Sci. Aging Knowl. Environ., 28 June 2006
Vol. 2006, Issue 10, p. pe18
[DOI: 10.1126/sageke.2006.10.pe18]

PERSPECTIVES

WRN's Tenth Anniversary

Fuki M. Hisama, Vilhelm A. Bohr, and Junko Oshima

The authors are in the Department of Neurology at Yale University, New Haven, CT 06520, USA (F.M.H.), the Laboratory of Molecular Gerontology at the National Institutes on Aging, the National Institutes of Health, Baltimore, MD 21224, USA (V.A.B.), and the Department of Pathology at the University of Washington, Seattle, WA 98195, USA (J.O.). E-mail: fuki.hisama{at}yale.edu (F.M.H.), vbohr{at}nih.gov (V.A.B.), picard{at}u.washington.edu (J.O.)

http://sageke.sciencemag.org/cgi/content/full/2006/10/pe18

Key Words: Werner syndrome • progeroid • WRN • RecQ helicase • exonuclease • telomere • genome instability • DNA repair

Introduction

Werner syndrome (WS) is a rare autosomal recessive disorder characterized by many features suggestive of accelerated aging (1-3) (see "Of Hyperaging and Methuselah Genes" and Online Mendelian Inheritance in Man entry). WS patients often suffer from bilateral ocular cataracts, type 2 diabetes mellitus, osteoporosis, various forms of arteriosclerosis including atherosclerosis, and hypogonadism at a relatively young age. The aged appearance is due to the short stature, premature graying and loss of hair, scleroderma-like skin changes, and regional atrophy of subcutaneous fat. Deep ulcerations around the Achilles tendon and ankles are common and virtually pathognomonic (Fig. 1). WS subjects have an elevated risk of various cancers, particularly sarcomas (4).


Figure 1
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Fig. 1. WS patients with homozygous WRN mutations. (Top) A Caucasian female with homozygous deletion of nucleotides 2089 to 2825 (c.2089-2825del; p.M696fsX705) in the region of the cDNA encoding the helicase domains resulting in a frameshift beginning with Met696 and in a premature stop codon in the protein. (Bottom)´┐ŻA Caucasian male with a homozygous mutation with deletion of eight nucleotides in exon 9 (c.867-874del AGAAAATC; p.I288fsX301) resulting in a protein frameshift beginning with Ile288and introducing a premature stop after codon 301. Notice the bandage for the ankle ulceration.

 
In 1996, the WRN gene was identified by positional cloning. Because this gene discovery method relies on mapping a disease gene based solely on its location, its function and relation to the disease are often mysterious (5). During the past 10 years, this previously unknown molecule has been intensely studied by researchers from different disciplines through regional and international collaborations. We look back and summarize this exciting experience and also raise some of the difficult questions left to be answered.

Biochemistry and Cell Biology of WRN Protein

Domain structure

The sequence alignment and structural analysis of the then-newly cloned WRN gene immediately predicted that the WRN protein would contain RecQ-type helicase domains in the central region and a nuclease domain in the N-terminal region (5, 6) (Fig. 2) (see Fry Review). During the first 4 or so years following the WRN cloning, much effort was devoted to understanding the protein chemistry of the enzymatic activities of WRN. Subsequent biochemical studies confirmed that the WRN protein displays both 3'->5' helicase and exonuclease activities. The most striking finding from these efforts is that alternative or unusual DNA structures, such as double-stranded DNA containing a single-stranded "bubble" region or G-quadruplex DNA (a four-stranded structure that has been shown to form in specific chromosomal regions such as telomeres and certain promoters) are the preferred substrates of the WRN protein (7, 8).


Figure 2
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Fig. 2. Functional domains of WRN protein. Bottom numbers indicate the positions in the protein sequence for the exonuclease and helicase regions. See text for details. Tx activation, putative transcriptional activation domain; NLS, nuclear localization signal.

