Abstract:Dietary restriction (DR), limiting certain factors in diet without causing malnutrition, delays the ageing process and extends lifespan in multiple organisms. The conserved life-extending effect of DR suggests a fundamental mechanism, though it remains a subject of debate. We established a web-accessible database (GenDR) of DR-essential genes, which if genetically altered interfere with the effect of DR to extend the lifespan in model organisms (yeast, worm, fly and mice), and then explored the mechanistic links among DR-essential genes. Gene-regulatory circuits reveal that nutrient-sensitive, stress-response and meiotic transcription factors govern the DR induced transcriptional signature in yeast. Our comparative study shows that DR-essential genes are more conserved on the molecular level and interact with each other more than expected by chance. We suggest that DR commonly suppresses translation, while stimulates chromatin reorganization, reproductive processes (meiosis) and stress responses in species separated billions of years in evolution.
Highlights
Introduction
Results
GenDR
Molecular Evolution
Molecular Interactions
Differentially Expression of DR-Essential Genes
Spermidine Affects DR-Essential and Differentially Genes
Comparing Interactomes
Transcription Factors Governing the DR-Signatures from Yeast to Mammals
Proteins and Chromatin Marks Associated to DR-Differentially Genes
Lipid Metabolism
Does DR Up- or Downregulate Fatty Acid Elongases?
Does DR and Spermidine Treatment Upregulate Lipid Catabolism?
Discussion
Molecular Conservation of DR-Essential Genes
Differential Expressed DR-essential Genes
PI3K
FOXOs Regulate Entry Into Gametogenesis
ZBTB16
eor-1
Conclusions
Methods
dNdS
Transcription Factor Analysis
Chromatin Mark Enrichment/Depletion
Interaction Integration
Chance of Differentially Expression
Tables & Figures
Table: Number of DR-essential genes and orthologs in GenDR
Table: Lifespan and vacuolar changes of novel DR-essential genes
Table: Induced and suppressed interactions common to yeast, worm and fly
Table: Induced and suppressed interactions common to yeast, worm, fly and mammals
Table: Meiotic transcription factor binding sites are enriched in the promoters of DR-differential genes
Table: Meiotic transcription factor binding sites are enriched in the promoters of DR-essential genes
Table: Reproductive development-related factors are enriched in the promoters of the mammalian DR-signature
Table: Reproductive development-related factors are enriched in the promoters of mammalian DR-essential genes
Table: Motifs enriched 500bp upstream of the transcriptional start site of the more than two-fold DR-suppressed genes
GenDR is a database on genes related to lifespan extension by dietary restriction
Applied a network approach which predicted eight novel DR-essential genes
Comparing interactomes of three species revealed induction of reproductive-genes
Spermidine treatment regulates many of the same genes as dietary restriction
Restriction of defined factors in the diet increases lifespan in diverse species, from yeast to rodents. Furthermore, there is evidence that DR affects also the ageing process in primates, including humans. The mechanisms by which DR retards ageing remain a subject of intense debate but have been shown in model organisms to be mediated by genetic pathways, many of which are evolutionary conserved and operate in humans.
Genes impacting on the ageing process and essential for the effect of DR, if genetically modified, interfere with specific signalling cascades and programs which lead to lifespan extension. There are over 100 of such DR-essential genes in various model organisms. In the ideal case manipulation of DR-essential genes should not alter the lifespan under ad libitum (AL) and cancel out the lifespan-extending effect of DR, or being a DR-mimetic, already extend lifespan under AL, but also be non-additive with DR. Furthermore, it is logical to assume that most DR-essential genes or their gene products change in some way under DR, for instance on the transcript, protein or activity level. DR-essential genes form an incomplete signalling web, consequently novel DR-essential gene candidates can potentially be identified by interactions with established DR-essential genes either on the physical, genetic or gene regulatory level. Understanding their interactions with each other in a systemic fashion would likely reveal the actually mechanism of DR-conferred lifespan extension. Therefore, studying the mechanism of DR is especially suited for a network-guided approach.
The concept of networks can be applied on a multitude of biological levels, from genes to organisms. Biological networks tend to be scale-free (i.e. only a small subset of nodes have many connections), modular in composition and hierarchically structured. Different types of biological networks can provide complementary information. For instance, interactions between genes derived from epistasis experiments and physical interactions between proteins give complementary information about functional association (e.g. signalling pathways, complexes and processes).
Ageing genes are conserved across taxa, for example C. elegans ageing genes are more likely to be functional in S. cereviase [Smith et al., 2008]. Longevity genes/proteins network hubs and centrally located nodes have higher likelihoods of being associated with ageing and longevity than do randomly selected nodes [Witten and Bonchev, 2007]. Numerous network studies on ageing have been conducted with fruitful results [Bell et al., 2009; Budovsky et al., 2007; de Magalhaes and Toussaint, 2004; Li et al., 2010; Lorenz et al., 2009; Promislow, 2004; Wang et al., 2009b]. However, networks studies on DR are rare, likely because of the lack of adequate datasets.
In order to address this, we constructed a database around genes essential for this phenomenon and found that those genes are like the lifespan extending effect of a restricted diet conserved and interact with each other, which enabled us to predict and validate novel ones pointing to one-carbon metabolism and its interlinked regulation of intracellular polyamine levels. Polyamines can themselves extend lifespan in multiple species and these molecules are abundant in the germline.
This study aimed to define and establish a database of DR-essential genes in order to investigate their evolutionary and network properties and identify common regulators of DR-induced lifespan extension across multiple species. We integrated other types of data, such as genomic, transcriptional and epigenetic data and performed a variety of network and systems biology analysis to reveal common elements of DR effects. By concentrating only on the essential processes of DR-induced lifespan extension we hope to be able to narrow down the various DR affected/involved processes, potentially explaining why DR slows down ageing in multiple species including our own.
Nutrient-sensing, stress responding and meiotic transcription factors were found associated with DR-induced transcriptional changes in yeast and reproduction-associated transcription factor were even found enriched in mammalian DR-essential and differential genes. By using the interactomes of multiple species and comparing the impact of gene expression changes upon DR we show that DR commonly suppresses translation, while stimulates chromatin restructuring and reproductive rejuvenation processes.
By employing large-scale data (interactomes and transcriptomes) on multiple organisms, we were able to condense this information to the most essential and eliminated species-specific adaptive responses. Thereby we found evidence that lifespan extension by a restricted diet commonly may exploit an ancient rejuvenation process derived from gametogenesis. A restricted diet triggers rejuvenation in 1 billion of years by evolution separated species.
A literature-curated list of genes which if genetically manipulated in model organisms cancel out the effect of DR to increase lifespan was compiled (DR-essential genes). Their respective homologs in model organisms and humans were retrieved from NCBI/HomoloGene, Ensembl/Biomart, InParanoid and OrthoMCL [Table: Number of DR-essential genes and orthologs in GenDR]. The list of DR essential genes, a subset of their homologs and the mammalian DR transcriptional signature were integrated and a database (GenDR) was created. These data is freely available for the research community at the Human Ageing Genomics Resources [http://genomics.senescence.info/diet/].
A DR-essential gene was defined as follows:
Mutation interferes with effect of DR to extend lifespan
Mutant is not sick and capable of lifespan extension
Evolutionary conserved / under evolutionary positive selection
Change in activity under DR
Interact on physical or genetic level with other DR-essential genes
Whereas only criteria number one is obligatory, the others are optional and are very useful to infer/identify further DR-essential genes. We will use gene expression profiles, interaction networks, molecular evolution data to further prioritize potential DR-essential genes and identify crucial regulators. The second criteria could be used to discriminate from false-positives. Mutants should still be capable exhibiting lifespan extension via other interventions which are assumed not to work via DR signalling. Thus, proving that the mutation does not just renders the animal general sick and unable to life longer. RNAi against genes such as pat-4 and pat-6 abrogated DR-induced lifespan, but as the animals appeared to be very unhealthy they were not classified as DR-essential [16103914].
Given that DR is evolutionary conserved across model organisms; we expected DR-essential genes to be evolutionary conserved at the molecular level.
Using GenDR we tested the proposed evolutionary conservation of DR on the molecular level. The presence of orthologs (in percentage) for all species is higher than expected by chance [Figure: DR–essential genes are molecular conserved and form a tight interaction network A]. The median non-synonyms (dN) as well as synonyms (dS) nucleotide mutations rates and their ratio (dN/dS), a measure for evolutionary selection pressure, for DR-essential gene orthologs in mammalian species pairs, were lower than expected by chance [Figure: DR–essential genes are molecular conserved and form a tight interaction network B]. As such it appears that indeed DR is evolutionary conserved at the genetic level and DR-essential genes may belong to the core-metabolism and regulation network.
Figure: DR–essential genes are molecular conserved and form a tight interaction network. A, The proportion of genes (shown as percentages) with orthologs from multiple species in the specified organisms is in all cases higher (by 10–30%) for DR-essential genes than expected by chance. B, The average degree (number of interactions) for DR-essential genes and DR-essential gene orthologs (the ortholog-complemented set) is higher than the interactome-wide average which serves as control. C, The average percentage of specificity in interconnectivity (100× specific interactions with seed genes/all interactions) of DR-essential genes to each other is higher than for other genes in interactomes, and complementation with orthologs of DR-essential genes from other species further increases the specificity of interconnectivity. ns, P>0.05; , P*](#id1)*, P
System Message: WARNING/2 (, line 77); backlink
Inline strong start-string without end-string.
