Divergent cellular phenotypes of human and mouse cells lacking the Werner syndrome RecQ helicase

Werner syndrome (WS) is a human autosomal recessive genetic instability and cancer predisposition syndrome with features of premature aging. Several genetically determined mouse models of WS have been generated, however, none develops features of premature aging or an elevated risk of neoplasia unless additional genetic perturbations are introduced. In order to determine whether differences in cellular phenotype could explain the discrepant phenotypes of Wrn^?/? mice and WRN-deficient humans, we compared the cellular phenotype of newly derived Wrn^?/? mouse primary fibroblasts with previous analyses of primary and transformed fibroblasts from WS patients and with newly derived, WRN-depleted human primary fibroblasts. These analyses confirmed previously reported cellular phenotypes of WRN-mutant and WRN-deficient human fibroblasts, and demonstrated that the human WRN-deficient cellular phenotype can be detected in cells grown in 5% or in 20% oxygen. In contrast, we did not identify prominent cellular phenotypes present in WRN-deficient human cells in Wrn^?/? mouse fibroblasts. Our results indicate that human and mouse fibroblasts have different functional requirements for WRN protein, and that the absence of a strong cellular phenotype may in part explain the failure of Wrn^?/? mice to develop an organismal phenotype resembling Werner syndrome.     

Differential stem cell aging kinetics in Hutchinson-Gilford progeria syndrome and Werner syndrome

Hutchinson-Gilford progeria syndrome (HGPS) and Werner syndrome (WS) are two of the best characterized human progeroid syndromes. HGPS is caused by a point mutation in lamin A (LMNA) gene, resulting in the production of a truncated protein product—progerin. WS is caused by mutations in WRN gene, encoding a loss-of-function RecQ DNA helicase. Here, by gene editing we created isogenic human embryonic stem cells (ESCs) with heterozygous (G608G/+) or homozygous (G608G/G608G) LMNAmutation and biallelic WRN knockout, for modeling HGPS and WS pathogenesis, respectively. While ESCs and endothelial cells (ECs) did not present any features of premature senescence, HGPS- and WS-mesenchymal stem cells (MSCs) showed aging-associated phenotypes with different kinetics. WS-MSCs had early-onset mild premature aging phenotypes while HGPS-MSCs exhibited late-onset acute premature aging characterisitcs. Taken together, our study compares and contrasts the distinct pathologies underpinning the two premature aging disorders, and provides reliable stem-cell based models to identify new therapeutic strategies for pathological and physiological aging.     

Werner综合征小鼠模型在早衰与肿瘤研究中的应用

Werner综合征(Werner syndrome, WS)是一种罕见的人类常染色体隐性遗传疾病, 一直以来该病作为研究人类早老综合征的典型病例而受到关注。Werner蛋白(WRN)是Werner综合征中突变的核蛋白, 最近的生化及遗传学研究证明WRN在DNA复制、DNA损伤修复以及端粒的维持方面起着重要的作用。文章综述了Werner综合征的分子遗传学机理及端粒和WRN在Werner综合征发病中的重要作用。通过双敲除Wrn与端粒酶基因建立的小鼠模型忠实地再现了人类Werner综合征, 这种Werner综合征小鼠模型因其同时具有早衰与肿瘤表型而在研究人类肿瘤及衰老的相关性中起到的独特作用。     

Increase in DNA synthesis in Werner''s syndrome cells by hybridization with normal human diploid and HeLa cells

The dominance or recessiveness of the senescent phenotype in cells from patients with Werner's syndrome (WS cells) was investigated using cell fusion. The [³H]thymidine labeling index of normal human diploid fibroblast cell X WS cell heterodikaryons was considerably lower than that of normal homodikaryons, but was significantly higher than that of WS homodikaryons. The labeling index of WS cell X HeLa cell heterodikaryons was the same as that of HeLa homodikaryons. The labeling indices of heterodikaryons obtained by fusion between various strains of premature aging cells were as low as those of parental homodikaryons. These results indicate: (1) the senescent phenotype of WS cells appears to be partially recessive to the phenotype of normal cells and completely recessive to that of HeLa cells; (2) the marked inhibition of DNA synthesis in normal nuclei in heterodikaryons with WS cells could be due to ‘senescent factor(s)’ in WS cells; and (3) no complementation phenomenon was observed among genetically different premature aging cells, probably due to ‘senescent factor(s)’.     

Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity

Werner syndrome (WS) is a human premature aging disorder characterized by chromosomal instability. The cellular defects of WS presumably reflect compromised or aberrant function of a DNA metabolic pathway that under normal circumstances confers stability to the genome. We report a novel interaction of the WRN gene product with the human 5' flap endonuclease/5'-3' exonuclease (FEN-1), a DNA structure-specific nuclease implicated in DNA replication, recombination and repair. WS protein (WRN) dramatically stimulates the rate of FEN-1 cleavage of a 5' flap DNA substrate. The WRN-FEN-1 functional interaction is independent of WRN catalytic function and mediated by a 144 amino acid domain of WRN that shares homology with RecQ DNA helicases. A physical interaction between WRN and FEN-1 is demonstrated by their co-immunoprecipitation from HeLa cell lysate and affinity pull-down experiments using a recombinant C-terminal fragment of WRN. The underlying defect of WS is discussed in light of the evidence for the interaction between WRN and FEN-1.     

Werner syndrome protein suppresses the formation of large deletions during the replication of human telomeric sequences

Werner syndrome (WS) is a disorder characterized by features of premature aging and increased cancer that is caused by loss of the RecQ helicase WRN. Telomeres consisting of duplex TTAGGG repeats in humans protect chromosome ends and sustain cellular proliferation. WRN prevents the loss of telomeres replicated from the G-rich strand, which can form secondary G-quadruplex (G4) structures. Here, we dissected WRN roles in the replication of telomeric sequences by examining factors inherent to telomeric repeats, such as G4 DNA, independently from other factors at chromosome ends that can also impede replication. For this we used the supF shuttle vector (SV) mutagenesis assay. We demonstrate that SVs with [TTAGGG]₆ sequences are stably replicated in human cells, and that the repeats suppress the frequency of large deletions despite G4 folding potential. WRN depletion increased the supF mutant frequency for both the telomeric and non-telomeric SVs, compared with the control cells, but this increase was much greater (27-fold) for telomeric SVs. The higher SV mutant frequencies in WRN-deficient cells were primarily due to an increase in large sequence deletions and rearrangements. However, WRN depletion caused a more dramatic increase in deletions and rearrangements arising within the telomeric SV (70-fold), compared with non-telomeric SV (8-fold). Our results indicate that WRN prevents large deletions and rearrangements during replication, and that this role is particularly important in templates with telomeric sequence. This provides a possible explanation for increased telomere loss in WS cells.     

Werner syndrome protein suppresses the formation of large deletions during the replication of human telomeric sequences

Werner syndrome (WS) is a disorder characterized by features of premature aging and increased cancer that is caused by loss of the RecQ helicase WRN. Telomeres consisting of duplex TTAGGG repeats in humans protect chromosome ends and sustain cellular proliferation. WRN prevents the loss of telomeres replicated from the G-rich strand, which can form secondary G-quadruplex (G4) structures. Here, we dissected WRN roles in the replication of telomeric sequences by examining factors inherent to telomeric repeats, such as G4 DNA, independently from other factors at chromosome ends that can also impede replication. For this we used the supF shuttle vector (SV) mutagenesis assay. We demonstrate that SVs with [TTAGGG]₆ sequences are stably replicated in human cells, and that the repeats suppress the frequency of large deletions despite G4 folding potential. WRN depletion increased the supF mutant frequency for both the telomeric and non-telomeric SVs, compared with the control cells, but this increase was much greater (27-fold) for telomeric SVs. The higher SV mutant frequencies in WRN-deficient cells were primarily due to an increase in large sequence deletions and rearrangements. However, WRN depletion caused a more dramatic increase in deletions and rearrangements arising within the telomeric SV (70-fold), compared with non-telomeric SV (8-fold). Our results indicate that WRN prevents large deletions and rearrangements during replication, and that this role is particularly important in templates with telomeric sequence. This provides a possible explanation for increased telomere loss in WS cells.     

