New molecular mechanisms of aging regulation
Sen, Ilke
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New molecular
mechanisms of aging
regulation
University of Groningen, The Netherlands
Graduate school for drug exploration GUIDE, Groningen, The Netherlands Copyright Ilke Sen 2018
Printed by: ProefschriftMaken || www.proefschriftmaken.nl ISBN (print) : 978-94-93019-85-0
New molecular mechanisms
of aging regulation
PhD thesis
to obtain the degree of PhD at the
University of Groningen
on the authority of the
Rector Magnificus Prof. E. Sterken
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on
Monday 22 October 2018 at 12.45 hours
by
Ilke Sen
born on 17 April 1987
in Izmir, Turkey
Supervisors Prof. G. de Haan Prof. P.M. Lansdorp Co-supervisor Dr. C.G. RiedelNew molecular mechanisms
of aging regulation
PhD thesis
to obtain the degree of PhD at the
University of Groningen
on the authority of the
Rector Magnificus Prof. E. Sterken
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on
Monday 22 October 2018 at 12.45 hours
by
Ilke Sen
born on 17 April 1987
in Izmir, Turkey
Supervisors Prof. G. de Haan Prof. P.M. Lansdorp Co-supervisor Dr. C.G. RiedelNew molecular mechanisms
of aging regulation
PhD thesis
to obtain the degree of PhD at the
University of Groningen
on the authority of the
Rector Magnificus Prof. E. Sterken
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on
Monday 22 October 2018 at 12.45 hours
by
Ilke Sen
born on 17 April 1987
in Izmir, Turkey
Supervisors Prof. G. de Haan Prof. P.M. Lansdorp Co-supervisor Dr. C.G. RiedelNew molecular mechanisms
of aging regulation
PhD thesis
to obtain the degree of PhD at the
University of Groningen
on the authority of the
Rector Magnificus Prof. E. Sterken
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on
Monday 22 October 2018 at 12.45 hours
by
Ilke Sen
born on 17 April 1987
in Izmir, Turkey
Supervisors Prof. G. de Haan Prof. P.M. Lansdorp Co-supervisor Dr. C.G. RiedelNew molecular mechanisms
of aging regulation
PhD thesis
to obtain the degree of PhD at the
University of Groningen
on the authority of the
Rector Magnificus Prof. E. Sterken
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on
Monday 22 October 2018 at 12.45 hours
by
Ilke Sen
born on 17 April 1987
in Izmir, Turkey
Supervisors Prof. G. de Haan Prof. P.M. Lansdorp Co-supervisor Dr. C.G. RiedelNew molecular mechanisms
of aging regulation
PhD thesis
to obtain the degree of PhD at the
University of Groningen
on the authority of the
Rector Magnificus Prof. E. Sterken
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on
Monday 22 October 2018 at 12.45 hours
by
Ilke Sen
born on 17 April 1987
in Izmir, Turkey
Supervisors Prof. G. de Haan Prof. P.M. Lansdorp Co-supervisor Dr. C.G. RiedelNew molecular mechanisms
of aging regulation
PhD thesis
to obtain the degree of PhD at the
University of Groningen
on the authority of the
Rector Magnificus Prof. E. Sterken
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on
Monday 22 October 2018 at 12.45 hours
by
Ilke Sen
born on 17 April 1987
in Izmir, Turkey
Supervisors Prof. G. de Haan Prof. P.M. Lansdorp Co-supervisor Dr. C.G. RiedelNew molecular mechanisms
of aging regulation
PhD thesis
to obtain the degree of PhD at the
University of Groningen
on the authority of the
Rector Magnificus Prof. E. Sterken
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on
Monday 22 October 2018 at 12.45 hours
by
Ilke Sen
born on 17 April 1987
in Izmir, Turkey
Supervisors Prof. G. de Haan Prof. P.M. Lansdorp Co-supervisor Dr. C.G. RiedelAssessment Committee
Prof. H.H. Kampinga Prof. E.A.A. Nollen Prof. B.M.T. Burgering
New molecular mechanisms
of aging regulation
PhD thesis
to obtain the degree of PhD at the
University of Groningen
on the authority of the
Rector Magnificus Prof. E. Sterken
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on
Monday 22 October 2018 at 12.45 hours
by
Ilke Sen
born on 17 April 1987
in Izmir, Turkey
Supervisors Prof. G. de Haan Prof. P.M. Lansdorp Co-supervisor Dr. C.G. Riedel
Nataly Puerta Cavanzo Jakub Wudarski
Chapter 1 Introduction 9
Chapter 2 Regulation of age-related decline by transcription factors 25
and their crosstalk with the epigenome Chapter 3 DAF-16/FOXO and HLH-30/TFEB function as 61
combinatorial transcription factors to promote stress resistance and longevity Chapter 4 DAF-16 requires Protein Phosphatase 4 to initiate the 105
transcription of stress resistance and longevity promoting genes Chapter 5 General discussion and future perspectives 159
Chapter 6 Summary 169
Chapter 7 Nederlandse samenvatting 173
Appendices: List of abbreviations 177
1
Chapter 1
Introduction
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1. Aging
During our lives we are exposed to multifarious environmental stressors such as UV, heat, or oxidizing agents which trigger tissue deterioration caused by the accumulation of damaged macromolecules within the cell. This in turn causes functional decline in our body over time - a process that we know as aging - and it makes us more prone to age-related diseases. Although once viewed as a haphazard process, the aging process has been shown subject to regulation by a number of essential signaling pathways, thus its rate can be accelerated as well as reduced. Targeting such mechanisms to reduce the rate of aging could be a new approach to defer age-related complications and thereby improve our quality of life.