System Message: WARNING/2 (, line 77); backlink
Inline emphasis start-string without end-string.
Our network analysis revealed that DR-essential genes interact more frequently with each other [Figure: DR–essential genes are molecular conserved and form a tight interaction network C].
In order to identify novel DR-essential gene candidates we used the guilt-by-association principle. Genes associated to a certain process can be identified via the guilt-by-association concept [de Magalhaes and Toussaint, 2004], which basically means that a gene having many interactions/associations to a certain set of genes (here DR-essential genes) is likely to belong to this group too.
To predict novel DR-essential candidate genes we used a guilt-by-association concept [de Magalhaes and Toussaint, 2004]. Essentially, a gene with more interactions with genes associated with a given process (i.e. DR) than expected by chance is likely to also play a role in this process (see Methods). Highly significantly connected candidate genes were for instance GAT1 (GATA transcription factor) in Saccharomyces cerevisiae, daf-28 (Insulin ortholog) in Caenorhabditis elegans, and Pi3K92E (PIK3CB ortholog) in Drosophila melanogaster. Pi3K92E is implicated in Drosophila diapause which is similar to the nematode dauer formation suggesting a conserved role for this kinase in the control of both reproduction-related traits and genes as well as developmental arrest in response to environmental cues [Williams et al., 2006]. Diapause doubles insect lifespan and post-diapause animals have age-related mortality rates to newly enclosed young flies [Tatar and Yin, 2001]. Interestingly, in C. elegans PI3K null mutants live up to ten-times longer than normal [Ayyadevara et al., 2008; Tazearslan et al., 2009]. After orthologous complementation, among the highly significant candidate genes were for example GLN3 (another GATA transcription factor) in S. cerevisiae, daf-15 (RAPTOR ortholog) in C. elegans, Ilp1 (Insulin-like peptide) in D. melanogaster, Kras (oncogenic GTPase) in Mus musculus, and ACD (involved in telomere maintenance and meiosis) in humans.
By Orthologs complementation it is meant adding for instance the yeast orthologs of DR-essential worm, fly and mouse genes to the seed list for yeast interaction network. This was the only way to investigate DR-essential networks in mammals and to improve the predictive power of the yeast, worm and fly networks.
Most DR essential genes interact tightly with each other as they were always top-ranked. It is some kind of proof of prediction because we could rediscover DR-essential genes as significant interaction partners.
In yeast we predicted 9 candidates of which 8 were found to be indeed DR-essential. Among them were three novel DR-mimetics, namely OPT2, FRE6 and RCR2, implicated in drug detoxification, transition metal ion homeostasis and vacuolar protein sorting/endocytosis, respectively.
The most significant interaction partners of DR-essential genes related to vacuole/autophagy were experimentally tested [Table: Lifespan and vacuolar changes of novel DR-essential genes]. 8 of 9 genes were found to be DR-essential as these mutants interfered with lifespan extension response to DR, hence had impaired lifespan extension on DR. FRE6, RCR2 and OPT2 mutants were already long-lived under AL. DAP2, SLM4 and YDL180 mutations cancelled out the lifespan extension effect of DR, while VPS20, FRE6, RCR2, OPT2 mutations even decreased lifespan under DR compared to AL [Figure: DR–essential vacuole-associated genes are required for DR lifespan extension and normal vacuolar morphology]. Shifting cells from AL to DR decreased the percentage of cells with only one vacuole, while increased the percentage of cells with 2-5 vacuoles in fre6, rcr2, dap2 and slm4. In contrast, to long-lived mutants (fre6 and rcr2), where the percentage of cells with more than 5 vacuoles decreased, in short-lived mutants (dap2 and slm4) the proportion of cells actually is increasing. Half of the long-lived rcr2 mutants had only a single vacuole under AL, while under DR the proportion of cells with 2-5 vacuoles is almost doubled to 80% [Figure: DR–essential vacuole-associated genes are required for DR lifespan extension and normal vacuolar morphology].
Figure: DR–essential vacuole-associated genes are required for DR lifespan extension and normal vacuolar morphology. A, Survival curves of wild-type and rcr2Δ mutant strains measured on AL/YEPD and DR media. The units of age (X axis) are generations. Mean and maximum lifespans as well as sample sizes are listed in Table: Lifespan and vacuolar changes of novel DR-essential genes. B, Vacuolar morphology on AL and DR media of wild-type and rcr2Δ. Left panel: DIC images showing the cells; middle panel: TRITC-fluorescent images showing the vacuoles; right panel: overlay of cell and vacuole images.
For the only strain (yol92w) that showed a longer lifespan on DR than on AL, the lifespan still did not reach the full capacity of wild type under DR. However this strain's lifespan under AL is already short. Thus, it is not surprising that its lifespan does not reach the some length as wild type upon DR.
Notably, OPT2 the gene which when delete extended to the lifespan to the greatest extent was also the one of the most strongly downregulated genes. Thus, we employed microarray data to validate our predicted DR-essential genes in yeast and other organisms.
As changes in gene activity of DR-essential genes via genetic manipulations mimic and/or abolish the lifespan extension conferred by DR, one assumption is that DR-essential genes are changing in their expression or activity level upon DR. As an approximation to investigate this, we employed large-scale microarray expression profiles of yeast, worm [Honjoh et al., 2009], fly [Bauer et al., 2010; Zid et al., 2009] and mammals [Hong et al., 2010; Swindell, 2008a, b, 2009].
DR-essential genes in yeast are more likely to be differential expressed than expected by chance. Although the statistical significance depends on the chosen threshold, it reached significance (hypergeometric p-value
We noted that in yeast spermidine treatment causes differential expression of DR-essential genes much more likely than expected by chance (hypergeometric p-value
The interactomes of yeast, worm and fly were integrated with respective gene expression profiles upon DR and the common functional enrichment of the induced and suppressed interactions determined [Figure: Comparing the influence of gene-expression changes upon DR, in multiple interactomes; Table: Induced and suppressed interactions common to yeast, worm and fly; Table: Induced and suppressed interactions common to yeast, worm, fly and mammals].
Figure: Comparing the influence of gene-expression changes upon DR, in multiple interactomes. The interactomes of yeast, worm, and fly were integrated with gene expression information upon DR and condensed interaction networks generated via the ExprEssence algorithm (Step 1). Interaction networks restricted to either suppressed or stimulated interactions for each species were created (Step 2). The suppressed and stimulated networks were then separately compared for common significant functional enrichments (Step 3). Functional terms which were common to suppressed and induced interactions upon DR in yeast, worm, and fly are listed with their respective p-values [Table: Induced and suppressed interactions common to yeast, worm and fly; Table: Induced and suppressed interactions common to yeast, worm, fly and mammals].
To extend our approach to mammals, the transcriptional signatures of DR in mammals [Hong et al., 2010; Swindell, 2008a, b, 2009], which are about 1348 genes, were utilized and pseudo-intensities generated of 2.0 and 0.5 corresponding to genes which are consistent across tissues and experimentally settings up- and downregulated (the default is set to 1.0). Among the terms identified to be consistent significantly (p-value
To ascertain the underlying cause of differential expression upon DR we utilized gene regulatory networks (transcription factor - target gene interactions [Abdulrehman et al., 2011; Balaji et al., 2006]) to identify candidate transcription factors responsible for the differential expression upon DR. Our criteria were, firstly, factors which regulate DR-differentially expressed genes with a high specificity, and secondly, interact with DR-essential genes either on the physical or genetic level.
In yeast, transcription factors which had very high specificity (i.e. specific / total interactions in %) for controlling DR-induced genes were in addition to nutrient-sensing, e.g. Msn2/4 (26%), Gis1 (53%), Mig1 (40%), and stress-responsive transcription factors, such as Hsf1 (34%), which are known to be important for DR-lifespan extension, strikingly also meiotic transcription factors, like Ime1 (41%), Ume1 (75%), Ume6 (38%) and Ndt80 (26%).
Sequences (500 bp upstream) of the promoter regions in yeast of the more than two-fold differentially expressed genes were scanned/examined for motif enrichment (hypergeometric test) and indeed upregulated genes are significant enriched for the STRE (stress-response element; p-value = 0.0034/5.6710^-4), PDS (post-diauxic shift) element (p-value = 3.89107/3.3710^-3), URS1 (upstream regulatory sequence; p-value = 1.0910^-3), MSE (middle-sporulation element; p-value = 3.8510^-3) including the motifs of Ume6 (p = 6.7110^-6), Ime1 and Ndt80 (p = 0.013324). Motifs of Ume6, Ime1 and Ndt80 were highly enriched in the promoters of DR-induced genes, while the motif of the repressor of meiosis initiation, Rme1, is significant enriched in the downregulated gene promoters [Table: Meiotic transcription factor binding sites are enriched in the promoters of DR-differential genes]. Interestingly, URS1 as well as motifs of Ume6, Sum1, Ndt80 and Ime1 are even enriched in the upstream sequences of DR-essential genes [Table: Meiotic transcription factor binding sites are enriched in the promoters of DR-essential genes]. Also in another signature of DR [Lin et al., 2002], meiotic binding motifs were significantly enriched in the 500 bp upstream regions of induced genes. For instance, YGNCACAAAW (NDT80) was present in 11 DR-induced genes of the 14 genes in the genome harbouring such a motif (p-value = 0.0025, q-value = 0.029). In this signature Ime1 and Ndt80 had also a high specificity to regulate DR-differentially expressed genes [Figure: Transcriptional regulation of DR in yeast]. NDT80 transcript level increase as function of the strength of restriction [Lee and Lee, 2008] and were found to be 2.6-fold elevated in our own DNA-microarray data at 0.5% glucose concentration.