 
In addition, there are two consensus domains in the C-terminal region of the WRN protein: the RecQ helicase C-terminal region (RQC) and the helicase-and-RnaseD-C-terminal conserved region (HRDC) (9). Three-dimensional structural analysis of Escherichia coli RecQ, a prototype for mammalian RecQ helicases, predicted that WRN binding to the interacting proteins and substrate DNA may occur via the RQC region (10) (see "Portrait of DNA's Protector"). The E. coli RCQ region contains a zinc finger motif and a winged helix (WH) motif (11), sequence motifs that are associated with DNA binding. The zinc finger motif may be involved in the regulation of helicase enzymatic activity by modulating DNA binding as well as protein folding of WRN helicase (12). Additional structural analysis suggests that the DNA- and protein-binding domain (DPBD), which includes the WH motif, may play a role in directing the WRN protein to the site of action (13). In fact, the DPBD region overlaps with a number of known protein-interacting regions of WRN (14). The HRDC region was proposed as a DNA binding site by structural analysis as well (15). Further cell biological study demonstrated that the WRN protein accumulates at sites of double-stranded DNA breaks via its HRDC domain (16). Independent biochemical studies demonstrated that substrate-specific DNA binding is mediated through the three domains: N-terminal, RQC, and HRDC (17).

Subcellular localization

Sequence elements that are required for the subcellular localization of WRN to the nucleus and nucleolus have been identified. There is a nuclear localization signal between amino acids 1369 and 1402 at the C-terminal region of the WRN protein, with Lys1371-Arg1372-Arg1373 being critical amino acids (18, 19), and the nucleolar localization depends on Arg1403-Lys1404 (20). An additional nucleolar localization signal resides between residues 949 to 1092 within the RecQ consensus domain (21). Indeed, the WRN protein has been localized primarily in the nucleoli in human cells and shown to relocate to form nucleoplasmic foci at sites of DNA damage (22).

Phenotype of WRN-/- cells

Limited replicative capacity and increased genomic instability of somatic cells from WS individuals were known for some decades before cloning WRN (23, 24). By using genetically confirmed WS cells, a number of genotoxic agents have been shown to differentiate WRN+/+ and WRN-/- cells. These reagents include 4-nitroquinoline-1-oxide (4-NQO) (25), topoisomerase I inhibitors (camptothecin) (26), and DNA cross-linking agents (27, 28).

Role of WRN

These studies further support the involvement of WRN in the removal or resolution of the unusual structures that block DNA processing, particularly during replication and possibly during RNA polymerase II transcription (29). The unstable genome of WS cells, and the associated limited replicative potential and cell death , may be of central importance to the pathogenesis of WS.

WRN Interacting Proteins

The studies on interacting proteins suggest that WRN protein is able to participate in multiple DNA metabolic pathways when recruited to the site of gene action by binding proteins (30) (see Cheng Perspective) (Fig. 3). At least 16 proteins that interact with WRN protein have been identified to date, many of which bind at the overlapping RQC and HRDC regions. Most noticeably, the WRN protein is involved in DNA double-strand break repair by nonhomologous end joining in concert with the DNA-protein kinase complex (DNA-protein kinase catalytic subunit, Ku70, and Ku80) (31-33) (see also "Twisted Logic"). It clearly also has a role in recombination in vitro and in vivo (34-36) (see Monnat Review). Physical and functional interactions with flap endonuclease 1 (FEN-1) (37, 38), DNA polymerase beta (37-40), poly (ADP-ribose) polymerase 1 (PARP-1) (41, 42), and apurinic/apyrimidinic endonuclease 1 (APE1) (39) suggest a role of WRN in base excision repair. WRN protein may modulate DNA replication by interaction with topoisomerase I (43) and DNA polymerase {delta} (44). Physical association of telomeric repeat binding factor 2 (TRF-2) and WRN protein (45), in conjunction with biochemical data demonstrating the disruption of D loops (structures formed at telomeres) by WRN protein (46, 47), suggests that WRN may participate in the maintenance of telomeres. WRN protein has been shown to be involved in the synthesis of the lagging strand of telomeres (48). Additionally, the tumor suppressor p53 binds to and inhibits WRN exonuclease (37) and helicase activity, while signaling downstream transcriptional activity that regulates the initiation of apoptosis following DNA damage (49).