1.1 Aging-regulatory signaling pathways:
There are several signaling pathways and downstream transcription factors regulating longevity. These pathways response to nutrient changes such as dietary restriction, or even hormonal signals from reproductive system, signaling a delay or lack of fertility as well as the environmental stressors mentioned before. Many of these pathways such as insulin/IGF-1 signaling (IIS) pathway and mechanistic target of rapamycin (mTOR) signaling, are responsible for the expression of specific gene sets which are generally involved in growth and reproduction of the organism under normal dietary conditions and less stressful environment. However, when there is impairment in insulin signaling, dietary restriction condition or more stressful environment, downstream transcription factors of these signaling pathways trigger the expression of the genes that become savior of the organism by protecting cells from harsh environmental conditions and even extending their lifespan. These aging-regulatory signaling pathways are comprehensively discussed in Chapter 2.
1.2 Insulin/IGF-1 is one of the most central aging-regulatory signaling pathways:
The first pathway that was shown to have an influence on lifespan regulation is Insulin/IGF-1 signaling (IIS) pathway. It was discovered by the observation that a single gene mutation in daf-2, the homolog of the insulin/IGF receptor in Caenorhabditis elegans, led to a two-fold lifespan extension of the organism (Kenyon et al., 1993). C. elegans is a wonderful model organism in the study of aging, since its mean lifespan is only 2-3 weeks and even a single gene manipulation may yield a significant lifespan extension, up to 3-fold (Kenyon et al., 1993). Since C. elegans is mostly comprised of post-mitotic cells, there was a possibility that lifespan effects of this pathway may not be preserved in more complex organisms. However, several studies revealed that mutations in the insulin/IGF receptor increased lifespan in different organisms as well, such as Drosophila melanogaster and mice (Tatar et al., 2001; Blüher, Kahn and Kahn, 2003; Holzenberger et al., 2003; Taguchi, Wartschow and White, 2007) , illustrating that the lifespan regulatory role of the IIS pathway is evolutionarily conserved.
In C. elegans, IIS is acting via phosphoregulatory signaling cascades, which negatively regulate several downstream transcription factors, such as DAF-16, a FOXO transcription factor, SKN-1, an NRF-like xenobiotic response factor (Tullet et al., 2008), and the
heat-1
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shock transcription factor HSF-1 (Hsu, Murphy and Kenyon, 2003; Morley and Morimoto, 2004). These transcription factors genetically synergize within the nucleus to activate the expression of several stress response factors, i.e. heat shock proteins, antimicrobial peptides, glutathione S-transferases and other antioxidant proteins, proteins related to metabolism, etc – most of which are aging-preventive and thereby also extend the lifespan of the organism. Under unstressed and well-fed conditions in C. elegans (Figure 1A), insulin is abundant and binds to the insulin/IGF-1 receptor DAF-2, which then becomes active and directly phosphorylates the phosphatidylinositol 3-kinase (PI(3)K) AGE-1that is responsible for the
conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol
3,4,5-trisphosphate (PIP3). Production of PIP3 activates 3‐phosphoinositide‐dependent kinase‐1
(PDK-1) which in turn places activatory phosphorylations on the downstream AKT family kinases AKT-1, AKT-2 and SGK-1 (Paradis and Ruvkun, 1998; Paradis et al., 1999; Hertweck, Göbel and Baumeister, 2004). When these AKT family kinases are active, they interact and phosphorylate DAF-16. As a result, phosphorylated DAF-16 is sequestered by the 14-3-3 proteins in the cytoplasm, which prevent DAF-16 from entering the nucleus and thus render it inactive – away from its target genes (Lin, 1997; Ogg et al., 1997; Paradis and Ruvkun, 1998).
When there is an impairment in IIS, for instance, the loss-of-function mutation of DAF-2, downstream kinases are no longer active and cease to phosphorylate DAF-16. This allows DAF-16 to evade sequestration by 14-3-3 proteins and to translocate into nucleus, where it binds to its target gene promoters in order to regulate transcription of stress response genes (Figure 1B). Besides, there also exist negative regulators of IIS, including the DAF-18/PTEN
lipid phosphatase and the serine/threonine phosphatase PP2APPTR-1, counteracting
AGE-1/PI3K and AKT-1 signaling, and thus assisting DAF-16 to transform to its active form (Ogg and Ruvkun, 1998; Padmanabhan et al., 2009).
In addition to DAF-16, the heat shock transcription factor HSF-1, is another downstream transcription factor required for low IIS to delay aging and extend the lifespan of C. elegans (Garigan et al., 2002; Hsu, Murphy and Kenyon, 2003) (Figure 1A, B). HSF-1 is a master regulator of heat shock protein (HSP) expression, which makes it essential for the survival under thermal stress against its resulting proteotoxic stress (Hsu, Murphy and Kenyon, 2003; Morley and Morimoto, 2004). It was also shown that in daf-2 mutants, expression of a subset of heat-shock response genes is increased under normal/unstressed conditions (Hsu, Murphy and Kenyon, 2003). These findings reveal a possibility that, to some extend, IIS pathway may affect longevity by regulating protein homeostasis.
Another transcription factor, Skinhead (SKN)-1/Nrf, is involved in the regulation of detoxification genes, particularly promoting resistance to oxidative stress in response to low IIS (Figure 1A, B). Interestingly, DAF-16 contributes to the induction of a subset of these SKN-1 target genes, pointing to an occasional combinatorial role of these two transcription factors (Tullet et al., 2008).
1.3 DAF-16/FOXO is a major mediator of the transcriptional effects of IIS DAF-16/FOXO is involved in many physiological responses to aging-regulatory and stress signals. Consistently, under low IIS DAF-16 becomes activated and drives the expression of
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downstream genes, conferring phenotypes such as increased longevity, stress resistance, or the developmental decision of dauer formation (Figure 2) (Murphy and Hu, 2013).
