Abstract:Dietary restriction without malnutrition delays ageing and extends lifespan, but the molecular mechanism is still unknown. Ageing gene expression changes originate from development and growth cessation, are modulated by diet, and manipulate circadian rhythmicity. A significant proportion of the genes differentially expressed under dietary restriction, during ageing and the juvenile growth period are under circadian control. Circadian clock genes are among the most significant interaction partners of dietary restriction differentially expressed genes in mouse and human interactome, while clock modulators interact highly significant with genes controlling nuclear lumen, ribosomal biogenesis and cell cycle. Canonical clock genes and clock modifiers itself exhibit differential expression upon DR, as well as during ageing and already during the juvenile period. Notable, for instance circadian mRNA expression of NAMPT was identified to increase during juvenile, while to decrease during ageing and is induced upon dietary restriction. Changing cycles in redox state and epigenetic marks could be possible mechanism to measure time over age.
Introduction
Intersections of Transcriptional Signatures
Common Set of Genes Regulated by Juvenile, Ageing, DR & Circadian Clock
Coexpression of Transcriptional Signature Intersection
Clock Gene Differential Expression
Functional Roles of Different Clock Modifier Types
Interactome of Clock Modifiers
DR-Essential Clock Modulators and Circadian DR-essential gene
Ageing Clock Genes
Longevity Associated Clock Modulators
Imprinting of Ageing, Juvenile, DR and Circadian Transcriptional Signatures
α-Lipoic Acid Modifies Clock Gene Expression
Discussion
Regulatory Programs Governing Changes Over Time
Metabolic Enzymes
Clock Systemic Factors
Cell Cycle Regulators
Transcription Factors
Juvenile, Ageing & DR Have Discrete Effects on the Circadian Clock
Clock Modulator Interactome Impacts on Nuclear Lumen and Ribosomal Biogenesis
α-Lipoic Acid Treatment blocks the Oscillatory State Change
Conclusion
Methods
Tables and Figures
Table: Genes exhibiting enrichment for differentially expression during juvenile growth, ageing, DR and are circadian
Table: Coexpressed genes of the juvenile up-, ageing down- and DR upregulated circadian genes
Table: Functional Enrichment of human Clock modifiers
Table: Ageing and DR clock modifiers
Table: Specific interactors of clock modifiers (GW-RNAi screen) in a human protein-protein interactions network
Table: Specific interactors of clock modifiers (GW-RNAi screen) in a mouse functional interaction network
Table: Longevity-associated clock modulators
Table: LA induced genes in liver of young rats (1.5-fold)
Dietary restriction (DR) in defined factors, without causing malnutrition, slows down the ageing process and extends healthy lifespan in multiple species, from yeast to primates. Different regimens can affect lifespan by restricting total calorie intake (calorie restriction), certain nutrients (sugars, amino acids) or time of feeding (intermittent fasting). It is totally unclear why various forms of restricted diets are working in so many diverse life forms.
Biological rhythmicity is generated by gene expression loops and metabolic cycles. The circadian clock is an ancient intrinsic timer, ubiquitous present in all species on earth. Thus, it governs timing in evolutionary far-related species, from unicellular organisms to mammals [Eelderink-Chen et al., 2010].
In D. melanogaster starvation, DR and many longevity mutants (like Rpd3, Sir2, chico, methusalem) all upregulate takeout (to). to is a secreted potential juvenile hormone binding protein and its induction by starvation is blocked by all arrhythmic central clock mutants [Bauer et al., 2010; Galikova and Flatt, 2010].
It is known that DR invokes hormonal changes which have systemic effects on the whole body physiology and decreases core body temperature. The circadian clock controls peripheral tissues also in an endocrine manner as well via the body temperature.
The light-responsive suprachiasmatic nucleus (SCN) of the hypothalamus harbours the circadian master clock. Besides this there is also a food-entrainable pacemaker, probably in the dorsomedial hypothalamic nucleus [Mieda et al., 2006] and the pineal gland appears to be ancient pacemaker too. The molecular clock is also present in all peripheral organs and tissues as well as in every single cell of the organism. Caloric restriction and maybe intermittent fasting reset the SCN clock and generates high amplitude circadian rhythms (similar to young ages) and increased lifespan [Froy and Miskin, 2010].
Many clock gene mutants (Clock-/-; Arntl-/-; Per2-/-; Dbp/Tef/Hlf triple knockout) display phenotypes of rapid ageing. Although in general clock disruption correlates often with accelerated ageing, disrupted circadian rhythms does not cause abnormal phenotypes per se (e.g. Cry1,2 double knockout) and specific clock manipulation are even associated with extended longevity (Csnk1e-/- under constant darkness, α-MUPA and to).
Juvenile growth appears to be controlled by non-cell autonomous endocrine mechanisms as well as cell-autonomous genetic program [Finkielstain et al., 2009; Lui et al., 2008; Lui et al., 2010].
We hypothesize here that the systemic and genetic program which regulates postnatal growth continues and cause ageing, is manipulated by diet (delayed by restriction) and in fact timed by biological clocks (like circadian rhythmicity).
Comparing gene expression molecular signatures can generate hypothesis which gene expression programs are shared between particular biological processes [Plaisier et al., 2010]. DNA-microarray meta-analysis of postnatal (juvenile) development/growth [Finkielstain et al., 2009; Lui et al., 2008; Lui et al., 2010], ageing [de Magalhaes et al., 2009; Hong et al., 2010; Swindell, 2009], dietary (caloric) restriction [Hong et al., 2010; Plank et al., 2012; 18155270; 18634819; Swindell, 2009] and circadian rhythmicity [Yan et al., 2008] lead to the identification of transcriptional signatures, hence genes which transcript expression commonly change in multiple tissues.
All living forms are programs executed by sequence of events encoded in the sequence of the genome. The complexity arises from chaos (stochastic interactions with the environment) which based on feedback loops and oscillations give rise to structured patterns of an organism.