Figure: Transcriptional regulation of DR in yeast. Transcription factors identified via guilt-by-association to regulate DR-differential genes.
In C. elegans the transcription factor p53-homolog CEP-1 is associated to reproduction and its motif (RCWWGYYY) is significant enriched (p-value = 0.048) in the regulatory regions (+800 to -500) of the DR-induced genes (glucose restriction [Lee et al., 2009], > 1.5-fold-change). The top cluster of terms enriched in the 907 DAF-16/FOXO target genes is multicellular organism reproduction (Benjamini p-value = 7.910^-5) [Schuster et al., 2010]. Among them is for instance the single lamin gene in C. elegans lmn-1* which is ubiquitous expressed in all cells except in cells undergoing spermatogenesis. The 98 progeric genes in C. elegans which shorten long-lived daf-12 mutants lifespan are significantly enriched for larval development (p-value = 4.5E-10; q-value = 8.4E-8), and reproductive developmental process (p-value = 3.3E-9; q-value = 2.5E-7).
dFOXO is a positive regulator of sexual reproduction related genes in an Insulin/IGF-like signalling mutant as evidenced by a high enrichment of sexual reproduction associated genes (Benjamini p-value = 4.7*10^-2) among those genes that are physically bound by dFOXO, downregulated in short-lived dFOXO-/- mutant and upregulated in long-lived dominant-negative insulin-receptor overexpression transgenic animals [Alic et al., 2011].
Next the mammalian promoter regions of DR-differentially expressed genes were scanned for enrichment of cis-regulatory elements and again factors interacting with DR-essential orthologs which are associated to reproductive processes had a high significant enrichment of their motifs in the regulatory sequences of the upregulated genes in the mammalian DR signature [Table: Reproductive development-related factors are enriched in the promoters of the mammalian DR-signature].
The target genes of Zbtb16 are mostly enriched for reproductive development process (p-value = 1.810^-2) and acetylation (p-value = 1.910^-2): Msh2, Pten, Nrip1, Atp5a1 (acetylated in liver mitochondria from fasted mice but not from fed mice), Plekhf1, Pura (transcription factor that actives c-Myc). The only significant target gene of Ppara with a perfect match of its consensus sequence is Narf (nuclear prelamin A recognition factor), which is interesting as the only nuclear proteins which are prenylated in mammalian cells are prelamin A and B encoded by LMNA, a gene associated to heterochromatin organization and premature ageing.
As we found enrichment for the motifs of reproduction-related transcription factors even in the promoter regions of DR-essential genes and lower dS values than expected by chance, we tested whether this is a conserved feature of DR-essentiality even in humans. Indeed, DR-essential orthologs were enriched for the motifs of defined factors [Table: Reproductive development-related factors are enriched in the promoters of mammalian DR-essential genes].
TP63 is a DR-essential ortholog and itself regulates DR-essential gene orthologs. PPARA and CITED2 are also direct target genes of p63, whereas PPARA regulates specific TP63 isoforms [Pozzi et al., 2009]. Heat shock factor 1 (HSF1) and FOXO3 are DR-essential orthologs while ZBTB16 is novel. The mammalian factors identified on the basis of sequence analysis are also significant interaction partners in the networks of DR-essential orthologs in mouse with Foxo3 (p-value = 7.1410^-9), Hsf1 (p-value = 1.4110^-7) and Zbtb16 (p-value = 7.36*10^-4) as well as in humans with HSF1 (p-value = 0.017), ZBTB16 (p-value = 0.020), TP63 (p-value = 0.041). ZBTB16 and TP63, both are targets of CDK2, the mammalian IME2 homolog [Figure: TP63 and ZBTB16 are significant interaction partners of DR-essential genes and targeted by CDK2/IME2].
Figure: TP63 and ZBTB16 are significant interaction partners of DR-essential genes and targeted by CDK2/IME2. Genes up- or downregulated upon DR are in red and green, respectively, while genes which were enriched for up- and downregulation are in yellow. DR-essential orthologs are marked by a golden halo.
ZBTB16 is downregulated by TP63 RNAi, indicating that it is either a direct or indirect target gene of TP63. DR-essential NNMT1 is right upstream of ZBTB16 in mouse and humans. ZBTB16 and TP63 are co-expressed in stem cells [Majo et al., 2008]. TP63, BMI1 and ZBTB16 mediate proliferation potential of cells via parallel pathways through various gene transcription and silencing programs [Senoo et al., 2007].
TP63 bound genes are enriched for response to nutrient levels (22 genes of 551; Benjamini p-value: 3.210^-4) and regeneration (p-value = 1.310^-3) as well as negative regulation of cell differentiation (Benjamini p-value = 8.110^-3) and reproductive developmental process (p-value = 1.810^-2) [Barton et al., 2010; Vigano et al., 2006]. Genes identified to harbour a TP63 consensus sequence within 800 bp of their transcriptional start sites (TSS) are enriched for reproductive developmental process p value = 2.710^-2 (CITED2, DDX25, CENPI, CEP57, EIF2B2, FOXA1, PDE3A as well as NOTCH1), vacule/lysosomal genes (ACP2, ADRB2, CTNS) and nucleolus. Zbtb16 target genes with a recognition sequence within 800 bp of the TSS are enriched for zing-finger (Benjamini p-value = 2.210^-2; p-value = 7.510^-5), RhGAP (p-value = 8.610&-4) and gamete generation / sexual reproduction (p-value = 4.7*10^-3) with for instance Oct4, Dnmt3l, Cctc, and Pparg.
We scanned DR-differentially expressed genes in yeast for enrichment and depletion of chromatin-associated proteins and chromatin modifications [Kurdistani et al., 2004; Pokholok et al., 2005; Xu et al., 2005] and found that indeed they are enriched and depleted for specific DNA-binding proteins as well as histone acetylation and methylation marks. For DNA-binding proteins we tested their over-representation in the two-fold differentially expressed genes with a hypergeometric test. DR- and spermidine-induced genes were most strongly enriched for Ume6 and Sum1, respectively.
Specifically for chromatin marks, we tested (by a binomial test) the comparisons up versus down and differential versus non-differential (two-fold). DR-induced genes were enriched and DR-suppressed genes were depleted for H3, H3K4me1/me2/K14ac/K14me3/K27ac/K56acH4K8ac (p-values: 2.110 2/2.010^-10/2.910^-4/8.710^-3/1.710^-15/9.610^-10), H2BK11ac/K16ac (p-values: 9.610^-10/5.610^-10) and Gcn4 binding (p-value = 7.310^-4), while it was vice versa for H3K9ac/K18ac/K36me3 (p-values: 5.310^-4/5.0310^-7/2.3310^-9) and H4K79me3 (p-value = 4.6*10^-4).
Spermidine treatment, which extends lifespan of multiple species, is also associated with specific chromatin marks on the differentially expressed genes, but it appears to oppose global histone mark changes on the up- vs. downregulated genes by DR in the opposite manner, while enriched or depleted in the same direction by the comparison differential vs. others. For instance, the differentially expressed genes (both up- and downregulated genes) by DR and spermidine are enriched for H3K56ac by 0.012 and 0.022 but greatly depleted on other non-differentially expressed genes by 0.0098 and -0.0026, respectively (binomial p-values: 0.002 and 0.03).
We identified an ortholougs group of fatty acid elongases which were commonly differentially expressed in DR-signatures from yeast to mammals with the following members: FEN1 Elovl5 CG5278 CG31523 bond elo-6 Elovl6 SUR4 CG31522 elo-2 CG8534 CG9458 CG5326
Yeast encodes at least three fatty acid elongases: ELO1, SUR4 and FEN1. While ELO1 (0.7) is only slightly downregulated, both the DR-essential SUR4 (0.05) and FEN1 (0.06) are greatly downregulated.
In C. elegans there are at least 9 fatty acid elongase orthologous. Intermittent fasting upregulates the SUR4 orthologs elo-2 (1.9) and elo-6 (2.2) as well as elo-1 (1.3) and elo-5 (1.4) and downregulated elo-3 (0.7). Glucose restriction upregulates elo-4 (1.3) and downregulates elo-2 (0.85) and elo-5 (0.6). CeMM upregulates elo-7 (3.8) and downregulates elo-2 (0.7), elo-3 (0.4), elo-5 (0.3) and elo-6 (0.4).
In D. melanogaster CG5326 is downregulated on the level of transcription, while on the level of translation CG8534 (33) was strongly upregulates and CG31522, CG6921, CG33110, Elo68beta, CG31522, CG5326, CG5326, CG5278 and James Bond were all downregulated to different extents.
In mammals Elovl6 is enriched for downregulation, whereas Elovl5 is enriched for upregulation.
Thus, DR always leads to downregulation of at least a few fatty acid elongases across species boundaries.
DR upregulates genes annotated with "cellular lipid catabolic process" (FOX2, PGC1, TGL3, ECI1, IDP3, SPS19, TGL4, POT1, TES1, PLB3, ISC1, POX1, DCI1) and "lipid catabolic process" (ATG15, YDL109C, TGL2, ISC1, POX1, YJR107W, FOX2, TGL4, ECI1, TGL3, IDP3, SPO1, IDP3, YOR022C, YOR059C, DCI1 and PGC1).
