Several recent gene expression studies identified hundreds of genes that are correlated with age in brain and other tissues in human. However, these studies used linear models of age correlation, which are not well equipped to model abrupt changes associated with particular ages. We developed a computational algorithm for age estimation in which the expression of each gene is treated as a dichotomized biomarker for whether the subject is older or younger than a particular age. In addition, for each age-informative gene our algorithm identifies the age threshold with the most drastic change in expression level, which allows us to associate genes with particular age periods. Analysis of human aging brain expression datasets from three frontal cortex regions showed that different pathways undergo transitions at different ages, and the distribution of pathways and age thresholds varies across brain regions. Our study reveals age-correlated expression changes at particular age points and allows one to estimate the age of an individual with better accuracy than previously published methods.
The Flory-Stockmayer theory for the polycondensation of branched polymers, modified for finite systems beyond the gel point, is applied to the connection (synapses) of neurons, which can be considered highly branched “monomeric” units. Initially, the process is a linear growth and tree-like branching between dendrites and axons of nonself-neurons. After the gel point and at the maximum “tree” size, the tree-like model prescribes, on average, one pair of twin synapses per neuron. About 13% of neurons, “unconnected” to the maximum tree, migrate to the surface to form cortical layers. The number of synapses in each neuron may reach 10000, indicating a tremendous amount of flexible, redundant, and neuroplastic loop-forming linkages which can be preserved or pruned by experience and learning.
We undertook genetic and non-genetic approaches to investigate the relationship between telomere maintenance and osteoblast differentiation, as well as to uncover a possible link between a known mediator of cellular aging and senile bone loss. Using mouse models of disrupted telomere maintenance molecules, including mutants in the Werner helicase (Wrn−/−), telomerase (Terc−/−) and Wrn−/− Terc−/− double mutants predisposed to accelerated bone loss, we measured telomere dysfunction-induced foci (TIFs) and markers of osteoblast differentiation in mesenchymal progenitor cells (MPCs). We found that telomere maintenance is directly and significantly related to osteoblast differentiation, with dysfunctional telomeres associated with impaired differentiation independent of proliferation state. Telomere-mediated defects in osteoblast differentiation are associated with increased p53/p21 expression and concomitant reduction in RUNX2. Conversely, MPCs from p53−/− mice do not have substantial telomere dysfunction and spontaneously differentiate into osteoblasts. These results suggest critical telomere dysfunction may be a prominent mechanism for age-related osteoporosis and limits MPC differentiation into bone-forming cells via the p53/p21 pathway.
Telomere; telomere dysfunction; aging; osteoporosis; mesenchymal stem cells
Sequences with the potential to form intramolecular G-quadruplexes (G4-structures) are found in highly nonrandom distributions in the genomes of diverse organisms. These sequences are associated with nucleic acid metabolic processes ranging from transcription and translation to recombination and telomere function. Here we review different computational methods for identifying potential G4-forming sequences and provide protocols for their implementation. We also discuss methods for assessing the significance and specificity of associations between the sequences and different biological functions.
G-quadruplex; G4-DNA; Bioinformatics; Computational biology
Bioinformatic approaches to the identification of genomic sequences having G-quadruplex forming potential (QFP) has enabled important tests of the structure of these sequences in vitro and of their behavior under conditions where the formation or function of G-quadruplexes is modulated in vivo. Several similar approaches to identifying intramolecular QFP (i.e. forming among G-runs on one strand of DNA) have been developed previously, but none appears to perfectly predict G-quadruplex formation. Here we describe a new approach, which complements and differs from prior approaches in that it identifies motifs containing G-runs on both strands of duplex DNA that could contribute to G-quadruplex structures. We call these motifs duplex-derived interstrand QFP (ddiQFP), and illustrate their potential applications by describing their genomic distribution and an example of their correspondence to loci targeted by a G-quadruplex-unwinding DNA helicase in yeast.
