transcription factors, and more generally developmental regulators, responding unidirectionally to Med19 loss with an expression collapse. Moreover, we show in vivo that the Notch-responsive wingless and the E(spl)-C genes require Med19 for their expression. Combined with experimental evidences suggesting that Med19 could function as a direct transcriptional effector of Notch signaling, our data support a model in which Med19 plays a critical role in the transcriptional activation of developmental genes in response to cell signaling pathways. Author summaryThe Mediator is a large evolutionarily conserved multisubunit complex that plays essential functions in gene expression by relaying cues emanating from enhancer-bound transcription factors to the RNA polymerase II machinery. The transcriptional landscapes regulated by each Mediator subunit, especially in vivo in the context of developmental processes, remains poorly characterized. We therefore sought to provide a comprehensive view of the genes directly regulated by Med19, an archetypal Mediator subunit, in the context of the development of the Drosophila wing. Our work has important methodological implications as to carry out our analysis in the best experimental conditions, we generated mutant flies in which we could trigger fast degradation of the Med19 subunit on demand in the tissues of the living animal. We found that only a small part of the genes expressed in the developing wing are controlled by Med19 showing that this Mediator component exercises specific functions. Another major finding is the strong involvement of Med19 in the regulation of the expression of the genes involved in developmental processes supporting the idea that Med19 is a Mediator component dedicated to highly regulated mode of gene expression. The most unanticipated discovery of our study is the fact that Med19 does not appear to play a role in the expression of the genes that are constitutively transcribed, among which the housekeeping genes. This raises the notion that in the context of simple --non-regulated-- mode of expression, part of the Mediator functionalities, among which Med19, otherwise dedicated to highly regulated type of transcription, may become dispensable.

developmental biology↗

bioRxiv2021

Tissue-specific versus pleiotropic enhancers within the bric-a-brac tandem gene duplicates display differential regulatory activity and evolutionary conservation

During animal evolution, de novo emergence and modifications of pre-existing transcriptional enhancers have contributed to biological innovations, by implementing gene regulatory networks. The Drosophila melanogaster bric-a-brac (bab) complex, comprising the tandem paralogous genes bab1-2, provides a paradigm to address how enhancers contribute and co-evolve to regulate jointly or differentially duplicated genes. We previously characterized an intergenic enhancer (named LAE) governing bab2 expression in leg and antennal tissues. We show here that LAE activity also regulates bab1. CRISPR/Cas9-mediated LAE excision reveals its critical role for bab2-specific expression along the proximo-distal leg axis, likely through paralog-specific interaction with the bab2 gene promoter. Furthermore, LAE appears involved but not strictly required for bab1-2 co-expression in leg tissues. Phenotypic rescue experiments, chromatin features and a gene reporter assay reveal a large "pleiotropic" bab1 enhancer (termed BER) including a series of cis-regulatory elements active in the leg, antennal, wing, haltere and gonadal tissues. Phylogenomics analyses indicate that (i) bab2 originates from bab1 duplication within the Muscomorpha sublineage, (ii) LAE and bab1 promoter sequences have been evolutionarily-fixed early on within the Brachycera lineage, while (iii) BER elements have been conserved more recently among muscomorphans. Lastly, we identified conserved binding sites for transcription factors known or prone to regulate directly the paralogous bab genes in diverse developmental contexts. This work provides new insights on enhancers, particularly about their emergence, maintenance and functional diversification during evolution. Author summaryGene duplications and transcriptional enhancer emergence/modifications are thought having greatly contributed to phenotypic innovations during animal evolution. However, how enhancers regulate distinctly gene duplicates and are evolutionary-fixed remain largely unknown. The Drosophila bric-a-brac locus, comprising the tandemly-duplicated genes bab1-2, provides a good paradigm to address these issues. The twin bab genes are co-expressed in many tissues. In this study, genetic analyses show a partial co-regulation of both genes in the developing legs depending on tissue-specific transcription factors known to bind a single enhancer. Genome editing and gene reporter assays further show that this shared enhancer is also required for bab2-specific expression. Our results also reveal the existence of partly-redundant regulatory functions of a large pleiotropic enhancer which contributes to co-regulate the bab genes in distal leg tissues. Phylogenomics analyses indicate that the Drosophila bab locus originates from duplication of a dipteran bab1-related gene, which occurred within the Brachycera (true flies) lineage. bab enhancer and promoter sequences have been differentially-conserved among Diptera suborders. This work illuminates how transcriptional enhancers from tandem gene duplicates (i) differentially interact with distinct cognate promoters and (ii) undergo distinct evolutionary changes to diversifying their respective tissue-specific gene expression pattern.

genetics↗

bioRxiv2020

Mediator complex subunit Med19 binds directly GATA DNA-binding zinc finger and functions with Med1 in GATA-driven gene regulation in vivo

The evolutionarily-conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Polymerase II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. However, binary MED subunit - TF partnerships are probably oversimplified models. Using Drosophila TFs of the GATA family - Pannier (Pnr) and Serpent (Srp) - as a model, we have previously established GATA cofactor evolutionarily-conserved function for the Med1 Mediator subunit. Here, we show that another subunit, Med19, is required for GATA-dependent gene expression and interacts physically with Pnr and Srp in cellulo, in vivo and in vitro through their conserved C-zinc finger (ZF), indicating general GATA co-activator functions. Interestingly, Med19 is critical for the regulation of all tested GATA target genes which is not the case for Med1, suggesting differential use of MED subunits by GATAs depending on the target gene. Lastly, despite their presumed distant position within the MED middle module, both subunits interact physically. In conclusion, our data shed new light first on the MED complex, engaging several subunits to mediate TF-driven transcriptional responses and second, on GATA TFs, showing that ZF DNA-binding domain also serves for transactivation.

developmental biology↗