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================================================ Comparative Interactomics of Dietary Restriction ================================================
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. ''')#Mention candidates tested, comparative interactomics and identified transcription factors.
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. signaling 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 likelyhoods 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 analyses 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:
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-positive. 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.
Cynthia Kenyon come up with the second criteria. 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 (http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.0010017).
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 1A). 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 1B). 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.
Our network analyses revealed that DR-essential genes interact more frequently with each other.
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. The top ten candidates of a more up-to-date assembled interactome for each species are given in Table S4.
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 the were always topranked. 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 are listed in Tab. 1. 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 (Fig. 1). 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% (Fig. 1).
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 suprising 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.
Paragraph('''As changes in gene activity of DR-essential genes via genetic manipulations mimic and/or abolishes 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 < 0.05) for a broad spectrum of cut-offs (1.1 to 1.2, 3.0 to 3.2, 3.5 as well as 5.1 to 24.7). Similar in worm, DR-essential genes were significant more likely to be differentially expressed than expected by chance from 2.5 to 25.6-fold change. In fly, the likelihood was more moderate as DR-essential genes are more likely to be differential expressed from 1.5 to 1.9-fold change cut-offs for transcript level and only at 1.1-fold threshold for protein translation.
In mammals DR-essential gene orthologs are more likely to be differentially expressed upon DR than expected by chance (30 of the 215 DR-essential orthologs were among the 1348 DR-differential expressed genes; hypergeometric p-value < 10-5) in the meta-analytic signatures of DR (Hong et al., 2010; Swindell, 2008a, b, 2009). Even if only restricted to our newly derived DR signature (Plank et al., in preparation), DR-essential genes (one out of one among 174 DR-differentially expressed genes; p-value = 0.008) and orthologs (5 of 174 DR-essential genes among 174 DR-differentially expressed genes; p-value = 0.02) are more likely to be differentially expressed than expected randomly.
'While the lack of correlation between lifespan and expression responses to DR by DR-essential genes at the genome-scale seems contradictory to the example of vacuolar DR-essential genes, post-transcriptional regulations of DR-essential genes triggered by DR treatment and noises caused by the combined analyses of multiple sets of microarray data may mask the regulatory effects of DR at the transcriptional level and made our analyses complicated. Alternatively, it might be simple due to the complication that there are different ageing paradigms in yeast and also many different versions of DR.
OPT2 is differential in numerous intervention which impact on lifespan (Aghajan et al., 2010; Chattopadhyay et al., 2009; Gonzalez et al., 2009; Lin et al., 2002; Reinke et al., 2006).
We noted that in yeast spermidine treatment causes differential expression of DR-essential genes much more likely than expected by chance (hypergeometric p-value < 0.05) on a whole spectrum of fold-changes from 1.6 ways up to 2.9 (for example at two-fold there were 15 of the 70 DR-essential genes among the 727 spermidine-differentially expressed genes; hypergeometric p-value = 0.003). This is quite specific as it was not significant for any fold-change by treatment with another polyamine, namely spermine which was not yet shown to affect lifespan (Supplemental Table 10).
Astonishing DR and spermidine signatures regulate the same genes (far more likely than expected by chance) above any cut-off over 1.7-fold (for instance at two-fold there were 323 of 727 spermidine-differentially expressed genes among the 2560 DR-differentially expressed genes; hypergeometric p value = 0.0004), whereas DR and spermine did not regulate the same genes than expected by chance at any cut-off at all (Supplemental Table 11).
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 (Table: Induced and suppressed interactions common to yeast, worm, fly and mammals; 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 down-regulated (the default is set to 1.0). Among the terms identified to be consistent significantly (p-value < 0.05) associated to the induced interactions in yeast, worm, fly and mammals were phosphorylation-related terms and reproductive developmental process [Table 4].
The DR-essential genes networks in S. cereviasae and C. elegans were enriched for sporulation/meiosis and meiosis respectively, although not significant the latter. Reproduction-associated genes such as mes-4 are also a significant interaction partners of DR-essential genes (total = 17; specific = 4; specificity = 0.235294118; p-value = 9.87E-05).
In C. elegans induced meiotic related kinases were air-2 (Aurora/Ipl1 Related kinase), kgb-1 (Kinase, GLH-Binding), mek-1 (MAP kinase kinase or Erk Kinase), plk-1 (POLO Kinase), zen-4 (Zygotic epidermal Enclosure defective).
