Aging is associated with a large number of both phenotypic and molecular changes, but for most of these, it is not known whether these changes are detrimental, neutral, or protective. We have identified a conserved Caenorhabditis elegans GATA transcription factor/MTA-1 homolog egr-1 (lin-40) that extends lifespan and promotes resistance to heat and UV stress when overexpressed. Expression of egr-1 increases with age, suggesting that it may promote survival during normal aging. This increase in expression is dependent on the presence of the germline, raising the possibility that egr-1 expression is regulated by signals from the germline. In addition, loss of egr-1 suppresses the long lifespan of insulin receptor daf-2 mutants. The DAF-16 FOXO transcription factor is required for the increased stress resistance of egr-1 overexpression mutants, and egr-1 is necessary for the proper regulation of sod-3 (a reporter for DAF-16 activity). These results indicate that egr-1 acts within the insulin signaling pathway. egr-1 can also activate the expression of its paralog egl-27, another factor known to extend lifespan and increase stress resistance, suggesting that the two genes act in a common program to promote survival. These results identify egr-1 as part of a longevity-promoting circuit that changes with age in a manner that is beneficial for the lifespan of the organism.
aging; Caenorhabditis elegans; GATA; gene regulation; insulin signaling; NuRD; stress
Supercentenarians (110 years or older) are the world’s oldest people. Seventy four are alive worldwide, with twenty two in the United States. We performed whole-genome sequencing on 17 supercentenarians to explore the genetic basis underlying extreme human longevity. We found no significant evidence of enrichment for a single rare protein-altering variant or for a gene harboring different rare protein altering variants in supercentenarian compared to control genomes. We followed up on the gene most enriched for rare protein-altering variants in our cohort of supercentenarians, TSHZ3, by sequencing it in a second cohort of 99 long-lived individuals but did not find a significant enrichment. The genome of one supercentenarian had a pathogenic mutation in DSC2, known to predispose to arrhythmogenic right ventricular cardiomyopathy, which is recommended to be reported to this individual as an incidental finding according to a recent position statement by the American College of Medical Genetics and Genomics. Even with this pathogenic mutation, the proband lived to over 110 years. The entire list of rare protein-altering variants and DNA sequence of all 17 supercentenarian genomes is available as a resource to assist the discovery of the genetic basis of extreme longevity in future studies.
Gene expression genetic studies in human tissues and cells identify cis- and trans-acting expression quantitative trait loci (eQTLs). These eQTLs provide insights into regulatory mechanisms underlying disease risk. However, few studies systematically characterized eQTL results across cell and tissues types. We synthesized eQTL results from >50 datasets, including new primary data from human brain, peripheral plaque and kidney samples, in order to discover features of human eQTLs.
We find a substantial number of robust cis-eQTLs and far fewer trans-eQTLs consistent across tissues. Analysis of 45 full human GWAS scans indicates eQTLs are enriched overall, and above nSNPs, among positive statistical signals in genetic mapping studies, and account for a significant fraction of the strongest human trait effects. Expression QTLs are enriched for gene centricity, higher population allele frequencies, in housekeeping genes, and for coincidence with regulatory features, though there is little evidence of 5′ or 3′ positional bias. Several regulatory categories are not enriched including microRNAs and their predicted binding sites and long, intergenic non-coding RNAs. Among the most tissue-ubiquitous cis-eQTLs, there is enrichment for genes involved in xenobiotic metabolism and mitochondrial function, suggesting these eQTLs may have adaptive origins. Several strong eQTLs (CDK5RAP2, NBPFs) coincide with regions of reported human lineage selection. The intersection of new kidney and plaque eQTLs with related GWAS suggest possible gene prioritization. For example, butyrophilins are now linked to arterial pathogenesis via multiple genetic and expression studies. Expression QTL and GWAS results are made available as a community resource through the NHLBI GRASP database [http://apps.nhlbi.nih.gov/grasp/].