G-quadruplex; G4 DNA; interstrand G-quadruplex; ddiQFP; Bioinformatics
Previous studies in mice have demonstrated antagonistic effects of telomerase loss on carcinogenesis. Telomere attrition can promote genome instability, thereby stimulating initiation of early-stage cancers, but can also inhibit tumorigenesis by promoting permanent cell growth arrest or death. Human cancers likely develop in cell lineages with low levels of telomerase, leading to telomere losses in early lesions, followed by subsequent activation of telomerase. Mouse models constitutively lacking telomerase have thus not addressed how telomere losses within telomerase-proficient cells have an impact on carcinogenesis. Using a novel transgenic mouse model, Begus-Nahrmann et al. demonstrate in this issue of the JCI that transient telomere dysfunction in telomerase-proficient animals is a potent stimulus of tumor formation.
The PinX1 protein inhibits telomerase, an enzyme that lengthens telomeres — the structures that protect the ends of chromosomes. Loss of PinX1 leads to increased telomere length along with defects in chromosome dynamics. In this issue of the JCI, Zhou et al. present novel evidence from human tumors and mouse models indicating that PinX1 is a clinically significant tumor suppressor. Importantly, the genome-destabilizing effects of PinX1 loss appear to depend on telomerase activity, raising new models and questions for how telomeres and telomerase contribute to the development of cancer.
Functional telomerase is essential to the replicative longevity of vascular cells. To gain insights into mechanisms by which intimal hyperplasia interferes with the repair process, expression and function of the telomerase catalytic subunit (TERT) were investigated following vascular injury.
METHODS AND RESULTS
We found that TERT was de novo activated in intima of the injured arteries, involving activation of the nuclear factor κB (NF-κB) pathway. Stimulation of the isolated intimal smooth muscle cell (SMC) by basic fibroblast growth factor or tumor necrosis factor α resulted in increased TERT activity. This depends on the activation of c-Myc signaling since mutation of the E-box in the promoter or over-expression of MAD1, a c-Myc competitor, abrogated the transcriptional activity. Inhibition of NF-κB in both intimal SMC and in the injured artery attenuated TERT transcriptional activity through reduction of c-Myc expression. Pharmacological blockade of TERT led to SMC senescence. Finally, depletion of telomerase function in mice resulted in severe intimal SMC senescence following vascular injury.
These results support a model whereby vascular injury induces de novo expression of TERT in intimal SMC via activation of NF-κB and up-regulation of c-Myc. The resumed TERT activity is critical for intimal hyperplasia.
telomerase; nuclear factor-κB; smooth muscle cell; intimal hyperplasia
Researchers usually employ orthogonal arrays or D-optimal designs with little or no attribute overlap in stated-choice surveys. The challenge is to balance statistical efficiency and respondent burden to minimize the overall error in the survey responses. This study examined whether simplifying the choice task, by using a design with more overlap, provides advantages over standard minimum-overlap methods. We administered two designs for eliciting HIV test preferences to split samples. Surveys were undertaken at four HIV testing locations in San Francisco, California. Personal characteristics had different effects on willingness to pay for the two treatments, and gains in statistical efficiency in the minimal-overlap version more than compensated for possible imprecision from increased measurement error.
stated-choice approach; experimental design; overlap; taste heterogeneity; USA; HIV testing
Telomere capping conceals chromosome ends from exonucleases and checkpoints, but the full range of capping mechanisms is not well defined. Telomeres have the potential to form G-quadruplex (G4) DNA, although evidence for telomere G4 DNA function in vivo is limited. In budding yeast, capping requires the Cdc13 protein and is lost at nonpermissive temperatures in cdc13-1 mutants. Here, we use several independent G4 DNA–stabilizing treatments to suppress cdc13-1 capping defects. These include overexpression of three different G4 DNA binding proteins, loss of the G4 DNA unwinding helicase Sgs1, or treatment with small molecule G4 DNA ligands. In vitro, we show that protein-bound G4 DNA at a 3′ overhang inhibits 5′→3′ resection of a paired strand by exonuclease I. These findings demonstrate that, at least in the absence of full natural capping, G4 DNA can play a positive role at telomeres in vivo.
Herpes simplex viruses exist as two major serotypes, type 1 (HSV-1) and type 2 (HSV-2). Determination of type, either HSV-1 or HSV-2, is important in accurate diagnosis and clinical control of transmission. Several tests are available for typing HSV, including a monoclonal antibody specific for glycoprotein G and several PCR assays.