Asymmetry of DNA replication fork progression in Werner's syndrome

Human aging is associated with accumulation of cells that have undergone replicative senescence. The rare premature aging Werner's syndrome (WS) provides a phenocopy of normal human aging and WS patient cells recapitulate the aging phenotype in culture as they rapidly lose the ability to proliferate or replicate their DNA. WS is associated with loss of functional WRN protein. Although the biochemical properties of WRN protein, which possesses both helicase and exonuclease activities, suggest an involvement in DNA metabolism, its action in cells is not clear. Here, we provide experimental evidence for a role of the WRN protein in DNA replication in normally proliferating cells. Most importantly, we demonstrate that in the absence of functional WRN protein, replication forks from origins of bidirectional replication fail to progress normally, resulting in marked asymmetry of bidirectional forks. We propose that WRN acts in normal DNA replication to prevent collapse of replication forks or to resolve DNA junctions at stalled replication forks, and that loss of this capacity may be a contributory factor in premature aging.     

WRN is required for ATM activation and the S-phase checkpoint in response to interstrand cross-link-induced DNA double-strand breaks

Werner syndrome (WS) is a human genetic disorder characterized by extensive clinical features of premature aging. Ataxia-telengiectasia (A-T) is a multisystem human genomic instability syndrome that includes premature aging in some of the patients. WRN and ATM, the proteins defective in WS and A-T, respectively, play significant roles in the maintenance of genomic stability and are involved in several DNA metabolic pathways. A role for WRN in DNA repair has been proposed; however, this study provides evidence that WRN is also involved in ATM pathway activation and in a S-phase checkpoint in cells exposed to DNA interstrand cross-link–induced double-strand breaks. Depletion of WRN in such cells by RNA interference results in an intra-S checkpoint defect, and interferes with activation of ATM as well as downstream phosphorylation of ATM target proteins. Treatment of cells under replication stress with the ATM kinase inhibitor KU 55933 results in a S-phase checkpoint defect similar to that observed in WRN shRNA cells. Moreover, γH2AX levels are higher in WRN shRNA cells than in control cells 6 and 16 h after exposure to psoralen DNA cross-links. These results suggest that WRN and ATM participate in a replication checkpoint response, in which WRN facilitates ATM activation in cells with psoralen DNA cross-link–induced collapsed replication forks.     

Premature aging in RecQ helicase-deficient human syndromes

The RecQ family of DNA helicases have potential roles in DNA repair, replication and/or recombination pathways. In humans, a defect in the RecQ family helicases encoded by the BLM, WRN and RECQ4 genes gives rise to Bloom's (BS), Werner's (WS) and Rothmund-Thomson (RTS) syndromes, respectively. These disorders are associated with cancer predisposition and/or premature aging. In Bloom's syndrome, affected individuals are predisposed to many types of cancer at an early age. Werner's syndrome is a premature aging disorder with a complex phenotype, which includes many age-related disorders that develop from puberty, including greying and thinning of the hair, bilateral cataract formation, type II diabetes mellitus, osteoporosis and atherosclerosis. The phenotype of Rothmund-Thomson syndrome patients also consists of some features associated with premature aging, as well as predispositon to certain cancers. Here, we discuss the molecular basis of these RecQ helicase-deficient disorders.     

Aberrant DNA methylation profiles in the premature aging disorders Hutchinson-Gilford Progeria and Werner syndrome

DNA methylation gradiently changes with age and is likely to be involved in aging-related processes with subsequent phenotype changes and increased susceptibility to certain diseases. The Hutchinson-Gilford Progeria (HGP) and Werner Syndrome (WS) are two premature aging diseases showing features of common natural aging early in life. Mutations in the LMNA and WRN genes were associated to disease onset; however, for a subset of patients the underlying causative mechanisms remain elusive. We aimed to evaluate the role of epigenetic alteration on premature aging diseases by performing comprehensive DNA methylation profiling of HGP and WS patients. We observed profound changes in the DNA methylation landscapes of WRN and LMNA mutant patients, which were narrowed down to a set of aging related genes and processes. Although of low overall variance, non-mutant patients revealed differential DNA methylation at distinct loci. Hence, we propose DNA methylation to have an impact on premature aging diseases.     