Expression QTLs inform the interpretation of human trait variability, and may account for a greater fraction of phenotypic variability than protein-coding variants. The synthesis of available tissue eQTL data highlights many strong cis-eQTLs that may have important biologic roles and could serve as positive controls in future studies. Our results indicate some strong tissue-ubiquitous eQTLs may have adaptive origins in humans. Efforts to expand the genetic, splicing and tissue coverage of known eQTLs will provide further insights into human gene regulation.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-532) contains supplementary material, which is available to authorized users.
eQTL; RNA; Gene expression; Genomics; Transcriptome; GWAS; Genome-wide; Tissue; Cis; Trans
Germline maintenance in the nematode C. elegans requires global repressive mechanisms that involve chromatin organization. During meiosis, the X chromosome in both sexes exhibits a striking reduction of histone modifications that correlate with transcriptional activation when compared with the genome as a whole. The histone modification spectrum on the X chromosome corresponds with a lack of transcriptional competence, as measured by reporter transgene arrays. The X chromosome in XO males is structurally analogous to the sex body in mammals, contains a histone modification associated with heterochromatin in other species and is inactivated throughout meiosis. The synapsed X chromosomes in hermaphrodites also appear to be silenced in early meiosis, but genes on the X chromosome are detectably expressed at later stages of oocyte meiosis. Silencing of the sex chromosome during early meiosis is a conserved feature throughout the nematode phylum, and is not limited to hermaphroditic species.
C. elegans; Germline; Silencing; X-inactivation; Histone modifications; Gametogenesis
Understanding the in vivo dynamics of protein localization and their physical interactions is important for many problems in Biology. To enable systematic protein function interrogation in a multicelluar context, we built a genome-scale transgenic platform for in vivo expression of fluorescent and affinity tagged proteins in Caenorhabditis elegans under endogenous cis regulatory control. The platform combines computer-assisted transgene design, massively parallel DNA engineering and next generation sequencing to generate a resource of 14637 genomic DNA transgenes, which covers 73% of the proteome. The multipurpose tag used allows any protein of interest to be localized in vivo or affinity purified using standard tag-based assays. We illustrate the utility of the resource by systematic chromatin immunopurification and automated 4D imaging, which produced detailed DNA binding and cell/tissue distribution maps for key transcription factor proteins
In conducting genome-wide association studies (GWAS), analytical approaches leveraging biological information may further understanding of the pathophysiology of clinical traits. To discover novel associations with estimated glomerular filtration rate (eGFR), a measure of kidney function, we developed a strategy for integrating prior biological knowledge into the existing GWAS data for eGFR from the CKDGen Consortium. Our strategy focuses on single nucleotide polymorphism (SNPs) in genes that are connected by functional evidence, determined by literature mining and gene ontology (GO) hierarchies, to genes near previously validated eGFR associations. It then requires association thresholds consistent with multiple testing, and finally evaluates novel candidates by independent replication. Among the samples of European ancestry, we identified a genome-wide significant SNP in FBXL20 (P = 5.6 × 10−9) in meta-analysis of all available data, and additional SNPs at the INHBC, LRP2, PLEKHA1, SLC3A2 and SLC7A6 genes meeting multiple-testing corrected significance for replication and overall P-values of 4.5 × 10−4–2.2 × 10−7. Neither the novel PLEKHA1 nor FBXL20 associations, both further supported by association with eGFR among African Americans and with transcript abundance, would have been implicated by eGFR candidate gene approaches. LRP2, encoding the megalin receptor, was identified through connection with the previously known eGFR gene DAB2 and extends understanding of the megalin system in kidney function. These findings highlight integration of existing genome-wide association data with independent biological knowledge to uncover novel candidate eGFR associations, including candidates lacking known connections to kidney-specific pathways. The strategy may also be applicable to other clinical phenotypes, although more testing will be needed to assess its potential for discovery in general.
A report on the Keystone Symposium 'Functional Genomics: Global Analysis of Complex Biological Systems', Santa Fe, USA, 20-24 February 2003.
A report on the Keystone Symposium 'Functional Genomics: Global Analysis of Complex Biological Systems', Santa Fe, USA, 20-24 February 2003.
