We present genome-wide microarray expression analysis of 11,000 genes in an aging potentially mitotic tissue, the liver. This organ has a major impact on health and homeostasis during aging. The effects of life-and health-span-extending caloric restriction (CR) on gene expression among young and old mice and between long-term CR (LT-CR) and short-term CR (ST-CR) were examined. This experimental design allowed us to accurately distinguish the effects of aging from those of CR on gene expression. Aging was accompanied by changes in gene expression associated with increased inflammation, cellular stress, and fibrosis, and reduced capacity for apoptosis, xenobiotic metabolism, normal cell-cycling, and DNA replication. LT-CR and just 4 weeks of ST-CR reversed the majority of these changes. LT-CR produced in young mice a pattern of gene expression that is a subset of the changes found in old LT-CR mice. It is possible that the early changes in gene expression, which extend into old age, are key to the life-and health-span-extending effects of CR. Further, ST-CR substantially shifted the ''normo-aging'' genomic profile of old control mice toward the ''slow-aging'' profile associated with LT-CR. Therefore, many of the genomic effects of CR are established rapidly. Thus, expression profiling should prove useful in quickly identifying CRmimetic drugs and treatments.P ublished microarray studies of mammalian aging have focused on the postmitotic tissues, muscle and brain (1-3). These studies found that aging was associated with changes in gene expression linked to the development of the characteristic age-related pathologies of these tissues. In contrast to muscle and brain, the liver is a potentially mitotic tissue that is thought to age well from a clinical perspective (4). The liver is the central organ for the regulation of glucose homeostasis, xenobiotic metabolism and detoxification, and steroid hormone biosynthesis and degradation. This organ also has a major impact on health and homeostasis through its control of serum protein composition. While differentiated hepatic functions are generally well maintained with age, changes do occur. Serum and biliary cholesterol rise, liver regeneration declines, hepatic drug clearance decreases, and liver volume and blood flow decrease (4). The resilience of the liver to aging, and its central role in the maintenance of whole body health and homeostasis make it an intriguing target for genome-wide expression analysis of aging.Caloric restriction (CR) is the only intervention shown to extend lifespan in mammals (5). It is also the most effective means known of reducing cancer incidence and increasing the mean age of onset of age-related diseases and tumors (6). Our studies made use of an experimental design that allowed us to clearly distinguish the effects of diet from those of age on genome-wide expression patterns. Another distinctive aspect of the study allowed us to resolve changes in gene expression induced directly by CR from those that arise over time as a consequence of ...

BackgroundSmall RNAs complex with proteins to mediate a variety of functions in animals and plants. Some small RNAs, particularly miRNAs, circulate in mammalian blood and may carry out a signaling function by entering target cells and modulating gene expression. The subject of this study is a set of circulating 30–33 nt RNAs that are processed derivatives of the 5′ ends of a small subset of tRNA genes, and closely resemble cellular tRNA derivatives (tRFs, tiRNAs, half-tRNAs, 5′ tRNA halves) previously shown to inhibit translation initiation in response to stress in cultured cells.ResultsIn sequencing small RNAs extracted from mouse serum, we identified abundant 5′ tRNA halves derived from a small subset of tRNAs, implying that they are produced by tRNA type-specific biogenesis and/or release. The 5′ tRNA halves are not in exosomes or microvesicles, but circulate as particles of 100–300 kDa. The size of these particles suggest that the 5′ tRNA halves are a component of a macromolecular complex; this is supported by the loss of 5′ tRNA halves from serum or plasma treated with EDTA, a chelating agent, but their retention in plasma anticoagulated with heparin or citrate. A survey of somatic tissues reveals that 5′ tRNA halves are concentrated within blood cells and hematopoietic tissues, but scant in other tissues, suggesting that they may be produced by blood cells. Serum levels of specific subtypes of 5′ tRNA halves change markedly with age, either up or down, and these changes can be prevented by calorie restriction.ConclusionsWe demonstrate that 5′ tRNA halves circulate in the blood in a stable form, most likely as part of a nucleoprotein complex, and their serum levels are subject to regulation by age and calorie restriction. They may be produced by blood cells, but their cellular targets are not yet known. The characteristics of these circulating molecules, and their known function in suppression of translation initiation, suggest that they are a novel form of signaling molecule.

