Biology subjects

Gagne, J.-P.

Publications and source records attributed to Gagne, J.-P..

6 recordsLinked to original sources

bioRxiv2025

Nucleoplasmic Lamin A/C controls replication fork restart upon stress by modulating local H3K9me3 and ADP-ribosylation levels

Mild replication interference is a consolidated strategy for cancer chemotherapy. Tolerance to mild replication stress (RS) relies on active fork slowing, mediated by transient fork reversal and RECQ1-assisted restart, and modulated by PARP1 and nuclear architectural components via yet-elusive mechanisms. We combined acute protein inactivation with cell biology and single-molecule approaches to investigate the role of Lamin A/C upon mild RS. We found that Lamin A/C dynamically interacts with replication factories throughout the nucleus and, together with its nucleoplasmic partner LAP2, is required to induce active fork slowing and maintain chromosome stability upon mild genotoxic treatments. Inactivating nucleoplasmic Lamin A/C reduces poly-ADP-ribosylation (PAR) levels at nascent DNA, triggering deregulated RECQ1-mediated restart of reversed forks. Moreover, we found that the heterochromatin mark H3K9me3, previously reported at stalled forks, also accumulates in response to mild RS. H3K9me3 accumulation requires Lamin A/C, which prevents its premature removal by the histone demethylase JMJD1A/KDM3A. H3K9me3 loss per se phenocopies Lamin A/C inactivation, reducing PAR levels and deregulating RECQ1 activity at forks. Hence, nucleoplasmic Lamin A/C, H3K9me3 and PARylation levels are crucial, mechanistically-linked modulators of fork slowing, remodelling and restart upon mild RS, with important implications for chemotherapy response and Lamin A/C deregulation in human disease.

molecular biology↗

bioRxiv2023

pADP-ribosylation Regulates the Cytoplasmic Localization, Cleavage and Pro-apoptotic Function of HuR

HuR (ElavL1) is one of the main posttranscriptional regulators that determines cell fate. While the role of HuR in apoptosis is well-established, the posttranslational modifications that governs this function remain elusive. In this study we show that PARP1/2-mediated poly(ADP)-ribosylation (PARylation) is instrumental in the pro-apoptotic function of HuR. During apoptosis or in cells depleted of PARP1/2 enzymes, a substantial reduction in HuR PARylation is observed. This results in the cytoplasmic accumulation and the cleavage of HuR, both of which are essential events for apoptosis. These effects are mediated by a pADP-ribose (PAR) binding motif within the HuR-HNS region (HuR PAR Binding Site (HuR-PBS)). Under normal conditions, the association of HuR-PBS with PAR is responsible for the nuclear retention of HuR. Mutations within this motif prevents the binding of HuR to its import factor TRN2, leading to its cytoplasmic accumulation and cleavage. Collectively, our findings underscore the role of PARylation in controlling the proapoptotic function of HuR, offering insight into the mechanism by which PARP1/2 enzymes regulate cell fate and adaptation to various assaults.

cell biology↗

bioRxiv2023

Poly(ADP-ribose) mediates bioenergetic defects and redox imbalance in neurons following oxygen and glucose deprivation

PARP-1 over-activation results in cell death via excessive PAR generation in different cell types, including neurons following brain ischemia. Glycolysis, mitochondrial function, and redox balance are key cellular processes altered in brain ischemia. Studies show that PAR generated after PARP-1 over-activation can bind hexokinase-1 (HK-1) and result in glycolytic defects and subsequent mitochondrial dysfunction. HK-1 is the neuronal hexokinase and catalyzes the first reaction of glycolysis, converting glucose to glucose-6-phosphate (G6P), a common substrate for glycolysis, and the pentose phosphate pathway (PPP). PPP is critical in maintaining NADPH and GSH levels via G6P dehydrogenase activity. Therefore, defects in HK-1 will not only decrease cellular bioenergetics but will also cause redox imbalance due to the depletion of GSH. In brain ischemia, whether PAR-mediated inhibition of HK-1 results in bioenergetics defects and redox imbalance is not known. We used oxygen-glucose deprivation (OGD) in mouse cortical neurons to mimic brain ischemia in neuronal cultures and observed that PARP-1 activation via PAR formation alters glycolysis, mitochondrial function, and redox homeostasis in neurons. We used pharmacological inhibition of PARP and adenoviral-mediated overexpression of wild-type HK-1 (wtHK-1) and PAR-binding mutant HK-1 (pbmHK-1). Our data show that PAR inhibition or overexpression of HK-1 significantly improves glycolysis, mitochondrial function, redox homeostasis, and cell survival in mouse cortical neurons exposed to OGD. These results suggest that PAR binding and inhibition of HK-1 during OGD drives bioenergetic defects in neurons due to inhibition of glycolysis and impairment of mitochondrial function.