FOX2 null mutants had decreased chronological lifespan. TGL3 mutant displayed decreased sporulation efficiency. IDP3 deletion increased replicative lifespan. SPS19 is a SPorulation-Specific transcript. ISC1 mutant had a decreased chronological lifespan and exhibit negligible sporulation induction. POX1 mutants also had decreased sporulation efficiency.
Spermidine induces the expression of "lipid catabolic process" related genes too (SPO14, TGL1, YJR107W, TES1, ROG1, YDR444W, ATG15, GDE1, PLC1, YOR022C, SPO1, TGL3 and CSR1). SPO14 and SPO1 are required for meiosis and spore formation. SPS19 is sequential activated during meiosis and spore-formation.
Common to these two interventions is the induction of ATG15, SPO1, YOR022C, SPS19 and TGL3.
Our findings show that DR-essential genes are conserved at the molecular level, interact with each other more than expected by chance and therefore allow identification of novel candidates via the guilt-by-association principle.
DR-essential genes interact with phosphorylation-related and ageing genes. Nutrient-sensing signalling is intrinsically coupled to growth regulation and a link between growth and DR signalling pathways is well-established [de Magalhaes et al., 2011]. Some growth programs are differentially important in various organisms that may be reflected in other effectors downstream DR in different species. In addition, terms associated with translation were suppressed in the comparison of the interactomes of multiple species [Table: Induced and suppressed interactions common to yeast, worm and fly], again in line with previous findings [de Magalhaes et al., 2011].
DR may be considered to act as a nutritional stressor. Gluconeogenesis genes located at subtelomeric regions are known to be upregulated by stress [Mak et al., 2009] via evolutionarily conserved epigenetic control [Smith et al., 2011]. In fact, key enzymes and factors of gluconeogenesis are induced by DR which may sequester TORC1 [Brown et al., 2010] away from its normal functioning membranes (i.e. vacuolar and endosome membrane, etc.).
Rpd3 deacetylation mediates UME6 transcriptional repression activity [Rundlett et al., 1998], which is converted to an activator by Ime1 binding and Ime2 phosphorylation. DR-essential HSF1 is already known to be involved in gametogenesis in mammals and even to be essential for meiosis [Akerfelt et al., 2010; Bierkamp et al., 2010; Le Masson et al., 2011; Metchat et al., 2009]. Inducible HSF1-binding site are associated with histone acetylation [Guertin and Lis, 2010].
The investigation of the molecular evolution of DR-essential genes showed conservation at the molecular level reflecting the observation that DR extends lifespan in various evolutionary distant related organisms (yeast, worm, fly, spider, fish, dog, hamster, mouse, rats, monkeys, etc.).
Via the guilt-by-association concept we identified novel DR-essential genes primarily implicated in drug detoxification, transition metal ion homeostasis and vacuolar-trafficking/endocytosis as well as a potential role of S-adenosylhomocysteinase as a common interactor of DR-essential genes in yeast, worm, fly and mammals. Several DR-essential genes change their expression level upon DR, some even across species, such as genes encoding the S-adenosylmethionine synthetase activity. DR-differentially expressed genes are associated with specific chromatin marks on AL, some are even shared with lifespan extending spermidine treatment.
We found that S-adenosylmethionine synthetase is commonly differentially expressed upon DR, while S adenosylhomocysteinase is commonly interacting specifically with DR-essential genes in multiple organisms.
Kinase signalling is dramatically suppressed in very long-lived PI3K mutant worms, and S-adenosylhomocysteinase, several insulin-like peptides, and DAF-15 all turn up in proteomics and phosphoproteomic studies.
It is clear that diet alters epigenetic marks (i.e. chemical tags such as acetyl or methyl groups) and results in an altered chromatin state. Stress alters the methylation state of histones in fruit fly with transgenerational inheritable effects [Seong et al., 2011]. The lifespan prolonging effect of DR is inherited to the next generation in rotifier [Kaneko et al., 2010]. Deficiencies in H3K4 trimethylase complex components ASH-2, WDR-5 or SET-2 in parental generation extend lifespan of descendants up until the third generation [Greer et al., 2011]. Moreover diet has also been shown to alter gene expression transgenerationally in mammalian species [Flintoft, 2011; Ng et al., 2010]. In humans the rate of mortality is affected in a transgenerational manner by food intake in the parental generation [Kaati et al., 2007]. Food intake in one generation influences genes expression [Benyshek et al., 2006], glucose and cholesterol metabolism [Benyshek et al., 2006; Carone et al., 2010] and β-cell function [Ng et al., 2010] in the subsequent generation in mammals. Thus, dietary intake affects chromatin marks that may not be completely erased between generations. The lifespan prolonging effect of DR can be mimicked or blocked by α-lipoic acid supplementation during AL, which exerts a memory effect of the previous feeding regimen in feeding switch experiments [Merry et al., 2008] and is associated with an altered proteome acetylation state, with a hyperacetylated state linked to longevity. This evidence indicates that an altered chromatin state in DR is its principle lifespan extending mechanism across species boundaries, but why and how?
Taken together this indicates that an altered chromatin state in DR is the principle major component for its lifespan extending mechanism across species boundaries.
A common theme of longevity mutants is the silencing of multiple signalling pathways which is also evident in an attenuation of total kinase activity resulting in proteome-wide reduced phosphorylation in, for instance, strong alleles of age-1 mutants [Shmookler Reis et al., 2009].
Numerous DR-essential genes and their orthologs exhibit the expected change already either on the transcriptional or translational level. However, this was not observed at the genome-scale at all cut-offs. For common-used cutoffs, DR-essential genes are not more likely to be differentially expressed than expected by chance. The problem here may be that for instance in yeast there are at least 3 different longevity definitions used and at least as many different versions of DR. If it would be focused on just one, say replicative lifespan (the bud-counting assay), and moderate glucose withdrawal (0.5%), a significant enrichment might be seen. Our microarray data showed that numerous DR-essential genes are differentially regulated by DR. Moreover, other longevity manipulations such as spermidine treatment also differentially regulate DR-essential genes. Thus we expanded our systems biology analysis to other organisms and predict that longevity might converge on influencing intracellular polyamine levels.
PI3K activity positively regulates organismal reproduction and developmental progression. However, on the cellular level it inhibits the expression of reproductive (i.e. germline) genes in the somatic cells. Thus ectopic downregulation of PI3K ceases organismal reproduction, but induces germline-trait-like expression in the soma. Fascinating, PI3K tightly regulates gametogenesis in mammals.
Foxos promote organismal longevity in invertebrates, and in humans single nucleotide polymorphisms in FOXO3 are associated with extreme longevity [Kenyon, 2010].
Foxo1 mediates some aspects of the DR-effect [Yamaza et al., 2010]. PI3K signalling regulates stability and subcellular localization of Foxos, including Foxo1 [Goertz et al., 2011]. Foxos control key aspects in stem cell maintenance as they regulate self-renewal in hematopoetic and neural stem cells [Paik et al. 2007; Tothova et al. 2007; Renault et al. 2009]. Foxos, particular Foxo3 co-ordinately regulates neural stem cell homeostasis through genes influencing stress response and oxygen metabolism [Renault et al. 2009; Paik et al. 2009]. Foxos regulate hematopotic stem cell differentiation and assist long-term maintenance by protecting against oxidative stress.
Foxo function in male and female germ-line shares some similarity. Foxos evolved to control gametogenesis within the gonad itself. Whereas FOxo1 is highly epressed in undifferentiated spermatogonia, Foxo3 is highly expressed in primordial oocytes, in which it serves to retrain their growth [John et al. 2008]. Foxo3 function in female germ line maintenance [Castrillon et al. 2003; John et al. 2007; John et al. 2008], while Foxo1 controls multiple steps of spermatogenesis, from SSC proliferation and self-renewal to progression of spermatogenesis, including meiosis. Spermatonia are capable of self-renewal and immortal growth. Foxo1 specifically marks mouse gonacytes and a spermatogonia subset with stem cell potential. Foxo1 was required for both spermatogonial stem cells (SSC) homeostasis and initiation of spermatogenesis. Combined deficiency of Foxo1, Foxo3, and Foxo4 resulted in a severe impairment of SSC self-renewal and a complete block of differentiation, indicating that Foxo3 and Foxo4 contribute to SSC function. PI3K controls both SSC maintenance and differentiation. PI3K-Akt pathway is principal pathway regulating FOxo1 subcellular localization and activity in the context of spermatogenesis. Foxos appear to control a network of genes unique to spermatogenesis. Foxos join the growing network of transcription factors - Zbtb16 and Taf4b - that control early steps of spermatogenesis, including SSC self-renewal [Buaas et al. 2004; Falender et al. 2005].
Forkhead transcription factors control crucial steps in embryongenesis and are essential for the development of all germ layers and organs [Lehmann et al. 2003]. Foxo3-/- mice exhibit signs of accelerated ageing and have global follicular activation, therefore become premature infertile too [Castrillon et al. 2003]. Primordial follicles (which are in pre-meiotic phase) might be regarded as being in a state of developmental arrest. To maintain this arrest, FoxO3a is apparently necessary. Forced FoxO activation induces also an reversible arrest in C. elegans, Drosophila [Kramer et al. 2003] and in mammalian cells [Kops et al. 2002]. In general FoxOs are capable of causing a reversible arrest. Stem cells are example of reversible arrested cells that can re-enter proliferation and/or differentiation. Foxo-regulated genes are required for the maintenance of satellite cells, which include p27kip1 and p130/Rb2, both of which are also required for muscle differentiation [Bois and Grosveld. 2003].