In fruit fly, reproduction associated induced genes were, for instance Axn (Axin), CDC2, cycE (Cyclin E), TOP1 (Topoisomerase 1) and dap (dacapo) as well as CDC2 (Cell division control protein 2 homolog), Ikb1 (DmeI_CG9374), baz (bazooka), koko (kokopelli), put (punt), sax (saxophone).
dDnmt2 (MT2), its interaction partner Ipod as well as psq and 3 isoforms of lola were translational upregulated upon DR. Rbf is downregulated on the transcriptional level.
There was even an enrichment for stem cell division (Benjamini p-value = 7.0*10-3) in the DR-induced nuclear kinases of Drosophila.
Even mammals induce expression of nuclear kinases which were enriched for response to DNA damage stimulus (Benjamini p-value = 2.4*10-2): Csnk1e (casein kinase 1, epsilon), Cdkn1a (p21), Foxo3h, Gtf2h1 (p62, which is related to autophagy) and Msh2 (mutS homolog 2).
DR induces the expression of genes annotated with embryogenesis (23 in mice); however it did not reached significance.
In the mammalian interaction network of DR-differentially expressed genes the most upregulated term was acetylation (p-value = 3.910-15,; Benajmini p-value = 8.0410-13). Reproductive developmental processes was among the terms of the induced interactions in mammals (p-value = 0.037; Benajmini p-value = 0.48). Among them were Cited2, Pten, Msh2, Dld (dihydrolipoamide dehydrogenase), Nrip1 (nuclear receptor interacting protein 1) and Hsd17b4 (hydroxysteroid (17-beta) dehydrogenase 4). To mention are two DR-induced transcription factors associated to reproductive development: ZBTB16 and FOXO3 (a DR-essential ortholog).
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%) (Supplemental table 22).
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) (Supplemental table 23-25). 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 down-regulated gene promoters (Table 6). Interestingly, URS1 as well as motifs of Ume6, Sum1, Ndt80 and Ime1 are even enriched in the upstream sequences of DR-essential genes (Supplemental table 25). 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 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. 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.
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 = 7.9*10-5) (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 signaling 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 5).
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 organisation 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 6).
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].
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 = 4.710 3) with for instance Oct4, Dnmt3l, Cctc, and Pparg.
TP63 is the founding ancestor of the p53-family members and it displays homology to the rejuvenation transcription factor Ndt80 (Figure 3). Ndt80 and TAp63? are to 21% (75/706) identical and to 32% (147/706) similar on the global level (Table 7), while exhibiting 23% (139/615) identity and 40% (243/615) similarity in a local alignment (Table 8).
Subsection("Proteins and Chromatin Marks Associated to DR-Differentially Genes") 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 overrepresentation 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 (Table S25).
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 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. down-regulated genes by DR in the opposite manner, while enriched or depleted in the same direction by the comparison differential vs. others (Supplemental table 26B). For instance, the differentially expressed genes (both up- and down-regulated 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).
Does DR Up- or Down-regulate Fatty Acid Elongases? ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 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.
Does DR and Spermidine Treatment Upregulate Lipid Catabolism? ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 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 4), 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 up-regulated 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 (Table 22) which may sequester TORC1 (Brown et al., 2010) away from its normal functioning membranes (i.e. vacuolar and endosome membrane, etc.).
Taken together this indicates that an altered chromatin state in DR is the principle major component for its lifespan extending mechanism across species boundaries.
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 (not published yet, but a fascinating convergence!).
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 ?-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 ?-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?
A common theme of longevity mutants is the silencing of multiple signaling 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.
Reproductive active females have a reduced lifespan [20 in (Brenkman and Burgering, 2003)]. For instance in Drosophila, high rates of egg production result in a decreased lifespan [21 in (Brenkman and Burgering, 2003)]. However, implanting young ovary in post-reproductive female mice extends lifespan.
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 [12 in (Goertz et al., 2011)].
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 [13-15 in (Goertz et al., 2011)]. Foxos, particular Foxo3 co-ordinately regulates neural stem cell homeostasis through genes influencing stress response and oxygen metabolism [15, 39 in (Goertz et al., 2011)]. 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 [18 in (Goertz et al., 2011)]. Foxo3 function in female germ line maintenance [16-18 in (Goertz et al., 2011)], 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 [21,22 in (Goertz et al., 2011)].