Here, we found that the transcriptional signatures of juvenile, ageing, DR and circadian rhythmicity significantly overlap and regulate a common set of a few genes which are themselves highly co-expressed with genes related to reactive oxygen generation (e.g. associated to mitochondria and peroxisome). Among the circadian genes in this common set which have the similar expression profile of being juvenile-induced, ageing-suppressed and DR-triggered is for instance NAMPT. Several clock genes are themselves differential expressed during the juvenile period, ageing and upon DR. On the genetic level juvenile growth appears to shorten the period, while ageing seems to reduce the amplitude and lengthening the period, whereas DR restores high amplitude rhythms. Defined classes of clock modifiers have enrichment in certain functions and in general are highly enriched for nuclear lumen association. The interactome of clock modifiers in mouse and humans are enriched for genes related to ribosome biogenesis. We identified specific DR-essential, ageing and longevity associated genes which are either circadian or function as clock modifiers. Moreover, imprinted genes are present in the signatures, although only to a moderate extent. Lastly, the transcriptional signature of α-lipoic acid (a DR-mimetic) is highly enriched for circadian clock-related terms.
As DR oppose age-related gene expression changes and age-related gene expression changes where shown to originate from development, transcriptional signature of DR, ageing and juvenile growth were intersected and indeed found to share more genes in common than expected by chance [Figure: Bidirectional comparisons of DR, ageing and juvenile signatures]. The overlap between DR and ageing was most significant. There was a strong enrichment in terms associated to Endosome/Vacuole/Lysosome, Heat shock 70 stress responses, immune response and extracellular matrix (ECM)/collagen/hydroxylation. Ageing and juvenile signatures also had ECM/Collagen and inflammation/immune system related terms overrepresented. The intersection between juvenile and DR signatures had Immune system and ECM/Collagen in common too, but notable as third important term circadian rhythm/rhythmic processes.
Figure: Bidirectional comparisons of DR, ageing and juvenile signatures.
DR as it retards ageing should be expected to act in opposite on ageing-gene expression alterations. The overlap of genes which are oppositely expressed during ageing and DR were more significant than those genes which change in the same direction [Figure: Genes oppositely and commonly differentially expressed during ageing and DR]. It was noted that a significant amount of genes were enriched for up- and downregulation both during ageing and under DR. Those genes which are regulated by ageing and DR in the same direction could represent protective mechanisms against ageing [de Magalhaes et al., 2009], which are further enhanced upon DR. This is important as it argues that it could be detrimental to simple restore aged gene expression patterns to young state. It would be much more of interest to identify the gene expression signature associated to longevity, i.e. ageing suppression.
Figure: Genes oppositely and commonly differentially expressed during ageing and DR.
Genes going commonly up in juvenile and ageing are dominated by immune system related clusters/terms [Figure: Genes oppositely and commonly affected by juvenile and ageing (juvenile impact on ageing)]. Of note, genes related to wound healing and cell cycle start to be downregulated already in the juvenile and continue this trend during ageing which could explain why the proliferative/regenerative potential declines with age.
Figure: Genes oppositely and commonly affected by juvenile and ageing (juvenile impact on ageing).
Juvenile and DR commonly downregulate collagen genes, actin-related genes and cell cycle genes, while juvenile and DR both upregulate Drug metabolism and positive regulators of Apoptosis [Figure: Impact of DR on juvenile gene expression changes].
Figure: Impact of DR on juvenile gene expression changes.
Next we asked whether there is an enrichment of circadian genes in the transcriptional signatures of DR, ageing and juvenile growth [Figure: Intersections of circadian genes with DR, ageing and juvenile]. Although more than half of the DR-differential expressed genes were circadian, the ageing and juvenile signature were also significant enriched for circadian genes.
Figure: Intersections of circadian genes with DR, ageing and juvenile.
To dissect which circadian process are differential regulated upon DR, during ageing and in the juvenile growth phase the circadian signature was intersected with the respective up- and downregulated genes [Figure: Intersections of circadian genes with the up- and downregulated genes during juvenile, ageing and DR].
Figure: Intersections of circadian genes with the up- and downregulated genes during juvenile, ageing and DR.
Finally, it was asked which genes are common to more than two signatures [Figure: Overlap of the transcriptional signatures common to juvenile growth, ageing, DR and circadian clock (CC)].
Figure: Overlap of the transcriptional signatures common to juvenile growth, ageing, DR and circadian clock (CC).
Among these genes several (Nampt, Inmt, Klf9, Pim3, Dbp, Gstm1) are directly bound by Arntl close to their transcription start site. Most of them exhibit the common expression pattern of being upregulated during juvenile, downregulated during ageing and upregulated upon DR, which could indicate that reduced Arntl transactivation accounts at least in part for this program.
In total 27 genes are common to the transcriptional signatures of juvenile, ageing, DR and circadian. Seven were metabolic enzymes, five transcription factors, three collagen genes, 7 immune related genes, 5 clock genes. At least five genes of this gene set have defined role in the clockwork. Dbp is an output transcriptional regulator. Nampt mediates an enzymatic feedback loop in the clock. Ccrn4l, chaperone Hspa8 and Cirbp are all three clock system entrained factors. Ccrn4l is a clock component and output gene and its knockdown has positive metabolic effects ("lean mice clock machine"). Based on the expression pattern, genes in this set can be grouped. One group consist of immune-related genes, which are increased in expression during juvenile and ageing, while DR suppresses their expression. Among these genes, the histocompatibility antigen (like HLA-DRB1) were identified in genome-wide association studies to correlate with human longevity in Sicilian [Listi et al., 2010], French [Soto-Vega et al., 2005] and Okinawa [Akisaka et al., 1997] populations. Another group is formed by three collagen genes, which bind growth factors, thus interacting selectively and non-covalently with growth factors, proteins or polypepides that stimulate a cell or organism to grow or proliferate. There is no gene upregulated during juvenile, which becomes downregulated during ageing which is also differentially expressed upon DR and exhibit circadian rhythmicity.
