**4. Discussion**

#### **4.1 The promoter regions of the shelterin-encoding genes coordinately respond to CR mimetic drugs**

In the present study, 5'-flanking regions of different human telomere-associated protein factor-encoding genes were isolated, and these Luc reporter plasmids were used for transient transfection assays. The shelterin- or telomere-associated protein-encoding genes, including *TERT*, *TERC*, *DKC1*, and double-stranded break repair protein-encoding genes,

Characterization of 5'-Flanking Regions of Various

**determinant of longevity** 

such as RNA interference.

are consistent with the concept of hormesis.

Human Telomere Maintenance Factor-Encoding Genes 593

species (ROS) generated by mitochondria and damages to DNA including telomeric regions of the chromosomes (Sahin & DePinho, 2010). Recently, it was shown that telomere dysfunction causes activation of p53 which directly represses PGC-1α and PGC-1β, leading to mitochondrial compromise (Sahin et al., 2011). Moreover, an experiment to reactivate telomerase in telomerase-deficient mice ameliorated DNA damage signaling and reversed neurodegeneration (Jaskelioff et al., 2011). These lines of evidence suggest that telomeres exert signals to affect mitochondria along with DNA repair systems. The concept that telomere length-associated signaling stimulates mitochondrial function might have combined the mitochondrial free radical theory with the molecular mechanism of chromosomal maintenance system against DNA damaging stresses. Rsv has been shown to have effect activation of PGC-1α to improve mitochondrial function in mouse brown adipose tissue and muscle (Lagouge et al., 2006). The present study indicates that shelterin protein-encoding gene promoters are simultaneously activated by Rsv treatment. Thereby, accumulation of shelterin proteins might

lead to stabilization of telomeric regions of the chromosome and activation of PGC-1α.

**4.3 Hormesis, the beneficial effects from low doses of toxic stresses, might be a** 

**4.4 Molecules that are involved in the regulation of the aging process** 

The deficiencies in RecQ DNA helicases, including WRN and BLM, are known to cause premature aging (Chu & Hickson, 2009). In the present study, we have observed that CR mimetic drug treatment activates promoters of the shelterin protein-encoding genes, suggesting that CR evokes functions of the telomere maintenance machinery. Hormesis is a phenomenon that generally refers to the beneficial effects from low level toxic or other harmful damage, such as irradiation, heat shock, or food restriction (Schumacher, 2009). High doses of 2DG and Rsv have harmful or toxic effects on cells, leading to cell death or apoptosis (Lin et al., 2003; Cosan, et al., 2010). In contrast, relatively low doses of these CR mimetic reagents, as used in the present study, have effects similar to hormesis. Therefore, resistance to stresses eventually provoked by prolonged low doses of CR mimetic reagents could promote the longevity of organisms. Thus, the results obtained in the present study

From studies of life spans of the *C. elegans*, it has been suggested that the insulin/IGF-1 pathway influences aging (Kenyon, 2010). In this signaling system, DAF-16 (FoxO transcription factor) plays a role in activating genes that act to extend life span (van der Horst & Burgering, 2007). AMP-activated protein kinase (AMPK), which is known to extend the life span of nematodes (Apfeld et al., 2004), phosphorylates FoxO, PGC-1α, and CREB to induce various genes encoding mitochondrial and oxidative metabolism regulating factors (Cantó & Auwerx, 2010). The other biologically important function of AMPK is that it blocks the mTOR (mammalian target of rapamycin) pathway (Cantó & Auwerx, 2010). A recent study suggested that mTOR is a prime target in the genetic control of aging to determine life span and aging in yeast, worms, flies, and mice (Zoncu et al., 2011). The mTOR pathway accelerates growth by regulating signals downstream of insulin/IGF-1 receptors (Zoncu et al., 2011). Activation of mTOR is thought to speed up aging in adulthood, and reduced mTOR signaling would have the opposite effect, acting downstream of dietary restriction. Thus the antiaging effect could be expected by mTOR inhibition, such as dietary restriction, rapamycin, introduction of the AMPK expression vector, and genetic inactivation of mTOR by techniques