Forkhead transcription factors control crucial steps in embryongenesis and are essential for the development of all germ layers and organs [reviewed in 1 in (Brenkman and Burgering, 2003)]. Foxo3-/- mice exhibit signs of accelerated ageing and have global follicular activation, therefore become premature infertile too [2 in (Brenkman and Burgering, 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 [12 in (Brenkman and Burgering, 2003)] and in mammalian cells [11 in (Brenkman and Burgering, 2003)]. 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 [15] and p130/Rb2 [16], both of which are also required for muscle differentiation [17 in (Brenkman and Burgering, 2003)].
Unique genetic requirement for FOxo1 in males and Foxo3 in females mirrors their high expression at discrete cellular stages in spermatogenesis or oogenesis.
How do other well-known SSC maintenance and differentiation factors such as Zbtb16 and retoinic acid interact with PI3K-FOxo?
Retinoic acid stimulates meiosis . #http://www.answers.com/topic/meiosis
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 [14,15,39 in (Goertz et al., 2011)].
The single FOxo gene in Hydra is expressed at high levels [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0011686].
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].
TP63 is the founding ancestor of the p53/p63/p73-family members and it displays homology to the rejuvenation transcription factor Ndt80 (Figure 7). Yeast NDT80 encodes a single gene product, C. elegans CEP-1 has two isoforms, D. melanogaster Dmp53 encodes three isoforms. In mammals, there are three different paralogs p53, p63 and p73 with p63 beeing the ancestor. p63 has two different transcriptional initiation sites generating proteins containing (TA) or lacing (DeltaN) an acidic transactivation domain. Alternative C-terminal splicing createds three splice variants (alpha,Beta or Gamma). These results in a minimum of six p63 isoforms at various levels of relative expression.
The process of meiotic recombination instigates programmed activation of Dmp53 in the germ line. Double-stranded breaks in DNA generated by the topoisomerase Spo11 provoked functional Dmp53 activity. Given the function of Dmp53 in meiosis it suggests that tumor-suppressive fucntions may have been co-opted form primordial activities linked to recombination.
p53/p63/p73-family members coordinate stress responses and promote genome stability [8-12 in (Lu et al., 2010)].
Organisms lacking NDT80 and Dmp53 are viable and fertile. Mice lacking all p63 isoforms die at birth and exhibit severe developmental abnormalities.
P53-family members in fly and mammals are like NDT80 transiently induced during gametogenesis. Appeared to peak between the leptonene and dzygotene stages and disappears after early pachytene stage. A defining step in sexual reproduction, meiosis, signals a programmed burst of p53-family activation. Activity is stimulated by the action of Spo11 in Drosophila females (rembination does not occur in males) and in mice. Activaiton in flies reaures jinase chk2 (which also has roles tater in oogenesis [22 in (Lu et al., 2010)].
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. down-regulated genes by DR in the opposite manner, while enriched or depleted in the same direction by the comparison differential vs. others.
Loss-of-function mutations in ZBTB16 also called promyelocytic leukemia zinc finger protein (PLZF) cause an age-dependent loss of germline stem cells leading to sterility. ZBTB16 is expressed in germline 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. Howerver, eor-1 is the C. elegans ortholog of mammalian ZBTB16 [Zhang et al., 1999 in (Howard and Sundaram, 2002)]. ZBTB16 does influence developmental patterning and Hox gene expression in mouse [Barna et al. 2000 in (Howard and Sundaram, 2002)].
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 muscel formation in ageing adults and delays accumulation of lipofuscin-enriched ageing pigments within intestinal cells. EGS 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 in (Howard and Sundaram, 2002) as well as (Howard and Sundaram, 2002). SynMuc gene products could antagonize Ras signalling in part by binding to and inhibiting activity of positively acting transcrpitonal regulators such as EOR-1. LIN-36 EOR1 interaction could also be involved in positive role of some SynMuv N genes [Chen and Han 2001 in (Howard and Sundaram, 2002)]. 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.
Hit me until I understand it. Hit me until I can see any sense in it.
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 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, up-regulated genes, down-regulated genes and all other were collected. Then the average values (actually the lists which give raise to these numbers) of them were compared with binomial test in order to find which marks are enriched in up vs. down and delta vs. other.
Protein sequences were retrieved from UniProt, Muscle was used for creating a multiple sequence alignment which was utilized for Jalview and alignment coloured with Clustalx. The global and local alignments between two amino acid sequences were determined with the Needleman-Wunsch (Needleman and Wunsch, 1970) and Smith-Waterman (Smith and Waterman, 1981) algorithm, respectively.