Coexpression [http://coxpresdb.jp/] among these genes in Table: Genes exhibiting enrichment for differentially expression during juvenile growth, ageing, DR and are circadian clusters them in at least three different groups. Collagen genes (Col1a1, Col5a1 and Col3a1), genes related to the immune system (Igh-6, Ccl6, Cd74, H2-Aa, H2-D1, H2-Eb1 and Psmb9) and others. Many of the immune related genes (H2-Aa, H2-D1, H2-Eb1 and Psmb9) are also clustered in close proximity on chromosome 17. This argues that these modules compromise distinctive regulatory programs. 8 genes (Nampt, Inmt, Glul, Dbp, Zbtb16, Klf9, Pim3 and Hspa8) were selected as they had similar expression pattern (up in juvenile, down in ageing and up in DR) and their coexpressed genes were investigated. 289 genes were found to be significantly coexpressed [Table: Coexpressed genes of the juvenile up-, ageing down- and DR upregulated circadian genes]. The strongest enriched clusters were terms related to control of the redox balance. Interestingly there were 4 antioxidant activities among them: Gpx1, Prdx5/6, Sod2. The PRDX5, the mitochondrial peroxidoxin, progressively declines with advanced age in the nervous system of humans and PRDX6 RNAi causes arrhythmicity.
Canonical core clock and modifier genes (indicated in bold) as well as non-canonical pacemakers were identified in the transcriptional signatures of juvenile, ageing and DR [Table: Genetic consequences of the transcriptional signatures]. Pacemakers which knockdown via RNAi in human cell causes arrythymicity (AR), long period (LP), short period (SP), high amplitude (HA) or low amplitude (LA) circadian phenotype were annotated. From this it would be expected that juvenile growth leads to shorter period, ageing in contrast would cause a long period and low amplitude, whereas DR would lead to high amplitude rhythmicity.
Remarkable, Wnt signalling which is crucial in development and stem cells was enriched in the overlaps of the transcriptional signatures as well as intersections of ageing and DR with clock genes/modifiers [Figure: Juvenile, ageing and DR clock genes].
Figure: Juvenile, ageing and DR clock genes.
Clock modifiers from the genome-wide RNAi screen in human cells [Zhang et al., 2009] were divided into groups according to their effect on cellular clocks (i.e. high amplitude and short/long period) were assessed for specific functional enrichments [Table: Functional Enrichment of human Clock modifiers]. Clock modifiers in general were enriched for terms related to nuclear lumen, stress response, RNA-binding and histone modification. High amplitude genes were associated to cell cycle control, H4 acetylation, apoptosis regulation and ion transport. The enrichment for ion transport could couple imprinted genes (also enriched for ion transport) and redox state. Short period genes are enriched for nucleolus, while long period genes are associated to nucleoplasm, RNA processing and Hedgehog signalling.
Genes associated to be required for circadian rhythmicity (here called Pacemakers) and differential expressed during ageing and DR were identified and checked for enrichment [Table: Ageing and DR clock modifiers]. Ageing clock modifiers are enriched for acetylation and DR clock modifiers for ribonucleoprotein.
In order to gather how the interactome is affected by the clock modifiers identified in the genome-wide RNAi screen in human cells, a human protein-protein interaction network in mouse functional interaction network were generated. Genes/proteins which were identified to interact with a higher specificity in connectivity with clock modifiers than expected by chance were analyzed for functional enrichment [Table: Specific interactors of clock modifiers (GW-RNAi screen) in a human protein-protein interactions network; Table: Specific interactors of clock modifiers (GW-RNAi screen) in a mouse functional interaction network]. Some clusters of functional terms were commonly enriched in both networks, such as nuclear lumen and ribosome biogenesis.
8 DR-essential gene orthologs [Ep300, Crebbp, Sirt1, Epas1 (long period), Foxa3 (long period), Mtor (long period), Tsc2 (long period) and Sfn (high amplitude)] are in fact clock modifiers (p-value = 0.005), 25 DR-essential gene orthologs (Atg7, Dlat, Ywhab, Irs2, Ghr, Elovl6, Slc25a30, Cryab, Sc5d, Eif4b, Hspb1, Prkdc, Gclc, Elovl5, Ywhaq, Ywhaz, Mtor, Rgl1, Trp53, Mat2a, Foxo3, Ywhah, Ywhag, Egln3 and Elovl1) exhibit circadian oscillation on transcript level in multiple tissues. Of these two sets only Mtor is in common. Also 13 of 27 circadian systemic entrained factors are differentially expressed under DR (not significant).
104 of 252 ageing-associated genes (derived from human genes in GenAge) were circadian (p-value 6 = 0.027). 3 of 11 positive ageing-suppressors (Cebpb, Pck1, and Igf1) were circadian (not significant). 1 of 2 negative gerontogenes was circadian [P(X) > 6 = 0.031].
Genetic variants linked to longevity in genome-wide association studies give clues which players might be associated to exceptional longevity. At least three clock modulators were found to be associated to human longevity [Table: Longevity-associated clock modulators].
Of note, thyroid hormone receptor activator and coactivator activity is one of the enriched molecular functions of ageing hypomethylated genes (Wuttke, unpublished).
Several genes which were differentially expressed in juvenile, ageing and DR are imprinted genes [Figure: Imprinted circadian, juvenile, ageing and DR genes]. This striking correlation might point to a causal relationship.
Figure: Imprinted circadian, juvenile, ageing and DR genes.
Imprinted genes are differentially expressed from the paternal or maternal copy duo to epigenetic regulation such as silencing DNA methylation. Imprints are established in the developing gametes and erased and reset in germ cells. The memory is set by DNA-methylation, post-translational histone modifications and protein/RNA factors. Paternally expressed genes encourage fetal growth (oncogenic), while maternally expressed genes are growth inhibitor (similar to tumour-suppressors).
The circadian imprinted gene Slc22a1 transports methylnicotinamide, metformin, spermidine in both directions, is inhibited by PKA, and induced by PPARA and PPARG. All these molecules were strongly implicated in ageing.
α-Lipoic acid (LA) supplementation in the ad libitum phase during feeding switch regimens can mimic or block the lifespan extending effect of DR [Merry et al., 2008]. Ageing suppresses hepatitic Dbp and Bhlhe41 expression. LA significantly modulated the expression of circadian clock genes and led to a high enrichment of clock-related terms in the induced genes (First cluster in DAVID analysis with an enrichment score of 3.54) [Table: LA induced genes in liver of young rats (1.5-fold)]. In young animals LA induced the expression of Naps2, Arntl and Cdkn1a, while it suppressed Dbp, Per2 and Per3 as well as Dact1 (dapper, antagonist of β-catenin, homolog 1). In old animals LA induced Npas2, Arntl and suppressed Tef and Jdp2, Dbp, Per2 and Per3 in the liver.