A clinical isolate was identified as herpes simplex virus, but tested negative for both HSV-1 and HSV-2 antigens using type-specific monoclonal antibody assays. The isolate was determined to be HSV-1 by PCR analysis. A mutation which likely caused the monoclonal antibody non-reactivity was found in glycoprotein G. Phylogenetic analysis revealed two groups of HSV, one with the mutation and one without. Three population studies examining mutations in HSV-1 glycoprotein G were analyzed by chi-squared test. To this point, the epitope which the monoclonal antibody recognizes was only found in HSV-1 isolates from human European populations (p < 0.0001).
These findings suggest that the PCR-based methods for HSV typing may be more useful than the standard monoclonal antibody test in areas of the world where the variant in glycoprotein G is more prevalent.
Herpes Simplex Virus; serotyping; glycoprotein G
The essential yeast protein Cdc13 facilitates chromosome end replication by recruiting telomerase to telomeres, and together with its interacting partners Stn1 and Ten1, it protects chromosome ends from nucleolytic attack, thus contributing to genome integrity. Although Cdc13 has been studied extensively, the precise role of its N-terminal domain (Cdc13N) in telomere length regulation remains unclear. Here we present a structural, biochemical, and functional characterization of Cdc13N. The structure reveals that this domain comprises an oligonucleotide/oligosaccharide binding (OB) fold and is involved in Cdc13 dimerization. Biochemical data show that Cdc13N weakly binds long, single-stranded, telomeric DNA in a fashion that is directly dependent on domain oligomerization. When introduced into full-length Cdc13 in vivo, point mutations that prevented Cdc13N dimerization or DNA binding caused telomere shortening or lengthening, respectively. The multiple DNA binding domains and dimeric nature of Cdc13 offer unique insights into how it coordinates the recruitment and regulation of telomerase access to the telomeres.
Aplastic anemia developed in 9 of 32 patients (28 percent) undergoing orthotopic liver transplantation for acute non-A, non-B hepatitis, at one to seven weeks after the procedure. No patient previously had evidence of hematologic dysfunction or conditions known to be associated with aplastic anemia. No other cases of aplastic anemia were identified among 1463 patients undergoing liver transplantation for all other indications at the four centers participating in the study (chi-square = 415, P<0.001; 95 percent confidence interval for the incidence of aplastic anemia after transplantation for non-A, non-B hepatitis, 13 to 44 percent, vs. 0.00 to 0.13 percent for all other indications).
The operative and postoperative treatment of these patients was not otherwise different, indicating that the aplastic anemia was a complication of the hepatitis, not of the transplantation procedure. Four of the nine patients died of complications due to infections. Three of the surviving patients have been followed for less than six months, one for one year, and one for two years. The two patients followed the longest have recovered marrow function to an appreciable degree, and two of the others have evidence of early recovery.
We conclude that patients undergoing orthotopic liver transplantation for non-A, non-B hepatitis are at a high risk for the development of aplastic anemia.
Recent data supports increased expression of PD-1, a negative regulator of immune function, is associated with T-cell exhaustion during chronic viral infection. However, PD-1 expression during acute infection and vaccination has not been studied in great detail in primates. Here we examine PD-1 expression on CD3+ T cells following DNA vaccination or lentiviral infection of macaques. Ex vivo peptide stimulation of PBMCs from DNA-vaccinated uninfected macaques revealed a temporal increase in PD-1 expression in proliferating antigen-specific CD8+ T cells. Following the initial increase, PD-1 expression steadily declined as proliferation continued with a concomitant increase in IFN-γ secretion. Subsequent examination of PD-1 expression on T cells from uninfected and lentivirus-infected non-vaccinated macaques revealed a significant increase in PD-1 expression with lentiviral infection, consistent with previous reports. PD-1 expression was highest on cells with activated memory and effector phenotypes. Despite their decreased telomere length, PD-1hi T-cell populations do not appear to have statistically significant uncapped telomeres, typically indicative of proliferative exhaustion, suggesting a different mechanistic regulation of proliferation by PD-1. Novelly, our data indicate PD-1 expression is increased as a result of T-cell activation during a primary immune response as well as during persistent immune activation in macaques.