Blast Zbtb16 and make a multiple alignment, maybe with Ndt80. Compare recognition sequences. Zbtb16 is homologus to Zap1 and Azf1.
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 (Plank et al., unpublished).
Further we thank Robert Shmookler Reis for his useful comments and feedback which significantly strengthened this work. Finally, we thank all of them for their thoughtful manuscript-shaping discussions.
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Table: Number of DR-essential genes and orthologs in GenDR ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 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.
Table: Induced and suppressed interactions common to yeast, worm, fly and mammals
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
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
Table: Induced and suppressed interactions common to yeast, worm, fly and mammals ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 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 binding site:ATP 1.50E-12 nucleotide phosphate-binding region:ATP 3.01E-12 phosphotransferase 3.94E-12 GO:0031981~nuclear lumen 1.74E-10
Table: Meiotic transcription factor binding sites are enriched in the promoters of DR-differential and -essential genes ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ DR-differential genes DR-essential genes Factor Motif Targets #Up #Down Up p-value Down p-value Factor Motif p-value Ume6 TSGGCGGCTAW 56 25 10 6.71106 0.32 URS1 GGCGGC 0.0009 URS1 DSGGCGGCND 216 68 31 3.57105 0.83 Ume6 SGCGGYWV 0.002 Ime1 TRGSCGSCKA 78 27 13 0.0008 0.42 Sum1 GYGWCASWAAW 0.009 MSE HDVKNCACAAAAD 122 35 13 0.0084 0.26 Ndt80 YGNCACAAAA 0.04 Ndt80 YGNCACAAAA 77 23 10 0.0133 0.76 Ime1 TRGSCGSCKA 0.04 Rme1 GWACCTCAARA 8 0 5 1 0.00041
Table: Reproductive development-related factors are enriched in the promoters of the mammalian DR-signature ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 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
Table: Reproductive development-related factors are enriched in the promoters of mammalian DR-essential genes ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 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
Table ~~~~~~ CEP-1 314 247 142 105 0.23047494 0.004765808 CEP-1 318 195 91 104 0.0478943264651 0.413199562179 0.0487762848055 0.0478943264651 0.413199562179 0.0487762848055
Table: Global Alignment (Needlemann-Wunsch) of p63-family members. In violate (left bottom) is percentage identity and in purple (right top) the percentage similarity ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Ndt80 CEP-1a CEP-1b Dmp53a Dmp53b Dmp53g TAp63a TAp63b TAp63g DNp63a DNp63b DNp63g Ndt80 100 20 32 29 32 29 32 31 31 32 31 30 CEP-1a 18 100 31 26 23 26 20 22 24 21 23 26 CEP-1b 24 31 100 30 32 30 31 34 32 35 32 31 Dmp53a 23 22 24 100 77 100 28 34 36 27 37 39 Dmp53b 22 21 22 77 100 77 29 32 37 29 33 33 Dmp53g 23 22 24 100 77 100 28 34 36 27 37 39 TAp63a 21 18 21 22 21 22 100 82 70 86 68 56 TAp63b 23 19 22 24 19 24 82 100 84 68 83 67 TAp63g 24 21 23 25 23 25 70 84 100 56 67 80 DNp63a 22 18 22 20 19 20 86 67 55 100 79 65 DNp63b 24 19 21 23 18 23 67 83 66 79 100 81 DNp63g 24 21 22 24 22 24 55 66 80 65 80 100
Table 8: Local Alignment (Smith-Waterman) of p63-family members. In violate (left bottom) is percentage identity and in purple (right top) the percentage similarity ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Ndt80 CEP-1a CEP-1b Dmp53a Dmp53b Dmp53g TAp63a TAp63b TAp63g DNp63a DNp63b DNp63g Ndt80 100 39 39 40 43 40 40 42 41 40 43 42 CEP-1a 26 100 100 42 44 42 42 42 42 43 43 43 CEP-1b 24 100 100 45 45 45 41 41 42 41 41 41 Dmp53a 24 24 24 100 100 100 46 46 47 46 46 46 Dmp53b 21 24 23 99 100 100 46 46 46 47 47 47 Dmp53g 24 24 24 100 99 100 46 46 47 46 46 46 TAp63a 23 28 23 25 25 25 100 100 98 100 100 97 TAp63b 22 28 23 25 25 25 100 100 98 100 100 97 TAp63g 23 28 24 26 26 26 97 97 100 97 97 100 DNp63a 23 28 22 25 25 25 100 100 96 100 100 97 DNp63b 22 28 22 25 25 25 100 100 96 100 100 97 DNp63g 23 28 23 26 26 26 96 96 100 96 96 100
Figure: Transcriptional regulation of DR in yeast. Transcription factors identified via guilt-by-association to regulate DR-differential genes ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Figure: TP63 and ZBTB16 are significant interaction partners of DR-essential genes and targeted by CDK2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Genes up-and down-regulated upon DR are in red and green, respectively, while genes which were enriched for up and down-regulation are in yellow. DR-essential orthologs are marked by a golden halo.