Figure 3
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Fig. 3. WRN at the junction of DNA metabolic pathways. This diagram represents a summary of the physical and functional protein interactions between WRN and the other proteins depicted. See text for details. BER, base excision repair; NHEJ, nonhomologous end joining; RPA, replication protein A; DNA PKcs, DNA-protein kinase catalytic subunit; BLM, Bloom helicase; Mre11, meiotic recombination 11; Nbs1, Nijmegen breakage syndrome 1.

 
Taken together, WRN exonuclease and helicase activities may be used for various DNA transactions, particularly during various modalities of DNA repair. The specifics of the DNA transaction may depend on the protein complexes that direct WRN protein to the site of DNA damage and coordinate its enzymatic activities. The RQC domain seems to have a central function in both protein and DNA transactions of the WRN protein (50). It is a challenge to understand how WRN can operate in a number of DNA transactions and DNA repair processes. One important aspect is the posttranslational modification of WRN protein. For example, phosphorylation of the protein by the catalytic subunit of DNA-dependent protein kinase can substantially alter its catalytic activity (51) and may function as a molecular switch that regulates the relative activity of the exonuclease and helicase throughout a metabolic process or DNA repair pathway. Other posttranslational modifications may play similar roles, an area of investigation of great future potential.

Clinical Update on Werner Syndrome

Symptoms of WS patients

Previous clinical reviews of WS cases have based the diagnosis on clinical signs and symptoms and did not include molecular data (1, 3, 52). We now have the means to reevaluate the known signs and symptoms of WS in genetically diagnosed WS patients. This is important because some of the WS cases are caused by mutations in LMNA, which appear to cause progeroid features similar to, but more severe than, WRN mutant WS cases (53) (see "Atypical Situation"). Mutations in LMNA are also associated with Hutchinson-Gilford progeria syndrome and a variety of other disorders (see "Pushing the Envelope"). A recent review published in 2006 by Huang et al. (54) summarized the demographics of 99 WS patients with documented WRN mutations. The patients were referred to the International Registry of Werner Syndrome (Seattle, WA) by the collaborating physicians who suspected a diagnosis of WS, with a median age of referral of 46.4 years. Four cardinal signs of clinical diagnosis were (i) bilateral ocular cataracts, (ii) characteristic dermatological pathology, (iii) short stature, and (iv) premature graying and/or thinning of scalp hair. Among these cardinal signs, cataracts were reported in all of the cases for which information was available. The other three signs were observed in more than 95% of the cases for which clinical information was available. Osteoporosis and diabetes mellitus were reported at rates of 91% and 71%, respectively. Frequencies of these other signs may depend on the stage of the disease at the time of referral and the degree to which these complications were investigated.

In this latest review, the median age of death of WS patients was reported to be 54.3 years, 7 years older than the median age of death reported by Epstein et al. (1) in 1966. The mean age of onset of cataracts was approximately 31 years, which is comparable to what was reported by Epstein et al. (1). This observation suggests that the extended life span of WS patients may reflect the longer life span of the general population, partly a result of improved health care, and not a change in the course of this disorder. Two major causes of death (atherosclerosis and neoplasia) were unchanged; in the registry patients for whom the information was available, atherosclerosis was reported in 44% and neoplasia were reported in 39.5% of the patients (54).

WS-associated mutations

The most common WRN mutation among non-Japanese WS patients was found to be Arg369->Term369 (due to a C>T transition in the reference cDNA at position 1105; c.1105C>T), seen in approximately 25 of the instances. This mutation generates a truncated WRN protein containing the entire exonuclease domain, which is presumably localized to the cytoplasm. The most common Japanese WRN mutation described in this report was the deletion of exon 26 (due to a G>C transversion at –1 nucleotide from position 3139 of the cDNA that results in a splice junction mutation; c.3139–1G>C) seen in approximately 65% of the Japanese cases. This mutant WRN mRNA is extremely labile (55), although it could generate a partial WRN protein truncated immediately after the RQC domain.

All the mutations so far identified in WS patients, except for the two missense mutations in the exonuclease domain, are nonsense mutations or insertion/deletion/substitution mutations that result in the truncation of protein translation before the nuclear localization signal. The two missense mutations in the exonuclease domain, Lys125->Asn125 and Lys135->Glu135, found in a German WS case, appear to cause protein instability. Therefore, these missense mutations may also act as null mutations (54).

