GroEL/S Overexpression Helps to Purge Deleterious Mutations and Reduce Genetic Diversity during Adaptive Protein Evolution

Chaperones are proteins that help other proteins fold. They also affect the adaptive evolution of their client proteins by buffering the effect of deleterious mutations and increasing the genetic diversity of evolving proteins. We study how the bacterial chaperone GroE (GroEL+GroES) affects the evolution of green fluorescent protein (GFP). To this end, we subjected GFP to multiple rounds of mutation and selection for its color phenotype in four replicate Escherichia coli populations, and studied its evolutionary dynamics through high-throughput sequencing and mutant engineering. We evolved GFP both under stabilizing selection for its ancestral (green) phenotype, and to directional selection for a new (cyan) phenotype. We did so both under low and high expression of the chaperone GroE. In contrast to previous work, we observe that GroE does not just buffer but also helps purge deleterious (fluorescence reducing) mutations from evolving populations. In doing so, GroE helps reduce the genetic diversity of evolving populations. In addition, it causes phenotypic heterogeneity in mutants with the same genotype, helping to enhance their fluorescence in some cells, and reducing it in others. Our observations show that chaperones can affect adaptive evolution in more than one way.     

A Genome-Wide Scan Reveals Important Roles of DNA Methylation in Human Longevity by Regulating Age-Related Disease Genes

It is recognized that genetic factors contribute to human longevity. Besides the hypothesis of existence of longevity genes, another suggests that a lower frequency of risk alleles decreases the incidence of age-related diseases in the long-lived people. However, the latter finds no support from recent genetic studies. Considering the crucial role of epigenetic modification in gene regulation, we then hypothesize that suppressing disease-related genes in longevity individuals is likely achieved by epigenetic modification, e.g. DNA methylation. To test this hypothesis, we investigated the genome-wide methylation profile in 4 Chinese female centenarians and 4 middle-aged controls using methyl-DNA immunoprecipitation sequencing. 626 differentially methylated regions (DMRs) were observed between both groups. Interestingly, genes with these DMRs were enriched in age-related diseases, including type-2 diabetes, cardiovascular disease, stroke and Alzheimer’s disease. This pattern remains rather stable after including methylomes of two white individuals. Further analyses suggest that the observed DMRs likely have functional roles in regulating disease-associated gene expressions, with some genes [e.g. caspase 3 (CASP3)] being down-regulated whereas the others [i.e. interleukin 1 receptor, type 2 (IL1R2)] up-regulated. Therefore, our study suggests that suppressing the disease-related genes via epigenetic modification is an important contributor to human longevity.     

The effect of superoxide dismutase alleles on aging inDrosophila

The effects of superoxide dismutase on aging were tested using two differt experimental approaches. In the first, replicated populations with postponed aging were compared with their controls for frequencies of electrophoretic alleles at the SOD locus. Populations with postponed aging had consistently greater frequencies of the allele coding for more active SOD protein. This allele was not part of a segregating inversion polymorphism. The second experimental approach was the extraction ofSOD alleles from different natural populations followed by the construction of differentSOD genotypes on hybrid genetic backgrounds. This procedure did not uncover any statistical effect ofSOD genotype on hybrid genetic backgrounds. This effects on longevity and fecundity due to the family from which a particularSOD genotype was derived. To detect the effects ofSOD genotypes on longevity with high probability would require a ten-fold increase in the number of families used.     