Figure: Evolutionary conservation of rejuvenation factors ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Multiple sequence alignment of Ndt80 (S. cerevisiae) with the p53/p63/p73-family members CEP-1 (C. elegans), dmp53 (D. melanogaster) and the TP63 isoform TAp63? (Homo sapiens).
Table: Motifs enriched 500bp upstream of the transcriptional start site of the more than two-fold DR-induced genes ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Motif Factor #Genes #Delta Delta p #Up Up p #Down Down p ATGTGAAAT INO4 INO2 2 2 0 1 0 1 0 CGGCN{6}GCCG DAL81 2 1 0 1 0 0 1 YCCNYTNRRCCGN SIP4 CAT8 1 1 0 1 0 0 1 TCTTTTTGCTG CUP2 1 1 0 1 0 0 1 CGGCN{6}CGGC STP1 2 1 0 1 0 0 1 TTWCCYAAWNNGGWAAWW MCM1 1 1 0 1 0 0 1 TGATTAATAATCA ARR1 1 1 0 1 0 0 1 TGACGTCA SKO1 HAC1 CST6 ACA1 1 1 0 1 0 0 1 ATCACGTGA EBOX 3 2 0 2 0 0 1 GACACAAAA NDT80 3 2 0 2 0 0 1 TAGCCGCCGA UME6 3 2 0 2 0 0 1 CACGTGGG PHO4 5 4 0 4 0 0 1 CCCCT MSN4 STRE MSN2 RPH1 NRG1 175 89 0.067041 66 4.64E-05 23 0.994054 CGGN{2,11}CCG PPR1 196 113 0.00026 71 0.000132 42 0.36981 HCCCCTWD GIS1 64 34 0.068491 28 0.000193 6 0.984551 TTAGGG TBF1 59 31 0.087897 26 0.000257 5 0.988025 TWAGGGAT GIS1 16 10 0.043084 9 0.001234 1 0.862321 GAGCCC CRZ1 52 23 0.463913 22 0.001269 1 0.999895 SGCGGYWV UME6 55 38 6.76E-05 23 0.001274 15 0.075273 CGGNNNNNNNNNCGG GSM1 24 16 0.007961 11 0.005412 5 0.351266 CGGN{9}CGG MAL63 24 16 0.007961 11 0.005412 5 0.351266 TCACGTT CBF1 27 18 0.005695 12 0.005789 6 0.294004 CGGN{6}CCG PPR1 27 17 0.016312 12 0.005789 5 0.470209 VKNCRCAAAWD MSE 48 29 0.009179 19 0.00589 10 0.376376 GNCRCAAAW SUM1 NDT80 33 20 0.020485 14 0.006172 6 0.510678 CCYWWWNNRG MCM1 185 87 0.324766 60 0.007622 27 0.980477 CGGN{3}TNRN{8,12}CCG OAF1 PIP2 25 16 0.014704 11 0.008161 5 0.391097 GGCGG URS1 155 82 0.024141 51 0.008171 31 0.530435 TCRTN{5}AYGA ABF1 56 29 0.112105 21 0.009191 8 0.828989 GWCACAAA MSE 20 11 0.118256 9 0.010882 2 0.798756 TRGSCGSCKA IME1 7 4 0.139964 4 0.01095 0 1 GCCGNNNNCGGC LEU3 5 5 0 3 0.012867 2 0.05821 RTCRYNNNNNACGR ABF1 68 33 0.224168 24 0.015089 9 0.908888 CGGN{10}CCG PUT3 CHA4 TEA1 27 16 0.040329 11 0.016802 5 0.470209 RTCRYNNNNNACG ABF1 90 41 0.40689 30 0.02122 11 0.96989 CTGTGGC MET32 MET31 16 10 0.043084 7 0.022255 3 0.40702 CGGADNAWH RGT1 129 67 0.050588 41 0.025359 26 0.490495 RTCRYBN{4}ACG ABF1 78 35 0.448362 26 0.027608 9 0.970191 TGACTGA GCN4 26 12 0.350747 10 0.032127 2 0.919384 YGNCACAAAA NDT80 6 3 0.235375 3 0.033038 0 1 GGCGGC URS1 49 29 0.013755 17 0.035147 12 0.1768 RTCRN{6}ACGNR ABF1 63 31 0.196719 21 0.038894 10 0.759453 TACTGTAC ZBTB16 9 5 0.153936 4 0.042593 1 0.56709 AAACTGTGG MET32 MET31 4 3 0 2 0.042665 1 0.180459 TAGCCGCCSA UME6 4 2 0.193021 2 0.042665 0 1 HDVGNCACAAAA MSE 4 2 0.193021 2 0.042665 0 1 HDVKNCACAAAAD MSE 15 9 0.069143 6 0.047972 3 0.356477 YGNCACAAAW NDT80 15 6 0.52642 6 0.047972 0 1