Four pedigrees among the Werner Registry families have been reported to carry wild-type WRN and heterozygous mutations at the LMNA locus (53). It is possible that other "atypical WS" cases with wild-type WRN and LMNA are caused by mutations in the various WRN interacting proteins. These atypical WS cases are followed by the International Registry of Werner Syndrome, and the patient materials can be made available to investigators upon request.

WRN Gene and Normal Aging

A compelling reason to study rare, monogenic diseases is that they present an opportunity to pinpoint their cause. The effect of a single, highly penetrant mutation is sufficient to produce a recognizable disease even in the background of the remarkable degree of genetic variation and differences in environment found among humans. These single genes are so powerful presumably because they are located at critical, physiological intersections. Thus, rare diseases are considered a window to potentially understanding the pathophysiology of the genetically more complex, common forms of disease. This model has proven useful, for example, for the study of neurodegenerative diseases, including Alzheimer's disease (see "Detangling Alzheimer's Disease"), Parkinson's disease (see Andersen Review), and amyotrophic lateral sclerosis, and has led to the creation of the first murine models for some diseases (56-60) (see entries in SAGE KE's Genetically Altered Mice section).

What is the potential role of WRN in normal human aging? Do persons who are heterozygous for WRN mutations display accelerated aging? Are there common sequence variants that exist in the normal population that affect the rate of aging?

WRN heterozygotes appear to be fairly common, with estimated frequencies in the United States and Japan of 1/200 to 1/500 (61). Parents and offspring of WS patients are obligate heterozygotes for WRN mutations, and siblings have a 66% chance of being heterozygous. Because heterozygotes have half of the normal levels of functional WRN protein, do they have a detectable phenotype? Lymphoblastoid cell lines from heterozygous individuals show sensitivity to the genotoxin 4-NQO that is intermediate between that of wild-type and homozygous mutant cell lines (62). The frequency of somatic mutations in peripheral red blood cells (measured by the glycophorin A assay, which detects the inactivation of alleles encoding variants of a blood-type antigen) was shown to be increased in 10 heterozygous carriers and 11 WS subjects with proven WRN mutations, as compared with normal individuals (63). What is not clear is whether this degree of increased risk of genetic instability corresponds to an increased lifetime risk of malignancy for heterozygous individuals. Although one early study (64) recorded the presence of juvenile cataracts, hypertension, diabetes mellitus, and malignancy among the relatives of WS patients, and suggested an increased risk of cancer in heterozygous carriers, this finding has not been reexamined since then in a large sample of mutation proven carriers. Despite these findings, there are no other systematic clinical studies of whether heterozygous carriers have an earlier age of onset or are at increased risk for cataracts, diabetes, atherosclerosis, malignancy, or osteoporosis as compared with the general population.

A few population studies have examined the effect of WRN polymorphisms on life span and health span. One examined a nonsynonymous coding single-nucleotide polymorphism (SNP) resulting in Arg or Cys at position 1367 in the protein sequence and found a decreased incidence of myocardial infarction in subjects in the Japanese population who had an Arg1367 allele (65). Another Japanese study found a correlation between Arg1367 and decreased myocardial infarction, but Bohr et al. (66) did not detect any such protective effect of the Arg1367 allele in a study of individuals from the Baltimore longitudinal study of aging. In that study (66), they also purified the recombinant polymorphic protein and did not find any functional changes as compared with the wild-type version. Another study examined the frequency of the Cys/Arg1367 polymorphism in Mexican newborns, Finnish newborns, Finnish centenarians, and North American adults (67). Although the allele frequencies differed in these three populations from the Japanese cohort reported by (65), these differences likely represent ethnic variation. There was no difference in the allele frequencies in Finnish newborns compared with Finnish centenarians, whereas, if one allele were protective, it would be expected to be enriched in the centenarian cohort.

A single study showed the association of a WRN polymorphism and longevity (68). However, association studies of the WRN polymorphisms and common age-related disorders have been inconclusive (66, 68-70). This is possibly because common disorders are associated with a number of covariates. Critical examination of large cohorts may reveal more consistent findings that will give us additional clues as to the function of the domains in which polymorphisms reside.