Figure 1. The IIS pathway uses several downstream transcription factors to regulate longevity.
A) DAF-16 and SKN-1 transcription factors are phosphorylated by AKTs and SGK-1 and remain in the cytoplasm in inactive forms under high insulin signaling conditions. HSF-1 is not regulated by the phosphorylation of these kinases but it also remains in the cytoplasm under such conditions. B) Under low insulin signaling, such as in daf-2 mutants, DAF-16 and SKN-1 cannot be phosphorylated by AKTs and SGK-1, hence they translocate into the nucleus where they bind to the promoter of stress response genes required for longevity. Similarly, HSF-1 translocates into nucleus under low IIS conditions. However, its activity is regulated by a different repression complex (Chiang et al., 2012).
C. elegans DAF-16 is a member of the Forkhead box O family of transcription factors. Its
key features, such as the forkhead DNA binding domain, its preferred DNA binding motif, its regulated sequestration by cytoplasmic 14-3-3 proteins at RxRxxS/T are all conserved from C. elegans to mammals (Obsil and Obsilova, 2008). Therefore, it is not surprising to observe an evolutionary conservation of DAF-16 functions, e.g. the regulation of growth, metabolism, stress resistance, and longevity across metazoans (Accili and Arden, 2004). Most relevant to this thesis is the conserved role of DAF-16/FOXO for lifespan regulation.
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Many studies testing for the lifespan phenotypes of mutants in the insulin/IGF–FOXO signaling axis confirm that the lifespan regulatory role of this pathway through DAF-16/FOXO is conserved in different organisms, even in humans.
In Drosophila, systemic inhibition of IIS or tissue specific increase in the activity of FOXO in adipose tissue leads to lifespan extension (Kenyon, 2010). Similarly in mice, low IGF1 levels were found to significantly associate with lifespan extension in inbred mouse strains (Yuan et al., 2009). Finally, inhibitions of the insulin and IGF1 receptors as well as their upstream regulators and downstream effectors give rise to longevity (Bartke, 2008; Kappeler
et al., 2008; Selman et al., 2008; Yuan et al., 2009).
Figure 2. DAF-16/FOXO controls a myriad of downstream events of IIS.
DAF-16/FOXO is the major downstream transcription factor of IIS regulating several metabolic, stress response and longevity related processes.
In human cohorts of Ashkenazi Jew, Japanese, Italians, Germans, Chinese, Californian, and New England centenarians, mutations impairing Insulin/IGF1 receptor function and variants of AKT and FOXO3A have been found associated with longevity (Kojima et al., 2004; Lunetta et al., 2007; Suh et al., 2008; Willcox et al., 2008; Anselmi et al., 2009; Flachsbart
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have been linked to longevity in American and Chinese cohorts (Lunetta et al., 2007; Li et al., 2009).
2. Regulators and co-factors of DAF-16 that modulate its transcriptional activity and specificity
As mentioned in the previous section, DAF-16 is under tight regulation by several kinases and co-factors in the cytoplasm, i.e. AKTs, SGK-1 and 14-3-3 proteins, which determine the phosphorylation status and localization of DAF-16 and in turn affect the activity of DAF-16. However, the regulation of DAF-16 activity is way beyond such mechanisms. For instance, although nuclear translocation of DAF-16 is essential for target gene expression, neither triggering nuclear localization nor overexpressing DAF-16 is sufficient to promote longevity (Henderson and Johnson, 2001; Lin et al., 2001). Moreover, the DAF-16 Binding Element (DBE), a DNA motif DAF-16 is known to bind to is present in the promoters of 78% of C. elegans’ genes (Furuyama et al., 2000; Kenyon and Murphy, 2006), whereas only a small fraction of these genes were found to be DAF-16 targets in young adults (Murphy et al., 2003; Niu et al., 2011). Hence, DBE is insufficient to predict DAF-16 target gene expression. These observations point to additional mechanisms that control or assist the transcriptional activities of DAF-16. Indeed, when DAF-16/FOXO enters the nucleus in response to reduced insulin/IGF-1 signaling, it interacts with several co-factors/regulators that are important to modulate its activity. These include regulators such as the suppressor of MEK (SMK-1/SMEK) and host cell factor-1 (HCF-1), and finally co-factors like the chromatin remodeler SWI/SNF (Wolff et al., 2006; Lapierre and Hansen, 2012; Riedel et al., 2013). These regulators are essential for the distinct downstream functions of DAF-16/FOXO.
2.1 SMK-1 is an essential regulator of DAF-16-mediated longevity under low IIS
SMK-1 is the C. elegans ortholog of the protein phosphatase 4 (PP4) regulatory subunit 3 which was first identified in Dictyostelium as a suppressor of the MEK1 null phenotype (Mendoza et al., 2005; Chen et al., 2008).
SMK-1/SMEK has several distinct roles in different organisms. It is important for DNA repair during DNA replication by dephosphorylating gamma-H2AX (Chowdhury et al., 2008), asymmetric cell division in Drosophila by modulating the localization of Miranda (Sousa-Nunes, Chia and Somers, 2009), suppression of brachyury expression in embryonic stem cells (ESCs) (Lyu, Jho and Lu, 2011), the regulation of hepatic glucose metabolism in mice via dephosphorylation of cAMP-response element binding protein regulated transcriptional coactivator 2 (CRTC2) (Yoon et al., 2010), neuronal differentiation of neuronal stem cell (NSC) by negatively regulating Par3 (Lyu et al., 2013) and prevention of the recruitment of Mbd3/NuRD complex to the target promoters of genes important for neuronal differentiation during cortical development by interacting and mediating the degradation of Mbd3 via the ubiquitin-proteasome system (Moon et al., 2017). In a recent study, PP4/SMEK1 complex was also shown to be important for promoting miRNA biogenesis, by antagonizing the MAPK cascade by dephosphorylating a core co-factor HYL1 (Hyponastic Leaves 1) in Arabidopsis (Su et al., 2017).