PD-1; HIV; SIV; exhaustion; vaccine
The identification of the cellular mechanisms responsible for the wide differences in species lifespan remains one of the major unsolved problems of the biology of aging. We measured the capacity of nuclear protein to recognize DNA double strand breaks (DSB) and telomere length of skin fibroblasts derived from mammalian species that exhibit wide differences in longevity. Our results indicate DNA DSB recognition increases exponentially with longevity. Further, an analysis of the level of Ku80 protein in human, cow, and mouse suggests that Ku levels vary dramatically between species and these levels are strongly correlated with longevity. In contrast mean telomere length appears to decrease with increasing longevity of the species, although not significantly. These findings suggest that an enhanced ability to bind to DNA-ends may be important for longevity. A number of possible roles for increased levels of Ku and DNA-PKcs are discussed.
Species life-span; DNA-end binding activity; telomere length; DNA double-strand break repair; Ku protein
The therapeutic effect of allogeneic hematopoietic stem cell transplantation (HSCT) for patients with myeloid malignancies has been attributed in part to a graft-versus-leukemia effect that is dependent on donor T lymphocytes. CD8+ T cell responses to MHC class I restricted tumor epitopes, not just allogeneic antigens, may help mediate anti-leukemia effects after HSCT, but the specificity and function of such cells are not completely understood.
We examined the diversity, phenotype, and functional potential of leukemia-associated antigen-specific CD8+ T cells in patients with myeloid leukemia following allogeneic HSCT. Screening for antigen-specific T cells was accomplished with a peptide/MHC tetramer library.
Patients with AML or CML in remission following HSCT exhibited significant numbers of peripheral blood CD8+ T cells that recognized varying combinations of epitopes derived from leukemia-associated antigens. However, these cells failed to proliferate, release cytokines, or degranulate in response to antigen-specific stimuli. As early as two months after HSCT, CD8+ T cells from patients were predominantly CD28neg CD57+ and had relatively short telomeres, consistent with cellular senescence.
Circulating leukemia-specific CD8+ T cells are prominent in myeloid leukemia patients after HSCT, but such cells are largely functionally unresponsive, most likely due to replicative senescence. These findings carry important implications for the understanding of the graft-versus-leukemia effect and for the rationale design of immunotherapeutic strategies for patients with myeloid leukemias.
Cellular senescence is a permanent growth arrest that occurs in response to cellular stressors, such as telomere shortening or activation of oncogenes. Although the process of senescence growth arrest is somewhat conserved between mouse and human cells, there are some critical differences in the molecular pathways of senescence between these two species. Recent studies in human fibroblasts have defined a cell signaling pathway that is initiated by repression of a specific Wnt ligand, Wnt2. This, in turn, activates a histone chaperone HIRA, and culminates in formation of specialized punctate domains of facultative heterochromatin, called Senescence-Associated Heterochromatin Foci (SAHF), that are enriched in the histone variant, macroH2A. SAHF are thought to repress expression of proliferation-promoting genes, thereby contributing to senescence-associated proliferation arrest. We asked whether this Wnt2-HIRA-SAHF pathway is conserved in mouse fibroblasts.
We show that mouse embryo fibroblasts (MEFs) and mouse skin fibroblasts, do not form robust punctate SAHF in response to an activated Ras oncogene or shortened telomeres. However, senescent MEFs do exhibit elevated levels of macroH2A staining throughout the nucleus as a whole. Consistent with their failure to fully activate the SAHF assembly pathway, the Wnt2-HIRA signaling axis is not overtly regulated between proliferating and senescent mouse cells.
In addition to the previously defined differences between mouse and human cells in the mechanisms and phenotypes associated with senescence, we conclude that senescent mouse and human fibroblasts also differ at the level of chromatin and the signaling pathways used to regulate chromatin. These differences between human and mouse senescence may contribute to the increased propensity of mouse fibroblasts (and perhaps other mouse cell types) to become immortalized and transformed, compared to human cells.
Alzheimer’s disease (AD) patients have been reported to have shorter telomeres in peripheral blood leukocytes (PBLs) than age-matched control subjects. However, it is unclear if PBL telomere length reflects brain telomere length, which might play a more direct role in AD pathogenesis. We examined the correlation between PBL and cerebellum telomere length in AD patients, and compared telomere lengths in cerebella from individuals with AD versus age-matched control subjects.