Table: Motifs enriched 500bp upstream of the transcriptional start site of the more than two-fold 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
Table: Global Alignment (Needlemann Wunsch)
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
ZBTB16b ZBTB16a Psq L Psq I Psq H Psq K Psq J Psq E Psq D Psq G Psq F Psq A Psq C Psq B
ZBTB16b 100 82 34 34 35 34 34 35 35 35 35 31 30 30
ZBTB16a 82 100 34 32 32 32 32 32 32 32 32 27 27 27
Psq L 23 22 100 34 34 34 34 34 34 34 34 48 48 48
Psq I 19 21 22 100 99 100 100 99 99 99 99 58 60 60
Psq H 19 21 23 99 100 99 99 100 100 100 100 59 61 61
Psq K 19 21 22 100 99 100 100 99 99 99 99 58 60 60
Psq J 19 21 22 100 99 100 100 99 99 99 99 58 60 60
Psq E 19 21 23 99 100 99 99 100 100 100 100 59 61 61
Psq D 19 21 23 99 100 99 99 100 100 100 100 59 61 61
Psq G 19 21 23 99 100 99 99 100 100 100 100 59 61 61
Psq F 19 21 23 99 100 99 99 100 100 100 100 59 61 61
Psq A 22 21 48 58 59 58 58 59 59 59 59 100 98 98
Psq C 22 21 47 60 61 60 60 61 61 61 61 98 100 100
Psq B 22 21 47 60 61 60 60 61 61 61 61 98 100 100
Table: Local Alignment (Smith Waterman)
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
ZBTB16b ZBTB16a Psq L Psq I Psq H Psq K Psq J Psq E Psq D Psq G Psq F Psq A Psq C Psq B
ZBTB16b 100 82 45 41 41 41 41 41 41 41 41 41 41 41
ZBTB16a 82 100 42 40 40 40 40 40 40 40 40 42 42 42
Psq L 24 22 100 42 42 42 42 42 42 42 42 90 90 90
Psq I 19 21 24 100 100 100 100 100 100 100 100 97 100 100
Psq H 19 21 24 100 100 100 100 100 100 100 100 97 100 100
Psq K 19 21 24 100 100 100 100 100 100 100 100 97 100 100
Psq J 19 21 24 100 100 100 100 100 100 100 100 97 100 100
Psq E 19 21 24 100 100 100 100 100 100 100 100 97 100 100
Psq D 19 21 24 100 100 100 100 100 100 100 100 97 100 100
Psq G 19 21 24 100 100 100 100 100 100 100 100 97 100 100
Psq F 19 21 24 100 100 100 100 100 100 100 100 97 100 100
Psq A 22 21 87 97 97 97 97 97 97 97 97 100 98 98
Psq C 22 21 87 100 100 100 100 100 100 100 100 98 100 100
Psq B 22 21 87 100 100 100 100 100 100 100 100 98 100 100
Figure: Direct Alignamnet of blmp-1 with ZBTB16 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Figure: Directed Alignment of Ndt80 with ZBTB16 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Figure: Zoom of the Directed Alignment of Ndt80 with ZBTB16 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Figure: Direct alignment of Ndt80 with TAp63a ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Figure: Multiple sequence alignment of Ndt80 with all p63 family members in worm, fly and humans ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
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