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Additionally and interestingly, in C. elegans SMK-1 was shown to be an essential positive regulator of DAF-16 (Wolff et al., 2006). It is required for DAF-16-driven longevity and resistance to oxidative stress, to ultraviolet radiation, and to pathogens. However, it is not required for DAF-16 to promote thermotolerance, to promote dauer formation or to delay reproduction in IIS mutants (Wolff et al., 2006). This suggests that SMK-1 genetically interacts with DAF-16/FOXO but regulates only a fraction of DAF-16/FOXO functions – likely by influencing the expression of only a specific subset of DAF-16’s target genes. Nevertheless, the mechanism by which SMK-1 controls this subset of DAF-16/FOXO target genes has remained entirely unclear (Murphy and Hu, 2013). Further studies should be conducted to understand by which mechanisms SMK-1 influences DAF-16 functions.
2.2 The helix-loop-helix (HLH) transcription factor HLH-30/TFEB collaborates with DAF-16/FOXO to provide stress resistance and longevity under low IIS
HLH-30 is a member of a group of 42 helix-loop-helix (HLH) transcription factors in C. elegans and the closest ortholog to the mammalian transcription factor EB (TFEB) (Rehli et al., 1999).
C. elegans HLH-30 was found to induce autophagy and in this regard to also be required for lifespan extension driven by a variety of longevity promoting pathways (Lapierre et al., 2013). Autophagy is a lysosome-dependent catabolic process, which is activated by starvation and negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1) (Sengupta, Peterson and Sabatini, 2010). The resulting breakdown products that emerge upon autophagy are used to produce energy and to create new cellular components (Settembre et al., 2013). Analogous to C. elegans, the role of TFEB in the transcriptional regulation of autophagy and lysosomal biogenesis genes is conserved in mammals (Settembre et al., 2011). Moreover, it has a conserved role in the mobilization of lipid stores in response to starvation (O’Rourke and Ruvkun, 2013; Settembre et al., 2013).
In addition to its functions in metabolism, HLH-30/TFEB was also found to be essential for host defense against infection, both in C. elegans and mammals (Visvikis et al., 2014). By virtue of its role in stress responses, several studies have been conducted to understand the beneficial effect of this nutritionally controlled stress-response factor as a potential therapeutic target in several lysosomal and protein aggregation disorders (Decressac et al., 2013; Pastore et al., 2013; Spampanato et al., 2013).
Interestingly, in a recent study, where DAF-16 co-factors were identified comprehensively by large scale IP and mass spectrometry (Riedel et al., 2013), HLH-30 was identified as a binding partner of DAF-16. This finding brings a new perspective to a possible synergy between these two transcription factors for the regulation of stress response and longevity related genes. However, further studies were required to understand the biological significance of this physical interaction.
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2.3. Other regulators and co-factors of DAF-16 in IIS longevity pathway DAF-16/FOXO is directly regulated by several kinases in addition to AKTs, such as AMP-dependent protein kinase (AMPK) (Apfeld et al., 2004), Ste20-like kinase CST-1/MST-1 (Lehtinen et al., 2006) and c-Jun N-terminal kinase JNK-1 (Oh et al., 2005). Further important regulators are the arginine methyltransferase PRMT-1 (Takahashi et al., 2011), the E3 ubiquitin ligase RLE-1 (Li et al., 2007), the ubiquiting hydrolase MATH-33 (Heimbucher et al., 2015), β-catenin (BAR-1) (Essers, 2005), and the NAD+-dependent deacetylase SIR-2.1 (Tissenbaum and Guarente, 2001) – just to mention the most prominent examples. 3. Role of the chromatin landscape in the regulation of longevity by aging-regulatory transcription factors
Aging-regulatory signaling pathways mainly confer their phenotypes by the hands of downstream transcription factors and their gene regulatory actions. Accessibility of these target genes is strongly influenced by the epigenome at these genomic loci, thus regulating the epigenome provides powerful means to influence aging. At the same time, aging-regulatory pathways can also actively shape the epigenome, to influence genome stability and manifest their transcriptional effects.
There are several key players that influence the chromatin landscape and are known to affect aging, such as chromatin remodelers, histone modifiers and histone variants, and eventually pioneer transcription factors which are essential for the recruitment of chromatin remodelers and/or histone modifiers target loci of particular importance. In the course of cellular and organismal aging, the epigenome undergoes substantial changes characterized by the general loss of heterochromatin as well as alterations in histone marks and DNA methylation (Booth and Brunet, 2016; Pal and Tyler, 2016). This results in a profound change in the chromosomal architecture, genomic integrity, and gene expression patterns. At the same time, it has been shown that manipulation of the epigenome, e.g. by mutations in histone modifying enzymes, has the ability to change the rate of aging in the organism. In the second chapter of this thesis, the major aging-regulatory signaling pathways and their synergy between transcriptional regulation and the epigenetic landscape will be discussed extensively.
4. Overview of the thesis 4.1 Aim
The aim of this thesis is to gain a better mechanistic understanding of specific transcriptional events that regulate the lifespan and stress resistance of the organism. We used one of the most convenient model organisms, the nematode C. elegans, for this kind of research, to study one of the most important aging-regulatory signaling pathways know to date: IIS with its downstream transcription factor DAF-16/FOXO. In particular, we investigated the roles and mechanisms of an important positive regulator of DAF-16, SMK-1, as well as an important downstream effector, HLH-30. Both proteins and their underlying mechanisms may eventually serve as new and powerful targets for therapeutic approaches to counteract aging process and age-related diseases.