There are discrete regulatory programs operating during the life history of an organism [Table: Regulatory programs controlling ageing and the determination of lifespan]. First of all, there are gene regulatory patterns established during development and maturation which exhibit the same trend after adulthood (drift). Secondly, the initiation of a growth subroutine ensures that the animal stops growing in height/size, but continues action of these changes in adults cause detrimental effects. Third, there are also protective responses to the negative effects of the continuation of development and growth cessation subroutine as well as to environmental influences and perhaps damage. Forth, as these programs are not sufficient to explain all observed changes there are indications for an active ageing program, also it nature remains controversial. Dietary restriction appears to counteract some but not all age-related changes.
It was totally elusive how all these regulatory programs are coordinated in time and in space at the whole organisms physiology. Here we present compiling evidence that biological timekeeping mechanism involving circadian cycles regulate not only daily rhythms, but also participate in controlling the development, growth and ageing of an organism [Figure: Changes in circadian clock rhythmicity as a mediator of development, juvenile growth, ageing and its modulation by diet].
Figure: Changes in circadian clock rhythmicity as a mediator of development, juvenile growth, ageing and its modulation by diet.
Further, the sexual dimorphism on lifespan by genetic mutations, DR or pharmacological interventions might be duo to imprinted genes and circadian hormones.
A specific set of genes was identified to be commonly regulated by the programs mediating juvenile growth, ageing, DR response and circadian rhythmicity. Besides the immune-related and collagen gene several of these are very interesting candidates and therefore will be discussed in the following in more detail and put into context. Among them were five clock genes (Nampt, Hspa8, Glul, Dbp and Ccrn4l). At least six genes encode metabolic enzymatic activities (Nampt, Inmt, Glul, Aldh1a1, Gstm1 and Gsta3). Three genes are clock systemic entrained factors, which encode a heat and cold shock protein, respectively (Hspa8 and Cirbp), as well as a deadenylase (Ccrn4l). Two have as primary function the regulation of cell cycle in common (Ccnd2 and Pim3). Five encode transcription factors (Zbtb16, Sox4, Klf9, Dbp and eventually Peg3). A few of all of them were implicated in ageing. For instance, Hspa8 and Glul are oxidatively modified in ageing rat brain [Perluigi et al., 2010].
Nampt (nicotinamid phosphoribosyltransferase) catalyses the condensation of nicotinamide with 5-phosphoribosyl-1-pyrophosphate to yield nicotinamide mononucleotide, which is crucial step in the biosynthesis of nicotinamide adenine dinucleotide (NAD). Nampt knockout is embryonic lethal. Replicative senescence is preceded by a marked decline in Nampt expression and activity. Pharmacological suppression of Nampt activity induces premature senescence, while introducing Nampt gene into ageing human cells delayed senescence and substantially lengthened cell lifespan [van der Veer et al., 2007]. The substrate of Nampt, nicotinamide, increases the period in Arabidopsis [Dodd et al., 2007] and mammalian cells [Asher et al., 2008], just as ageing does [Table: Genetic consequences of the transcriptional signatures]. Its product NAD is considered to function as a metabolic oscillator of ageing [Imai, 2010]. Intracellular NAD+ levels and the NAD:NADH ratio decline in multiple organs during the progression from young to middle age [Braidy et al., 2011]. Ageing decreases systemic NAD biosynthesis via lowering of plasma NMN (nicotinamid adenine dinucleotide) levels (5 months vs. 19 months old) in BESTO (beta-cell specific Sirt1-overexpressing) mice [Ramsey et al., 2008]. Systemic NAD biosynthesis is mediated by intra- and extracellular Nampt functions as driver that keeps up the pace of metabolism in multiple tissues/organs. NAD-dependent enzymes like Sirt1 serve as universal mediators that execute metabolic effects in a tissue-dependent manner in response to changes in systemic NAD biosynthesis [Imai, 2009]. SIRT1 itself regulates amplitude and duration of circadian gene expression.
Inmt (Indolethylamine N-methyltransferase; a.k.a. Temt) catalyses the N-methylation of amines such as tryptamine (derived from e.g. tryptophan) and serotonin via the usage of S-adenosyl-L-methionine (S-adenosyl-L-methionine + amine = S-adenosyl-L-homocysteine + a methylated amine) Thus, it participated in tryptophan metabolism and couples it with the one-carbon source metabolism of (S-adeno-)methionine and related structures.
Glul (Glutamine synthetase) is a ubiquitously expressed enzyme that converts glutamate (a signalling molecule) to glutamine and glutamine into 4-aminobutanoate (gamma-aminobutyric acid, GABA). It is involved in ammonium detoxification and interorgan nitrogen flux [Haberle et al., 2006] as well as essential for cell proliferation. It transcription increases with development [Vardimon et al., 1993].
Aldh1a1 (aldehyde dehydrogenase family 1 member A1) was implicated in stem cell function as it is highly expressed in adult stem cells but changes during age. Aldh1a1 knockout mice develop cataracts at 6-9 months if age and had decreased proteosomal activity, increased protein oxidation and increased glutathione levels [Lassen et al., 2007]. Aldh1a1 participates in retinoic acid (RA) synthesis in vivo (Retinal + NAD+ + H2O -> retinoate + NADH). RA signalling is necessary for development and RA synthesis is catalysed by multiple enzymes including Aldh1a1 [Fan et al., 2003]. Retinoic signalling represses Wnt signalling via induction of Pitx2 and Dkk2 [Kumar and Duester, 2010].
Glutathione S-transferases are multifunctional enzymes important in the metabolism of xenobiotics, but catalyse also important reaction in hormone synthesis. For instance, Gsta3 catalyses the double bond isomerization of precursors for progesterone and testosterone during the biosynthesis of steroid hormones.
Hspa8 and Cirbp as well as Ccna2 (only suppressed during juvenile) are cellular stress genes [Li et al., 2010]. Meal time feeding induced Hspa8, Cirbp and Ccna2 transcription ten-fold. Physiological temperature fluctuations drive rhythmic DNA binding of HSF1 (heat shock factor-1), which regulates HSP transcription [Reinke et al., 2008].