Unique genetic requirement for FOxo1 in males and Foxo3 in females mirrors their high expression at discrete cellular stages in spermatogenesis or oogenesis.
FOxos together with ZBTB16 control early steps of spermatogenesis. Foxo1 and ZBTB16 were always coexpressed during spermatogenesis, where their expression was restricted to undifferentiated spermatogonia. ZBTB16 indirectly regulates mTOR activity [45].
Foxo1 transcriptome in male germ cells was distinct from that in other cell types, for which genes regulating oxidative stress resistance were rendered as major targets [17254970; 19896443; 19896444].
GnRH regulates FOXOs. FOXOs regulate LHB subunit expression. Foxo targets GADD45, FAsL, p21Cip1 and p27Kip1. Foxo3a mediates GADD45 and FasL expression in response to GnRH. GnRH is an essential regulator of the reproductive processes by stimulating the synthesis of LH and FSH in pituitary gonadotropes, thereby regulating gametogenesis and steroidogenesis [http://www.era.lib.ed.ac.uk/handle/1842/5686].
Loss-of-function mutations in ZBTB16 also called promyelocytic leukemia zinc finger protein (PLZF) cause an age-dependent loss of germ-line stem cells leading to sterility. ZBTB16 is expressed in germ-line stem cells and is required for their self-renewal. ZBTB16 facilitates transcriptional repression through recruitment of chromatin modifying enzymes to specific DNA sequences. SSCs have a unique epigenetic methyl-histone modification that are distinct from their progeny [http://research.jax.org/faculty/bob_braun.html].
Blasting ZBTB16 identified blmp-1 as the closest sequence homolog, which directly regulates cep-1/p63. However, eor-1 is the C. elegans ortholog of mammalian ZBTB16 [Zhang et al., 1999]. ZBTB16 does influence developmental patterning and Hox gene expression in mouse [Barna et al. 2000].
Epidermal growth factor (EGF) promotes cell division and cellular differentiation in developing animals. EGF acts through phospholipase Cy and IP3 receptor signalling maintains pharyngeal and body wall muscle formation in ageing adults and delays accumulation of lipofuscin-enriched ageing pigments within intestinal cells. EGF also acts through RAS/ERK pathway to regulate protein homeostasis by promoting antioxidants gene expression, stimulating activity of the ubiquitin proteasome system (UPS) and repressing expression of small heat shock protein chaperons. Effects of EGF signaling on lifespan is largely independent of IIS as effect of EGF signalling mutants on lifespan are largely independent of DAF-2 and DAF-16 mutants. However these two signalling pathways have multiple points of overlap, coordination and cross talk. IIS and EGF signalling respond to environment and to developmental timing, respectively. let-23 loss-of-function mutant had a decreased lifespan when raised at 20 degree Celsius. eor-1 is oocyte enriched. eor-1 and eor-2 act downstream or in parallel to SynMuv/Rb pathway to promote Ras/Erk-dependent transcription. EOR-1 interacts with SynMuv B pathway component LIN-36, a possible histone deacetylase complex component [Thomas and Horvitz, 1999; Walhout et al. 2000; Howard and Sundaram, 2002]. SynMuc gene products could antagonize Ras signalling in part by binding to and inhibiting activity of positively acting transcriptonal regulators such as EOR-1. LIN-36 EOR-1 interaction could also be involved in positive role of some SynMuv N genes [Chen and Han 2001]. eor-1 regulates cooperatively Hox-dependent patterning [Howard and Sundaram, 2002].
As discoveries are made in different model organisms and the lifespan-extending effect of DR as well as DR essential genes are conserved across taxa it makes sense to also consider their orthologs, therefore GenDR was built.
Invertebrates were not studied because of the difficulty to obtain dN and dS values.
For the microarray data from worms and flies, GSE files were retrieved from the GEO database, replicates were averaged and fold-changes calculated via python scripts (GSE9217 (different levels of glucose for yeast), GSE9682 (intermittent fasted nematodes), GSE18563 (glucose restricted nematodes), GSE6057 (nematodes in CeMM), GSE26726 (fly transcriptional) and GSE16738 (fly translational)).
The +500 bp sequences of yeast genes, +1500 bp to -500 bp for murine genes and +1000 bp to 500 bp for human genes, relative to the transcription start site were retrieved from Ensembl. Whole genomes (sacCer3, ce6, dm3, mm9, hg19) were retrieved from UCSC via worldbase.
Data about chromatin modifications (histone acetylation and methylation) were obtained from [Kurdistani et al., 2004; Pokholok et al., 2005; Xu et al., 2005]. Differentially expressed genes (> 2-fold) were assigned to their value for each chromatin mark. A binomial test between the average upregulated and downregulated genes as well as between the average differentially-expressed and non-differentially expressed genes was performed. The signal strength for all differentially expressed genes, upregulated genes, downregulated genes and all other were collected. Then the average values (actually the lists which give raise to these numbers) of them were compared with a binomial test in order to find which marks that are enriched in up vs. down and delta vs. other.
Via a python script each dataset gene/protein names/identifiers, experimental system type (genetic or physical), interaction type (e.g. association, complex formation, etc.) experimental system (e.g. yeast-two hybrid), post-translational modifications events (e.g. phosphorylation), pmids, and source databases were extracted first converted to a unified schema (unique_id_a, unique_id_b, experimental_system_type, interaction_type, experimental_system, modification, pmid, taxid, source_database), sorted according to taxonomy identification (taxid) and then subsequently fused.
Whether DR-essential genes are more likely to be differentially expressed than expected by change was calculated by the hypergeometric test. In mammals we employed a signature derived from genome-wide meta-analysis of DNA-microarrays [22327899].
Abdulrehman, D., Monteiro, P.T., Teixeira, M.C., Mira, N.P., Lourenco, A.B., dos Santos, S.C., Cabrito, T.R., Francisco, A.P., Madeira, S.C., Aires, R.S., et al. (2011). YEASTRACT: providing a programmatic access to curated transcriptional regulatory associations in Saccharomyces cerevisiae through a web services interface. Nucleic acids research 39, D136-140.
Alic, N., Andrews, T.D., Giannakou, M.E., Papatheodorou, I., Slack, C., Hoddinott, M.P., Cocheme, H.M., Schuster, E.F., Thornton, J.M., and Partridge, L. (2011). Genome-wide dFOXO targets and topology of the transcriptomic response to stress and insulin signalling. Molecular systems biology 7, 502.
Balaji, S., Babu, M.M., Iyer, L.M., Luscombe, N.M., and Aravind, L. (2006). Comprehensive analysis of combinatorial regulation using the transcriptional regulatory network of yeast. Journal of molecular biology 360, 213-227.
Barton, C.E., Johnson, K.N., Mays, D.M., Boehnke, K., Shyr, Y., Boukamp, P., and Pietenpol, J.A. (2010). Novel p63 target genes involved in paracrine signaling and keratinocyte differentiation. Cell death & disease 1, e74. Bauer, J., Antoch, M., Chang, C., Schorl, C., Kolli, S., Neretti, N., and Helfand, S.L. (2010). Comparative transcriptional profiling identifies takeout as a gene that regulates life span. Ageing 2, 298-310.
Bell, R., Hubbard, A., Chettier, R., Chen, D., Miller, J.P., Kapahi, P., Tarnopolsky, M., Sahasrabuhde, S., Melov, S., and Hughes, R.E. (2009). A human protein interaction network shows conservation of ageing processes between human and invertebrate species. PLoS genetics 5, e1000414.
Benyshek, D.C., Johnston, C.S., and Martin, J.F. (2006). Glucose metabolism is altered in the adequately-nourished grand-offspring (F3 generation) of rats malnourished during gestation and perinatal life. Diabetologia 49, 1117-1119.
Bodai, L., Pardi, N., Ujfaludi, Z., Bereczki, O., Komonyi, O., Balint, E., and Boros, I.M. (2007). Daxx-like protein of Drosophila interacts with Dmp53 and affects longevity and Ark mRNA level. The Journal of biological chemistry 282, 36386-36393.
Brenkman, A.B., and Burgering, B.M. (2003). FoxO3a eggs on fertility and ageing. Trends in molecular medicine 9, 464-467.
Budovsky, A., Abramovich, A., Cohen, R., Chalifa-Caspi, V., and Fraifeld, V. (2007). Longevity network: construction and implications. Mechanisms of ageing and development 128, 117-124.
Carone, B.R., Fauquier, L., Habib, N., Shea, J.M., Hart, C.E., Li, R., Bock, C., Li, C., Gu, H., Zamore, P.D., et al. (2010). Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals. Cell 143, 1084-1096.
Castrillo, J.I., Zeef, L.A., Hoyle, D.C., Zhang, N., Hayes, A., Gardner, D.C., Cornell, M.J., Petty, J., Hakes, L., Wardleworth, L., et al. (2007). Growth control of the eukaryote cell: a systems biology study in yeast. Journal of biology 6, 4.
Cheng, C., Fabrizio, P., Ge, H., Longo, V.D., and Li, L.M. (2007). Inference of transcription modification in long-live yeast strains from their expression profiles. BMC genomics 8, 219.
Curtis, R., O'Connor, G., and DiStefano, P.S. (2006). Ageing networks in Caenorhabditis elegans: AMP-activated protein kinase (aak-2) links multiple ageing and metabolism pathways. Ageing cell 5, 119-126. de Magalhaes, J.P., and Toussaint, O. (2004). GenAge: a genomic and proteomic network map of human ageing. FEBS letters 571, 243-247.
