During the reporting period, we provided evidence for a functional link between nucleotide excision repair (NER), chromatin architecture and the developmental silencing of imprinted genes during mammalian development. Using an innovative in vivo biotinylation tagging knock-in approach in mice, along with animals defective in NER and mouse strains that allow the parental genes to be distinguished, we provide in vivo and functional evidence that the NER endonuclease ERCC1-XPF interacts with the insulator binding protein CTCF, the cohesin subunits SMC1A and SMC3 and with the methyl binding protein MBD2. We find that the complex assembles with the ATP-dependent helicase ATRX at the promoters and control regions (ICRs) of imprinted genes in the developing liver. Disruption of Ercc1 in mice or exposure of cells to the DNA crosslinker mitomycin C triggers the re-localization of CTCF and ATRX to heterochromatin, the dissociation of the CTCF-cohesin complex and ATRX from promoters and ICRs, altered histone marks and the aberrant expression of imprinted genes in the developing liver. Importantly, the response is cell autonomous; it requires ATM and it is instigated in a DNA lesion-specific manner. On the basis of these observations, we proposed that ERCC1-XPF cooperates with CTCF and the cohesin subunits to facilitate the developmental silencing of imprinted genes and that persistent DNA damage signaling triggers chromatin changes that affect gene expression programs associated with NER developmental disorders. Further work has revealed that infiltrating macrophages carrying an inborn NER defect mediate exosome-based metabolic reprogramming upon DNA damage. Indeed, DNA damage and metabolic adaptations are intimately linked in mammals but the underlying mechanisms remain unresolved. In this part of the work, we show that persistent DNA damage accumulation in tissue-infiltrating macrophages carrying an ERCC1-XPF DNA repair defect triggers Golgi dispersal, dilation of endoplasmic reticulum, autophagy and exosome biogenesis leading to the secretion of extracellular vesicles (EVs) in vivo and ex vivo. Lys2-Ercc1f/- (ErF/-) macrophage-derived EVs are enriched in RAS GTPases, they accumulate in ErF/-animal sera and are secreted in the macrophage media after DNA damage. The ErF/- EV cargo is taken up by tissues and cells leading to an increase in insulin-independent glucose transporter levels, enhanced cellular glucose uptake and greater tolerance to glucose challenge in mice. In turn, EV-mediated glucose uptake activates mTOR and pro-inflammatory signals in a cell-autonomous and rapamycin-dependent manner leading to chronic inflammation and tissue pathology with important ramifications for NER progeria and ageing.
