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...
The Fanconi anemia (FA) core complex member FANCM remodels synthetic replication forks and recombination intermediates. Thus far, only one FA patient with FANCM mutations has been described, but the relevance of these mutations for the FA phenotype is uncertain. To provide further experimental access to the FA-M complementation group we have generated Fancm-deficient mice by deleting exon 2. FANCM deficiency caused hypogonadism in mice and hypersensitivity to cross-linking agents in mouse embryonic fibroblasts (MEFs), thus phenocopying other FA mouse models. However, Fancm(Delta2/Delta2) mice also showed unique features atypical for FA mice, including underrepresentation of female Fancm(Delta2/Delta2) mice and decreased overall and tumor-free survival. This increased cancer incidence may be correlated to the role of FANCM in the suppression of spontaneous sister chromatid exchanges as observed in MEFs. In addition, FANCM appeared to have a stimulatory rather than essential role in FANCD2 monoubiquitination. The FA-M mouse model presented here suggests that FANCM functions both inside and outside the FA core complex to maintain genome stability and to prevent tumorigenesis.
Fanconi anaemia (FA) is a rare autosomal recessive or X-linked inherited disease characterised by an increased incidence of bone marrow failure (BMF), haematological malignancies and solid tumours. Cells from individuals with FA show a pronounced sensitivity to DNA interstrand crosslink (ICL)-inducing agents, which manifests as G2-M arrest, chromosomal aberrations and reduced cellular survival. To date, mutations in at least 15 different genes have been identified that cause FA; the products of all of these genes are thought to function together in the FA pathway, which is essential for ICL repair. Rapidly following the discovery of FA genes, mutant mice were generated to study the disease and the affected pathway. These mutant mice all show the characteristic cellular ICL-inducing agent sensitivity, but only partially recapitulate the developmental abnormalities, anaemia and cancer predisposition seen in individuals with FA. Therefore, the usefulness of modelling FA in mice has been questioned. In this Review, we argue that such scepticism is unjustified. We outline that haematopoietic defects and cancer predisposition are manifestations of FA gene defects in mice, albeit only in certain genetic backgrounds and under certain conditions. Most importantly, recent work has shown that developmental defects in FA mice also arise with concomitant inactivation of acetaldehyde metabolism, giving a strong clue about the nature of the endogenous lesion that must be repaired by the functional FA pathway. This body of work provides an excellent example of a paradox in FA research: that the dissimilarity, rather than the similarity, between mice and humans can provide insight into human disease. We expect that further study of mouse models of FA will help to uncover the mechanistic background of FA, ultimately leading to better treatment options for the disease.
Blood homeostasis is maintained by a rare population of quiescent hematopoietic stem cells (HSC), which self-renew and differentiate to give rise to all lineages of mature blood cells. In contrast to most other blood cells, HSCs are preserved throughout life and maintenance of their genomic integrity is therefore paramount to ensure normal blood production and prevent leukemic transformation. HSCs are also one of the few blood cells that truly age and exhibit severe functional decline in old organisms, resulting in impaired blood homeostasis and increased risk for hematological malignancies. In this review, we present the strategies used by HSCs to cope with the many genotoxic insults that they commonly encounter. We briefly describe the DNAdamaging insults that can affect HSC function, and the mechanisms that are employed by HSCs to prevent, survive, and repair DNA lesions. We also discuss an apparent paradox in HSC biology, in which the genome maintenance strategies used by HSCs to protect their function in fact render them vulnerable to the acquisition of damaging genetic aberrations.
Syndromes of disordered 'chromatin remodeling' are unique in medicine because they arise from a general deregulation of DNA transcription caused by mutations in genes encoding enzymes which mediate changes in chromatin structure. Chromatin is the packaged form of DNA in the eukaryotic cell. It consists almost entirely of repeating units, called nucleosomes, in which short segments of DNA are wrapped tightly around a disk-like structure comprising two subunits of each of the histone proteins H2A, H2B, H3 and H4. Histone proteins are covalently modified by a number of different adducts (i.e. acetylation and phosphorylation) that regulate the tightness of the DNA-histone interactions. Mutations in genes encoding enzymes that mediate chromatin structure can result in a loss of proper regulation of chromatin structure, which in turn can result in deregulation of gene transcription and inappropriate protein expression. In this review we present examples of representative genetic diseases that arise as a consequence of disordered chromatin remodeling. These include: alpha-thalassemia/mental retardation syndrome, X-linked (ATR-X); Rett syndrome (RS); immunodeficiency-centromeric instability-facial anomalies syndrome (ICF); Rubinstein-Taybi syndrome (RSTS); and Coffin-Lowry syndrome (CLS).
To identify the gene underlying Fanconi anemia (FA) complementation group I we studied informative FA-I families by a genome-wide linkage analysis, which resulted in 4 candidate regions together encompassing 351 genes. Candidates were selected via bioinformatics and data mining on the basis of their resemblance to other FA genes/proteins acting in the FA pathway, such as: degree of evolutionary conservation, presence of nuclear localization signals and pattern of tissue-dependent expression. We found a candidate, KIAA1794 on chromosome 15q25-26, to be mutated in 8 affected individuals previously assigned to complementation group I. Western blots of endogenous FANCI indicated that functionally active KIAA1794 protein is lacking in FA-I individuals. Knock-down of KIAA1794 expression by siRNA in HeLa cells caused excessive chromosomal breakage induced by mitomycin C, a hallmark of FA cells. Furthermore, phenotypic reversion of a patient-derived cell line was associated with a secondary genetic alteration at the KIAA1794 locus. These data add up to two conclusions. First, KIAA1794 is a FA gene. Second, this gene is identical to FANCI, since the patient cell lines found mutated in this study included the reference cell line for group I, EUFA592.
Hematopoietic aging is marked by a loss of regenerative capacity and skewed differentiation from hematopoietic stem cells (HSC) leading to impaired blood production. Signals from the bone marrow (BM) niche tailor blood production, but the contribution of the old niche to hematopoietic aging remains unclear. Here, we characterize the in ammatory milieu that drives both niche and hematopoietic remodeling. We nd decreased numbers and functionality of osteoprogenitors (OPr) and expansion of pro-in ammatory perisinusoidal mesenchymal stromal cells (MSC) with deterioration of the sinusoidal vasculature, which together create a degraded and in amed old BM niche. Niche in ammation, in turn, drives chronic activation of emergency myelopoiesis pathways in old HSCs and multipotent progenitors (MPP), which promotes myeloid differentiation at the expense of lymphoid and erythroid commitment and hinders hematopoietic regeneration. Remarkably, niche deterioration, HSC dysfunction and defective hematopoietic regeneration can all be ameliorated by blocking IL-1 signaling. Our results demonstrate that targeting IL-1 as a key mediator of niche in ammation is a tractable strategy to improve blood production during aging. HighlightsBoth endosteal and central marrow niche populations are remodeled with age Old niche populations show disruption of cell identity and enrichment of in ammatory response genes Emergency myelopoiesis pathways are chronically activated in response to niche in ammation Targeting niche-mediated IL-1 signaling attenuates stromal and blood aging Etoc BlurbPassegué and colleagues examine the aged bone marrow niche microenvironment to understand its contribution to blood aging and identify targetable factor(s) for functional anti-aging interventions. They show that crosstalk between the in amed niche and the in amed hematopoietic system leads to degraded blood production both at steady state and during regeneration, and identify IL-1 as a major targetable driver of age-related niche and blood system deterioration.
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