Ellisen, L.W., Ramsayer, K.D., Johannessen, C.M., Yang, A., Beppu, H., Minda, K., Oliner, J.D., McKeon, F., and Haber, D.A. (2002). REDD1, a developmentally regulated transcriptional target of p63 and p53, links p63 to regulation of reactive oxygen species. Molecular cell 10, 995-1005. Ewen-Campen, B., Schwager, E.E., and Extavour, C.G. (2010). The molecular machinery of germ line specification. Molecular reproduction and development 77, 3-18.
Fabrizio, P., Battistella, L., Vardavas, R., Gattazzo, C., Liou, L.L., Diaspro, A., Dossen, J.W., Gralla, E.B., and Longo, V.D. (2004). Superoxide is a mediator of an altruistic ageing program in Saccharomyces cerevisiae. The Journal of cell biology 166, 1055-1067.
Flintoft, L. (2011). Epigenetics: Dad's diet lives on. Nature reviews Genetics 12, 80.
Goertz, M.J., Wu, Z., Gallardo, T.D., Hamra, F.K., and Castrillon, D.H. (2011). Foxo1 is required in mouse spermatogonial stem cells for their maintenance and the initiation of spermatogenesis. The Journal of clinical investigation 121, 3456-3466.
Greer, E.L., Maures, T.J., Ucar, D., Hauswirth, A.G., Mancini, E., Lim, J.P., Benayoun, B.A., Shi, Y., and Brunet, A. (2011). Transgenerational epigenetic inheritance of longevity in Caenorhabditis elegans. Nature.
Hong, S.E., Heo, H.S., Kim, D.H., Kim, M.S., Kim, C.H., Lee, J., Yoo, M.A., Yu, B.P., Leeuwenburgh, C., and Chung, H.Y. (2010). Revealing system-level correlations between ageing and calorie restriction using a mouse transcriptome. Age (Dordr) 32, 15-30.
Honjoh, S., Yamamoto, T., Uno, M., and Nishida, E. (2009). Signalling through RHEB-1 mediates intermittent fasting-induced longevity in C. elegans. Nature 457, 726-730.
Howard, R.M., and Sundaram, M.V. (2002). C. elegans EOR-1/PLZF and EOR-2 positively regulate Ras and Wnt signaling and function redundantly with LIN-25 and the SUR-2 Mediator component. Genes & development 16, 1815-1827.
Hur, J.H., and Walker, D.W. (2009). p53, sex, and ageing: lessons from the fruit fly. Ageing 1, 881-883.
Kaati, G., Bygren, L.O., Pembrey, M., and Sjostrom, M. (2007). Transgenerational response to nutrition, early life circumstances and longevity. European journal of human genetics : EJHG 15, 784-790.
Kahana, S., Pnueli, L., Kainth, P., Sassi, H.E., Andrews, B., and Kassir, Y. (2010). Functional dissection of IME1 transcription using quantitative promoter-reporter screening. Genetics 186, 829-841.
Klutstein, M., Siegfried, Z., Gispan, A., Farkash-Amar, S., Zinman, G., Bar-Joseph, Z., Simchen, G., and Simon, I. (2010). Combination of genomic approaches with functional genetic experiments reveals two modes of repression of yeast middle-phase meiosis genes. BMC genomics 11, 478.
Kurdistani, S.K., Tavazoie, S., and Grunstein, M. (2004). Mapping global histone acetylation patterns to gene expression. Cell 117, 721-733.
Lee, J.H., Lee, E., Park, J., Kim, E., Kim, J., and Chung, J. (2003). In vivo p53 function is indispensable for DNA damage-induced apoptotic signaling in Drosophila. FEBS letters 550, 5-10.
Lee, S.J., Murphy, C.T., and Kenyon, C. (2009). Glucose shortens the life span of C. elegans by downregulating DAF-16/FOXO activity and aquaporin gene expression. Cell metabolism 10, 379-391.
Lee, Y.L., and Lee, C.K. (2008). Transcriptional response according to strength of calorie restriction in Saccharomyces cerevisiae. Mol Cells 26, 299-307.
Li, Y.H., Dong, M.Q., and Guo, Z. (2010). Systematic analysis and prediction of longevity genes in Caenorhabditis elegans. Mechanisms of ageing and development.
Lin, S.J., Kaeberlein, M., Andalis, A.A., Sturtz, L.A., Defossez, P.A., Culotta, V.C., Fink, G.R., and Guarente, L. (2002). Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration. Nature 418, 344-348.
Lin, Z., and Li, W.H. (2011). Evolution of 5' Untranslated Region Length and Gene Expression Reprogramming in Yeasts. Molecular biology and evolution.
Longo, V.D., Mitteldorf, J., and Skulachev, V.P. (2005). Programmed and altruistic ageing. Nature reviews Genetics 6, 866-872.
Lorenz, D.R., Cantor, C.R., and Collins, J.J. (2009). A network biology approach to ageing in yeast. Proceedings of the National Academy of Sciences of the United States of America 106, 1145-1150.
Lu, W.J., Chapo, J., Roig, I., and Abrams, J.M. (2010). Meiotic recombination provokes functional activation of the p53 regulatory network. Science 328, 1278-1281.
Lunardi, A., Di Minin, G., Provero, P., Dal Ferro, M., Carotti, M., Del Sal, G., and Collavin, L. (2010). A genome-scale protein interaction profile of Drosophila p53 uncovers additional nodes of the human p53 network. Proceedings of the National Academy of Sciences of the United States of America 107, 6322-6327.
Maier, B., Gluba, W., Bernier, B., Turner, T., Mohammad, K., Guise, T., Sutherland, A., Thorner, M., and Scrable, H. (2004). Modulation of mammalian life span by the short isoform of p53. Genes & development 18, 306-319.
Mair, W., Goymer, P., Pletcher, S.D., and Partridge, L. (2003). Demography of dietary restriction and death in Drosophila. Science 301, 1731-1733.
Majo, F., Rochat, A., Nicolas, M., Jaoude, G.A., and Barrandon, Y. (2008). Oligopotent stem cells are distributed throughout the mammalian ocular surface. Nature 456, 250-254.
Merry, B.J., Kirk, A.J., and Goyns, M.H. (2008). Dietary lipoic acid supplementation can mimic or block the effect of dietary restriction on life span. Mechanisms of ageing and development 129, 341-348.
Mock, D.M. (1999). Biotin status: which are valid indicators and how do we know? The Journal of nutrition 129, 498S-503S.
Natsume-Kitatani, Y., Shiga, M., and Mamitsuka, H. (2011). Genome-wide integration on transcription factors, histone acetylation and gene expression reveals genes co-regulated by histone modification patterns. PloS one 6, e22281.
Needleman, S.B., and Wunsch, C.D. (1970). A general method applicable to the search for similarities in the amino acid sequence of two proteins. Journal of molecular biology 48, 443-453.
Ng, S.F., Lin, R.C., Laybutt, D.R., Barres, R., Owens, J.A., and Morris, M.J. (2010). Chronic high-fat diet in fathers programs beta-cell dysfunction in female rat offspring. Nature 467, 963-966.
Niu, W., Lu, Z.J., Zhong, M., Sarov, M., Murray, J.I., Brdlik, C.M., Janette, J., Chen, C., Alves, P., Preston, E., et al. (2011). Diverse transcription factor binding features revealed by genome-wide ChIP-seq in C. elegans. Genome research 21, 245-254.
Ortt, K., and Sinha, S. (2006). Derivation of the consensus DNA-binding sequence for p63 reveals unique requirements that are distinct from p53. FEBS letters 580, 4544-4550.
Ouyang, Y., Song, Y., and Lu, B. (2011). dp53 Restrains Ectopic Neural Stem Cell Formation in the Drosophila Brain in a Non-Apoptotic Mechanism Involving Archipelago and Cyclin E. PloS one 6, e28098.
Perez, C.A., Ott, J., Mays, D.J., and Pietenpol, J.A. (2007). p63 consensus DNA-binding site: identification, analysis and application into a p63MH algorithm. Oncogene 26, 7363-7370.
Pinkston, J.M., Garigan, D., Hansen, M., and Kenyon, C. (2006). Mutations that increase the life span of C. elegans inhibit tumor growth. Science 313, 971-975.
Pokholok, D.K., Harbison, C.T., Levine, S., Cole, M., Hannett, N.M., Lee, T.I., Bell, G.W., Walker, K., Rolfe, P.A., Herbolsheimer, E., et al. (2005). Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122, 517-527.
Pozzi, S., Boergesen, M., Sinha, S., Mandrup, S., and Mantovani, R. (2009). Peroxisome proliferator-activated receptor-alpha is a functional target of p63 in adult human keratinocytes. The Journal of investigative dermatology 129, 2376-2385.
Promislow, D.E. (2004). Protein networks, pleiotropy and the evolution of senescence. Proceedings Biological sciences / The Royal Society 271, 1225-1234.
Sanchez-Roman, I., Gomez, A., Gomez, J., Suarez, H., Sanchez, C., Naudi, A., Ayala, V., Portero-Otin, M., Lopez-Torres, M., Pamplona, R., et al. (2011). Forty percent methionine restriction lowers DNA methylation, complex I ROS generation, and oxidative damage to mtDNA and mitochondrial proteins in rat heart. Journal of bioenergetics and biomembranes.