Mean telomere lengths were measured using quantitative telomere polymerase chain reaction of genomic DNA prepared from matched PBL and cerebellum samples from 29 individuals with pathologically confirmed sporadic AD. Telomere length was also measured in cerebellum samples of 30 AD patients versus 22 unaffected age-matched control subjects.
The PBL and cerebellum telomere lengths were directly correlated in individuals with AD (r = 0.42, P = 0.023). Nonetheless, cerebellum telomere lengths were not significantly different in AD patients and age-matched control subjects.
Reduced PBL telomere length in AD might not reflect reduced telomere length in bulk brain tissue, but may be a marker of changes in a subset of brain tissues or other tissues that affect the pathogenesis of AD.
Alzheimer’s disease; Telomere; Cerebellum; Aging
Latent infection by Epstein-Barr virus (EBV) requires both replication and maintenance of the viral genome. EBV nuclear antigen 1 (EBNA1) is a virus-encoded protein that is critical for the replication and maintenance of the genome during latency in proliferating cells. We have previously demonstrated that EBNA1 recruits the cellular origin recognition complex (ORC) through an RNA-dependent interaction with EBNA1 linking region 1 (LR1) and LR2. We now show that LR1 and LR2 bind to G-rich RNA that is predicted to form G-quadruplex structures. Several chemically distinct G-quadruplex-interacting drugs disrupted the interaction between EBNA1 and ORC. The G-quadruplex-interacting compound BRACO-19 inhibited EBNA1-dependent stimulation of viral DNA replication and preferentially blocked proliferation of EBV-positive cells relative to EBV-negative cell lines. BRACO-19 treatment also disrupted the ability of EBNA1 to tether to metaphase chromosomes, suggesting that maintenance function is also mediated through G-quadruplex recognition. These findings suggest that the EBNA1 replication and maintenance function uses a common G-quadruplex binding capacity of LR1 and LR2, which may be targetable by small-molecule inhibitors.
Telomeres play critical roles in protecting genome stability, and their dysfunction contributes to cancer and age-related degenerative diseases. The precise architecture of telomeres, including their single-stranded 3′ overhangs, bound proteins, and ability to form unusual secondary structures such as t-loops, is central to their function and thus requires careful processing by diverse factors. Furthermore, telomeres provide unique challenges to the DNA replication and recombination machinery, and are particularly suited for extension by the telomerase reverse transcriptase. Helicases use the energy from NTP hydrolysis to track along DNA and disrupt base pairing. Here we review current findings concerning how helicases modulate several aspects of telomere form and function.
telomere; helicase; senescence; replication; recombination; G-quadruplex
Understanding patient-specific differences in risk tolerance for new treatments that offer improved efficacy can assist in making difficult regulatory and clinical decisions for new treatments that offer both the potential for greater effectiveness in relieving disease symptoms, but also risks of disabling or fatal side effects. The aim of this study is to elicit benefit-risk trade-off preferences for hypothetical treatments with varying efficacy and risk levels using a stated-choice (SC) survey. We derive estimates of “maximum acceptable risk” (MAR) that can help decisionmakers identify welfare-enhancing alternatives. In the case of children, parent caregivers are responsible for treatment decisions and their risk tolerance may be quite different than adult patients' own tolerance for treatment-related risks. We estimated and compared the willingness of Crohn's disease (CD) patients and parents of juvenile CD patients to accept serious adverse event (SAE) risks in exchange for symptom relief. The analyzed data were from 345 patients over the age of 18 and 150 parents of children under the age of 18. The estimation results provide strong evidence that adult patients and parents of juvenile patients are willing to accept tradeoffs between treatment efficacy and risks of SAEs. Parents of juvenile CD patients are about as risk tolerant for their children as adult CD patients are for themselves for improved treatment efficacy. SC surveys provide a systematic method for eliciting preferences for benefit-risk tradeoffs. Understanding patients' own risk perceptions and their willingness to accept risks in return for treatment benefits can help inform risk management decision making.
