Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age1,2. Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation2. While many drivers of HSC ageing have been proposed2–5, the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated γH2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar γH2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies.
FANCM is a component of the Fanconi anemia (FA) core complex and one FA patient (EUFA867) with biallelic mutations in FANCM has been described. Strikingly, we found that EUFA867 also carries biallelic mutations in FANCA. After correcting the FANCA defect in EUFA867 lymphoblasts, a "clean" FA-M cell line was generated. These cells were hypersensitive to mitomycin C, but unlike cells defective in other core complex members, FANCM Ϫ/Ϫ cells were proficient in monoubiquitinating FANCD2 and were sensitive to the topoisomerase inhibitor camptothecin, a feature shared only with the FA subtype D1 and N. In addition, FANCM Ϫ/Ϫ cells were sensitive to UV light. IntroductionFanconi anemia (FA) is a recessive genetic instability syndrome that has uncovered a cellular pathway involved in the protection against replication-blocking lesions. Inactivation of this pathway, as seen in FA patients, results in hypersensitivity to DNA crosslinking agents and cancer susceptibility. 1 Defects in 13 different genes have been found in FA patients, 2 and the proteins encoded by these genes cooperate in a pathway that can be subdivided in an upstream and downstream part based upon the monoubiquitination of FANCD2 and FANCI. 1 The upstream part of the pathway consists of a nuclear core complex formed by the FA proteins FANCA, -B, -C, -E, -F, -G, -L, and -M and 2 FA-associated proteins FAAP100 and FAAP24. This complex monoubiquitinates FANCD2 through the E3-ubiquitin ligase FANCL in conjunction with the ubiquitin-conjugating enzyme. 3,4 The FA core complex, UBE2T, and FANCD2 are independently recruited to the stalled replication fork. 5 For FANCD2, this relies on the ATR-mediated phosphorylation of its binding partner FANCI, 6 whereas the recruitment of the FA core complex seems to depend on FANCM. 7 Like FANCD2, FANCI is also monoubiquitinated by the FA core complex and these modified proteins colocalize with Rad51 and BRCA1 in nuclear foci. 8,9 The link between FA and BRCA proteins was further strengthened by the discovery of FA patients with mutations in BRCA2, 10 and in the BRCA1-and BRCA2-interacting proteins BRIP1 11,12 and PALB2. 13,14 FA patients with a defect in any of these genes have normal FANCD2 monoubiquitination and therefore these proteins are considered as downstream players in the FA pathway.Despite the identification of the various components of the FA core complex, its role in the maintenance of genome stability remains unclear, because of the absence of functional domains in most of the core complex members. A notable exception is FANCM, an ortholog of the archaeal DNA repair protein HEF, which contains 2 conserved domains: a DEAH helicase domain in the N-terminus and an endonuclease domain in the C-terminus. 15,16 The helicase domain is shared with yeast orthologs MPH1 (Saccharomyces cerevisiae) and FML1 (Schizosaccharomyces pombe), which play a regulatory role in homologous recombination repair by replication fork reversal and D-loop disruption. [17][18][19] HEF and MPH1 possess helicase activity, 20,21 whereas for F...
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