Destabilizing domains are conditionally unstable protein domains that can be fused to a protein of interest resulting in degradation of the fusion protein in the absence of stabilizing ligand. These engineered protein domains enable rapid, reversible and dose-dependent control of protein expression levels in cultured cells and in vivo. To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20–25°C. This raises the possibility that our technology could be adapted for use at any temperature. We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans. These data reinforce that DD can function in virtually any organism and temperature.
The normal aging process is associated with stereotyped changes in gene expression, but the regulators responsible for these age-dependent changes are poorly understood. Using a novel genomics approach, we identified HOX co-factor unc-62 (Homothorax) as a developmental regulator that binds proximal to age-regulated genes and modulates lifespan. Although unc-62 is expressed in diverse tissues, its functions in the intestine play a particularly important role in modulating lifespan, as intestine-specific knockdown of unc-62 by RNAi increases lifespan. An alternatively-spliced, tissue-specific isoform of unc-62 is expressed exclusively in the intestine and declines with age. Through analysis of the downstream consequences of unc-62 knockdown, we identify multiple effects linked to aging. First, unc-62 RNAi decreases the expression of yolk proteins (vitellogenins) that aggregate in the body cavity in old age. Second, unc-62 RNAi results in a broad increase in expression of intestinal genes that typically decrease expression with age, suggesting that unc-62 activity balances intestinal resource allocation between yolk protein expression and fertility on the one hand and somatic functions on the other. Finally, in old age, the intestine shows increased expression of several aberrant genes; these UNC-62 targets are expressed predominantly in neuronal cells in developing animals, but surprisingly show increased expression in the intestine of old animals. Intestinal expression of some of these genes during aging is detrimental for longevity; notably, increased expression of insulin ins-7 limits lifespan by repressing activity of insulin pathway response factor DAF-16/FOXO in aged animals. These results illustrate how unc-62 regulation of intestinal gene expression is responsible for limiting lifespan during the normal aging process.
The normal aging process in Caenorhabditis elegans involves coordinated changes in expression of about a thousand genes. In order to find upstream regulators responsible for these aging genes, we used a genomics approach to screen for transcription factors that bind to them. We focused on one such regulator, unc-62/Homothorax, that encodes a co-factor for a Hox transcription factor. Although essential for development, expression of unc-62 in adults limits lifespan. We find that unc-62 is a transcriptional activator of yolk protein genes, which are synthesized in the intestine and encode essential nutrients for progeny but accumulate to toxic levels with age. Additionally, analysis of unc-62 knockdown indicates that when vitellogenin transcription is decreased, transcription of intestinal genes with somatic functions increases. Thus, activation of yolk protein gene expression by unc-62 is both a burden on maternal health and a necessary resource for embryonic growth. Surprisingly, we also found that the intestine of old worms has novel expression of non-intestinal proteins that are toxic when expressed in the intestine and are activated by unc-62 in old age. Our work on transcriptional regulation of intrinsic gene expression differences during normal aging has revealed insights into the mechanisms that limit lifespan.
Stress is a fundamental aspect of aging, as accumulated damage from a lifetime of stress can limit lifespan and protective responses to stress can extend lifespan. In this study, we identify a conserved Caenorhabditis elegans GATA transcription factor, egl-27, that is involved in several stress responses and aging. We found that overexpression of egl-27 extends the lifespan of wild-type animals. Furthermore, egl-27 is required for the pro-longevity effects from impaired insulin/IGF-1 like signaling (IIS), as reduced egl-27 activity fully suppresses the longevity of worms that are mutant for the IIS receptor, daf-2. egl-27 expression is inhibited by daf-2 and activated by pro-longevity factors daf-16/FOXO and elt-3/GATA, suggesting that egl-27 acts at the intersection of IIS and GATA pathways to extend lifespan. Consistent with its role in IIS signaling, we found that egl-27 is involved in stress response pathways. egl-27 expression is induced in the presence of multiple stresses, its targets are significantly enriched for many types of stress genes, and altering levels of egl-27 itself affects survival to heat and oxidative stress. Finally, we found that egl-27 expression increases between young and old animals, suggesting that increased levels of egl-27 in aged animals may act to promote stress resistance. These results identify egl-27 as a novel factor that links stress and aging pathways.