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4.2 Outline
In the first part of the thesis in Chapter 2, we give a general overview about aging regulatory signaling pathways, their downstream transcription factors, and the interplay between their transcriptional events and the epigenome.
In Chapter 3, we characterize the distinct and combinatorial roles of the two transcription factors, DAF-16/FOXO and the helix-loop-helix transcription factor HLH-30/TFEB, under low IIS and also other aging-regulatory stimuli. We show that while fulfilling some distinct and non-overlapping functions, DAF-16/FOXO and HLH-30/TFEB can also form a complex and jointly bind and regulate a subset of their target genes. This results in sophisticated combinatorial gene regulation, yielding perfectly tuned responses to promote stress resistance, longevity, and regulate certain aspects of development.
In Chapter 4, we focus on one of the essential positive regulators of DAF-16/FOXO, SMK-1/SMEK, and illustrate one of the mechanisms through which SMK-1 regulates DAF-16-mediated downstream processes. In order to determine this mechanism, we identified the binding partners of SMK-1 by large scale IP followed by mass spectrometry and observed that SMK-1 is part of a specific Protein Phosphatase 4 (PP4) complex. By genetic
and biochemical analyses we then show that PP4SMK-1 promotes Pol II recruitment and
subsequent transcriptional initiation at the promoter regions of a subset of DAF-16-activated genes via dephosphorylating the transcription initiation/elongation factor SPT-5/ SUPT5H and thereby mediating stress resistance and longevity under conditions of low IIS. In Chapter 5, we give an overall summary of the studies in this thesis, discuss them in the greater context of aging research, and highlight important remaining questions in the field that still need to be addressed in future studies.
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Chapter 2
Regulation of age-related decline by
transcription factors and their
crosstalk with the epigenome
Xin Zhou, Ilke Sen, Xin Xuan Lin, Christian Riedel
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Abstract
Aging is a complex phenomenon, where damage accumulation, increasing deregulation of biological pathways, and loss of cellular homeostasis lead to the decline of organismal functions over time. Interestingly, aging is not entirely a stochastic process and progressing at a constant rate, but it’s subject to extensive regulation, in the hands of an elaborate and highly interconnected signaling network. This network can integrate a variety of aging-regulatory stimuli, i.e. fertility, nutrient availability, or diverse stresses, and relay them via signaling cascades into gene regulatory events – mostly of genes that confer stress resistance and thus help protect from damage accumulation and homeostasis loss. Transcription factors have long been perceived as the pivotal nodes in this network. Yet, it is well known that the epigenome substantially influences eukaryotic gene regulation, too. A growing body of work has recently underscored the importance of the epigenome also during aging, where it not only undergoes drastic age-dependent changes but also actively influences the aging process. In this review, we introduce the major signaling pathways that regulated age-related decline and discuss the synergy between transcriptional regulation and the epigenetic landscape.
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Introduction
With the advent of modern medicine, acute diseases have become increasingly treatable, leaving aging and age-related diseases as major determinants of our health and longevity. Aging is a highly complex process, thought to result from the exposure to intrinsic and extrinsic stresses that lead to accumulation of molecular damage, deregulation of signaling pathways, loss of cellular homeostasis, stem cell depletion, tissue breakdown and thereby a functional decline of the entire organism over time. Additionally, old individuals are more likely to exhibit age-related diseases, in particular metabolic disorders (e.g. diabetes), neurodegenerative protein aggregation diseases (e.g. Alzheimer’s and Parkinson’s disease), cardiovascular disorders and cancer (1).
The rate at which an organism ages can be highly variable among different environmental conditions, species, or even genotypes, implying that age-related decline is a highly plastic process and subject to extensive regulation, giving hope for pharmaceutical interventions for aging and age-related disorders. However, to identify such interventions we must first acquire a thorough understanding of the underlying mechanisms that can slow aging down. There is an ongoing debate about whether aging itself is regulated or only the mechanisms and pathways that can interfere with it. For simplicity, this review uses the term “aging regulation” for any events that accelerate or decelerate the age-dependent decline of the organism.
Metazoans have evolved sophisticated mechanisms to sense adverse conditions that put their survival and reproduction at risk, such as lack of nutrients, exposure to toxins, irradiation, pathogens, heat, or changes in fertility. Such information is then relayed by various signaling pathways to trigger compensatory measures, to large extent the activation of stress responses and repair pathways (Figure 1). Some of the best-characterized signaling pathways include the nutrient sensing insulin/IGF signaling (IIS), mechanistic target of rapamycin (mTOR) signaling, AMP kinase signaling, the unfolded protein response, and fertility-related signals from the germline (2, 3). The centerpieces of these signaling pathways are downstream transcription factors, the activation of which converts environmental or physiological cues into a wide range of cellular responses that help to combat unfavorable conditions. On the other hand, loss of such transcription factors tends to profoundly impair the crucial gene expression changes needed for these responses and leads to a shorter lifespan (2, 3).
It is well established that transcriptional regulation is conferred not only by transcription factors, but also by the epigenetic landscape at their target genes. The latter controls accessibility of these genomic loci by transcription factors and the transcription machinery. The epigenetic landscape is regulated through a variety of mechanisms on different levels. First, DNA can be covalently modified, in particular by the methylation of cytosines, commonly leading to inaccessibility and gene repression. DNA is then folded around histone octamers to form nucleosomes, the smallest chromatin entities. Nucleosomes can be covalently modified by a plethora of posttranslational histone modifications or the deposition of histone variants. Furthermore, nucleosomes can be reorganized (assembled, disassembled or repositioned) by ATP-dependent chromatin remodelers (4).