such as *ATM* and *ATR*, are conserved among human, mouse and yeast (Stern & Bryan, 2008). Given that these telomere-associated proteins are localized to the telomere t-loop to protect the specific structure, and appear to act in co-operation with each other (O'Sullivan & Karlseder, 2010), their gene expression should be regulated synchronously when the telomeric region needs to be protected. Aging or cellular senescence are thought to be controlled by a genomic maintenance regulatory system (Vieg, 2007). Our hypothesis is that aging or longevity affecting reagents might have an effect on the expression of the telomere-associating proteinencoding genes. The results (Tables 2 and 3) indicate that promoter activities of the shelterinencoding genes are simultaneously up-regulated by Rsv and 2DG in HeLa-S3 cells when they are compared with *PIF1* promoter activity. Previously, we have reported that multiple GCboxes are commonly located in the human *TERT* and *WRN* promoter regions and that might play a role in the positive response to Rsv and 2DG in HeLa-S3 cells (Uchiumi et al., 2010c). Although there are no canonical roles of transcription factor binding elements or their order in these promoter regions, Sp1 binding elements or GC-boxes are found in all of them except 5' upstream of the *POT1* and *TANK2* genes (Fig. 1). Therefore, GC-box binding factors may upregulate this telomere-associated gene expression. However, there are no GC-box like motifs in the 300-bp up-stream regions of the *POT1* and *TANK2* genes, which are relatively AT-rich and contain Oct-1 binding sites. This observation suggests that POU family proteins might also be involved in the positive regulation of these genes. Apparent up-regulation of promoter activities by Rsv and 2DG treatment was observed in the cells transfected with the Luc reporter plasmids containing 200-bp 5'-upstream regions of the *TERT* and *TERC* genes (Tables 2 and 3). It is noteworthy that the duplicated GGAA-motifs are found in both promoter regions (Uchiumi et al., 2010a, Uchiumi et al., 2011b), suggesting that the GGAA-motif binding factors, including Ets family proteins, might be involved in the positive response to the aging or longevity affecting signals.

Previously, we observed elevation of the human *WRN* promoter activity in accordance with activation of telomerase after Rsv and 2DG treatment of HeLa-S3 cells (Uchiumi et al., 2011; Zhou et al., 2009). 2DG suppresses glucose metabolism to establish a limit for the usage or uptake of glucose into cells (Roth et al., 2001). On the other hand, Rsv is known to activate sirtuin family protein deacetylases (Kaeberlein, 2010). It is thought that both Rsv and 2DG are CR mimetic drugs (Stefani et al., 2007; Roth et al., 2001), and that CR can extend the mean and maximum life spans of numerous organisms (Carvallini et al., 2008; Roth et al., 2001). The present study suggests that induction of telomerase activity in concert with upregulation of the telomere-associated protein- or shelterin-encoding gene expression may play a role in regulating the aging process through the telomere maintainance system.

#### **4.2 A possible role for telomere maintenance system in aging/senescence regulation**

Aging or senescence is a complicated biological process involving various regulatory factors (Campisi & d'Adda di Fagagna, 2007; Kuningas et al., 2008; Sanz & Stefanatos, 2008). Aging could be explained by a mitochondrial free radical theory (Benz & Yau, 2008). On the other hand, cellular senescence could be caused by DNA damage or the associated signals on chromosomes (Vieg, 2007). It is well known that cellular senescence is correlated with the cell growth arrest (Campisi & d'Adda di Fagagna, 2007). DNA damage signals activate ATM or ATR, and then phosphrylate p53 to induce transcription of the *CDKN1A* gene that encodes cyclin-dependent kinase inhibitor 1A (p21). These sequentially occurring events arrest the cell cycle at G1-phase (Meek, 2009). Repair of DNA damage will occur at this stage, unless the cell has initiated apoptosis. Thus, aging is thought to be controlled through both reactive oxygen

such as *ATM* and *ATR*, are conserved among human, mouse and yeast (Stern & Bryan, 2008). Given that these telomere-associated proteins are localized to the telomere t-loop to protect the specific structure, and appear to act in co-operation with each other (O'Sullivan & Karlseder, 2010), their gene expression should be regulated synchronously when the telomeric region needs to be protected. Aging or cellular senescence are thought to be controlled by a genomic maintenance regulatory system (Vieg, 2007). Our hypothesis is that aging or longevity affecting reagents might have an effect on the expression of the telomere-associating proteinencoding genes. The results (Tables 2 and 3) indicate that promoter activities of the shelterinencoding genes are simultaneously up-regulated by Rsv and 2DG in HeLa-S3 cells when they are compared with *PIF1* promoter activity. Previously, we have reported that multiple GCboxes are commonly located in the human *TERT* and *WRN* promoter regions and that might play a role in the positive response to Rsv and 2DG in HeLa-S3 cells (Uchiumi et al., 2010c). Although there are no canonical roles of transcription factor binding elements or their order in these promoter regions, Sp1 binding elements or GC-boxes are found in all of them except 5' upstream of the *POT1* and *TANK2* genes (Fig. 1). Therefore, GC-box binding factors may upregulate this telomere-associated gene expression. However, there are no GC-box like motifs in the 300-bp up-stream regions of the *POT1* and *TANK2* genes, which are relatively AT-rich and contain Oct-1 binding sites. This observation suggests that POU family proteins might also be involved in the positive regulation of these genes. Apparent up-regulation of promoter activities by Rsv and 2DG treatment was observed in the cells transfected with the Luc reporter plasmids containing 200-bp 5'-upstream regions of the *TERT* and *TERC* genes (Tables 2 and 3). It is noteworthy that the duplicated GGAA-motifs are found in both promoter regions (Uchiumi et al., 2010a, Uchiumi et al., 2011b), suggesting that the GGAA-motif binding factors, including Ets family proteins, might be involved in the positive response to the aging

