Recent Advances in Understanding Werner Syndrome

Aging, the universal phenomenon, affects human health and is the primary risk factor for major disease pathologies. Progeroid diseases, which mimic aging at an accelerated rate, have provided cues in understanding the hallmarks of aging. Mutations in DNA repair genes as well as in telomerase subunits are known to cause progeroid syndromes. Werner syndrome (WS), which is characterized by accelerated aging, is an autosomal-recessive genetic disorder. Hallmarks that define the aging process include genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. WS recapitulates these hallmarks of aging and shows increased incidence and early onset of specific cancers. Genome integrity and stability ensure the normal functioning of the cell and are mainly guarded by the DNA repair machinery and telomeres. WRN, being a RecQ helicase, protects genome stability by regulating DNA repair pathways and telomeres. Recent advances in WS research have elucidated WRN’s role in DNA repair pathway choice regulation, telomere maintenance, resolution of complex DNA structures, epigenetic regulation, and stem cell maintenance.


Introduction
Werner syndrome (WS) is a segmental progeria. It belongs to a small group of disorders characterized by accelerated aging. WS patients in their 20s and 30s display features similar but not identical to those of normal older individuals, including skin atrophy, graying and loss of hair, wrinkles, loss of fat, cataracts, atherosclerosis, and diabetes (reviewed in Yokote et al. 1 ). WS is caused by mutations in the WRN gene and has an estimated global incidence ranging between 1 in 1,000,000 and 1 in 10,000,000 births; however, the incidence is higher in Japan at 1 in 100,000 births 2 . WS is inherited in an autosomal-recessive manner. To date, a total of 83 different mutations in WRN have been identified and catalogued by the International Registry of WS (Seattle, WA, USA) and the Japanese Werner Consortium (Chiba, Japan) 2 . Because of its resemblance to normal aging, WS is widely studied in the field of aging, and many consider WS the best example of an accelerated aging syndrome.
Diagnostic criteria for WS were proposed in 1994 3 and recently updated 4 . Individuals with WS develop normally until their first decade, and the first clinical sign of the syndrome appears as lack of the pubertal growth spurt during their teen years. Affected individuals in their 20s and 30s begin to manifest skin atrophy and loss and graying of hair. Bilateral cataracts, abnormal glucose and lipid metabolism, hypogonadism, skin ulcers, and bone deformity appear by the fourth decade. Fatty liver, osteoporosis, and calcification of the Achilles tendon are also predominantly observed. WS may be a good model to study sarcopenia 5 . Malignancy and atherosclerotic vascular diseases such as myocardial infarction are the major causes of death among patients with WS.
The WRN gene codes for the WRN protein. WRN is a member of the RecQ helicase family of proteins and is unique in that it possesses both helicase and exonuclease domains 6 . WRN also has strand annealing activity, but its in vivo role remains unclear. Recently, López-Otín et al. created a list of pathways that are changed during aging 7 . These hallmarks of aging pathways have been widely considered the key processes affected during aging. Since WS clinical features include many aspects of normal aging, it is not surprising that WRN functions in, or its loss impacts, many of these pathways. In this review, we survey the literature and compare each aging hallmark against patients with WS (Table 1). We go on to describe a few key areas of recent WRN-related advances and then point out areas for future research.

Telomere attrition
Progressive decrease in telomere length over multiple cell divisions. Telomere attrition mainly occurs owing to the end-replication problem and the lack of telomerase enzyme.
WRN interacts with Pot1 and TRF2 components of the shelterin complex to promote telomere maintenance. Telomere length in older patients with WS (40-60 years) is markedly shorter than in younger patients with WS (~30 years) and age-matched non-WS individuals.

Epigenetic alterations
Involves alterations in the DNA methylation patterns, post-translational modification of histones, and chromatin remodeling Patients with WS show an increased DNA methylation age with an average of 6.4 years. WRN interacts with methylation complex consisting of SUV39H1, HP1α, and LAP2β, which is responsible for the epigenetic histone mark H3K9 trimethylation (H3K9me3). In response to DNA damage, WRN recruits chromatin assembly factor 1 (CAF-1) to alter chromatin structure.

Loss of proteostasis
Impairment of protein homeostasis due to accumulation of misfolded proteins and deregulation of proteolytic system. Chronic expression of misfolded, unfolded, or aggregation of proteins contributes to the development of agerelated pathologies such as Alzheimer's disease and cataracts.

Stem cell exhaustion
A decline in the proliferation of stem and progenitor cells, which are required for tissue regeneration WRN-deficient mesenchymal stem cells showed progressive disorganization of heterochromatin and premature senescence.

