Unrestrained ESCRT-III drives chromosome fragmentation and micronuclear catastrophe

Abstract

The ESCRT-III membrane fission machinery 1,2 restores nuclear envelope integrity during mitotic exit 3,4 and interphase 5,6 . Whereas primary nuclei resealing takes minutes, micronuclear envelope ruptures appear irreversible and result in catastrophic collapse associated with chromosome fragmentation and rearrangements (chromothripsis), thought to be a major driving force in cancer development 7-10 . Despite its importance 11-13 , the mechanistic underpinnings of nuclear envelope sealing in primary nuclei and the defects observed in micronuclei remain largely unknown. Here we show that CHMP7, the nucleator of ESCRT-III filaments at the nuclear envelope 3,14 , and the inner nuclear membrane protein LEMD2 15 act as a compartmentalization sensor detecting the loss of nuclear integrity. In cells with intact nuclear envelope, CHMP7 is actively excluded from the nucleus to preclude its binding to LEMD2. Nuclear influx of CHMP7 results in stable association with LEMD2 at the inner nuclear membrane that licenses local polymerization of ESCRT-III. Tight control of nuclear CHMP7 levels is critical, as induction of nuclear CHMP7 mutants is sufficient to induce unrestrained growth of ESCRT-III foci at the nuclear envelope, causing dramatic membrane deformation, local DNA torsional stress, single-stranded DNA formation and fragmentation of the underlying chromosomes. At micronuclei, membrane rupture is not associated with repair despite timely recruitment of ESCRT-III. Instead, micronuclei inherently lack the capacity to restrict accumulation of CHMP7 and LEMD2. This drives unrestrained ESCRT-III recruitment, membrane deformation and DNA defects that strikingly resemble those at primary nuclei upon induction of nuclear CHMP7 mutants. Preventing ESCRT-III recruitment suppresses membrane deformation and DNA damage, without restoring nucleocytoplasmic compartmentalization. We propose that the ESCRT-III nuclear integrity surveillance machinery is a double-edged sword, as its exquisite sensitivity ensures rapid repair at primary nuclei while causing unrestrained polymerization at micronuclei, with catastrophic consequences for genome stability 16-18 .The formation of dynamic endosomal sorting complex required for transport (ESCRT)-III filaments competent for membrane fission is exquisitely sensitive to balanced levels of the factors that control these filaments 1,2,19 . This is exemplified by depletion of the essential subunit CHMP2A that results in persistent accumulation of CHMP4B, the main component of ESCRT-III filaments, at the reforming nuclear envelope (NE) without successful sealing 3 . We found

BAF facilitates interphase nuclear membrane repair through recruitment of nuclear transmembrane proteins

“…In our model, membrane recruitment could accelerate GFP-BAF removal by either passively increasing kinase concentration by nucleus resealing or actively targeting regulatory proteins to BAF-associated chromatin, similar to the correlation between membrane spreading and BAF release from chromatin during mitotic exit (Samwer et al, 2017). An alternative or additional hypothesis is that BAF remodeling at rupture sites reflects the membrane remodeling activity of the ESCRT-III complex, which drives LEMD2 spreading over the chromatin after micronucleus rupture (Vietri et al, 2019), similar to what we observe for BAF ( Figure 1F). We observed that large BAF foci are frequently unable to disassemble completely after nuclear integrity is restored, and consistent with previous observations of lamin A (Denais et al, 2016), we find these ruptureinduced BAF "scars" are refractory to re-rupture.…”

“…One possibility is that over-recruitment of LEMD2, or other NETs, could inhibit nuclear recompartmentalization through overactivation of ESCRT-III or excessive membrane recruitment. These conditions are both correlated with either the loss of nuclear membrane integrity or the prevention of membrane sealing after cell division or rupture (Appen et al, 2019;Penfield et al, 2019;Thaller et al, 2019;Vietri et al, 2019). Thus, the release of BAF from exposed chromatin during nuclear membrane rupture may be as critical as its recruitment to maintaining nucleus integrity.…”

CDK1 controls CHMP7-dependent nuclear envelope reformation

“…We reasoned that spatiotemporal regulation of the LEM2/CHMP7 interaction would be essential both during mitotic exit and in the subsequent interphase. CHMP7 contains predicted Nuclear Export Sequences (NESs) in Helices 5 and 6 that are thought to limit its exposure to INM proteins, including LEM2, during interphase [10,18]. We confirmed the presence of active NESs in helices 5 and 6 of CHMP7 ( Figure S2A-S2E) and demonstrated that transient perturbation of exportin function resulted in nuclear accumulation of GFP-CHMP7 ( Figure S2D).…”

“…However, by transient transduction of cells with retroviruses driving weak GFP-CHMP7 2xNESexpression, we could demonstrate the inappropriate capture of LEM2 during interphase in GFP-CHMP7 2xNES--positive nuclear envelope and cytoplasmic clusters ( Figure S2H). These data highlight the essential nature of the trans-nuclear envelope segregation of LEM2 and CHMP7, suggesting the existence of a surveillance system poised to monitor the integrity of this barrier [10,18]. The second NES in Helix6 overlaps with a predicted type-1 MIM ( [19], Figure S2A), suggesting that this region may regulate both nucleocytoplasmic compartmentalisation and the ability of CHMP7 to engage the AAA-ATPase, VPS4.…”

APOBEC3B-dependent kataegis and TREX1-driven chromothripsis in telomere crisis

“…2 are generally resolved before the connected daughter cells enter the next S phase. Bridge resolution is accelerated by the exonucleolytic activity of TREX1, which accumulates on the DNA bridge after nuclear envelope rupture 4,[7][8][9] and results in formation of RPA-coated single-stranded (ss) DNA. Rearranged clonal cell lines isolated after progression through this in vitro telomere crisis showed frequent chromothripsis in a pattern similar to cancer: the chromothripsis events were limited to (parts of) chromosome arms rather than whole chromosomes 4,10 .…”