Role of Chromatin Bridges in Activating Innate Immune Signaling following Failed Mitosis

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PROJECT SUMMARY/ABSTRACT Abnormal chromatin structures produced by errors in cell division can cause cell death, propagate genetic instability and, potentially, induce inflammatory signaling. Micronuclei and chromatin bridges (CBs) are two types of abnormal structures which are frequently observed in cells from cancers and cancer-related diseases such as Blooms Syndrome (BS). Aberrant chromatin structures can expose genomic self-DNA to the cytoplasm which activates anti-viral inflammatory signaling. Cytoplasmic self-DNA (cyDNA) triggers type-1 interferon (IFN) signaling through multiple innate immune pathways, of which the cGAS-STING axis is most prominent. However, only micronuclei have been studied for their potential to induce IFN signaling. Prior reports suggested that micronuclei spontaneously rupture and produce cyDNA which then activates cGAS. Surprisingly, I have identified that CBs, not micronuclei, are responsible for cGAS activation and IFN induction after failed mitosis. I propose to investigate the molecular and physical mechanisms underlying cGAS activation by CBs. I will use drug-induced mitotic failure or cellular models of BS to study CBs. I will investigate how cGAS-activating CBs arise from errors in cell division, how CBs activate cGAS and whether cGAS- activating CBs are generated in cellular models of BS. I hypothesize that inflammatory CBs are formed by unresolved catenations or merotelic attachments and activate cGAS through a tension-dependent mechanism. To test this hypothesis, I first will analyze CBs produced by drug-induced mitotic failure to determine their formation mechanism. I then will generate CBs through alternative methods, such as topoisomerase inhibition, to determine whether these CBs similarly activate cGAS. In my second aim I will investigate whether actin- mediated tension across the CB is required for cGAS activation. I will test my hypothesis that tension on CBs facilitates cGAS activation through the extrusion of chromatin-bound proteins. In aim 3 I will study cellular models of BS. BS is an inherited cancer pre-disposition syndrome characterized by chromosomal instability and frequent CBs. BS is caused by loss of function mutations in the BLM helicase and cells with a defective BLM helicase exhibit a high number of unresolved sister chromatid catenations during anaphase. I will study whether these catenations turn into cGAS-activating CBs, and if this occurs through the tension-dependent mechanism identified in aim 2. The Mitchison lab provides me an excellent space to explore the nexus of mitotic mechanisms and innate immune signaling. We have made important discoveries in the biology of mammalian cell division and the mechanisms of anti-mitotic chemotherapy. I propose to partake in this tradition by studying the inflammatory consequences of mitotic failure and its potential role in the pathology of Blooms Syndrome.