Genetics of healthy aging and longevity

Longevity and healthy aging are among the most complex phenotypes studied to date. The heritability of age at death in adulthood is approximately 25 %. Studies of exceptionally long-lived individuals show that heritability is greatest at the oldest ages. Linkage studies of exceptionally long-lived families now support a longevity locus on chromosome 3; other putative longevity loci differ between studies. Candidate gene studies have identified variants at APOE and FOXO3A associated with longevity; other genes show inconsistent results. Genome-wide association scans (GWAS) of centenarians vs. younger controls reveal only APOE as achieving genome-wide significance (GWS); however, analyses of combinations of SNPs or genes represented among associations that do not reach GWS have identified pathways and signatures that converge upon genes and biological processes related to aging. The impact of these SNPs, which may exert joint effects, may be obscured by gene-environment interactions or inter-ethnic differences. GWAS and whole genome sequencing data both show that the risk alleles defined by GWAS of common complex diseases are, perhaps surprisingly, found in long-lived individuals, who may tolerate them by means of protective genetic factors. Such protective factors may ‘buffer’ the effects of specific risk alleles. Rare alleles are also likely to contribute to healthy aging and longevity. Epigenetics is quickly emerging as a critical aspect of aging and longevity. Centenarians delay age-related methylation changes, and they can pass this methylation preservation ability on to their offspring. Non-genetic factors, particularly lifestyle, clearly affect the development of age-related diseases and affect health and lifespan in the general population. To fully understand the desirable phenotypes of healthy aging and longevity, it will be necessary to examine whole genome data from large numbers of healthy long-lived individuals to look simultaneously at both common and rare alleles, with impeccable control for population stratification and consideration of non-genetic factors such as environment.     

High-throughput Interpretation of Killer-cell Immunoglobulin-like Receptor Short-read Sequencing Data with PING

The killer-cell immunoglobulin-like receptor (KIR) complex on chromosome 19 encodes receptors that modulate the activity of natural killer cells, and variation in these genes has been linked to infectious and autoimmune disease, as well as having bearing on pregnancy and transplant outcomes. The medical relevance and high variability of KIR genes makes short-read sequencing an attractive technology for interrogating the region, providing a high-throughput, high-fidelity sequencing method that is cost-effective. However, because this gene complex is characterized by extensive nucleotide polymorphism, structural variation including gene fusions and deletions, and a high level of homology between genes, its interrogation at high resolution has been thwarted by bioinformatic challenges, with most studies limited to examining presence or absence of specific genes. Here, we present the PING (Pushing Immunogenetics to the Next Generation) pipeline, which incorporates empirical data, novel alignment strategies and a custom alignment processing workflow to enable high-throughput KIR sequence analysis from short-read data. PING provides KIR gene copy number classification functionality for all KIR genes through use of a comprehensive alignment reference. The gene copy number determined per individual enables an innovative genotype determination workflow using genotype-matched references. Together, these methods address the challenges imposed by the structural complexity and overall homology of the KIR complex. To determine copy number and genotype determination accuracy, we applied PING to European and African validation cohorts and a synthetic dataset. PING demonstrated exceptional copy number determination performance across all datasets and robust genotype determination performance. Finally, an investigation into discordant genotypes for the synthetic dataset provides insight into misaligned reads, advancing our understanding in interpretation of short-read sequencing data in complex genomic regions. PING promises to support a new era of studies of KIR polymorphism, delivering high-resolution KIR genotypes that are highly accurate, enabling high-quality, high-throughput KIR genotyping for disease and population studies.     

The PKB/FOXO switch in aging and cancer

Aging is characterized by the general decline in tissue and body function and the increased susceptibility to age-related pathologies, such as cancer. To maintain optimal tissue and body function, organisms have developed complex mechanisms for tissue homeostasis. Importantly, it is becoming apparent that these same mechanisms when deregulated also result in the development of age-related disease. The build in failsafe mechanisms of homeostasis, which prevent skewing toward disease, themselves contribute to aspects of aging. Thus, longevity is limited by an intrinsic trade-off between optimal tissue function and disease. Consequently, aging and age-related diseases, such as cancer and diabetes are driven by the same genetic determinants. Illustrative in this respect is the insulin/IGF-1 signaling pathway acting through PI3K/PKB and FOXO. Loss of PKB signaling contributes to diabetes, whereas gain of function of PKB drives cancer. Enhanced FOXO activity, at least in model organism contributes to extended lifespan and acts as a tumor suppressive mechanism. Here, we focus on the linkage between PKB and FOXO as a central switch in contributing to tissue homeostasis and age-related diseases in particular cancer. This article is part of a Special Issue entitled: P13K-AKT-FoxO axis in cancer and aging.     