A retarded rate of DNA replication and normal level of DNA repair in Werner's syndrome fibroblasts in culture

We investigated the cloning efficiency, DNA repair, and the rate of DNA replication in the skin fibroblasts from patients with Werner's syndrome (WS) of an autosomal recessive premature aging disease. Five WS strains exhibited normal levels of sensitivity toward X-ray and UV killings and repair of X-ray induced single strand breaks of DNA (rejoining) and UV damage to DNA (unscheduled DNA synthesis). The sedimentation of newly synthesizing DNA in alkaline sucrose gradients demonstrated a characteristic feature that only the elongation rate of DNA chains, estimated by the molecular weight increase, was significantly slower during early passages in WS cells than in normal Hayflick Phase II fibroblasts. In addition, plating efficiencies as well as the replicative potentials of five WS strains were more limited than those of normal cells under the identical culture conditions. It seems therefore that at least in the WS cells tested, the slow rate of DNA replication may be more related to the shortened lifespan and enhanced cell death, as manifestation of premature senescence at the cellular level, than be the DNA repair ability.     

Hypothesis: Werner syndrome and biological ageing: A molecular genetic hypothesis

Werner syndrome (WS) is an inherited disorder that produces somatic stunting, premature ageing and early onset of degenerative and neoplastic diseases. Cultured fibroblasts derived from subjects with WS are found to undergo premature replicative senescence and thus provide a cellular model system to study the disorder. Recently, several overexpressed gene sequences isolated from a WS fibroblast cDNA library have been shown to possess the capacity to inhibit DNA synthesis and disrupt many normal biochemical processes. Because a similar constellation of genes is overexpressed in WS and senescent normal fibroblasts, these data suggest the existence of a common molecular genetic pathway for replicative senescence in both types of cell. We propose that the primary defect in WS is a mutation in a gene for a trans-acting repressor protein that reduces its binding affinity for shared regulatory regions of several genes, including those that encode inhibitors of DNA synthesis (IDS). The mutant WS repressor triggers a sequence of premature expression of IDS and other genes, with resulting inhibition of DNA synthesis and early cellular senescence, events which occur much later in normal cells.     

Genetic correction of Werner syndrome gene reveals impaired pro‐angiogenic function and HGF insufficiency in mesenchymal stem cells

WRN mutation causes a premature aging disease called Werner syndrome (WS). However, the mechanism by which WRN loss leads to progeroid features evident with impaired tissue repair and regeneration remains unclear. To determine this mechanism, we performed gene editing in reprogrammed induced pluripotent stem cells (iPSCs) derived from WS fibroblasts. Gene correction restored the expression of WRN. WRN^+/+ mesenchymal stem cells (MSCs) exhibited improved pro‐angiogenesis. An analysis of paracrine factors revealed that hepatocyte growth factor (HGF) was downregulated in WRN^?/? MSCs. HGF insufficiency resulted in poor angiogenesis and cutaneous wound healing. Furthermore, HGF was partially regulated by PI3K/AKT signaling, which was desensitized in WRN^?/? MSCs. Consistently, the inhibition of the PI3K/AKT pathway in WRN^+/+ MSC resulted in reduced angiogenesis and poor wound healing. Our findings indicate that the impairment in the pro‐angiogenic function of WS‐MSCs is due to HGF insufficiency and PI3K/AKT dysregulation, suggesting trophic disruption between stromal and epithelial cells as a mechanism for WS pathogenesis.     

