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Common fragile sites (CFSs) are genomic regions frequently involved in cancer-associated rearrangements. Most CFSs lie within large genes, and their instability involves transcription- and replication-dependent mechanisms. Here, we uncover a role for the mitochondrial stress response pathway in the regulation of CFS stability in human cells. We show that FANCD2, a master regulator of CFS stability, dampens the activation of the mitochondrial stress response and prevents mitochondrial dysfunction. Genetic or pharmacological activation of mitochondrial stress signaling induces CFS gene expression and concomitant relocalization to CFSs of FANCD2. FANCD2 attenuates CFS gene transcription and promotes CFS gene stability. Mechanistically, we demonstrate that the mitochondrial stress-dependent induction of CFS genes is mediated by ubiquitin-like protein 5 (UBL5), and that a UBL5-FANCD2 dependent axis regulates the mitochondrial UPR in human cells. We propose that FANCD2 coordinates nuclear and mitochondrial activities to prevent genome instability., Fernandes et al. discover a connection between the mitochondrial stress response and genomic stability. They find that transcription of common fragile site (CFS) genes is induced by mitochondrial stress, whereas a regulator of CFS stability, FANCD2, acts to dampen the mitochondrial stress response and transcription-associated replication stress. These findings suggest a FANCD2-mediated coordination of nuclear and mitochondrial responses to stress.

Macropinocytosis allows cells to take up extracellular material in a non-selective manner into large vesicles called macropinosomes. After internalization, macropinosomes acquire phosphatidylinositol 3-phosphate (PtdIns3P) on their limiting membrane as they mature into endosomal-like vesicles. The molecular mechanisms that underlie recycling of membranes and transmembrane proteins from these macropinosomes still need to be defined. Here, we report that JIP4 (officially known as SPAG9), a protein previously described to bind to microtubule motors, is recruited to tubulating subdomains on macropinosomes by the PtdIns3P-binding protein Phafin2 (officially known as PLEKHF2). These JIP4-positive tubulating subdomains on macropinosomes contain F-actin, the retromer recycling complex and the retromer cargo VAMP3. Disruption of the JIP4–Phafin2 interaction, deletion of Phafin2 or inhibition of PtdIns3P production by VPS34 impairs JIP4 recruitment to macropinosomes. Whereas knockout of JIP4 suppresses tubulation, its overexpression enhances tubulation from macropinosomes. JIP4-knockout cells display increased retention of macropinocytic cargo in both early and late macropinosomes. Collectively, these data identify JIP4 and Phafin2 as components of a tubular recycling pathway that operates from macropinosomes. This article has an associated First Person interview with the first author of the paper., Summary: The motor adaptor JIP4 is recruited to macropinosomes by the PtdIns3P-binding protein Phafin2 to promote recycling to the plasma membrane via tubular extensions of macropinosomes.

International audience; Common fragile sites (CFSs) are loci that are hypersensitive to replication stress and hotspots for chromosomal rearrangements in cancers. CFSs replicate late in S phase, are cell-type specific and nest in large genes. The relative impact of transcription-replication conflicts versus a low density in initiation events on fragility is currently debated. Here we addressed the relationships between transcription, replication, and instability by manipulating the transcription of endogenous large genes in chicken and human cells. We found that inducing low transcription with a weak promoter destabilized large genes, whereas stimulating their transcription with strong promoters alleviated instability. Notably, strong promoters triggered a switch to an earlier replication timing, supporting a model in which high transcription levels give cells more time to complete replication before mitosis. Transcription could therefore contribute to maintaining genome integrity, challenging the dominant view that it is exclusively a threat.

Common fragile sites (CFSs) are chromosome regions prone to breakage upon replication stress known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription could elicit their instability; however, the underlying mechanisms remain elusive. Genome-wide replication timing analyses here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, replicated by long-travelling forks. Forks that travel long in late S phase explains CFS replication features, whereas formation of sequence-dependent fork barriers or head-on transcription–replication conflicts do not. We further show that transcription inhibition during S phase, which suppresses transcription–replication encounters and prevents origin resetting, could not rescue CFS stability. Altogether, our results show that transcription-dependent suppression of initiation events delays replication of large gene bodies, committing them to instability., Common Fragile Sites (CFSs) are chromosome regions prone to breakage upon replication stress known to drive chromosome rearrangements during oncogenesis. Here the authors use genome-wide and single cell techniques to assess how replication timing and transcriptional activity correlate with genome stability.

The endosomal sorting complex required for transport (ESCRT) machinery mediates cargo sorting, membrane deformation and membrane scission on the surface of endosomes, generating intraluminal vesicles (ILVs) to degrade signaling receptors. By live-cell imaging of individual endosomes in human cells, we find that ESCRT proteins are recruited in a repetitive pattern: ESCRT-0 and -I show a gradual and linear recruitment and dissociation, whereas ESCRT-III and its regulatory ATPase VPS4 display fast and transient dynamics. Electron microscopy shows that ILVs are formed consecutively, starting immediately after endocytic uptake of cargo proteins and correlating with the repeated ESCRT recruitment waves, unraveling the timing of ILV formation. Clathrin, recruited by ESCRT-0, is required for timely ESCRT-0 dissociation, efficient ILV formation, correct ILV size and cargo degradation. Thus, cargo sorting and ILV formation occur by concerted, coordinated and repetitive recruitment waves of individual ESCRT subcomplexes and are controlled by clathrin., Intraluminal vesicles are formed by the endosomal sorting complex required for transport (ESCRT) machinery. Here, the authors unravel the timing of vesicle budding, and that endosomal clathrin regulates concerted recruitment of ESCRT subcomplexes, required for efficient membrane remodeling.

Cytokinesis is the final stage of cell division and is concluded by abscission of the intercellular bridge to physically separate the daughter cells. Timing of cytokinetic abscission is monitored by a molecular machinery termed the abscission checkpoint. This machinery delays abscission in cells with persistent chromatin in the intercellular bridge. Recent work has also uncovered its response to high membrane tension, nuclear pore defects, and DNA replication stress. Although it is known that the abscission checkpoint depends on persistent activity of the Aurora B protein kinase, we have only recently begun to understand its molecular basis. We propose here a molecular framework for abscission checkpoint signaling and we discuss outstanding questions relating to its function and physiological relevance.

Common fragile sites (CFSs) are genomic regions frequently involved in cancer-associated rearrangements. Most CFSs lie within large genes, and their instability relies on transcription- and replication-dependent mechanisms. Here, we uncover a role for the UBL5-dependent branch of the unfolded protein response pathway (UPR) in the maintenance of CFS stability. We show that genetic or pharmacological UPR activation induces CFS gene expression and concomitant relocalization of FANCD2, a master regulator of CFS stability, to CFSs. Furthermore, a genomic analysis of FANCD2 binding sites identified an enrichment for mitochondrial UPR transcriptional response elements in FANCD2 bound regions. We demonstrated that depletion of FANCD2 increases CFS gene transcription and their instability while also inducing mitochondrial dysfunction and triggering the activation of the UPR pathway. Depletion of UBL5, a mediator of the UPR, but not ATF4, reduces CFS gene expression and breakage in FANCD2-depleted cells. We thus demonstrate that FANCD2 recruitment and function at CFSs depends on transcription and UPR signaling, and in absence of transcription or UBL5, FANCD2 is dispensable for CFS stability. We propose that FANCD2 coordinates nuclear and mitochondrial activities by tuning the UPR to prevent genome instability.