Sbisa, E., Catalano, D., Grillo, G., Licciulli, F., Turi, A., Liuni, S., Pesole, G., De Grassi, A., Caratozzolo, M.F., D'Erchia, A.M., et al. (2007). p53FamTaG: a database resource of human p53, p63 and p73 direct target genes combining in silico prediction and microarray data. BMC bioinformatics 8 Suppl 1, S20.
Schuster, E., McElwee, J.J., Tullet, J.M., Doonan, R., Matthijssens, F., Reece-Hoyes, J.S., Hope, I.A., Vanfleteren, J.R., Thornton, J.M., and Gems, D. (2010). DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO. Molecular systems biology 6, 399.
Schwartz, Y.B., Kahn, T.G., Nix, D.A., Li, X.Y., Bourgon, R., Biggin, M., and Pirrotta, V. (2006). Genome-wide analysis of Polycomb targets in Drosophila melanogaster. Nature genetics 38, 700-705.
Senoo, M., Pinto, F., Crum, C.P., and McKeon, F. (2007). p63 Is essential for the proliferative potential of stem cells in stratified epithelia. Cell 129, 523-536.
Seong, K.H., Li, D., Shimizu, H., Nakamura, R., and Ishii, S. (2011). Inheritance of Stress-Induced, ATF-2-Dependent Epigenetic Change. Cell 145, 1049-1061.
Shmookler Reis, R.J., Bharill, P., Tazearslan, C., and Ayyadevara, S. (2009). Extreme-longevity mutations orchestrate silencing of multiple signaling pathways. Biochimica et biophysica acta 1790, 1075-1083.
Smith, E.D., Tsuchiya, M., Fox, L.A., Dang, N., Hu, D., Kerr, E.O., Johnston, E.D., Tchao, B.N., Pak, D.N., Welton, K.L., et al. (2008). Quantitative evidence for conserved longevity pathways between divergent eukaryotic species. Genome research 18, 564-570.
Smith, E.M., Hoi, J.T., Eissenberg, J.C., Shoemaker, J.D., Neckameyer, W.S., Ilvarsonn, A.M., Harshman, L.G., Schlegel, V.L., and Zempleni, J. (2007). Feeding Drosophila a biotin-deficient diet for multiple generations increases stress resistance and lifespan and alters gene expression and histone biotinylation patterns. The Journal of nutrition 137, 2006-2012.
Smith, T.F., and Waterman, M.S. (1981). Identification of common molecular subsequences. Journal of molecular biology 147, 195-197.
Somel, M., Liu, X., Tang, L., Yan, Z., Hu, H., Guo, S., Jiang, X., Zhang, X., Xu, G., Xie, G., et al. (2011). MicroRNA-Driven Developmental Remodeling in the Brain Distinguishes Humans from Other Primates. PLoS biology 9, e1001214.
Swindell, W.R. (2008a). Comparative analysis of microarray data identifies common responses to caloric restriction among mouse tissues. Mechanisms of ageing and development 129, 138-153.
Swindell, W.R. (2008b). Genes regulated by caloric restriction have unique roles within transcriptional networks. Mechanisms of ageing and development 129, 580-592.
Swindell, W.R. (2009). Genes and gene expression modules associated with caloric restriction and ageing in the laboratory mouse. BMC genomics 10, 585. Unal, E., Kinde, B., and Amon, A. (2011). Gametogenesis eliminates age-induced cellular damage and resets life span in yeast. Science 332, 1554-1557.
Vigano, M.A., Lamartine, J., Testoni, B., Merico, D., Alotto, D., Castagnoli, C., Robert, A., Candi, E., Melino, G., Gidrol, X., et al. (2006). New p63 targets in keratinocytes identified by a genome-wide approach. The EMBO journal 25, 5105-5116. Wang, J., Zhang, S., Wang, Y., Chen, L., and Zhang, X.S. (2009). Disease-ageing network reveals significant roles of ageing genes in connecting genetic diseases. PLoS Comput Biol 5, e1000521.
Werner, H. (2011). Tumor suppressors govern insulin-like growth factor signaling pathways: implications in metabolism and cancer. Oncogene.
Witten, T.M., and Bonchev, D. (2007). Predicting ageing/longevity-related genes in the nematode Caenorhabditis elegans. Chemistry & biodiversity 4, 2639-2655.
Xu, F., Zhang, K., and Grunstein, M. (2005). Acetylation in histone H3 globular domain regulates gene expression in yeast. Cell 121, 375-385.
Yamaza, H., Komatsu, T., Wakita, S., Kijogi, C., Park, S., Hayashi, H., Chiba, T., Mori, R., Furuyama, T., Mori, N., et al. (2010). FoxO1 is involved in the antineoplastic effect of calorie restriction. Ageing cell 9, 372-382.
Zid, B.M., Rogers, A.N., Katewa, S.D., Vargas, M.A., Kolipinski, M.C., Lu, T.A., Benzer, S., and Kapahi, P. (2009). 4E-BP extends lifespan upon dietary restriction by enhancing mitochondrial activity in Drosophila. Cell 139, 149-160.
Kenyon CJ. The genetics of ageing. Nature. 2010; 464(7288):504–512.
Paik JH, et al. FoxOs are lineage-restricted redundant tumor suppressors and regulate endothelial cell homeostasis. Cell. 2007;128(2):309–323.
Tothova Z, et al. FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress. Cell. 2007;128(2):325–339.
Renault VM, et al. FoxO3 regulates neural stem cell homeostasis. Cell Stem Cell. 2009;5(5):527–539.
Paik JH, et al. FoxOs cooperatively regulate diverse pathways governing neural stem cell homeostasis. Cell Stem Cell. 2009;5(5):540–553.
John GB, Gallardo TD, Shirley LJ, Castrillon DH. Foxo3 is a PI3K-dependent molecular switch controlling the initiation of oocyte growth. Dev Biol. 2008;321(1):197–204.
Castrillon DH, Miao L, Kollipara R, Horner JW, DePinho RA. Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science. 2003;301(5630):215–218.
John GB, Shirley LJ, Gallardo TD, Castrillon DH. Specificity of the requirement for Foxo3 in primordial follicle activation. Reproduction. 2007;133(5):855–863.
Buaas FW, et al. Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet. 2004;36(6):647–652.
Falender AE, et al. Maintenance of spermatogenesis requires TAF4b, a gonad-specific subunit of TFIID. Genes Dev. 2005;19(7):794–803.
Lehmann, O.J. et al. (2003) Fox’s in development and disease. Trends Genet. 19, 339 – 344
Castrillon, D.H. et al. (2003) Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science 301, 215 – 218
Kramer, J.M. et al. (2003) Expression of drosophila FOXO regulates growth and can phenocopy starvation. BMC Dev. Biol. 3, 5
Kops, G.J. et al. (2002) Control of cell cycle exit and entry by protein kinase B-regulated forkhead transcription factors. Mol. Cell. Biol. 22, 2025– 2036
Bois, P.R. and Grosveld, G.C. (2003) FKHR (FOXO1a) is required for myotube fusion of primary mouse myoblasts. EMBO J. 22, 1147 – 1157
Zhang, T., Xiong, H., Kan, L.X., Zhang, C.K., Jiao, X.F., Fu, G., Zhang, Q.H., Lu, L., Tong, J.H., Gu, B.W., et al. 1999. Genomic sequence, structural organization, molecular evolution, and aberrant rearrangement of promyelocytic leukemia zinc finger gene. Proc. Natl. Acad. Sci. 96: 11422–11427.
Barna, M., Hawe, N., Niswander, L., and Pandolfi, P.P. 2000. Plzf regulates limb and axial skeletal patterning. Nat. Genet. 25: 166–172.
Thomas, J.H. and Horvitz, H.R. 1999. The C. elegans gene lin-36 acts cell autonomously in the lin-35 Rb pathway. Development 126: 3449–3459
Walhout, A.J., Sordella, R., Lu, X., Hartley, J.L., Temple, G.F., Brasch, M.A., Thierry-Mieg, N., and Vidal, M. 2000. Protein interaction mapping in C. elegans using proteins involved invulval development. Science 287: 116–122.
Chen, Z. and Han, M. 2001. C. elegans Rb, NuRD, and Ras regulate lin-39-mediated cell fusion during vulval fate specification. Curr. Biol. 11: 1874–1879.