Hspa8 (heat shock 70kDa protein 8; a.k.a. Hsc70 or Hsp73) is a ubiquitous expressed chaperone, which is localized in cytoplasmic mRNP granules. Melanosome related terms were also identified in the enrichments such as in the intersection of DR and circadian [Figure: Intersections of circadian genes with DR, ageing and juvenile]. Upon heat stress Hspa8 translocates rapidly from the cytoplasm to the nuclei (especially to the nucleoli). HSPA8 contributes to CMA (chaperone mediated autophagy) from both sides of the lysosomal membrane. The cytosolic form binds substrates and targets them to lysosomes, while the form in the lysosomal lumen is required to complete translocation across the lysosomal membrane, probably by actively pulling in [Hubbard et al., 2012]. Hspa8 was found to be upregulated in an in vitro model of DR [de Cabo et al., 2003].
Cirbp (Cold-inducible mRNA binding protein) is ubiquitous expressed and has protective role in the genotoxic stress response by stabilizing transcripts of genes involved in cell survival. It acts as a translational activator and is essential in cold-induced suppression of cell proliferation. Various cytoplasmic stresses, such as osmotic and heat shocks cause Cirbp translocation from the nucleus to the cytoplasm into stress granules. Moderate low temperature preserves stemness of neural stem cells and prevented apoptosis via stimulation of CIRPB [Saito et al., 2010] Cirbp-knockout mice exhibit neither gross abnormality nor defect in fertility, but had a decreased number of undifferentiated spermatogonia. Cirbp accelerates cell-cycle progression from G0 to G1 as well as from G1 to S phase in cultured mouse embryonic fibroblasts.
Ccrn4l expression increases during juvenile and further during ageing, while DR counteracts this trend as it suppresses it. Ccrn4l, the Carbon catabolite repressor protein 4 homolog (a.k.a. Ccr4 or nocturnin) is an ubiquitous expressed component of the circadian clock or downstream effector of clock function. Ccrn4l homozygous knockout mice are extraordinary healthy as they were lean, resistant to diet-induced obesity and fatty liver development, showed increased circulating glucose levels and increased insulin sensitivity on a standard diet as well as impaired glucose tolerance on a high fat diet [17517647]. Ccrn4l was described as a molecular factor of senescence regulated by chromatin and methylation status during ageing [Puech et al., 1997].
Ccnd2 is a direct Arntl circadian output target and essential for G1/S transition. It also participates in Wnt signalling.
Pim3 is a serine/threonine-protein kinase which is widely expressed, able to phosphorylate CDKN1, and involved in cell cycle progression and suppression of apoptosis.
Zbtb16 (alias PLZF) is enriched for upregulation during juvenile growth and DR, while it is enriched for downregulation during aging. Zbtb16 is enriched for circadian rhythmicity and coexpressed with Per1,2. Zbtb16 maintains the undifferentiated state of spermatogonial progenitor cells (SPCs). It inhibits mTORC1 pathway activity in SPCs by inducing Ddit4. Increased mTORC1 activity inhibits the response of SPCs to the niche-derived factor GDNF [Ellisen, 2005]. Ddit4 was greatly induced after-short term DR in male mice liver [Estep et al., 2009].
Sox4 knockout mice die at embryonic day 14 [8614465]. Sox4 was found here to be progressively suppressed during juvenile and ageing but being also induced upon DR. It regulates cell differentiation, proliferation and survival in multiple essential processes [Penzo-Mendez, 2010]. Sox4 enhances beta-catenin/TCF activity and proliferation.
Klf9 (Krueppel like factor 9) can activate transcription and also repress gene expression by directly binding to Sin3A a scaffold co-repressor. Its expression is sensitive to thyroid hormone and serotonin signalling. Klf9 was implicated in stem cell maintenance and differentiation of T- and B-lymphocytes and was found to be necessary for adult neurogenesis [Scobie et al., 2009]. Klf9 affects keratinocyte proliferation/differentiation by controlling the expression of target genes in a daytime-dependent manner. Kl9 is substantially upregulated in a cortisol and differentiation-state-dependent manner. Klf9 exhibits strong anti-proliferative effects and it putative target genes include proliferation/differentiation markers that also exhibit circadian expression in vivo.
Dbp (Albumin D-element binding protein) is classical circadian transcription factor which controls genes that peak in expression at puberty [Yano et al., 1992].
Peg3 (Paternally-expressed gene 3 protein) belongs to the krueppel C2H2-type zinc-finger protein family and inhibits Wnt signalling. It alters growth and development as well as regulates apoptosis [Jiang et al., 2010].
Ageing is accompanied by a decrease in circadian amplitude and a shift in the period. In contrast, DR slows down ageing and increases the amplitude of circadian oscillations. The causality of these correlations was not yet clear; i.e. if ageing impacts on clock or vice versa. Here it was shown that this trend was reflected on the genetic and transcriptional level as aging tend overall to decrease the amplitude and prolong the period, while DR counteracted these effects by increasing the amplitude (i.e. clock resetting). DR induces the expression of the first interconnected negative limb (Periods and Cryptochromes), which likely contributes to the higher amplitude. Furthermore, during juvenile the gene expression changes cause a shortening in the period. Period prolongation is therefore part of the growth cessation subroutine which becomes initiated as an organism reaches maturity [Figure: Changes in circadian clock rhythmicity as a mediator of development, juvenile growth, ageing and its modulation by diet].
How the cellular circadian clock changes during the juvenile period is not studied, but during the adolescent there are enormous changes/alterations in circadian behaviour and sleep/wake cycle [21302851].
Juvenile growth tends to be mediated by a shortening of the period, while ageing prolongs the period and lowers the amplitude. DR counteracts this trend by showing a tendency to shorten the period and increase the amplitude (thus clock resetting). These trends are consistence with the observation that DR and α-MUPA increase clock gene expression amplitude and reset the clock.
As the DR-essential Mtor modifies clock function and is itself circadian on transcript level, it represents a novel feedback loop coupling Mtor signalling to the molecular clock and vice versa.