How closely do the downstream cellular changes resulting from WRN mutations resemble those observed in normal aging? An expression profiling study of 6912 genes found that over 91% of annotated genes showed similar expression changes in WS and normal aging, 3% were specific to WS, and 6% were specific to normal aging (71) (see "Maturity Mimic"). In addition, there are similarities in WS and old individuals' aberrant transcriptional response to DNA damaging gamma or ultraviolet irradiation (72). Overall, there have been surprisingly few studies of the role of WRN in normal aging. In part, this may be because of the large size (5.2 kb) of the gene and the difficulties in performing well-controlled population-based genetic studies. With the outpouring of SNPs and haplotype data into readily accessible databases, and the declining costs of genotyping, further population studies to determine the prevalence and penetrance of sequence variants in the WRN gene are becoming more feasible.

The most stringent test of WRN as a regulator of the rate of aging would be if there were "super" alleles of WRN with enhanced function that were sufficient to prolong maximum or median life span. Thus far, no such alleles have been reported, but in this case, the absence of proof is not proof of absence.

Future Goals of Werner Syndrome Research

Considerable progress in understanding WRN function and identifying interacting proteins has been made during the past 10 years. Looking ahead, a number of difficult, but more refined, questions remain. So far, there has been solid evidence to support that WRN protein does play a part in nonhomologous end joining (73), homologous recombination (74), and base excision repair (75), based mostly on separate in vitro examination of individual repair pathways. There are also some in vivo results supporting the presence of DNA repair defects, for example, and increased accumulation of oxidative lesions in cells from WS patients versus cells from normal individuals (76). However, more work is needed to understand the role of WRN in DNA repair in cells. Accumulating evidence (46, 77) suggests that WRN function may be of particular importance in telomeric processing, and further elucidation of this process is of great interest, also in relation to the general aging process (see Aviv Perspective). One study (78) showed that the Bloom helicase (another RecQ helicase, which is defective in Bloom syndrome) and Werner helicase interact functionally. It would be of interest to understand whether more of the members of the highly conserved family of RecQ helicases (from organisms ranging from bacteria to human) interact functionally and coordinate to resolve specific DNA structures forming in the genome.

Another yet-to-be answered question is to what extent WRN is involved in determining longevity, and through which mechanism. Double knockout mice lacking WRN and telomerase exhibited various accelerated aging phenotypes in an additive manner, which further supports the hypothesis that WRN is involved in mammalian aging (77). What other genes are mutated in Werner-like progeroid syndrome? The genes encoding the interacting proteins or the modifying enzymes of WRN are one class of candidate genes. Mutations in genes that are seemingly unrelated to WRN but cause other types of segmental progeroid features (such as LMNA) (53) are also good candidates.

Finally, based on our current understanding, what are promising avenues for the treatment of WS? The premature replicative senescence of WS fibroblasts can be bypassed by transfection with telomerase without evidence of malignant transformation (79, 80), and mouse models support the role of telomere length in modulating the WRN phenotype (77). Although these findings open up the telomere as a possible therapeutic target in theory, there might be more practical approaches to consider, such as a recently reported cytokine-suppressive antiinflammatory drug (81). Primary WS fibroblasts derived from three patients were treated with 10 µM of SB203580, a selective inhibitor of p38 mitogen-activated protein kinase (which mediates growth arrest; see "Heart, Heal Thyself"), and showed improvements in growth rate, life span, and senescence-associated morphological changes. Because p38 inhibitors are being tested in Phase II and Phase III clinical trials for inflammatory disease, information on their safety, efficacy, and side-effect profiles in humans will become available (82). Although there is an enormous gap between in vitro studies in fibroblasts and clinical trials of treatment for WS itself, several of the necessary intermediate steps are in place. To link them together, as well to answer remaining questions, will require the collaborative effort of investigators in areas that might not have been previously associated with this syndrome.


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  83. Supported by NIH grant R24 CA78088 (J.O.). This work was in part supported by funds from the NIA/NIH intramural research program (V.A.B.). F.M.H was supported by a Paul Beeson Physician Faculty Scholar Award from the American Federation for Aging Research (AFAR).
Citation: F. M. Hisama, V. A. Bohr, J. Oshima, WRN's Tenth Anniversary. Sci. Aging Knowl. Environ. 2006 (10), pe18 (2006).








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