Genetics and epigenetics of aging and longevity

Evolutionary theories of aging predict the existence of certain genes that provide selective advantage early in life with adverse effect on lifespan later in life (antagonistic pleiotropy theory) or longevity insurance genes (disposable soma theory). Indeed, the study of human and animal genetics is gradually identifying new genes that increase lifespan when overexpressed or mutated: gerontogenes. Furthermore, genetic and epigenetic mechanisms are being identified that have a positive effect on longevity. The gerontogenes are classified as lifespan regulators, mediators, effectors, housekeeping genes, genes involved in mitochondrial function, and genes regulating cellular senescence and apoptosis. In this review we demonstrate that the majority of the genes as well as genetic and epigenetic mechanisms that are involved in regulation of longevity are highly interconnected and related to stress response.     

Replicative and chronological aging in Saccharomyces cerevisiae

Saccharomyces cerevisiae has directly or indirectly contributed to the identification of arguably more mammalian genes that affect aging than any other model organism. Aging in yeast is assayed primarily by measurement of replicative or chronological life span. Here, we review the genes and mechanisms implicated in these two aging model systems and key remaining issues that need to be addressed for their optimization. Because of its well-characterized genome that is remarkably amenable to genetic manipulation and high-throughput screening procedures, S. cerevisiae will continue to serve as a leading model organism for studying pathways relevant to human aging and disease.     

Genome-Wide Scan Informed by Age-Related Disease Identifies Loci for Exceptional Human Longevity

We developed a new statistical framework to find genetic variants associated with extreme longevity. The method, informed GWAS (iGWAS), takes advantage of knowledge from large studies of age-related disease in order to narrow the search for SNPs associated with longevity. To gain support for our approach, we first show there is an overlap between loci involved in disease and loci associated with extreme longevity. These results indicate that several disease variants may be depleted in centenarians versus the general population. Next, we used iGWAS to harness information from 14 meta-analyses of disease and trait GWAS to identify longevity loci in two studies of long-lived humans. In a standard GWAS analysis, only one locus in these studies is significant (APOE/TOMM40) when controlling the false discovery rate (FDR) at 10%. With iGWAS, we identify eight genetic loci to associate significantly with exceptional human longevity at FDR < 10%. We followed up the eight lead SNPs in independent cohorts, and found replication evidence of four loci and suggestive evidence for one more with exceptional longevity. The loci that replicated (FDR < 5%) included APOE/TOMM40 (associated with Alzheimer’s disease), CDKN2B/ANRIL (implicated in the regulation of cellular senescence), ABO (tags the O blood group), and SH2B3/ATXN2 (a signaling gene that extends lifespan in Drosophila and a gene involved in neurological disease). Our results implicate new loci in longevity and reveal a genetic overlap between longevity and age-related diseases and traits, including coronary artery disease and Alzheimer’s disease. iGWAS provides a new analytical strategy for uncovering SNPs that influence extreme longevity, and can be applied more broadly to boost power in other studies of complex phenotypes.     

Nonadditive indirect effects of group genetic diversity on larval viability in Drosophila melanogaster imply key role of maternal decision-making

