BAF restricts cGAS on nuclear DNA to prevent innate immune activation

The appearance of DNA in the cytosol is perceived as a danger signal that stimulates potent immune responses through cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS). How cells regulate the activity of cGAS toward self-DNA and guard against potentially damaging autoinflammatory responses is a fundamental biological question. Here, we identify barrier-to-autointegration factor 1 (BAF) as a natural opponent of cGAS activity on genomic self-DNA. We show that BAF dynamically outcompetes cGAS for DNA binding, hence prohibiting the formation of DNA-cGAS complexes that are essential for enzymatic activity. Upon acute loss of nuclear membrane integrity, BAF is necessary to restrict cGAS activity on exposed DNA. Our observations reveal a safeguard mechanism, distinct from physical separation, by which cells protect themselves against aberrant immune responses toward genomic DNA.

Exploring the mechanisms governing nuclear cGAS localization and activity

Marilena Wischnewski

Recognition of pathogen-derived molecules through germline-encoded receptors is a fundamental principle of innate immunity. Pattern recognition receptors detect specific intracellular danger signals to trigger potent immune responses. The DNA sensor cyclic GMP-AMP synthase (cGAS) detects double-stranded DNA derived from viruses and microbes in the cytoplasm. However, due to its ability to recognize DNA independent of sequence, aberrant detection of self-DNA in various settings can lead to autoinflammatory diseases. Under physiological conditions, genomic DNA is sequestered in the nucleus and shielded from cytosolic molecules by the nuclear envelope (NE). Nevertheless, cGAS gains access to the nuclear compartment in some instances, and the mechanisms controlling its activity on genomic DNA are not fully known.In this study, we investigate the regulation of nuclear cGAS localization and activity in different contexts. We first describe how cGAS activity against nuclear DNA is restricted in instances of spontaneous loss of nucleo-cytoplasmic compartmentalization. Upon NE rupture, cGAS enters the nucleus, but is prevented from activation by Barrier-to-autointegration factor 1 (BAF). BAF dynamically competes with cGAS for DNA binding, thereby preventing the formation of stable cGAS-DNA complexes and cGAS enzymatic activity. We thus identify a cellular safeguard mechanism, beyond physical separation, that restricts cGAS activity against genomic self-DNA.In addition, we investigate cGAS activation in conditions associated with a disrupted NE. Instability of the NE is a hallmark of laminopathies, diseases of the nuclear lamina that can manifest in premature aging phenotypes. While we find no contribution of basal cGAS signaling to the pathogenesis of two different progeria syndromes, we detect a decreased capacity to respond to DNA challenge on the cellular level, suggesting that cGAS may be sequestered inside the nucleus in these cells.Lastly, because cGAS is increasingly recognized to reside inside the nucleus at steady state, we delineate cGAS nuclear localization in time and space. In cycling cells, nuclear cGAS levels oscillate with the cell cycle and are highest after mitosis followed by a sharp decrease in G1 phase. We report that cGAS is not exported during G1, but rather subjected to degradation inside the nucleus by the nuclear proteasome. Moreover, we establish a technique to study potential interaction partners of nuclear cGAS, which opens up intriguing possibilities for further investigation.Together, our findings contribute to a better understanding of the regulatory processes that govern nuclear cGAS localization and activity, and will help to uncover potential disease-causing aberrations in the future.

EPFL2022

cGAS regulation and activity during genotoxic stress

Sélène-Véronique Glück

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

Elucidating the mechanisms of paclitaxel-loaded-nanoparticle-induced immune response

Efthymia Vokali

Dentritic cells (DC) are the most potent antigen-presenting cells and play a key role in the induction of effective immune responses, suggesting a novel target for anti-tumor immunotherapy. Paclitaxel (PXL) is a widely used chemotherapeutic, initially characterized as a mitotic inhibitor, that has been shown to enhance maturation and function of DCs as well as up-regulate the production of inflammatory cytokines. Thus, modulation of DC function by the approved-for-human-use PXL could constitute a novel approach for reshaping immune responses against a tumor. For targeted delivery of PXL to DCs in the lymph nodes (LNs), where they are present in high concentrations and apt for antigen uptake, poly(propylene) sulfide core nanopatricles (NPs) developed by our laboratory have been used. PXL loaded-NP (PXL-NP) treatment induces massive immune cell infiltration into the draining LN 24h after intradermal injection of PXL-NPs in mice, suggesting that PXL-NP treatment induces a local and robust immune response (Thomas et al., in preparation). We hypothesized that this effect stems directly from the action of PXL-NPs on LN-resident DCs. We demonstrate that PXL-NPs are able to induce maturation and cytokine production in murine and human DCs in vitro, which may explain the in vivo observations of robust cell recruitment to the draining LN and generation of adaptive immune responses. Furthermore, the striking cell infiltration in the LN upon PXL-NP treatment was abrogated in complement protein 3 (C3) knockout mice in vivo (Thomas et al. in preparation). Interestingly, we show that the presence of C3 regulates IL-12p40 expression and, in turn, plays a crucial role in the activation of mature, functional DCs in response to PXL-NPs. Thus, the absence of functional DCs in case of C3 deficiency might account for the hindered infiltration. Together, this data suggests that PXL-NPs may effectively direct the immunomodulatory action of PXL to LN-resident DCs to alter immune responses.

2011