Biology subjects

Bernasconi, L.

Publications and source records attributed to Bernasconi, L..

5 recordsLinked to original sources

bioRxiv2025

Pluripotency factors enhance the firing efficiency of late DNA replication origins in mouse embryonic stem cells

DNA replication initiates at genomic loci known as origins, which fire according to a spatiotemporal program, still not yet well-defined in mammals. Here, we investigated whether pluripotent mouse embryonic stem cells (mESCs), which proliferate rapidly and have very short G1 phases, have a different spatiotemporal origin firing program than differentiated cells. Using EdU-seq combined with various cell cycle synchronization methods, we identified DNA replication initiation zones (IZs) in mESCs, mesenchymal stem cells and mouse embryo fibroblasts (MEFs). Similar profiles of IZs were present in the early-replicating genomic domains in all cell types. Uniquely in mESCs, we observed, within 1-2 hours of entry into S phase, origin firing at IZs that mapped to the mid and late-replicating genomic domains. This change in the replication timing program, which did not lead to shortening of the length of S phase, was driven by pluripotency transcription factors, notably OCT4, as documented by examining mESCs in which OCT4 expression was transiently suppressed. We propose that pluripotency factors in mESCs bind to IZs at mid and late-replicating genomic domains to enhance chromatin accessibility, which in turn leads to changes in the replication timing program. These results provide an example of how transcription factors control DNA replication in a cell type-specific manner.

molecular biology↗

bioRxiv2023

Hsf1 and the molecular chaperone Hsp90 support a "rewiring stress response" leading to an adaptive cell size increase in chronic stress

Cells are exposed to a wide variety of internal and external stresses. Whereas many studies have focused on cellular responses to acute and severe stresses, little is known about how cellular systems adapt to sublethal chronic stresses. Using mammalian cells in culture, we discovered that they adapt to chronic mild stresses of up to two weeks, notably proteotoxic stresses such as heat, by increasing their size and translation, thereby scaling the amount of total protein. These adaptations render them more resilient to persistent and subsequent stresses. We demonstrate that Hsf1, well known for its role in acute stress responses, is required for the cell size increase, and that the molecular chaperone Hsp90 is essential for coupling the cell size increase to augmented translation. We term this translational reprogramming the "rewiring stress response", and propose that this protective process of chronic stress adaptation contributes to the increase in size as cells get older, and that its failure promotes aging.

cell biology↗

bioRxiv2023

Therapeutic and vaccine-induced cross-reactive antibodies with effector function against emerging Omicron variants

Currently circulating SARS-CoV-2 variants acquired convergent mutations at receptor-binding domain (RBD) hot spots1. Their impact on viral infection, transmission, and efficacy of vaccines and therapeutics remains poorly understood. Here, we demonstrate that recently emerged BQ.1.1. and XBB.1 variants bind ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1 and XBB.1 RBDs bound to human ACE2 and S309 Fab (sotrovimab parent) explain the altered ACE2 recognition and preserved antibody binding through conformational selection. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1, the variant displaying the greatest loss of neutralization. Moreover, in several donors vaccine-elicited plasma antibodies cross-react with and trigger effector functions against Omicron variants despite reduced neutralizing activity. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring persistent immune imprinting. Our findings suggest that this previously overlooked class of cross-reactive antibodies, exemplified by S309, may contribute to protection against disease caused by emerging variants through elicitation of effector functions.

immunology↗

bioRxiv2022

Translational reprogramming in response to accumulating stressors ensures critical threshold levels of Hsp90 for mammalian life

The cytosolic molecular chaperone Hsp90 is essential for eukaryotic life1, 2. It is involved in multiple branches of proteostasis2, 3, and as a molecular capacitor in morphological evolution4. Although reduced Hsp90 levels cause phenotypic variations5, 6 and correlate with aging7, whether eukaryotic cells and organisms can tune the basal Hsp90 protein levels to alleviate physiologically accumulated stress is unknown. To begin to explore this question, we investigated whether and how mice adapt to the deletion of three out of four alleles encoding cytosolic Hsp90, one Hsp90{beta} allele being the only remaining one. While the vast majority of such mouse embryos die during gestation, survivors apparently manage to increase their Hsp90{beta} protein to at least wild-type levels. Further mechanistic studies revealed an internal ribosome entry site in the 5UTR of the Hsp90{beta} mRNA allowing translational reprogramming to compensate for the genetic loss of Hsp90 alleles and in response to stress. We found that the minimum amount of total Hsp90 that is required to support viability of mammalian cells and organisms is 50-70% of what is normally there. Those that fail to maintain a threshold level are subject to accelerated senescence, proteostatic collapse, and ultimately death. Therefore, considering that Hsp90 levels can be reduced [≥]100-fold in the unicellular budding yeast, critical threshold levels of Hsp90 have been markedly increased during eukaryotic evolution. The incompressible part of the steady-state levels of Hsp90 may have increased to accommodate the ever-growing complexity of the proteome8 on the path towards mammals.

molecular biology↗

bioRxiv2019

The Hsp70/Hsp90 Co-Chaperone Hop/Stip1 Shifts the Proteostatic Balance from Folding Towards Degradation

Hop/Stip1/Sti1 is thought to be essential as a co-chaperone to facilitate substrate transfer between the Hsp70 and Hsp90 molecular chaperones. Despite this proposed key function for protein folding and maturation, it is not essential in a number of eukaryotes and bacteria lack an ortholog. We set out to identify and to characterize its eukaryote-specific function. Human cell lines and the budding yeast with deletions of the Hop/Sti1 gene display reduced proteasome activity due to inefficient capping of the core particle with regulatory particles. Unexpectedly, knock-out cells are more proficient at preventing protein aggregation and at promoting protein refolding. Without the restraint by Hop, a more efficient folding activity of the prokaryote-like Hsp70/Hsp90 complex, which can also be demonstrated in vitro, compensates for the proteasomal defect and ensures an alternate proteostatic equilibrium. Thus, cells may act on Hop to shift the proteostatic balance between folding and degradation.

cell biology↗