Finally, the spatial organization of chromatin in the nucleus is controlled by dynamic interactions between chromatin regions and their interaction with the nuclear lamina, a filamentous protein meshwork lining the inner nuclear membrane (5).
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Figure 1. Overview of the major aging-regulatory signaling pathways and their downstream transcription factors, relaying distress signals into aging-preventive transcriptional responses.
The various pathways and transcription factors shown are mentioned in the section of ‘transcription factors as central components of aging regulatory signaling pathways’.
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All of the aforementioned alterations in chromatin compose the epigenome, since the primary DNA sequence remains unchanged. And they confer an additional layer of tightly regulated, dynamic, and reversible gene regulation that interconnects with transcription factor activities. The epigenome participates in both, short-term and long-term regulatory events, ranging from the response to acute environmental stresses to the involvement in chronic pathologies. Therefore it comes as no surprise that a number of epigenetic changes have been documented in the context of cellular and organismal aging (6–8). Aged mammalian cells suffer from a general loss of heterochromatin, characterized by loss of repressive histone marks, reduction of nucleosome occupancy, and DNA hypomethylation. Meanwhile, DNA hypermethylation emerges within the active genomic regions (6, 7). These epigenetic alterations result in significant shifts in gene expression, including the expression of components and targets of aging regulatory signaling pathways. Interestingly, while being heavily influenced by the epigenome, aging regulatory pathways also actively shape the epigenome, often through the activities of their transcription factors that can either recruit chromatin remodelers/modifiers or alter their expression. In this review, we take the opportunity to discuss the current understanding of transcriptional aging regulation with its orchestration of signaling pathways, transcription factors, and epigenetic mechanisms.
Transcription factors as central components of aging-regulatory signaling pathways
Insulin/IGF1-like signaling
Aging is subject to regulation by a broad network of signaling pathways, many of which involve downstream transcription factors that convert signals of unfavorable conditions into appropriate gene expression changes, i.e. of genes involved in metabolism and stress responses, to protect the organism from these conditions, slow down aging, and thereby ensure its survival. Notably, when triggered under favorable conditions, these pathways can also dramatically extend the lifespan of an animal. The first aging-associated pathway, namely the highly conserved insulin/IGF signaling (IIS) pathway, was discovered in the nematode Caenorhabditis elegans (9–11). Mutations in several IIS components, such as the insulin/IGF receptor homolog daf-2 (11) and the PI3 kinase age-1 (9, 10), led to dramatic lifespan extension and increased stress resistance. Similar effects were later confirmed in other metazoan species (12). The main lifespan-regulatory output of the IIS pathway is conferred by forkhead box protein O (FOXO) transcription factors and their gene regulatory activities, as FOXO loss-of-function mutations in diverse organisms, e.g. in C. elegans (where the only FOXO transcription factor is called DAF-16), suppress most of the longevity phenotypes (13). In line with the FOXO/DAF-16-dependent effects observed in C. elegans, population studies of various human centenarian cohorts revealed a strong association between IIS polymorphisms and longevity (14–21). In particular, several genetic variants of FOXO3 are more frequently found in centenarians than in younger individuals and are linked to longevity-related traits (19). Briefly, under favorable conditions that maintain high insulin/IGF signaling, the insulin receptor tyrosine kinase recruits and phosphorylates insulin receptor substrate (IRS) proteins, which lead to the activation of AKT kinases through a PI3 kinase-PDK1-AKT cascade (22). Eventually, FOXOs are phosphorylated by AKT (23–25) and SGK (26, 27) kinases, which sequesters the transcription factors by binding to 14-3-3 proteins in the cytoplasm, away from their target genes (28–30). Such effects are negatively regulated by various phosphatases, most notably PTEN that reverts the effects of PI3 kinase (31). Under low IIS, these phosphorylation events on FOXO no longer take place, leading to
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FOXOs’ release from cytoplasmic sequestration and permitting its entry into the nucleus for transcriptional regulation of aging-preventive and thus longevity-promoting genes. FOXOs predominantly activate gene expression by promoter binding, promotion of HDAC activity, and the recruitment of co-activators (32). A study in C. elegans identified a broad variety of FOXO/DAF-16 binding partners, including co-activators, involved in diverse biological processes (33), which illustrates the potential complexity of the mechanisms used by FOXO/DAF-16.
In addition to FOXOs, IIS also regulates other transcription factors involved in aging regulation. In C. elegans, such transcription factors include the heat shock transcription factor HSF-1 (34–36) and the Nrf family transcription factor SKN-1 (37). Both transcription factors contribute to the lifespan extension induced by low IIS. Along with FOXO/DAF-16, HSF-1 activates the expression of small heat shock proteins (38) and other stress resistance genes, such as PAT-10 (39), while SKN-1 mainly regulates oxidative stress response and is required for lifespan extension induced by dietary restriction (40, 41). Notably, the two factors seem to act in distinct manners: 1) HSF-1, but not SKN-1, extends lifespan in a DAF-16-dependent manner (35, 37). 2) High IIS prevents HSF-1 and SKN-1 from nuclear entry through different mechanisms: For HSF-1 it promotes formation of an inactive DDL-1/DDL-2/HSF-1 protein complex in the cytoplasm (36), while SKN-1 undergoes phosphorylation by AKT kinases and 14-3-3-dependent sequestration in a manner similar to FOXOs (37).
