Sélène-Véronique Glück

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cGAS regulation and activity during genotoxic stress

The innate immune system has evolved to detect pathogens through germlineencoded pattern recognition receptors (PRRs). Nucleic acid sensors, a defined class of PRRs, bind to microbial and viral nucleic acids and trigger the production of type I interferons and cytokines which mediate the first line of host defense. In the past, these sensors have been primarily studied in the context of infections. Yet, the DNA sensor cyclic GMP-AMP synthase (cGAS) has been recently described to detect not only microbial and viral derived dsDNA but also self-DNA in a sequenceindependent manner. At steady state, the organization of nuclear self-DNA is tightly controlled by a complex network of multiple factors that can become imbalanced during genotoxic stress. Whether and how this stress can be sensed by cGAS in the absence of infection are the main questions of this thesis. Observing the fate of cGAS in space and time in the presence of several genotoxic insults including chemotherapeutics allowed me to gain novel insights into processes regulating the localization and activation of cGAS. In this thesis, I demonstrate that cGAS localizes to both nuclear and cytosolic compartments of the cell. We show that cGAS is inactive in the nucleus due to binding to the acidic patch on H2A-H2B heterodimers. Upon genotoxic stress, cGAS is removed from the nucleus in a manner that is dependent on histones and remains inactive. Moreover, with time cGAS transcription is repressed resulting in a gradual decrease of cGAS levels. On the other hand, cGAS-activating DNA substrates such as cytosolic chromatin fragments accumulate in the cytosol of senescent cells deficient in Lamin B1 when cGAS levels are already decreased. Along with the occurrence of these chromatin fragments, cGAS mediates an inflammatory response dependent on STING (Stimulator of interferon genes), referred to as the senescence-associated secretory phenotype (SASP). Overall, we conclude that acute genotoxic stress does not result in cGAS-STING signaling, whereas the chronic DNA damage response, senescence, activates the cGAS-STING pathway. Moreover, the results presented in this work suggest a new role for cGAS-STING signaling during senescence beyond the recognition of pathogens.

EPFL2020

Structural mechanism of cGAS inhibition by the nucleosome

Rudolf Hovius, Beat Fierz, Andrea Ablasser, Baptiste Claude André Guey, Alexiane Clara Decout, Pauline Ambre Melenec, Joscha Weiss, Simone Cavadini, Kristina Makasheva, Sélène-Véronique Glück

The DNA sensor cGAS initiates innate immune responses following microbial infection, cellular stress, and cancer1. Upon activation by double-stranded DNA, cytosolic cGAS produces 2’3’ cyclic GMP-AMP and triggers inflammatory cytokine and type I interferon (IFN) induction2–7. cGAS is also present inside the cell nucleus replete with genomic DNA8, where chromatin has been implicated in restricting its enzymatic activity9. However, the structural basis for cGAS inhibition by chromatin has remained unknown. Here we present the cryo-electron microscopy structure of human cGAS bound to nucleosomes at 3.1 Å resolution. cGAS makes extensive contacts with both the acidic patch of the histone H2A-H2B heterodimer and nucleosomal DNA. The structural and complementary biochemical analysis also finds cGAS engaged to a second nucleosome in trans. Mechanistically, nucleosome binding locks cGAS in a monomeric state, in which steric hindrance suppresses spurious activation by genomic DNA. We find that mutations to the cGAS-acidic patch interface are necessary and sufficient to abolish the inhibitory effect of nucleosomes in vitro and to unleash cGAS activity on genomic DNA in living cells. Our work uncovers the structural basis of cGAS interaction with chromatin and defines a compelling mechanism that permits self-nonself discrimination of genomic DNA by cGAS.

2020

cGAS regulation and activity during genotoxic stress

Structural mechanism of cGAS inhibition by the nucleosome

Innate immunosensing of DNA in cellular senescence

Innate immunosensing of DNA in cellular senescence

cGAS regulation and activity during genotoxic stress

Structural mechanism of cGAS inhibition by the nucleosome