Genetic variation can have important consequences for populations: high population genetic diversity is typically associated with ecological success. Some mechanisms that account for these benefits assume that local social groups with high genetic diversity are more successful than low‐diversity groups. At the same time, active decision‐making by individuals can influence group genetic diversity. Here, we examine how maternal decisions that determine group genetic diversity influence the viability of Drosophila melanogaster larvae. Our groups contained wild‐type larvae, whose genetic diversity we manipulated, and genetically marked ‘tester’ larvae, whose genotype and frequency were identical in all trials. We measured wild‐type and tester viability for each group. Surprisingly, the viability of wild‐type larvae was neither augmented nor reduced when group genetic diversity was altered. However, the viability of the tester genotype was substantially depressed in large, high‐diversity groups. Further, not all high‐diversity groups produced this effect: certain combinations of wild‐type genotypes were deleterious to tester viability, while other groups of the same diversity—but containing different wild‐type genotypes—were not deleterious. These deleterious combinations of wild‐type genotypes could not be predicted by observing the performance of the same tester and wild‐type genotypes in low‐diversity groups. Taken together, these results suggest that nonadditive interactions among genotypes, rather than genetic diversity per se, account for between‐group differences in viability in D. melanogaster and that predicting the consequences of genetic diversity at the population level may not be straightforward.     

Seed aging and survival mechanisms

Aging and death are universal to living systems. In temperate climate latitudes the mature seeds of higher plants are exposed to aging and have developed resistance mechanisms allowing survival and plant propagation. In addition to the physicochemical properties of the seed that confer stress resistance, the protein metabolism contributes importantly to longevity mechanisms. Recently, genetic studies have demonstrated the occurrence of the Protein L-isoaspartyl methyltransferase repair enzyme in controlling age-related protein damages and seed survival. These protective mechanisms by protein repair are widespread in all kingdoms, so that the use of seeds as models to study these controlling processes offers the prospect of understanding longevity mechanisms better.     

CRISPR/Cas9 genome editing demonstrates functionality of the autoimmunity-associated SNP rs12946510

Genome-wide association studies (GWAS) map genetic associations of complex traits with precision limited to a linkage disequilibrium group. To translate GWAS results into new understanding of disease mechanisms, individual causative polymorphisms and their target genes should be identified. CRISPR/Cas9 genome editing can be used to create isogenic cell lines bearing alternative genotypes of candidate single-nucleotide polymorphisms to test their causality and to reveal gene targets. An intergenic polymorphism rs12946510 is associated with multiple sclerosis, inflammatory bowel disease and asthma. We created sublines of the T-helper cell line bearing alternative genotypes of rs12946510 and showed that its risk (“T”) allele is associated with lower expression of IKZF3 and ORMDL3 genes and reduced cell activation. Our editing procedure can become an effective tool for discovering new genes involved in pathogenesis of complex diseases.     

The genetics of aging

Once thought to be an extremely complex conundrum of weak genetic and environmental effects, exceptional longevity is beginning to yield genetic findings. Numerous lower organism and mammalian models demonstrate genetic mutations that increase life-span markedly. These variations, some of them evolutionarily conserved, inform us about biochemical pathways that significantly impact upon longevity. Centenarian studies have also proven useful as they are a cohort that, relative to younger age groups, lacks genotypes linked to age-related lethal diseases and premature mortality. Pedigree studies have demonstrated a significant familial component to the ability to survive to extreme old age and a recent study demonstrates a locus on chromosome 4 linked to exceptional longevity indicating the likely existence of at least one longevity enabling gene in humans. Thus, a number of laboratories are making substantial and exciting strides in the understanding of the genetics of aging and longevity which should lead to the discovery of genes and ultimately drugs that slow down the aging process and facilitate people's ability to delay and perhaps escape age-associated diseases.     

Possible role of ABO system in age-related diseases and longevity: a narrative review

ABO blood group antigens are expressed either on the surface of red blood cells either on a variety of other cells. Based on the available knowledge of the genes involved in their biosynthesis and their tissue distribution, their polymorphism has been suggested to provide intraspecies diversity allowing to cope with diverse and rapidly evolving pathogens. Accordingly, the different prevalence of ABO group genotypes among the populations has been demonstrated to be driven by malaria selection. In the similar manner, a particular ABO blood group may contribute to favour life-extension via biological mechanisms important for surviving or eluding serious disease. In this review, we will suggest the possible association of ABO group with age-related diseases and longevity taking into account the biological role of the ABO glycosyltransferases on some inflammatory mediators as adhesion molecules.     