Further, as the molecular interaction network of clock modifiers is strongly enriched for functional terms related to ribosome biogenesis, both in mouse and human interactome, this further supports the notion that cell growth and circadian time keeping are tightly coupled.
Mtor signaling is known to be decreased under DR, it could therefore provide one pathway how DR impacts on circadian rhythmicity and especially its ability to reset the SCN.
A conformation of the role of circadian genes in the control of ageing and especially its modulation by diet stems from the fact that α-lipoic acid treatment (which is able to mimic or block the effect of DR by fixing the mortality tractor of the previous feeding regimen in feeding swap experiments) was found to primarily alter clock genes regardless of age.
In this work we intersected the transcriptional signatures of juvenile growth, ageing, dietary restriction and circadian rhythmicity and found a higher overlap of these processes as expected by chance. Among a common set of 26 genes around 12 genes with a similar expression profile (induced during juvenile, suppressed during ageing and upregulated by DR), were strongly coexpressed with processes related to reactive oxygen generation, although they all have no obvious primary function in this process. Clock genes (modifiers and pacemakers) are themselves differentially expressed during juvenile, ageing and upon DR and from genetic studies their expression suggests that juvenile phase is associated with shorting in the period, but ageing reduces the amplitude and prolongs the period, while DR increases the amplitude. Interestingly different type of clock modifiers have discrete functional enrichment and were in the interaction network of pacemakers strongly enriched for the terms related to the nucleolus (in both mouse and humans), which fragmentation was proposed to be a cause of ageing. Many DR-essential, ageing and some longevity associated genes were circadian or even clock modulators, providing some explanation for the here found overlap.
Several of the circadian genes commonly regulated by juvenile, ageing and DR can be associated with Wnt signalling [Table: Genes exhibiting enrichment for differentially expression during juvenile growth, ageing, DR and are circadian] and the intersections between clock-modulators with ageing and DR-differential genes were also enriched for Wnt signalling. A common cross-point might be GSK3 which is pivot component of Wnt signalling [19884009] as well as the circadian clock [Harms et al., 2003; Li et al., 2012] and the age-related changes of stem cells [Brack et al., 2007; 22190524]. Progeroid LaminA (leads to premature ageing in humans) shuts down Wnt signalling which alters the extracellular matrix synthesis and therefore impairs stem cell function [Hernandez et al., 2010]. Interestingly LaminA is circadian expressed and increases during ageing, Zmpste24 (which processes LaminA) is under clock control too. Furthermore, the huntingtin (Htt) gene is also circadian and ageing induced, providing an explanation for the late onset of this disease.
We here propose that the regulatory programs governing juvenile growth, ageing (and its modulation by diet) as well as circadian rhythmicity are interconnected by a common timing mechanism which probably operates on the genome via long-term epigenetic changes and on the level of oscillatory metabolites such as ROS and NAD.
Thus, ageing is here hypothesised to be caused by continuation of the development program and the growth cessation program both lead to changes in chromatin structure. The location and structure of chromatin is highly dynamic and changes in a rhythmic manner. Juvenile and ageing gene expression changes are established for instance by DNA methylation and demethylation, histone modifications as well as reposition of whole chromosomal locus to the nuclear periphery (LMNA). Differentially methylated genes are the product of cyclic (de)methylation reactions and targeted by defined factors, such as transcription factors and non-coding RNAs among them are imprinted genes. Some specific loci become hypermethylated while other become hypomethylated. This is controlled by an instructive program. Circadian imprinted genes are related to ion homeostasis. Altered ion transport leads change in the rhythms of cellular respiration and therefore changed redox cycles with aeging drive circadian transcriptional cycles.
If developmental progression as well as ageing and its modulation by diet is mediated by changes in the oscillatory state of metabolites and the decoration of the genome, α-lipoic acid might for instance block a switch in this transitions (e.g. to a higher amplitude).
Mammalian transcriptional signatures (commonly differential expressed genes in multiple tissues) of ageing [de Magalhaes et al., 2009; Hong et al., 2010; Swindell, 2009], DR [Hong et al., 2010; Plank et al., 2012; 18155270; 18634819; Swindell, 2009], juvenile growth [Finkielstain et al., 2009; Lui et al., 2008; Lui et al., 2010], and circadian rhythms were merged if representing the same category and intersected if belonging to different categories. Genes were mapped to unique Entrez gene identifiers and the overlap between any two signatures determined via the hypergeomtric test.
Overlaps were visualized with Venn diagrams of coloured circles via the use of python's SDL (Simple Directmedia Layer) bindings, with the size of a circle being proportional to the number of genes it represents. Functional enrichment of gene sets was obtained from DAVID [Huang et al., 2009] at [http://david.abcc.ncifcrf.gov/].
Coexpression were retrieved from the coexpression database [Obayashi and Kinoshita, 2011] at [http://coxpresdb.jp/].
The interactome information was integrated from various sources as described elsewhere [22912585].
Gene expression profile of α-lipoic acid treatment were derived from the GEO database, GSE27625 [Finlay et al., 2012].