Stress is a fundamental aspect of aging, but it is unclear whether the molecular mechanisms underlying stress response become altered during normal aging and whether these alterations can affect the aging process. In this study, we found a GATA transcription factor called egl-27, whose targets are significantly enriched for age-dependent genes and stress response genes, and whose expression increases with age. In contrast to previous work describing factors that are causal for aging, we found that egl-27 activity is likely beneficial for survival since egl-27 overexpression extends lifespan. egl-27 promotes longevity by enhancing stress response; specifically, increased levels of egl-27 protect animals against heat stress, while reduced egl-27 activity impairs survival following heat and oxidative stress. These results suggest that aging is not simply a process of constant decline. Some factors, such as egl-27, are more active in old animals, working to restore organismal function and to improve survival.
There has been a recent explosion in the wealth of genomic data available to C. elegans researchers, as efforts to characterize gene expression and its regulators at a molecular level have borne significant fruit. Detailed measurement of gene expression at a variety of developmental stages, and in numerous individual tissues, has dramatically increased our understanding of cell-type-specific gene expression networks. Characterization of the targets of transcription factors, chromatin-binding proteins, and miRNAs has provided genome-wide insights into the mechanisms governing gene expression. Development of new techniques have allowed this characterization to begin to shift from whole-organism studies to tissue-, and even single-cell-level profiling, creating a first glimpse into gene regulatory circuits at the single-cell level in a living organism. Integration of these datasets has yielded novel insights into evolution, gene expression regulation, and the link between sequence and phenotype.
We have taken an engineering approach to extending the lifespan of Caenorhabditis elegans. Aging stands out as a complex trait, because events that occur in old animals are not under strong natural selection. As a result, lifespan can be lengthened rationally using bioengineering to modulate gene expression or to add exogenous components. Here, we engineered longer lifespan by expressing genes from zebrafish encoding molecular functions not normally present in worms. Additionally, we extended lifespan by increasing the activity of four endogenous worm aging pathways. Next, we used a modular approach to extend lifespan by combining components. Finally, we used cell- and worm-based assays to analyze changes in cell physiology and as a rapid means to evaluate whether multi-component transgenic lines were likely to have extended longevity. Using engineering to add novel functions and to tune endogenous functions provides a new framework for lifespan extension that goes beyond the constraints of the worm genome.
We used bioengineering to extend the lifespan of C. elegans by expressing genes acting in critical aging pathways. We overexpressed five genes that act in endogenous worm aging pathways, as well as two genes from zebrafish encoding molecular functions not normally present in worms. For example, we used zebrafish genes to alter mitochondrial function and innate immunity in ways not normally available to C. elegans and extended worm lifespan by ∼40%. Next, we used a modular approach to extend lifespan by 130% by combining up to four components in the same strain. These results provide a platform to build worms having progressively longer lifespans. This project is conceptually similar to using engineering to increase the useful lifespan of a primitive machine (1931 Model T) using both parts from the model T as well as parts from a more advanced machine (2012 Toyota Corolla). Our results open the door to use engineering to go beyond the constraints of the C. elegans genome to extend its lifespan by adding non-native components.
Chronic kidney disease (CKD) is an important public health problem with a genetic component. We performed genome-wide association studies in up to 130,600 European ancestry participants overall, and stratified for key CKD risk factors. We uncovered 6 new loci in association with estimated glomerular filtration rate (eGFR), the primary clinical measure of CKD, in or near MPPED2, DDX1, SLC47A1, CDK12, CASP9, and INO80. Morpholino knockdown of mpped2 and casp9 in zebrafish embryos revealed podocyte and tubular abnormalities with altered dextran clearance, suggesting a role for these genes in renal function. By providing new insights into genes that regulate renal function, these results could further our understanding of the pathogenesis of CKD.