Caloric restriction (CR), the consumption of fewer calories while avoiding malnutrition, decelerates the rate of aging and the development of age-related diseases. CR has been viewed as less effective in older animals and as acting incrementally to slow or prevent age-related changes in gene expression. Here we demonstrate that CR initiated in 19-month-old mice begins within 2 months to increase the mean time to death by 42% and increase mean and maximum lifespans by 4.7 (P ‫؍‬ 0.000017) and 6.0 months (P ‫؍‬ 0.000056), respectively. The rate of age-associated mortality was decreased 3.1-fold. Between the first and second breakpoints in the CR survival curve (between 21 and 31 months of age), tumors as a cause of death decreased from 80% to 67% (P ‫؍‬ 0.012). Genome-wide microarray analysis of hepatic RNA from old control mice switched to CR for 2, 4, and 8 weeks showed a rapid and progressive shift toward the gene expression profile produced by long-term CR. This shift took place in the time frame required to induce the health and longevity effects of CR. Shifting from long-term CR to a control diet, which returns animals to the control rate of aging, reversed 90% of the gene expression effects of long-term CR within 8 weeks. These results suggest a cause-and-effect relationship between the rate of aging and the CR-associated gene expression biomarkers. Therefore, therapeutics mimicking the gene-expression biomarkers of CR may reproduce its physiological effects.C aloric restriction (CR), the consumption of fewer calories while avoiding malnutrition, is a robust method of decelerating aging and the development of age-related diseases (1). The effects of CR are conserved in nearly every species tested, perhaps including humans (2). CR delays the onset and reduces the incidence and severity of age-related diseases, including cancer (2, 3).Quantitative changes in the activity of genes can control the rate of aging and the development of age-related diseases in invertebrates and mammals (4, 5). Numerous cross-sectional studies of the relationship between CR and gene expression have been published (6). Almost without exception, they have been interpreted as though they were performed longitudinally. This has led to the widespread view that the major effect of CR is to prevent agerelated changes in gene expression. Funding and publication bias has reinforced this notion (6).We previously found that in old mice, a 4-week shift from long-term control (LT-CON) to short-term CR (ST-CR) reproduced 55% of the gene-expression changes induced by long-term CR (LT-CR; ref. 7). ST-CR reversed Ϸ70% of the age-related changes in gene expression seemingly prevented by LT-CR (7). These results suggested for the first time that many of the gene expression changes produced by LT-CR respond rapidly to diet.The studies described above left several important questions unanswered. First, which of the changes in gene expression are related to the health and longevity effects of CR? Further, what are the kinetics of the changes in gene exp...

Small noncoding RNAs circulating in the blood may serve as signaling molecules because of their ability to carry out a variety of cellular functions. We have previously described tRNA- and YRNA-derived small RNAs circulating as components of larger complexes in the blood of humans and mice; the characteristics of these small RNAs imply specific processing, secretion, and physiological regulation. In this study, we have asked if changes in the serum abundance of these tRNA and YRNA fragments are associated with a diagnosis of cancer. We used deep sequencing and informatics analysis to catalog small RNAs in the sera of breast cancer cases and normal controls. 5′ tRNA halves and YRNA fragments are abundant in both groups, but we found that a breast cancer diagnosis is associated with changes in levels of specific subtypes. This prompted us to look at existing sequence datasets of serum small RNAs from 42 breast cancer cases, taken at the time of diagnosis. We find significant changes in the levels of specific 5′ tRNA halves and YRNA fragments associated with clinicopathologic characteristics of the cancer. Although these findings do not establish causality, they suggest that circulating 5′ tRNA halves and YRNA fragments with known cellular functions may participate in breast cancer syndromes and have potential as circulating biomarkers. Larger studies with multiple types of cancer are needed to adequately evaluate their potential use for the development of noninvasive cancer screening.