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Genes exhibiting differential expression during juvenile growth (GH), ageing (AG), DR and are expressed in a circadian manner. Symbol JG AG DR Name Chr Peak Nampt ↑ ↓ ↑ Nicotinamide phosphoribosyltransferase 12 14.26 Inmt ↑ ↓ ↑ Indoletyhlamine N-methyltransferase 6 12.51 Glul ↑ ↓ ↑/↕ Glutamine synthetase 1 16.95 Klf9 ↑ ↓ ↑/↕ Krueppel-like factor 9 19 8.73 Zbtb16 ↑ ↓ ↑/↕ Zinc finger and BTB domain containing 16 9 19.03 Pim3 ↑ ↓ ↑/↕ Serine/threonine-protein kinase pim-3 15 8.08 Hspa8 ↑ ↓/↕ ↕ Heat shock cognate 71 kDa protein 9 17.08 Cirbp ↓ ↓ ↑/↕ Cold-inducible RNA-binding protein 10 5.81 Peg3 ↓ ↕ ↑/↕ Paternally-expressed gene 3 protein 7 4.58 Ccnd2 ↓ ↓/↕ ↓/↕ G1/S-specific cyclin-D2 6 13.08 Tmsb10 ↓ ↓/↕ ↓/↕ Thymosin beta-10 6 3.1 Sox4 ↓ ↓ ↓/↕ Transcription factor SOX-4 13 0.45 Col3a1 ↓ ↓ ↓/↕ Collagen alpha-1(III) chain 1 1.66 Col1a1 ↓ ↓ ↓ Collagen alpha-1(I) chain 11 2.67 Col5a1 ↓ ↓ ↓ Collagen alpha-1(V) chain 2 1.83 Aldh1a1 ↑ ↑ ↑ Aldehyde dehydrogenase 1 family, member A1 19 14.58 Igh-6 ↑ ↑/↕ ↓/↕ Ig mu chain C region secreted form 12 21.19 Ccl6 ↑ ↑/↕ ↓ C-C motif chemokine 6 11 3.57 Cd74 ↑ ↑ ↓ HLA class II histocompatibility antigen gamma chain 6 4.58 H2-Aa ↑ ↑/↕ ↓ H-2 class II histocompatibility antigen, A-K alpha 17 3.31 H2-D1 ↑ ↑/↕ ↓ H-2 class I histocompatibility antigen, D-B alpha chain 17 6.03 H2-Eb1 ↑ ↑ ↓ H-2 class II histocompatibility antigen, I-A beta chain 17 2.42 Psmb9 ↑ ↑ ↕ Proteasome subunit beta type-9 17 5.42 Dbp ↑ ↑ ↑/↓/↕ D site of albumin promoter (Albumin D-box) binding 7 9.18 Ccrn4l ↑ ↑/↕ ↓ Nocturnin 3 12.32 Gstm1 ↑ ↑/↕ ↕ Glutathione S-transferase Mu 1 3 10.04 Gsta3 ↑ ↑ ↑ Glutathione S-transferase A3 1 17.82
Functional enriched terms of the genes in the intersection of juvenile, ageing DR and circadian. Term Genes Score p-value mitochondrion 91 16.04 1.3E-035 peroxisome 15 5.81 2.2E-010 nad 16 5.26 5.4E-08 cofactor metabolic process 17 5.13 2.9E-08 fatty acid metabolism 10 4.164 3.6E-07 Short-chain dehydrogenase/reductase SDR 8 3.74 4.6E-05 hydrogen ion transmembrane transporter activity 11 3.48 3.6E-07 regulation of apoptosis 24 3.37 2.1E-05 cellular amide metabolic process 8 3.16 3.4E-04 mitochondrial lumen 14 2.83 2.5E-06 electron carrier activity 15 2.8 2.0E-06 Citrate cycle (TCA cycle) 7 2.77 2.6E-05 vitamin binding 10 2.56 8.1E-05 biological rhythms 6 2.33 2.6E-04 hydrogen ion transmembrane transporter activity 11 2.3 3.6E-07
System Message: ERROR/3 (, line 447)
Error parsing content block for the "table" directive: exactly one table expected.
.. table:: Table: Genetic consequences of the transcriptional signatures.
+----------------+------------------------------------------------------+----+----+----+----+----+
|Process |Gene (RNAi phenotype) |AR |LP |SP |HA |LA |
+========+=======+======================================================+====+====+====+====+====+
|Juvenile|Up |**Nampt** (LP), **Dbp**, **Ccrn4l**, |0 |0 |2 |0 |0 |
| | |**Nr1d1** (LP), **Nr1d2**, **Tef** | | | | | |
| +-------+------------------------------------------------------+----+----+----+----+----+
| |Down |**Arntl** (AR), **Npas2**, (SP), **Cry1** (SP), |1 |2 |3 |0 |0 |
| | |**Timeless**, **Peg3** (LP), Mad2l1 (LP), B4galt2 (SP)| | | | | |
| +-------+------------------------------------------------------+----+----+----+----+----+
| |Effect |3 SP -> short period |1 |2 |5 |0 |0 |
+--------+-------+------------------------------------------------------+----+----+----+----+----+
|Ageing |Up |**Dbp**, **Ccrn4l**, **Rorb**, **Csnk1d** (LP), |0 |3 |4 |0 |1 |
| | |Rrbp1 (SP), Antrx1 (LP), Pfkp (SP), Spr (HA), | | | | | |
| | |Cx3cl1 (LP), Blnk (SP), Gfap (LP) | | | | | |
| +-------+------------------------------------------------------+----+----+----+----+----+
| |Down |**Nampt** (LP), **Clock** (AR/LA), **Npas2** (SP), |2 |9 |4 |3 |2 |
| | |**Per1** (AR/LA), **Csnk1d** (LP), **Csnk2a1**, | | | | | |
| | |**Rora**, Ces1d (SP), Fbxw11 (LP), Brwd1 (HA), | | | | | |
| | |Ccnot2 (LP), Chid1 (SP), Actl6a (LP), Krr1 (LP), | | | | | |
| | |Srprb (LP), Cx3cl1 (LP), Lbx1 (SP), Cidec | | | | | |
| +-------+------------------------------------------------------+----+----+----+----+----+
| |Up \\ |**Csnk1a1** (LP), Scamp2 (HA), Ppm1b (SP), Ddx3x (LP) |0 |1 |1 |1 |1 |
| |Down | | | | | | |
| +-------+------------------------------------------------------+----+----+----+----+----+
| |Effect |1 LA, 6 LP -> low amplitude & long period |2 |12 |9 |4 |4 |
+--------+-------+------------------------------------------------------+----+----+----+----+----+
|DR |Up |**Nampt** (LP), **Dbp**, **Per1** (AR/LA), Per2 |0 |5 |8 |2 |0 |
| | |(LP/LA), Cry1 (SP), Cry2 (LP), Ccrn4l, Wdr5, Rora, | | | | | |
| | |Ppargc1a, Csnk1e (LP), Klf10 (SP) Peg3 (LP), Ces1d | | | | | |
| | |(SP), Mrpl18 (SP), Serinc1 (LP), Uba3 (LP), Rps4x | | | | | |
| | |(LP), Cops (SP) | | | | | |
| +-------+------------------------------------------------------+----+----+----+----+----+
| |Down |**Arntl**, **Dbp**, **Nr1d1** (LP), **Csnk1d** (LP), |0 |6 |3 |2 |0 |
| | |**Bhlhe40**, **Timeless**, Fbxw11 (LP), Brwd1 (HA), | | | | | |
| | |Rrbp1 (SP), Zbtb20 (SP), Tuba1b (LP), Spr (HA), Cx3cl1| | | | | |
| | |(LP), Cox4b (SP), Ddx3x (LP) | | | | | |
| +-------+------------------------------------------------------+----+----+----+----+----+
| |Up \\ |**Nfil3**, **Csnk1a1** (LP), Eny2 (LP), Snrpb2 (LP) |0 |3 |3 |0 |0 |
| |Down | | | | | | |
| +-------+------------------------------------------------------+----+----+----+----+----+
| |Effect |4 HA -> high amplitude |0 |13 |13 |4 |0 |
+--------+-------+------------------------------------------------------+----+----+----+----+----+
AR = ARrhythmic; LP = Long Period; SP = Short Period; HA = High Amplitude; LA = Low Amplitude.