Altered intercellular communication
Enhanced activation of nuclear factor kappa B (NF-κB) and increased production of tumor necrosis factor (TNF), interleukin-1 beta (IL-1β), and cytokines resulting in age-associated alteration in intercellular communication. Accumulation of pro-inflammatory tissue damage, failure of immune system to clear pathogens and dysfunctional host cells, and occurrence of defective autophagy response. Bystander effect in which senescent cells induce senescence in neighboring cells via gap junction-mediated cell-cell contacts and ROS. Aging research has enumerated nine hallmarks of aging: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication (Table 1) 7 . Patients with WS have defects in DNA repair machinery and show genomic instability 8,9 . WRN, in association with the telomere-protecting shelterin complex, promotes telomere maintenance, and loss of WRN, as seen in vitro and in patients with WS, results in the rapid decline of telomere length 10,11 . A progressive increase in DNA methylation is considered an aging biomarker, and, consistent with this, patients with WS display increased epigenetic age 12 . Increased DNA damage accumulation, genomic instability, telomere attrition, and histone methylation are contributing factors for cellular senescence and stem cell exhaustion in WS 13,14 . Although extensive research is required to sort out the molecular functions of WRN in regulating proteostasis, nutrient sensing, and mitochondria, WS is phenotypically associated with a loss in proteostasis and mitochondrial dysfunction 15,16 . WRN protects cells from starvation-induced autophagy, which is deregulated by an imbalance in nutrient-sensing mechanisms 17 . Inflammation alters intercellular communication owing to the accumulation of cytokines increasing with aging. Patients with WS have elevated cytokine levels of interleukin-2 (IL-2), IL-4, IL-6, tumor necrosis factor alpha (TNF-α), interferon gamma (IFNγ), and monocyte chemoattractant protein-1 (MCP-1) 18 .
In the past five years, there have been a large number of studies on WS, covering areas including those discussed in Table 1. Here, we discuss some areas of particular relevance where significant insight has been gathered in recent years. These include the role of WRN in DNA double-strand break (DSB) repair, telomere maintenance, senescence and heterochromatin stabilization, and cancer. We will discuss these selected areas in depth below.

WRN regulates double-strand break repair pathway choice
The human genome is under constant exposure to exogenous and endogenous agents. DSBs are among the most potent and deleterious forms of cellular DNA damage, causing mutagenic changes, developmental defects, gross chromosomal rearrangements, cell death, and malignancy 19 . Approximately 10 to 50 DSBs are being formed per cell per day 20 . DSBs are mainly detected, processed, and repaired by two pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). The choice of DNA repair pathway is tightly regulated and associated with the cell cycle. While NHEJ is active throughout the cell cycle, DSBs in S and G 2 phases are preferably repaired by HR using the intact sister chromatid. WRN recruits to DSB sites in G 1 as well as in S and G 2 phases 21 .  48,49 . Interestingly, the phosphorylation of WRN at S1133 by cyclin-dependent kinase 1 (CDK1), which occurs during late S/G 2 and M phases, regulates DSB repair pathway choice between HR and NHEJ 50 . Taken together, WRN plays a major role in DSB repair pathway choice ( Figure 1).

Telomere maintenance
Chromosome ends, the telomeres, are unique DNA structures that must be replicated and protected. Human telomeres are approximately 11-15 kilobases in length and composed of about 2,500 repeats of TTAGGG sequence followed by a single-stranded 3′ tail region of the same sequence. With age, telomere length is reduced mainly owing to end-replication problems. Telomeres are packed into protein-DNA complexes with the aid of shelterin proteins. We and others have previously shown that WRN interacts with TRF1, TRF2, and POT1 components of the shelterin complex 11,51-53 .
Several lines of evidence suggest that telomere dysfunction contributes to WS pathology. Cells from WS patients and WRN-deficient cells undergo early replicative senescence and display telomere loss and chromosomal rearrangements. Telomere fusions and chromosome translocations are also well documented in WS patient and WRN-deficient mouse cells 54-58 . Importantly, re-introduction of telomerase activity into WS cells prevents senescence and telomere loss 54,59 . Additionally, although the WRN protein is ubiquitously expressed, WS patient cells preferentially display premature aging of mesenchymal cells 60 . Reprogramming of induced pluripotent WS stem cells has reinforced the importance of the roles WRN plays in telomere maintenance because differentiation into any cell which naturally expresses telomerase extends the proliferative capacity of WS cells 61 . A Wrn-null mouse model further substantiates the importance of WRN in telomere maintenance. These mice failed to show significant pathology until bred with late-generation telomerase-deficient mice, demonstrating that short telomeres were critical to revealing WS-like premature aging features 62,63 .
WRN acts at telomeres to promote replication and suppress recombination. Cells use telomerase, a reverse transcriptase enzyme with an RNA component, Terc, to replicate and lengthen telomeres. It is thought that WRN's helicase activity contributes to telomere replication through the resolution or dissolution of complex DNA structures found at telomeres such as T-loops, D-loops, and G-quartets (G4s) 11,64-66 . G4s are formed by four guanines associated through Hoogsteen base pairing. They are thought to arise in areas of singlestranded DNA, in regions undergoing replication and transcription, and preferentially in the telomeric G-rich strand. G4s may promote genomic instability; therefore, enzymes, like WRN, exist to unwind them, thereby suppressing recombination [66][67][68][69] .
In the absence of telomerase, cells maintain their telomeres via recombination mechanisms termed alternative lengthening of telomeres (ALT). ALT cells and cells without WRN protein show increases in telomere-sister chromatid exchanges 21,56,70 . This increase has been, in part, attributed to a rise in alt-NHEJ in WRN-deficient cells 21 . Interestingly, knockdown of WRN in three different ALT cell lines demonstrated variable dependence on WRN to prevent telomere loss 71 . This suggests that there are multiple telomeraseindependent mechanisms that contribute to telomere maintenance.
Although it is routinely reported that skin cells from patients with WS have shorter telomere length 10 , it is still debated whether this is true in all organs and how it contributes to the pathology found in patients with WS. In one study of two patients with WS at autopsy, the authors did not find substantially shorter telomeres from the liver relative to controls 72 . However, the liver is considered a regenerative organ and therefore may have the capacity to re-activate telomerase in this tissue, negating the impact of WRN loss. In another recent study, younger WS patients with intractable ulcers had normal terminal restriction fragment lengths, suggesting that telomere length was not likely driving this phenotype 10 . While the prevailing theory is that telomere-driven replicative senescence promotes pathology in WS, this may need to be revised as we learn more about telomeres in different organs from patients with WS.