Chronic kidney disease (CKD) is an important public health problem with a hereditary component. We performed a new genome-wide association study in up to 130,600 European ancestry individuals to identify genes that may influence kidney function, specifically genes that may influence kidney function differently depending on sex, age, hypertension, and diabetes status of individuals. We uncovered 6 new loci associated with estimated glomerular filtration rate (eGFR), the primary measure of renal function, in or near MPPED2, DDX1, SLC47A1, CDK12, CASP9, and INO80. CDK12 effect was stronger in younger and absent in older individuals. MPPED2, DDX1, SLC47A1, and CDK12 loci were associated with eGFR in African ancestry samples as well, highlighting the cross-ethnicity validity of our findings. Using the zebrafish model, we performed morpholino knockdown of mpped2 and casp9 in zebrafish embryos and revealed podocyte and tubular abnormalities with altered dextran clearance, suggesting a role for these genes in renal function. These results further our understanding of the pathogenesis of CKD and provide insights into potential novel mechanisms of disease.
Using DNA microarrays to generate transcriptional profiles of the aging process is a powerful tool for identifying biomarkers of aging. In C. elegans, a number of whole-genome profiling studies identified genes that change expression levels with age. High-throughput RNAi screens in worms determined a number of genes that modulate lifespan when silenced. Transcriptional profiling of the fly head identified a molecular pathway, the ‘response to light’ gene set, that increases expression with age and could be directly related to the tendency for a reduction in light levels to extend fly lifespan. In mouse, comparing the gene expression profiles of several drugs to the gene expression profile of caloric restriction identified metformin as a drug whose action could potentially mimic caloric restriction in vivo. Finally, genes in the mitochondrial electron transport chain group decrease expression with age in the human, mouse, fly, and worm.
In C. elegans, the LET-23 receptor tyrosine kinase is localized to the basolateral membranes of polarized vulval epithelial cells. lin-2, lin-7, and lin-10 are required for basolateral localization of LET-23, since LET-23 is mislocalized to the apical membrane in lin-2, lin-7, and lin-10 mutants. Yeast two-hybrid, in vitro binding, and in vivo coimmunoprecipitation experiments show that LIN-2, LIN-7, and LIN-10 form a protein complex. Furthermore, compensatory mutations in lin-7 and let-23 exhibit allele-specific suppression of apical mislocalization and signaling-defective phenotypes. These results present a mechanism for basolateral localization of LET-23 receptor tyrosine kinase by direct binding to the LIN-2/LIN-7/LIN-10 complex. Each of the binding interactions within this complex is conserved, suggesting that this complex may also mediate basolateral localization in mammals.
We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor–binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor–binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.
A common property of aging in all animals is that chronologically and genetically identical individuals age at different rates. To unveil mechanisms that influence aging variability, we identified markers of remaining lifespan for Caenorhabditis elegans. In transgenic lines, we expressed fluorescent reporter constructs from promoters of C. elegans genes whose expression change with age. The expression levels of aging markers in individual worms from a young synchronous population correlated with their remaining lifespan. We identified eight aging markers, with the superoxide dismutase gene sod-3 expression being the best single predictor of remaining lifespan. Correlation with remaining lifespan became stronger if expression from two aging markers was monitored simultaneously, accounting for up to 49% of the variation in individual lifespan. Visualizing the physiological age of chronologically-identical individuals allowed us to show that a major source of lifespan variability is different pathogenicity from individual to individual and that the mechanism involves variable activation of the insulin-signaling pathway.
One of the long-standing mysteries in aging is that some individuals die early whereas others die late. The age at which a specific individual will die is difficult or impossible to predict, and thus a fundamental aspect of aging in all animals is that it is stochastic. Aging stochasticity is particularly interesting in model organisms such as C. elegans because they are genetically inbred, can have the exact same chronological age, and can be grown under standard lab conditions. In this paper, we uncover a major mechanism underlying stochasticity in aging. To do this, we first developed a fluorescent aging marker (sod-3::GFP) whose expression declines with age and can be used to measure physiological age. In young animals, the level of expression of this fluorescent marker indicates which animals will live longer and which will die earlier. We used this fluorescent aging marker to show that variable pathogenicity from individual to individual is a major source of lifespan variability and that the mechanism involves variable activation of the insulin-signaling pathway.