343 Clock modifiers 53 High amplitude 87 Short period 203 Long period nuclear lumen 5.28, 60, 2.010^-7; photoreceptor protein 2.38, 5, 2.910^-7; PIRSF000564: kinase-related transforming protein 1.78, 7, 1.910^-7; cellular response to stress 1.66, 24, 1.410^-3; domain:OPR 1.6, 3, 1.910^-2; rna-binding 1.44, 19, 7.810^-3; transcription cofactor activity 1.44, 15, 1.210^-2; histone modification 1.4, 9, 3.610^-3 regulation of cell cycle 1.13, 6, 4.210-3 (BTC, BRCA2, DDB1, GMNN, SFN, TRRAP1); histone H4 acetylation 1.12, 3, 1.910^-3 (DNMA1, IRF4, TRRP); regulation of apoptosis 0.89, 7, 4.410^-2 (NMNAT1); ion transport 0.8, 6, 2.010^-2; hemopoiesis 0.69, 3, 1.810^-1 aminoglycan catabolic process 1.64, 3, 4.610^-3; intracellular organelle lumen 1.37, 16, 1.810^-2; nucleolus 0.85, 8, 4.810^-2; positive regulation of I-kappaB kinase/NF-kappaB cascade 0.87, 3, 8.110^-2; cellular response to stress 0.65, 6, 1.410^-1; endoplasmic reticulum part 0.6, 5, 8.410^-2; mitosis 0.44, 3, 1.910^-1 nucleoplasm part 4.56, 25, 1.110^-7; PIRSF000564: kinase-related transforming protein 2.59, 7, 1.510^-5; photoreceptor protein 2.4, 5, 3.710^-6; RNA processing 2.12, 16, 2.810^-3; domain:OPR 2.03, 3, 6.810^-3; g-protein coupled receptor 1.9, 21, 3.910^-4; protein modification by small protein conjugation or removal 1.73, 10, 1.510^-4; Hedgehog signaling pathway 1.45, 5, 7.810^-3
26 Ageing Pacemaker 22 DR Pacemaker intracellular organelle lumen 0.66, 6, 1.310^-1; acetylation 0.49, 8, 4.610^-2; nucleus 0.42, 0, 1.710^-1 ribonucleoprotein complex 1.04, 4, 2.510^-2 (MRL18, RPS4C, RRBP1, SNRPB2); intracellular organelle lumen 1.03, 6, 6.0210^-2; endoplasmic reticulum part 0.79, 3, 6.910^-2; nucleotide-binding 0.7, 6, 3.210^-2; regulation of transcription 0.62, 7, 1.410^-2
Out of 216 genes Enrichment score # Genes p-value ribosome biogenesis 10.47 24 8.210^-19 repeat:WD 3 5.78 17 3.810^-7 proteolysis involved in cellular protein catabolic process 2.25 20 3.210^-4 Cyclin-like F-box 2.06 6 1.910^-3 domain:Helicase C-terminal 1.69 7 3.6*10^-3
Out of 952 genes Enrichment score # Genes p-value Serine/threonine protein kinase-related 175.1 243 6.610^-224 nuclear lumen 33.76 152 2.410^-64 DNA/RNA helicase, C-terminal 39.17 62 3.710^-51 ribosome biogenesis 26.28 49 1.110^-29 AGC-kinase, C-terminal 19.7 30 7.5*10^-23
LA = Longevity-associated; CM = Clock modulator; CMO = Clock modulator ortholog; LP = Long period; PM = Pacemaker. Symbol Name Classification TSHB Thyroid stimulating hormone, beta LA CM PM LP TH tyrosine hydroxylase LA CM PM LP PPARG peroxisome proliferator-activated receptor gamma G CM CMO LA
Functional enrichment terms which are upregulated in the liver by lipoic acid treatment in young rats. Term Count % p-Value Benjamini IPR013655:PAS fold-3 4 25 4.44E-07 1.29E-05 rno04710:Circadian rhythm 4 25 5.47E-07 9.29E-06 biological rhythms 4 25 7.59E-07 3.57E-05 SM00086:PAC 4 25 8.05E-07 4.02E-06 SM00091:PAS 4 25 1.18E-06 5.90E-06 IPR001610:PAC motif 4 25 2.15E-006 6.23E-05 IPR000014:PAS 4 25 3.15E-06 9.13E-05 rhythmic process 5 31.25 1.28E-05 0.003 circadian rhythm 4 25 2.73E-05 0.006 domain:PAS 2 3 18.75 1.10E-04 0.005 domain:PAS 1 3 18.75 1.10E-04 0.005 domain:PAC 3 18.75 2.84E-04 0.012
Table: Regulatory programs controlling ageing and the determination of lifespan.
Routine Juvenile Ageing DR
Continuation of development ↑ ↑ (↓)
↓ ↓ (↑)
Growth Cessation Subroutine ↑ ↓ (↑)
↓ ↑ (↓)
Protective response effects ↑ ↑ (↑)
↓ ↓ (↓)
↑ (↓)
↓ (↑)
Ageing program?
↑ (↓)
↓ (↑)