Benefit-risk analysis; Crohn's disease; maximum acceptable risk; stated-choice survey
Cells undergoing developmental processes are characterized by persistent non-genetic alterations in chromatin, termed epigenetic changes, represented by distinct patterns of DNA methylation and histone post-translational modifications. Sirtuins, a group of conserved NAD+-dependent deacetylases or ADP-ribosylases, promote longevity in diverse organisms; however, their molecular mechanisms in aging regulation remain poorly understood. Yeast Sir2, the founding member of the family, establishes and maintains chromatin silencing by removing H4 lysine 16 acetylation and bringing in other silencing proteins. Here we show an age-associated decrease in Sir2 protein abundance accompanied by an increase in H4 lysine 16 acetylation and loss of histones at specific subtelomeric regions in replicatively old yeast cells, which results in compromised transcriptional silencing at these loci. Antagonizing activities of Sir2 and Sas2, a histone acetyltransferase, regulate the replicative lifespan through histone H4 lysine 16 at subtelomeric regions. This pathway, distinct from existing aging models for yeast, may represent an evolutionarily conserved function of Sirtuins in regulation of replicative aging by maintenance of intact telomeric chromatin.
The human Werner and Bloom syndromes (WS and BS) are caused by deficiencies in the WRN and BLM RecQ helicases, respectively. WRN, BLM and their Saccharomyces cerevisiae homologue Sgs1, are particularly active in vitro in unwinding G-quadruplex DNA (G4-DNA), a family of non-canonical nucleic acid structures formed by certain G-rich sequences. Recently, mRNA levels from loci containing potential G-quadruplex-forming sequences (PQS) were found to be preferentially altered in sgs1Δ mutants, suggesting that G4-DNA targeting by Sgs1 directly affects gene expression. Here, we extend these findings to human cells. Using microarrays to measure mRNAs obtained from human fibroblasts deficient for various RecQ family helicases, we observe significant associations between loci that are upregulated in WS or BS cells and loci that have PQS. No such PQS associations were observed for control expression datasets, however. Furthermore, upregulated genes in WS and BS showed no or dramatically reduced associations with sequences similar to PQS but that have considerably reduced potential to form intramolecular G4-DNA. These findings indicate that, like Sgs1, WRN and BLM can regulate transcription globally by targeting G4-DNA.
Minute virus of canines (MVC) is a member of the genus Bocavirus in the family Parvoviridae. We have molecularly cloned and sequenced the 5′- and 3′-terminal palindromes of MVC. The MVC genome, 5,404 nucleotides (nt) in length, shared an identity of 52.6% and 52.1% with that of human bocavirus and bovine parvovirus, respectively. It had distinct palindromic hairpins of 183 nt and 198 nt at the left-end and right-end termini of the genome, respectively. The left-end terminus was also found in two alternative orientations (flip or flop). Both termini shared extensive similarities with those of bovine parvovirus. Four full-length molecular clones constructed with different orientations of the left-end terminus proved to be infectious in Walter Reed canine cell/3873D (WRD) canine cells. Both MVC infection and transfection of the infectious clone in WRD cells revealed an identical RNA transcription profile that was similar to that of bovine parvovirus. Mutagenesis of the infectious clone demonstrated that the middle open reading frame encodes the NP1 protein. This protein, unique to the genus Bocavirus, was essential for MVC DNA replication. Moreover, the phospholipase A2 motif in the VP1 unique region was also critical for MVC infection. Thus, our studies revealed important information about the genus Bocavirus that may eventually help us to clone the human bocavirus and study its pathogenesis.
Certain guanine-rich sequences are capable of forming higher order structures known as G-quadruplexes. Moreover, particular genomic regions in a number of highly divergent organisms are enriched for such sequences, raising the possibility that G-quadruplexes form in vivo and affect cellular processes. While G-quadruplexes have been rigorously studied in vitro, whether these structures actually form in vivo and what their roles might be in the context of the cell have remained largely unanswered questions. Recent studies suggest that G-quadruplexes participate in the regulation of such varied processes as telomere maintenance, transcriptional regulation and ribosome biogenesis. Here we review studies aimed at elucidating the in vivo functions of quadruplex structures, with a particular focus on findings in yeast. In addition, we discuss the utility of yeast model systems in the study of the cellular roles of G-quadruplexes.
G-quadruplex; yeast; telomere; transcription; chromatin