Direct and indirect genetic effects of sex-specific mitonuclear epistasis on reproductive ageing

Mitochondria are involved in ageing and their function requires coordinated action of both mitochondrial and nuclear genes. Epistasis between the two genomes can influence lifespan but whether this also holds for reproductive senescence is unclear. Maternal inheritance of mitochondria predicts sex differences in the efficacy of selection on mitonuclear genotypes that should result in differences between females and males in mitochondrial genetic effects. Mitonuclear genotype of a focal individual may also indirectly affect trait expression in the mating partner. We tested these predictions in the seed beetle Callosobruchus maculatus, using introgression lines harbouring distinct mitonuclear genotypes. Our results reveal both direct and indirect sex-specific effects of mitonuclear epistasis on reproductive ageing. Females harbouring coadapted mitonuclear genotypes showed higher lifetime fecundity due to slower senescence relative to novel mitonuclear combinations. We found no evidence for mitonuclear coadaptation in males. Mitonuclear epistasis not only affected age-specific ejaculate weight, but also influenced male age-dependent indirect effects on traits expressed by their female partners (fecundity, egg size, longevity). These results demonstrate important consequences of sex-specific mitonuclear epistasis for both mating partners, consistent with a role for mitonuclear genetic constraints upon sex-specific adaptive evolution.     

免疫基因多态性与衰老和增龄相关疾病关系

衰老是进行性的、多细胞普遍存在的、不可逆的功能减退状态。免疫衰老主要表现为造血干细胞再生和淋巴系分化能力下降、机体对感染和疫苗的反应减弱、对炎症反应的放大和自身的免疫反应增加, 与衰老和增龄相关疾病密切相关。免疫基因变异, 影响机体免疫反应, 可加速或延缓衰老和增龄相关疾病。获得性免疫基因, 如对自身免疫性疾病起保护性作用的HLA II 抗原基因DRB1*11和DRB*16相关的单倍型在长寿老人频率增加。抗炎因子IL-10-1082G等位基因频率和TGFβ1单倍型cnd10T/C、cnd25G/G、-988C/C、-800G/A频率的下降, 促炎因子TNFα低表达相关的扩展的TNF-A基因型-1031C/C、-863C/A、-857C/C、IL-6-174 CC基因型, 和IFN-γ+874 T等位基因频率减少与免疫炎症反应易感性, 衰老相关疾病的发病率和死亡率正相关。固有免疫基因, 如高频表达抗炎的+896 G KIR4等位基因、CCR5Δ32突变、-765 C Cox-2等位基因、-1708 G和21 C 5-Lox等位基因多见于长寿老人。KIR 单倍型 KIR2DS5、A1B10减少, MBL2表达缺乏的单倍型LYPB、LYQC 和HYPD增加的老年人常伴有较高血清CMV抗体滴度。高频出现的CRP ATG单倍型和CFH 402 His 等位基因预示老年人高死亡率风险。文章对固有和获得性免疫基因多态性、单倍体与衰老及衰老相关疾病关系进展进行综述。加强分析扩展的单倍型、表观遗传学和造血干细胞衰老的遗传学研究将有助于更好地理解衰老和长寿的免疫遗传学基础。     

Oxidative stress and aging: Learning from yeast lessons

The yeast Saccharomyces cerevisiae has played a vital role in the understanding of the molecular basis of aging and the relationship of aging process with oxidative stress (non-homeostatic accumulation of Reactive Oxygen Species, ROS). The mammalian and yeast antioxidant responses are similar and over 25 % of human-degenerative disease related genes have close homologues in yeast. The reduced genetic redundancy of yeast facilitates visualization of the effect of a deleted or mutated gene. By manipulating growth conditions, yeast cells can survive only fermenting (low ROS levels) or respiring (increased ROS levels), which facilitates the elucidation of the mechanisms involved with acquisition of tolerance to oxidative stress. Furthermore, the yeast databases are the most complete of all eukaryotic models. In this work, we highlight the value of S. cerevisiae as a model to investigate the oxidative stress response and its potential impact on aging and age-related diseases.     