neuroscience↗

bioRxiv2022

Proteome-wide microarray-based screening of PAR-binding proteins

Poly(ADP-ribose) (PAR) plays a crucial role in intracellular signaling and scaffolding through covalent modification or non-covalent binding to target proteins. The non- covalent binding PARylome has not been extensively characterized. Here we performed a PAR-binding screen using a human protein microarray that covers most of the human proteome to characterize the non-covalent binding PARylome. A total of 356 PAR- binding proteins were identified. The PAR-binding PARylome suggests that PAR- binding regulates a variety of biological processes beyond well-characterized DNA damage signaling and DNA repair. Proteins that may be reprogrammed by PAR-binding include signaling molecules, transcription factors, nucleic acid binding proteins, calcium binding proteins, ligases, oxidoreductases, enzymes, transferases, hydrolases, and receptors. The global database of PAR-binding proteins that we established will be a valuable tool for further in-depth analysis of the role of PARylation in a wide range of biological contexts.

biochemistry↗

bioRxiv2020

Aurora-A phosphorylates splicing factors and regulates alternative splicing

Aurora-A kinase is well known to regulate progression through mitosis. However, the kinase also performs additional functions that could explain the failure of its inhibitors to be effective in cancer treatments. To identify these functions, we applied a proteomics approach to search for interactors of Aurora-A. We found a large number of proteins involved in pre-mRNA splicing, strongly suggesting an important role for Aurora-A in this biological process. Consistently, we first report the subcellular localization of Aurora-A in nuclear speckles, the storehouse of splicing proteins. We also demonstrate direct interaction of Aurora-A with RRM domain-containing splicing factors such as hnRNP and SR proteins and their phosphorylation in vitro. Further, RNA-sequencing analysis following pharmacological inhibition of Aurora-A resulted in alternative splicing changes corresponding to 505 genes, including genes with functions regulated by Aurora-A kinase. Finally, we report enrichment of RNA motifs within the alternatively spliced regions affected by Aurora-A kinase inhibition which are bound by Aurora-A interacting splicing factors, suggesting that Aurora-A regulates alternative splicing by modulating the activity of these interacting splicing factors. Overall our work identified Aurora-A as a novel splicing kinase and for the first time, describes a broad role of Aurora-A in regulating alternative splicing.

molecular biology↗

bioRxiv2020

Drosophila dTBCE recruits tubulin around chromatin to promote mitotic spindle assembly

Proper assembly of mitotic spindles requires microtubule nucleation at centrosomes but also around chromatin. In this study, we reveal a novel mechanism by which an enrichment of tubulin in the nuclear space following nuclear envelope breakdown promotes nucleation of spindle microtubules. This event mediated by the tubulin-specific chaperone dTBCE, depends on its tubulin binding CAP-Gly motif and is regulated by Ran. Live imaging, proteomic and biochemical analyses suggest that dTBCE is enriched in the nucleus at nuclear envelope breakdown and interacts with nuclear pore proteins and the Ran machinery to create an environment that facilitates subsequent tubulin enrichment. We propose that dTBCE-dependent increase in tubulin concentration in the nuclear space is an important mechanism for microtubule nucleation in organisms where compartmentalization prevents free diffusion of tubulin.

cell biology↗