Mechanistic target of rapamycin (mTOR) signaling
The mTOR pathway was first linked to aging in the yeast S. cerevisiae, in which deletion of a major downstream factor, a ribosomal protein S6 kinase (S6K) homolog, doubled chronological lifespan (42). mTOR is a serine/threonine kinase that regulates metabolism through nutrient and hormone sensing (43). It functions in two protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (44), promoting anabolic processes (e.g. protein, lipid, and nucleotide synthesis) and repressing catabolic processes such as autophagy, when activated by nutrients and growth factor signals (44–46). mTORC1 can be activated by amino acids through RAG GTPases, which allows it to respond to the availability of nutrients across eukaryotes. However in metazoans, mTOR receives and coordinates yet additional upstream growth signals, including insulin/IGF, EGF, and multiple cytokines (47). Notably, mTOR signaling and IIS are intertwined (48), with IIS regulating mTORC1 (49) and with mTORC1/2 influencing IIS at multiple points (44, 48, 50). Under high IIS, AKT inhibits tuberous sclerosis protein 2 (TSC2), a suppressor of the mTORC1 activator RHEB, and thereby allows for the activation of mTORC1 (49). Most effects of mTOR activity are conferred by two means: 1) direct impact on protein synthesis, and 2) transcriptional regulation. Regarding protein synthesis, mTORC1 activation leads to the phosphorylation of the translation repressor protein, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), thus derepressing the eukaryotic translation initiation factor 4E (eIF4E) to promote protein synthesis (51). The full picture of transcriptional regulation by mTOR, however, remains elusive. Nevertheless, mTOR pathway, like IIS, deploys transcription factors to regulate downstream target genes. We give some examples in the later parts of this section.
Inhibition of mTORC1 by rapamycin and mutations in mTOR or the mTORC1 component Raptor exhibit lifespan extending effects in yeast (52, 53), C. elegans (54, 55), D.
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melanogaster (56) and mice (57–60), establishing mTORC1 as a major regulator of aging. Knockdown of the mTORC2 component Rictor also extends lifespan in C. elegans (61). Dietary restriction (DR), an environmental influence that can extend lifespan in multiple organisms (62, 63), largely acts through mTORC1 signaling, as ablation of mTOR or S6K attenuates the lifespan extending effect of dietary restriction in several species (53, 56). Much of the longevity by mTOR inhibition has been attributed to the global reduction of protein synthesis, as conditions that inhibit translation (e.g. reduced levels of ribosomal proteins or treatment with protein synthesis inhibitor) are sufficient to induce lifespan extension in C. elegans (64). Interestingly, while overall protein synthesis is reduced under mTOR inhibition, subsets of stress response and metabolic genes have been shown to be differentially translated at higher efficiency in multiple species (65), suggesting selectivity in translation regulation by mTOR.
In addition to protein synthesis, mTOR inhibition confers transcriptional regulation of various genes, many of which have the ability to combat stress conditions. For example, inhibition of mTORC1 helps to activate the heat shock transcription factor HSF1 upon heat stress in mammalian cells (66) as well as Nrf2/SKN-1 in mice and C. elegans (61), whereas mTORC1 activates the hypoxic response transcription factor HIF1 (67). Moreover, in C. elegans lifespan extension by mTOR inhibition was found to depend on the activities of Nrf2/SKN-1 (61) and FOXA/PHA-4, the latter being a forkhead transcription factor (68) that engages in dietary restriction-mediated longevity and promotes autophagy in response to TOR inhibition and germline removal(69, 70).
AMP-activated protein kinase (AMPK) signaling
AMP-activated protein kinase (AMPK), as indicated by its name, is triggered when the intracellular ATP/AMP ratio is low. It is crucial for energy metabolism as well as stress responses. The activity of AMPK can be stimulated by dietary restriction and has been found to decline with age, implying a connection between AMPK and aging regulation (71). Indeed, AMPK counteracts aging via an integrated signaling network that involves several well-known aging regulatory pathways. A prime example is the lifespan extending effect of AMPK through mTOR inhibition. Activated AMPK confers phosphorylation of the mTORC1 component Raptor and the mTORC1 inhibitor tuberous sclerosis protein 2 (TSC2), which entails repression of mTOR signaling (72, 73). Also among the various AMPK targets, direct or indirect, are several transcription factors. The mammalian FOXO3 transcription factor of the IIS pathway as well as its C. elegans ortholog DAF-16 have been identified as direct targets of AMPK. Under signals that activate FOXO/DAF-16, phosphorylation by AMPK at multiple regulatory sites activates FOXO/DAF-16-mediated transcription without affecting its nuclear localization (74, 75). In addition to such direct regulation, AMPK can also influence regulators of FOXO. For example, in liver, where AMPK plays a slightly different role, it can be activated in response to the presence of adipokines and as a consequence phosphorylates and thereby inhibits class II histone deacetylases (HDACs), which prevents their nuclear entry and activation of FOXO by deacetylation (76). In C. elegans, AMPK also directly phosphorylates CRTC, a co-activator of the transcription factor cAMP response element–binding protein CREB/CRH-1. This detains CRTC in the cytoplasm and thereby prevents CREB/CRH-1 from transcriptional regulation, leading to lifespan-extending gene expression changes (77).
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Finally, AMPK activates Nrf2/SKN-1 (78, 79) and represses NF-kB (80), the latter being a transcription factor involved in inflammaging – a phenomenon that we will discuss in a later paragraph.