Ageing in humans is typified by the decline of physiological functions in various organs and tissues leading to an increased probability of death. Some individuals delay, escape or survive much of this age-related decline and live past age 100. Studies comparing centenarians to average-aged individuals have found polymorphisms in genes that are associated with long life, including APOE and FOXOA3, which have been replicated many times. However, the associations found in humans account for small percentages of the variance in lifespan and many other gene associations have not been replicated in additional populations. Therefore, ageing is probably a highly polygenic trait. In humans, it is important to also consider differences in age-related decline that occur within and among tissues. Longitudinal data of age-related traits can be used in association studies to test for polymorphisms that predict how an individual will change over time. Transcriptional and genetic association studies of different tissues have revealed common and unique pathways involved in human ageing. Genomic convergence is a method that combines multiple types of functional genomic information such as transcriptional profiling, expression quantitative trait mapping and gene association. The genomic convergence approach has been used to implicate the gene MMP20 in human kidney ageing. New human genetics technologies are continually in development and may lead to additional breakthroughs in human ageing in the near future.
human ageing; centenarians; tissue ageing; longitudinal studies; genomic convergence
We have built a digital nuclear atlas of the newly hatched, first larval stage (L1) of the wild type hermaphrodite of C. elegans at single cell resolution from confocal image stacks of 15 individuals. The atlas quantifies the stereotypy of the locations and provides for other statistics on the spatial patterns of the 357 nuclei that could be faithfully segmented and annotated of the 558 present at this developmental stage. Given this atlas we then developed an automated approach to assign cell names to each nucleus in a 3D image of an L1 worm. We achieve 86% accuracy in identifying the 357 nuclei automatically. This computational method is essential for high-throughput single cell analyses of the worm at post-embryonic stages, such as determining the expression of every gene in every cell during development from the L1 onward, or ablating or stimulating cells under computer control in a high-throughput functional screen.
This paper takes a close look at balanced permutations, a recently developed sample reuse method with applications in bioinformatics. It turns out that balanced permutation reference distributions do not have the correct null behavior, which can be traced to their lack of a group structure. We find that they can give p-values that are too permissive to varying degrees. In particular the observed test statistic can be larger than that of all B balanced permutations of a data set with a probability much higher than 1/(B + 1), even under the null hypothesis.
functional genomics; gene expression; genomics; statistics
Transcription factors are key components of regulatory networks that control development, as well as the response to environmental stimuli. We have established an experimental pipeline in Caenorhabditis elegans that permits global identification of the binding sites for transcription factors using chromatin immunoprecipitation and deep sequencing. We describe and validate this strategy, and apply it to the transcription factor PHA-4, which plays critical roles in organ development and other cellular processes. We identified thousands of binding sites for PHA-4 during formation of the embryonic pharynx, and also found a role for this factor during the starvation response. Many binding sites were found to shift dramatically between embryos and starved larvae, from developmentally regulated genes to genes involved in metabolism. These results indicate distinct roles for this regulator in two different biological processes and demonstrate the versatility of transcription factors in mediating diverse biological roles.
The C. elegans transcription factor PHA-4 is a member of the highly conserved FOXA family of transcription factors. These factors act as master regulators of organ development by controlling how genes are turned off and on as tissues are formed. Additionally they regulate genes in response to nutrient levels and control both longevity and survival of the organism. However, the extent to which these factors control similar or distinct gene targets for each of these functions is unknown. For this reason, we have used the technique of chromatin immunoprecipitation followed by deep sequencing (ChIP–Seq), to define the target binding sites of PHA-4 on a genome-wide scale, when it is either functioning as an organ identity regulator or in response to environmental stress. Our data clearly demonstrate distinct sets of biologically relevant target genes for the transcription factor PHA-4 under these two different conditions. Not only have we defined PHA-4 targets, but we established an experimental ChIP–Seq pipeline to facilitate the identification of binding sites for many transcription factors in the future.