Number of genes reported in different model organisms to be essential for DR and their orthologs identified by the use of Ensembl, HomoloGene, InParanoid and OrthoMCL. Species Genes Genes+Orthologs Fission yeast 6 54 Budding yeast 70 170 Nematode 42 137 Fruit fly 19 187 House mouse 1 218 Norway rat 0 217 Rhesus monkey 0 200 Human 0 226
Vac = Vacuole; r = round; f = fragmented. Mutant GbA p-value Mean / max. AL lifespan, (N) Mean / max. DR lifespan, (N) AL, mutant vs. WT, log-rank p-value DR vs. AL, log-rank p-value Vac AL Vac DR Vac Δ Ratio of mRNA levels (DR/AL) in WT wild-type NA 26.4/47 (51) 31/54 (50) NA 0.002 r r NA NA vps20Δ 0.02 22.07/38 (30) 18.27/34 (30) 0.09 0.052 NA NA NA 1.56 fre6Δ 0.022 30.07/46 (30) 28/42 (30) 0.043 0.173 r f 29 -> 14 0.85 rcr2Δ 0.019 31.27/45 (30) 24.96/45 (25) 0.008 0.048 r f/r 52 -> 18 3.22 ydl180wΔ 0.043 27.67/44 (30) 28.2/46 (25) 0.262 0.596 f f/r NA 2.25 opt2Δ 0.048 32.6/51 (15) 25.43/39 (30) 0.007 0.015 f f NA 0.05 gtr1Δ 0.037 16.93/23 (15) 18.73/29 (15) 4.50E-009 0.208 r f NA 2.77 dap2Δ 0.013 21.27/30 (15) 19.93/32 (15) 0.001 0.969 r f 60 -> 1 1.22 slm4Δ 0.049 23.87/48 (15) 23.4/36 (15) 0.793 0.597 r f 44 -> 16 3.28 yol092wΔ 0.043 20.8/34 (15) 27.4/42 (15) 0.087 0.062 f f NA 0.33
The functional enriched terms for either up- or downregulated interaction associated to the DR-induced gene expression changes of multiple species (budding yeast, nematode, fruit fly) aer listed. Terms associated to stimulated interactions p-value Terms associated to suppressed interactions p-value phosphoprotein 1.55E-51 phosphoprotein 5.26E-97 nucleus 1.72E-46 ribonucleoprotein 5.15E-87 nucleotide-binding 5.61E-42 GO:0030529~ribonucleoprotein complex 1.55E-86 atp-binding 1.48E-39 cytoplasm 7.21E-74 GO:0007049~cell cycle 5.82E-36 nucleus 1.54E-45 kinase 5.5E-35 nucleotide-binding 2.46E-42 serine/threonine-protein kinase 9.92E-33 ribosome 3.68E-37 GO:0022402~cell cycle process 6.53E-30 rna-binding 7.55E-32 GO:0003006~reproductive developmental process 1.65E-22 GO:0005198~structural molecule activity 3.92E-28 chromatin regulator 5.85E-14 atp-binding 6.94E-27 GO:0016568~chromatin modification 1.35E-13 ATP 9.92E-17 GO:0051276~chromosome organization 3.07E-13 GO:0000166~nucleotide binding 4.7E-09 ATP 1.92E-11 ubl conjugation 9.5E-09 GO:0048610~reproductive cellular process 4.51E-11 repressor 1.33E-08 coiled coil 2.82E-08 GO:0033554~cellular response to stress 3.88E-08 GO:0044257~cellular protein catabolic process 6.82E-08 GO:0010629~negative regulation of gene expression 4.17E-07
The functional enriched terms for either up- or downregulated interactions common the DR-induced gene expression changes in budding yeast, nematode, fruit fly and mammals (meta-analytic signature). Common terms of induced interactions p-value Common terms of suppressed interactions p-value phosphoprotein 7.30E-64 phosphoprotein 2.32E-116 nucleotide-binding 3.51E-50 GO:0043228 ~non-membrane-bounded organelle 2.49E-89 atp-binding 1.31E-45 GO:0043232~intracellular non-membrane-bounded organelle 2.49E-89 active site:Proton acceptor 4.27E-32 cytoplasm 4.85E-84 GO:0003006~reproductive developmental process 8.89E-30 nucleotide-binding 1.14E-54 transcription regulation 1.88E-24 nucleus 6.20E-53 transferase 6.69E-23 GO:0032268~regulation of cellular protein metabolic process 4.92E-46 phosphotransferase 7.58E-17 rna-binding 2.00E-37 GO:0010605~negative regulation of macromolecule metabolic process 2.40E-13 GO:0005198~structural molecule activity 2.73E-37 GO:0010629~negative regulation of gene expression 1.82E-12 atp-binding 3.01E-36 GO:0045934~negative regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process 1.64E-11 ATP 1.10E-23 GO:0051172~negative regulation of nitrogen compound metabolic process 1.73E-11 GO:0000166~nucleotide binding 5.09E-23 GO:0016481~negative regulation of transcription 2.23E-11 kinase 2.32E-18 GO:0010558~negative regulation of macromolecule biosynthetic process 4.77E-11 stress response 5.07E-17 mutagenesis site 1.34E-10 GO:0032555~purine ribonucleotide binding 9.49E-15 GO:0006357~regulation of transcription from RNA polymerase II promoter 1.78E-10 GO:0032553~ribonucleotide binding 9.49E-15 ubl conjugation 2.70E-06 GO:0017076~purine nucleotide binding 1.38E-13 NA NA binding site:ATP 1.50E-12 NA NA nucleotide phosphate-binding region:ATP 3.01E-12 NA NA phosphotransferase 3.94E-12 NA NA GO:0031981~nuclear lumen 1.74E-10
The binding site of meiotic transcription factors are significantly enriched in the promoter region of DR-differential expressed genes. Factor Motif Targets #Up #Down Up p-value Down p-value Ume6 TSGGCGGCTAW 56 25 10 6.71106 0.32 URS1 DSGGCGGCND 216 68 31 3.57105 0.83 Ime1 TRGSCGSCKA 78 27 13 0.0008 0.42 MSE HDVKNCACAAAAD 122 35 13 0.0084 0.26 Ndt80 YGNCACAAAA 77 23 10 0.0133 0.76 Rme1 GWACCTCAARA 8 0 5 1 0.00041
The binding sites of meiotic transcription factors are significantly enriched in the promoter region of DR-essential genes. Factor Motif p-value URS1 GGCGGC 0.0009 Ume6 SGCGGYWV 0.002 Sum1 GYGWCASWAAW 0.009 Ndt80 YGNCACAAAA 0.04 Ime1 TRGSCGSCKA 0.04
The binding sites of transcription factors involved in reproductive development are significantly enriched in the promoter regions of DR-upregulated genes in mammals. Factor Motif # All motif # Delta motif # Up # Down Up p-value Down p-value Delta p-value Foxo3 TTGTTTAC 1182 58 26 23 0.006 0.300 0.005 Zbtb16 TACTGTAC 548 27 14 8 0.007 0.632 0.028 Ppara AGGTCAWAGGTCA 13 1 1 0 0.012 1 0.071 Nr5a1 YCAAGGYC 6006 268 100 135 0.030 0.178 0.110 FOXA1 TGTTTGC 4271 169 78 77 0.0001 0.211 0.002
The binding sites of transcription factors involved in reproductive development are significantly enriched in the promoter regions of mammalian DR-essential gene orthologs. Factor Motif # All motif # Delta motif p-value TP63 RRRCWYGYYY 7652 67 0.003 FOXA1 TGTTTGC 4271 37 0.007 HSF1 ATGGAABD 5807 49 0.008 FOXO3 TTGTTTAC 768 9 0.010 ZBTB16 TACTGTAC 351 4 0.042
Motifs corresponding to transcription factors were used to scan the genome for the presence within the 500 bp upstream regions of DR-suppressed genes. Motif Factor # Genes # Delta Delta p # Up Up p # Down Down p ATGTGAAAT INO4 INO2 2 2 0 1 0 1 0 GGCGAGATCT SKN7 2 1 0 0 1 1 0 RMMAWSTGKSGYGSC MET4 1 1 0 0 1 1 0 CCGTTAACGG LEU3 1 1 0 0 1 1 0 YYYATTGTTCTC ROX1 2 2 0 0 1 2 0 TYTTCACATGY INO4 2 1 0 0 1 1 0 KTTSAAYKGTTYASA IXR1 1 1 0 0 1 1 0 CGGNNNTANCGG HAP1 1 1 0 0 1 1 0 TTTGCN{97}GCAAA MSS11 FLO8 1 1 0 0 1 1 0 GAATGGCTG CRZ1 2 1 0 0 1 1 0 CGGN{3}TANCGG HAP1 1 1 0 0 1 1 0 CCGGNNCCGG LEU3 2 2 0 0 1 2 0 AAAAWTTTT SFP1 101 46 0.414097 10 0.999853 36 0.000108 GGCCGGC SKN7 10 7 0.024195 1 0.737021 6 0.000884 CAGCGTG HAC1 7 5 0.02925 1 0.533902 4 0.004738 TTGCGTGA GCN4 7 5 0.02925 1 0.533902 4 0.004738 TCCGCGCA PDR1 PDR3 5 3 0.10673 0 1 3 0.006486 AYCCRTACAY SFP1 16 10 0.043084 3 0.565801 7 0.007214 TCGTATA ECM22 UPC2 43 25 0.025538 10 0.481214 15 0.007331 CACCTCTA ARG81 11 6 0.161187 1 0.785572 5 0.011904 TCCGCGGG PDR1 PDR3 6 3 0.235375 0 1 3 0.017131 TGACTAA YAP1 32 17 0.121483 6 0.689786 11 0.017653 NCCDTYNVNCCGN SIP4 CAT8 9 6 0.044544 2 0.375955 4 0.019927 WWWTTTGCTCR MAC1 9 5 0.153936 1 0.679395 4 0.019927 AAAAGAAA AZF1 178 93 0.030026 46 0.345985 47 0.025333 TCGGCGGCTD URS1 4 2 0.193021 0 1 2 0.025866 TCGGCGGCTDW URS1 4 2 0.193021 0 1 2 0.025866 GTMCGGGTAA REB1 4 2 0.193021 0 1 2 0.025866 TCGGCGGCT URS1 4 2 0.193021 0 1 2 0.025866 CCGN{4}CGG LEU3 35 19 0.090106 8 0.485888 11 0.036324 CGGCTC STP1 STP2 39 23 0.023767 11 0.219962 12 0.037791