Sirtuin activity
Sirtuins are NAD-dependent protein deacetylases that regulate a wide range of biological functions. They first caught attention as anti-aging factors in yeast, where the founding member of this protein family, SIR2, extends lifespan by suppressing the generation of toxic extrachromosomal rDNA circles (81). The pro-longevity effects of sirtuins were confirmed in metazoan species, in spite of distinct mechanisms as well as some discordances in C. elegans and D. melanogaster (82, 83). Nevertheless, some evidence suggests that overexpression of sirtuins in C. elegans and D. melanogaster promotes longevity, too (82). The dependence on NAD suggests a link between sirtuin activity and the metabolic state of the organism. Indeed, in yeast, nematodes, and flies dietary restriction leads to increased sirtuin activity, which is required for dietary restriction to extend lifespan. Sirtuins regulate a variety of protein substrates, including several transcription factors involved in aging-related signaling pathways. In C. elegans, overexpression of the sirtuin gene sir-2.1 is thought to increase lifespan by activating FOXO/DAF-16 (29, 84). SIR-2.1 directly interacts with 14-3-3 and FOXO/DAF-16, and by NAD-dependent deacetylation of DAF-16 it promotes expression of its target genes – an energy- and DAF-16-dependent longevity control mechanism independent of IIS (85, 86). In mammals, the closest SIR2 ortholog SIRT1 deacetylates several transcription factors involved in stress response and metabolism: By deacetylation of p53 and FOXOs, SIRT1 activity leads to reduced apoptosis and activation of DNA repair genes (87–90). Furthermore, by deacetylation of PGC1α, SIRT1 promotes mitochondrial biogenesis and gluconeogenesis (91). The hypoxic response factor HIF1α, on the contrary, is suppressed by SIRT1 activity. This suppression is lost in hypoxia due to NAD depletion, allowing the activation of HIF-1 in such conditions (92). Yet another negatively regulated SIRT1 target is NF-kB (93). Beyond SIRT1, mammals express six additional sirtuins (SIRT2-7). Although less understood than SIRT1, they confer aging-related functions, too. In mitochondria, SIRT3, 4, and 5 deacetylate and activate enzymes involved in energy metabolism and oxidative stress resistance (94). SIRT6 targets several sites on the histone H3 (K9, K18 and K56) and non-histone proteins to maintain telomere stability and promote chromatin changes required for DNA repair (95). Overexpression of SIRT6 extends the lifespan of male mice (96).
Signals from the gonad
Removal of the germline has lifespan extending effects in C. elegans and Drosophila (97, 98). However, the connection between reproduction and longevity reaches beyond the commonly speculated competition between germline and soma for the same resources. First evidence came from the nematode C. elegans, where elimination of the germ cells, genetically (by glp-1 or mes-1 mutation) or physically (by laser ablation), significantly extends lifespan (98, 99). However, removal of the entire gonad does not extend lifespan, suggesting that the somatic parts of the gonad are required for longevity induced by germline removal. Additionally in mice, transplantation of ovaries from young females prolongs lifespan of old recipients (100). These phenomena imply that the reproductive system
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produces aging-regulatory signals which eventually influence the lifespan of the whole organism. In search for the gonadal cell types contributing to such signals in C. elegans, surprisingly, sperm and oocyte-deficient mutant animals exhibited no lifespan changes, whereas genetic manipulations of germline stem cells (GSCs) were found sufficient to change lifespan (99). GLP-1, the C. elegans homologe of Notch, is required for the maintenance of a normal GSC identity (101). glp-1 loss-of-function mutants have arrested GSCs and live longer than wild type, while glp-1 gain-of-function mutations can cause GSC overexpansion and shorten lifespan. Such observations directly connect GSCs and presumably their crosstalk with the somatic gonad to organismal aging regulation. In the tissues receiving these signals from the germline, two transcription factors are essential for the gonad-mediated longevity: FXR/DAF-12 (98, 102, 103) and FOXO/DAF-16 (98, 104, 105). FXR/DAF-12 is a nuclear hormone receptor downstream of TGFβ signaling (reviewed in (106)), which is consistent with the existence of an endocrine signaling axis between the gonad and other parts of the organism. Search for the signal in C. elegans eventually showed that loss of components in the synthesis pathway of dafachronic acid (DA), a known activator to DAF-12, completely abolishes the longevity of germline-less worms (102). DA supplementation restores the negative effects of DA synthesis pathway mutations in a DAF-12 dependent manner. Likewise, DAF-12 dependent lifespan extension is observed with DA supplementation in worms with whole gonad loss (103). It could be concluded that DA biosynthesis and DAF-12 are essential for the longevity mediated by absence of the germline, and that the source of the DA signal is likely to be the somatic gonad, secreting DA in the absence of GSCs in their vicinity. One of the identified downstream mechanisms goes through FOXO/DAF-16 and the transcription elongation factor TCERG1/TCER-1. In C. elegans, particularly intestinal DAF-16 and TCER-1 work together to enhance the expression of genes involved in lipid synthesis and breakdown that are important for promoting longevity of germline-less animals (104, 105). Consistently, nuclear translocation of DAF-16 is observed specifically in the intestine in adult glp-1 mutant animals, and expression of a constitutively nuclear DAF-16 protein in the intestine is sufficient to fully restore longevity in germline-less DA-deficient glp-1; daf-16; daf-9 animals. In short, DA-DAF-12 signaling promotes the nuclear translocation of DAF-16 in response to germline removal (107–109). However, expression of a constitutive nuclear DAF-16 protein is unable to restore the longevity in glp-1; daf-16; daf-12 mutant animals (109), suggesting that DAF-12 has an additional role, either in parallel or downstream of DAF-16, to achieve longevity upon germline loss.
Signals regulating mitochondrial maintenance
Mitochondria, as the power plants of the cell, not only govern the energy metabolism, but also play an important role during aging. Their dysfunction is considered one of the hallmarks and may even be a driver of aging (110). Thus it is important for every cell to maintain a pool of healthy mitochondria. Several mechanisms are in place to achieve this, regulating the biogenesis of mitochondria, the structure of the mitochondrial network (111), the maintenance of a healthy mitochondrial proteome, and the removal of old and damaged mitochondria. In this paragraph we will discuss the mechanisms controlling mitochondrial biogenesis and removal, while we focus on the mitochondrial protein homeostasis in a later section of this review.