In this work we present a method for the differential analysis of gene co-expression networks and apply this method to look for large-scale transcriptional changes in aging. We derived synonymous gene co-expression networks from AGEMAP expression data for 16-month-old and 24-month-old mice. We identified a number of functional gene groups that change co-expression with age. Among these changing groups we found a trend towards declining correlation with age. In particular, we identified a modular (as opposed to uniform) decline in general correlation with age. We identified potential transcriptional mechanisms that may aid in modular correlation decline. We found that computationally identified targets of the NF-ΚB transcription factor decrease expression correlation with age. Finally, we found that genes that are prone to declining co-expression tend to be co-located on the chromosome. Our results conclude that there is a modular decline in co-expression with age in mice. They also indicate that factors relating to both chromosome domains and specific transcription factors may contribute to the decline.
There is mounting evidence that mammalian aging is marked by increased gene transcriptional variation. This trend was shown not only by studying gene expression in single cells (Bahar et al. 2006), but at the coarse tissue resolution as well (Somel et al. 2006; Li et al. 2009). These led us to believe that looking at absolute changes in expression level alone may not tell the whole story of transcriptional changes in age. Instead the story may be in the more subtle changes in the coordination of expression among multiple genes. For this reason, we decided to look at changes in co-expression relationships with age. To this end, we developed a methodology for differential co-expression network analysis for the comparison gene co-expression on a global scale. We applied this methodology to compare co-expression between young (16-month) and old (24-month) mice. This allowed us to find both gene groups whose coordination appear to be affected by age and to propose potential mechanisms for the change. We believe our work is of broad importance because it represents a different paradigm for looking not only at aging but also at any complex condition or disease—away from changes in individual genes towards changes in gene relationships.
Kidneys age at different rates, such that some people show little or no effects of aging whereas others show rapid functional decline. We sequentially used transcriptional profiling and expression quantitative trait loci (eQTL) mapping to narrow down which genes to test for association with kidney aging. We first performed whole-genome transcriptional profiling to find 630 genes that change expression with age in the kidney. Using two methods to detect eQTLs, we found 101 of these age-regulated genes contain expression-associated SNPs. We tested the eQTLs for association with kidney aging, measured by glomerular filtration rate (GFR) using combined data from the Baltimore Longitudinal Study of Aging (BLSA) and the InCHIANTI study. We found a SNP association (rs1711437 in MMP20) with kidney aging (uncorrected p = 3.6×10−5, empirical p = 0.01) that explains 1%–2% of the variance in GFR among individuals. The results of this sequential analysis may provide the first evidence for a gene association with kidney aging in humans.
Although family studies have shown that genes play a role in longevity, it has proven difficult to identify the specific genetic variants involved. We developed a sequential transcriptional profiling and eQTL mapping approach to find genes associated with aging in the kidney. First, we used genome-wide transcriptional profiling to determine which genes change expression with age in kidney tissue. Next, we used two methods to determine which of these age-regulated genes contain SNPs that associate with expression level. The allele-specific expression method, which compares the mRNA levels of the two alleles within heterozygous individuals, was more sensitive than the total expression method. We tested the eQTLs for association with kidney aging in two populations. One gene that encodes an extracellular matrix protein, MMP20, significantly associated with kidney aging, providing the first gene association with kidney aging. Our approach of combining both expression and genotype data can be applied to any phenotype of interest to increase the power to find genetic associations.
