Periodic Reporting for period 3 - DeFiNER (Nucleotide Excision Repair: Decoding its Functional Role in Mammals)
Reporting period: 2019-01-01 to 2020-06-30
The links between defects in DNA repair and age-related diseases, including cancer are well established. However, little is known about the functional links between DNA repair factors, DNA damage signaling and mammalian development. The latter has recently attracted keen scientific interest for two major reasons: i) because of the growing appreciation of a link between DNA repair deficiencies and severe developmental defects in patients and ii) because of the increasing evidence that events governing the earliest stages of an organism's existence are now known to affect disease onset at later stages in life. During the first period of the proposed work, we have provided evidence that i. NER factors interact with proteins involved in chromatin architecture and are instrumental for vital processes during mammalian development and that inborn defects in NER lead to the accumulation of irreparable DNA lesions that trigger ii. an exosome-based metabolic reprogramming and iii. chronic inflammation with important ramifications for NER progeria and natural aging.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
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.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
Random or programmed transcription-associated DNA damage events may occur aimlessly or deliberately in the mammalian genome compromising or supporting cell viability. Cells employ nucleotide excision repair (NER), a highly-conserved DNA repair pathway, to detect and remove a wide range of structurally diverse DNA lesions in their genome. With the exception of skin cancer, however, the links between inborn defects in NER and developmental abnormalities remain obscure. Recent evidence suggests that NER factors are involved, in addition to the canonical DNA repair mechanism, in processes that facilitate mRNA synthesis or shape the 3D chromatin architecture. Our current work puts forward a working model that explains the puzzling heterogeneity of NER syndromes highlighting the relevance of transcription-associated DNA damage and NER to mammalian development and disease.