Epigenetic modification and senescence
In vitro, premature cellular senescence is a striking feature of WRN-deficient cells 73 . Senescent cells are defined as viable growth-arrested cells. Senescence and exhaustion of stem cells are thought to contribute to tissue degeneration and aging. There are many actions which induce cellular senescence: extended cellular division, oncogene activation, telomere attrition, and exposure to DNA-damaging agents 74 . Senescent cells typically have an altered appearance and elevated secretion of pro-inflammatory cytokines which promote inflammation 75 . Until recently, cellular senescence in WRN-deficient cells was believed to be due to telomere issues and the accumulation of replication-associated endogenous DNA damage.
The genome-wide distribution of histone methylation marks changes during aging 76 . Consistent with WS as an aging model, patients with WS display increased epigenetic age as measured by DNA methylation of known aging biomarkers 12 . In humans, H3K9 trimethylation (H3K9me3) denotes constitutive heterochromatin and is mainly methylated by SUV39H1/2 histone methyltransferase (HMTase) 77 . Though not fully characterized, loss of heterochromatin is considered to increase the susceptibility of genomic DNA to mutations and reduce transcriptional precision, both of which promote genomic instability during aging 76 . Interestingly, Zhang et al. 14 reported that WRN exists in complex with SUV39H1, HP1α, and LAP2β, which together are responsible for the epigenetic histone mark H3K9me3. WRN also interacts with the chromatin remodeling chaperone chromatin assembly factor 1 (CAF-1) 78 , which deposits histones H3 and H4 onto newly replicated DNA 79 . In response to DNA damage, WRN recruits CAF-1 and participates in chromatin structure restoration 78 .
In stem cells, the histone methylation pattern is preserved over generations and is associated with the maintenance of stem cell potential. CPT-induced WRN degradation, but not Top1 degradation, was found specifically in CPT-sensitive cells 30 . Thus, it is possible that WRN expression or degradation (or both) could be used as a biomarker for personalized chemotherapy, and further research should explore this potential.

Conclusions and future perspectives
As shown in Table 1, many of the hallmarks of aging are found in patients with WS and altered as a direct consequence of WRN loss. Although there is strong evidence for a role for WRN in several of the pathways (Figure 2), others show a weak association and need further investigation. Patients with WS display many aging features, but the initiating pathology for most is still not known. For example, patients with WS suffer from severe intractable foot ulcers; however, the underlying pathology has yet to be understood. Cataracts are another cardinal feature found in patients with WS, but the mechanism or mechanisms instigating cataracts in patients with WS have to be identified. Age-associated cataracts occur because of an imbalance in the proteostasis in the lens cells 94 ; perhaps a similar mechanism is at play in WS. Recent studies suggest that WRN regulates cellular functions, like DSB repair, via catalytic and non-catalytic functions. Further investigations into its catalytic and non-catalytic functions may help elucidate disease pathology. Additional research is also needed to further define the specific functions for the exonuclease and helicase domains and to understand why these two activities are present in the same protein.
They may cooperate in some pathways such as base excision repair 95 or telomere maintenance 11 or they may not. Model organisms may be of benefit in this research because not all species express both the helicase and the exonuclease from one gene. In Caenorhabditis elegans and Drosophila, the two domains are expressed from separate genes. Additionally, the analysis of WRN functions in model organisms will help identify conserved and divergent WRN roles over an organism's life span.
Mammals have five RecQ helicases; it is important to dissect out why and how they cooperate in genome maintenance. Mutations in three of the five human RecQ helicases cause unique syndromes, indicating that they have non-overlapping functions. Patients with WS are affected by certain types of cancers compared with patients with Bloom and Rothmund-Thomson syndrome. Studies identifying the mechanisms behind the susceptibility of these patients to certain type of cancers are still needed. Furthermore, the reason why patients with WS are prone to non-epithelial malignant tumors remains to be determined.

Competing interests
The authors declare that they have no competing interests.

Grant information
This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute on Aging.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.