BackgroundA feature common to all DNA sequencing technologies is the presence of base-call errors in the sequenced reads. The implications of such errors are application specific, ranging from minor informatics nuisances to major problems affecting biological inferences. Recently developed "next-gen" sequencing technologies have greatly reduced the cost of sequencing, but have been shown to be more error prone than previous technologies. Both position specific (depending on the location in the read) and sequence specific (depending on the sequence in the read) errors have been identified in Illumina and Life Technology sequencing platforms. We describe a new type of systematic error that manifests as statistically unlikely accumulations of errors at specific genome (or transcriptome) locations.ResultsWe characterize and describe systematic errors using overlapping paired reads from high-coverage data. We show that such errors occur in approximately 1 in 1000 base pairs, and that they are highly replicable across experiments. We identify motifs that are frequent at systematic error sites, and describe a classifier that distinguishes heterozygous sites from systematic error. Our classifier is designed to accommodate data from experiments in which the allele frequencies at heterozygous sites are not necessarily 0.5 (such as in the case of RNA-Seq), and can be used with single-end datasets.ConclusionsSystematic errors can easily be mistaken for heterozygous sites in individuals, or for SNPs in population analyses. Systematic errors are particularly problematic in low coverage experiments, or in estimates of allele-specific expression from RNA-Seq data. Our characterization of systematic error has allowed us to develop a program, called SysCall, for identifying and correcting such errors. We conclude that correction of systematic errors is important to consider in the design and interpretation of high-throughput sequencing experiments.

Disrupted growth hormone/insulin-like growth factor-1 signaling (DF) and caloric restriction (CR) extend life span and delay the onset of age-related diseases in rodents. In combination, these interventions additively extend life span. To investigate the molecular basis for these effects, we performed genome-wide, microarray expression analysis of liver from homozygous and heterozygous Ames dwarf mice fed ad libitum or CR. CR and DF additively affected a group of 95 genes. Individually and together, DF and CR independently affected the expression of 212 and 77 genes, respectively. These results indicate that DF and CR affect overlapping sets of genes and additively affect a subset of genes. Together, the interventions produced changes in gene expression consistent with increased insulin, glucagon and catecholamine sensitivity, gluconeogenesis, protein turnover, lipid beta-oxidation, apoptosis, and xenobiotic and oxidant metabolism; and decreased cell proliferation, lipid and cholesterol synthesis, and chaperone expression. These data suggest that the additive effects of DF and CR on life span develop from their additive effects on the level of expression of some genes and from their independent effects on other genes. These results provide a novel and focused group of genes closely associated with the regulation of life span in mammals.

Cytosine methylation in the genome of Drosophila melanogaster has been elusive and controversial: Its location and function have not been established. We have used a novel and highly sensitive genomewide cytosine methylation assay to detect and map genome methylation in stage 5 Drosophila embryos. The methylation we observe with this method is highly localized and strand asymmetrical, limited to regions covering ∼1% of the genome, dynamic in early embryogenesis, and concentrated in specific 5-base sequence motifs that are CA- and CT-rich but depleted of guanine. Gene body methylation is associated with lower expression, and many genes containing methylated regions have developmental or transcriptional functions. The only known DNA methyltransferase in Drosophila is the DNMT2 homolog MT2, but lines deficient for MT2 retain genomic methylation, implying the presence of a novel methyltransferase. The association of methylation with a lower expression of specific developmental genes at stage 5 raises the possibility that it participates in controlling gene expression during the maternal-zygotic transition.

To facilitate the development of assays for the discovery of pharmaceuticals capable of mimicking the effects of caloric restriction (CR) on life- and healthspan (CR mimetics), we evaluated the effectiveness of glucoregulatory and putative cancer chemopreventatives in reproducing the hepatic gene expression profile produced by long-term CR (LTCR), using Affymetrix microarrays. We have shown that CR initiated late in life begins to extend lifespan, reduce cancer as a cause of death, and reproduce approximately three-quarters of the genomic effects of LTCR in 8 wk (CR8). Eight weeks of metformin treatment was superior to CR8 at reproducing LTCR-like gene expression changes, maintaining a superior number of such changes over a broad range of statistical stringencies, and producing more Gene Ontology terms overlapping those produced by LTCR. Consistent with these results, metformin has been shown to reduce cancer incidence in mice and humans. Phenformin, a chemical cousin of metformin, extends lifespan and reduces tumor incidence in mice. Taken together, these results indicate that gene expression biomarkers can be used to identify promising candidate CR mimetics.