Endless paces of degeneration—applying comparative genomics to study evolution's moulding of longevity

Why do mice and humans have such different lifespans? Genome sequencing efforts are allowing researchers to pick apart the genetic foundations of longevity, with some promising results beginning to emerge.Why can mice not live more than five years and dogs not more than 30, yet bats can live over 40 years and humans over a century? Differences in longevity between closely related species are one of the greatest mysteries in biology, and identifying the processes responsible could ultimately presage the development of therapies against a multitude of age-related diseases. The variation in mammalian longevity must have a genomic basis, with recent genome sequencing efforts opening up exciting opportunities to decipher it; some promising results are beginning to emerge. Analysis of two bat genomes revealed that a high proportion of genes in the DNA damage checkpoint–DNA repair pathway, including ATM, TP53, RAD50 and KU80, are under selection in bats [1]. This finding is exciting because these genes have been directly associated with ageing in model systems and, therefore, it points towards a potential role for averting DNA damage in longevity assurance mechanisms; a notion dating back several decades that remains contentious. In addition, the report of a systematic scan for proteins with accelerated evolution in mammalian lineages in which longevity increased over the course of their evolution, hinted that some repair systems, such as the ubiquitin–proteasome pathway and a few proteins related to DNA damage repair, might have been selected for in long-lived lineages [2]. However, much work remains to improve the signal-to-noise ratio of this and similar methods.With decreasing costs of sequencing, the growing number of genomes of species with diverse lifespans is expected to facilitate studies in this area. As such, we can make an increasing number of comparisons such as those described above. Put simply, if we study long-lived species and find that they share genetic adaptations—for example in DNA damage response pathways—then we might assume that those adaptations are important to increase longevity. There are major intrinsic difficulties with this type of analysis, however, that one must keep in mind. Perhaps the best illustration is that despite the dramatic phenotypic divergence between humans and chimpanzees, only a relatively small number of genetic adaptations that are probably responsible for such divergence have thus far been identified [3]. One difficulty is that the genomic elements underlying species differences remain controversial. Possible processes include mutations in coding and non-coding sequences, gene family expansion and contraction, and copy number variation, all of which we think must be explored in the context of longevity adaptations. Whilst changes in regulatory regions might be important, standard methods are lacking for the detection of selection on functional non-coding sequences on a genome-wide scale and this, we think, is a limitation for progress in this area. Another limitation is that experimental validation of promising candidates is often extremely difficult to obtain.Applying comparative genomics to study the evolution of longevity also has unique challenges. For one, the force of natural selection weakens with age, indicating that, although under low-hazard conditions selection favours genes and pathways conferring longevity, selective pressure for longevity is significantly less than for other traits. Furthermore, we think that the integration of additional data—for example gene expression and age-related phenotypic data—is crucial to link genotypes to phenotypes and identify physiological adaptations that are required for extended longevity. Unfortunately, such data and even the necessary samples to generate it are as yet only available for a subset of species. In our opinion, another crucial issue is the extent to which common mechanisms underlie the extension of longevity by evolution in different species. Just as rare variants contribute to missing human heritability, taxa-specific adaptations might contribute to longevity. It can be assumed that the environment—for example, diet—of each species will influence the physiological and biochemical pathways that must be optimized to fend off ageing and age-related diseases. However, the ageing process, despite progressing at different rates, is remarkably similar across most mammals studied [4], hinting that retarding ageing might involve adaptations in similar pathways. The degree of overlap between longevity assurance mechanisms is, in our view, a crucial determinant of how much we can expect to learn about species differences in ageing in the foreseeable future. If common pathways do indeed underlie longevity evolution in multiple species, even if involving different genetic elements in different taxa, then it is reasonable to expect that they can be identified by using comparative genomics as more genomes of short- and long-lived species are sequenced. We hope to live long enough to help unravel this age-old problem.