Telomerase serves a critical role in stem cell function and tissue homeostasis. This role depends on its ability to synthesize telomere repeats in a manner dependent on the reverse transcriptase (RT) function of its protein component telomerase RT (TERT), as well as on a novel pathway whose mechanism is poorly understood. Here, we use a TERT mutant lacking RT function (TERTci) to study the mechanism of TERT action in mammalian skin, an ideal tissue for studying progenitor cell biology. We show that TERTci retains the full activities of wild-type TERT in enhancing keratinocyte proliferation in skin and in activating resting hair follicle stem cells, which triggers initiation of a new hair follicle growth phase and promotes hair synthesis. To understand the nature of this RT-independent function for TERT, we studied the genome-wide transcriptional response to acute changes in TERT levels in mouse skin. We find that TERT facilitates activation of progenitor cells in the skin and hair follicle by triggering a rapid change in gene expression that significantly overlaps the program controlling natural hair follicle cycling in wild-type mice. Statistical comparisons to other microarray gene sets using pattern-matching algorithms revealed that the TERT transcriptional response strongly resembles those mediated by Myc and Wnt, two proteins intimately associated with stem cell function and cancer. These data show that TERT controls tissue progenitor cells via transcriptional regulation of a developmental program converging on the Myc and Wnt pathways.
Stem cells and progenitor cells within a tissue are required to maintain tissue homeostasis and to repair tissues after injury by giving rise to differentiated daughter cells. Many progenitor cells express telomerase, a reverse transcriptase enzyme that adds DNA repeats to telomeres, the protective structures that cap chromosome ends. Telomere addition by telomerase is important for normal progenitor cell function and is crucial for enabling cancer cells to divide an unlimited number of times. In addition to its telomere-lengthening function, telomerase reverse transcriptase (TERT) can directly activate quiescent epidermal stem cells. However, the mechanism underlying this novel function for TERT is still not understood. In this study, we demonstrate that the catalytic activity of TERT is dispensable for its ability to activate tissue progenitor cells in vivo. Furthermore, using gene microarrays, we show that TERT controls a developmental program that overlaps the natural transcriptional program of hair follicle cycling in mouse skin. Using pattern-matching algorithms, we find that the TERT-controlled genetic program significantly resembles programs regulated by Myc and Wnt, two pathways critical for stem cell function and tumorigenesis. This paper reveals critical new insights into novel mechanisms of non-telomerase functions of TERT, identifying TERT as a developmental regulator linked to control of transcriptional responses.
We present the AGEMAP (Atlas of Gene Expression in Mouse Aging Project) gene expression database, which is a resource that catalogs changes in gene expression as a function of age in mice. The AGEMAP database includes expression changes for 8,932 genes in 16 tissues as a function of age. We found great heterogeneity in the amount of transcriptional changes with age in different tissues. Some tissues displayed large transcriptional differences in old mice, suggesting that these tissues may contribute strongly to organismal decline. Other tissues showed few or no changes in expression with age, indicating strong levels of homeostasis throughout life. Based on the pattern of age-related transcriptional changes, we found that tissues could be classified into one of three aging processes: (1) a pattern common to neural tissues, (2) a pattern for vascular tissues, and (3) a pattern for steroid-responsive tissues. We observed that different tissues age in a coordinated fashion in individual mice, such that certain mice exhibit rapid aging, whereas others exhibit slow aging for multiple tissues. Finally, we compared the transcriptional profiles for aging in mice to those from humans, flies, and worms. We found that genes involved in the electron transport chain show common age regulation in all four species, indicating that these genes may be exceptionally good markers of aging. However, we saw no overall correlation of age regulation between mice and humans, suggesting that aging processes in mice and humans may be fundamentally different.
This work studies the aging process in mice using DNA microarrays to identify genes that change expression during aging. The entire set of age-regulated genes constitutes a transcriptional profile that can be used to measure ages of different individuals. Furthermore, the aging expression profile highlights genetic and metabolic pathways that change with age, providing key insights about possible molecular changes that may contribute to cell senescence and physiological decline. This aging study is massive in scope, involving gene expression measurements from 16 different tissues at four different ages. Expression data for genes of interest can be queried over the web by researchers interested in aging. Different tissues were found to have strikingly different levels of age-related change, and could be divided into three groups based on their patterns of aging: a neural group, a vascular group, and a steroid-responsive group. The electron transport chain pathway stands out because it is the only genetic pathway that shows a similar pattern of age-related change in mice, humans, worms, and flies. However, there is little overall similarity between changes in gene expression during aging of humans and mice, consistent with evolutionary theories suggesting that aging lies outside the force of natural selection.