综述: 人类早衰症的发病机制及干预方法

衰老是一种在细胞和组织水平逐渐发生功能衰退的过程.早衰症是一类罕见的人类遗传性疾病,以加速衰老为特征.对早衰症的研究有助于理解人类衰老的生理过程,对衰老相关疾病的防治具有借鉴意义.成人早衰症和儿童早衰症是两种著名的人类早衰症,本文将综述这两种早衰症的发病机制及干预方法.     

The Saccharomyces cerevisiae WRN homolog Sgs1p participates in telomere maintenance in cells lacking telomerase

Werner syndrome (WS) is marked by early onset of features resembling aging, and is caused by loss of the RecQ family DNA helicase WRN. Precisely how loss of WRN leads to the phenotypes of WS is unknown. Cultured WS fibroblasts shorten their telomeres at an increased rate per population doubling and the premature senescence this loss induces can be bypassed by telomerase. Here we show that WRN co-localizes with telomeric factors in telomerase-independent immortalized human cells, and further that the budding yeast RecQ family helicase Sgs1p influences telomere metabolism in yeast cells lacking telomerase. Telomerase-deficient sgs1 mutants show increased rates of growth arrest in the G2/M phase of the cell cycle as telomeres shorten. In addition, telomerase-deficient sgs1 mutants have a defect in their ability to generate survivors of senescence that amplify telomeric TG1-3 repeats, and SGS1 functions in parallel with the recombination gene RAD51 to generate survivors. Our findings indicate that Sgs1p and WRN function in telomere maintenance, and suggest that telomere defects contribute to the pathogenesis of WS and perhaps other RecQ helicase diseases.     

Reduced 5-hydroxymethyluracil-DNA glycosylase activity in Werner's syndrome cells.

Werner's syndrome (WS) is an autosomal recessive disease marked by early symptoms of accelerated aging. There is evidence indicating accumulation of oxidized DNA bases to be a major factor in cellular aging. The first step of excision repair of such bases in human cells is their removal from DNA by glycosylases. 5-Hydroxymethyluracil (HMU)-DNA glycosylase excises HMU from DNA; another glycosylase removes many non-aromatic pyrimidine derivatives. Levels of glycosylases that excise oxidized pyrimidines from DNA were compared between confluent and proliferating populations of WS cells, age-matched controls, and young control cells. They were assayed by measurements of direct release of free bases from their respective DNA substrates. Specific activities of the glycosylase that releases various modified pyrimidines and of uracil-DNA glycosylase (which removes uracil from DNA) were essentially the same in all cell lines. Cell cycle variations of these enzymes also did not differ between WS and control cells. HMU-DNA glycosylase specific activity was reduced in WS cells. Reduction of HMU-DNA glycosylase has been described in senescent human WI-38 cells. Therefore, while neither WS nor senescent cells have overall deficiencies of DNA glycosylase activities, they both might have reduced excision of HMU from DNA. This indicates a possible role of HMU accumulation in the aging process.     

A novel protein interacts with the Werner's syndrome gene product physically and functionally

Werner's syndrome (WS) is a rare autosomal recessive disorder characterized by premature aging. The gene responsible for WS encodes a protein homologous to Escherichia coli RecQ. Here we describe a novel Werner helicase interacting protein (WHIP), which interacts with the N-terminal portion of Werner protein (WRN), containing the exonuclease domain. WHIP, which shows homology to replication factor C family proteins, is conserved from E. coli to human. Ectopically expressed WHIP and WRN co-localized in granular structures in the nucleus. The functional relationship between WHIP and WRN was indicated by genetic analysis of yeast cells. Disruptants of the SGS1 gene of Saccharomyces cerevisiae, which is the WRN homologue in yeast, show an accelerated aging phenotype and high sensitivity to methyl methanesulfonate as compared with wild-type cells. Disruption of the yeast WHIP (yWHIP) gene in wild-type cells and sgs1 disruptants resulted in slightly accelerated aging and enhancement of the premature aging phenotype of sgs1 disruptants, respectively. In contrast, disruption of the yWHIP gene partially alleviated the sensitivity to methyl methanesulfonate of sgs1 disruptants.