Previously, we observed elevation of the human *WRN* promoter activity in accordance with activation of telomerase after Rsv and 2DG treatment of HeLa-S3 cells (Uchiumi et al., 2011; Zhou et al., 2009). 2DG suppresses glucose metabolism to establish a limit for the usage or uptake of glucose into cells (Roth et al., 2001). On the other hand, Rsv is known to activate sirtuin family protein deacetylases (Kaeberlein, 2010). It is thought that both Rsv and 2DG are CR mimetic drugs (Stefani et al., 2007; Roth et al., 2001), and that CR can extend the mean and maximum life spans of numerous organisms (Carvallini et al., 2008; Roth et al., 2001). The present study suggests that induction of telomerase activity in concert with upregulation of the telomere-associated protein- or shelterin-encoding gene expression may play a role in regulating the aging process through the telomere maintainance system.

**4.2 A possible role for telomere maintenance system in aging/senescence regulation**  Aging or senescence is a complicated biological process involving various regulatory factors (Campisi & d'Adda di Fagagna, 2007; Kuningas et al., 2008; Sanz & Stefanatos, 2008). Aging could be explained by a mitochondrial free radical theory (Benz & Yau, 2008). On the other hand, cellular senescence could be caused by DNA damage or the associated signals on chromosomes (Vieg, 2007). It is well known that cellular senescence is correlated with the cell growth arrest (Campisi & d'Adda di Fagagna, 2007). DNA damage signals activate ATM or ATR, and then phosphrylate p53 to induce transcription of the *CDKN1A* gene that encodes cyclin-dependent kinase inhibitor 1A (p21). These sequentially occurring events arrest the cell cycle at G1-phase (Meek, 2009). Repair of DNA damage will occur at this stage, unless the cell has initiated apoptosis. Thus, aging is thought to be controlled through both reactive oxygen

or longevity affecting signals.

species (ROS) generated by mitochondria and damages to DNA including telomeric regions of the chromosomes (Sahin & DePinho, 2010). Recently, it was shown that telomere dysfunction causes activation of p53 which directly represses PGC-1α and PGC-1β, leading to mitochondrial compromise (Sahin et al., 2011). Moreover, an experiment to reactivate telomerase in telomerase-deficient mice ameliorated DNA damage signaling and reversed neurodegeneration (Jaskelioff et al., 2011). These lines of evidence suggest that telomeres exert signals to affect mitochondria along with DNA repair systems. The concept that telomere length-associated signaling stimulates mitochondrial function might have combined the mitochondrial free radical theory with the molecular mechanism of chromosomal maintenance system against DNA damaging stresses. Rsv has been shown to have effect activation of PGC-1α to improve mitochondrial function in mouse brown adipose tissue and muscle (Lagouge et al., 2006). The present study indicates that shelterin protein-encoding gene promoters are simultaneously activated by Rsv treatment. Thereby, accumulation of shelterin proteins might lead to stabilization of telomeric regions of the chromosome and activation of PGC-1α.

#### **4.3 Hormesis, the beneficial effects from low doses of toxic stresses, might be a determinant of longevity**

The deficiencies in RecQ DNA helicases, including WRN and BLM, are known to cause premature aging (Chu & Hickson, 2009). In the present study, we have observed that CR mimetic drug treatment activates promoters of the shelterin protein-encoding genes, suggesting that CR evokes functions of the telomere maintenance machinery. Hormesis is a phenomenon that generally refers to the beneficial effects from low level toxic or other harmful damage, such as irradiation, heat shock, or food restriction (Schumacher, 2009). High doses of 2DG and Rsv have harmful or toxic effects on cells, leading to cell death or apoptosis (Lin et al., 2003; Cosan, et al., 2010). In contrast, relatively low doses of these CR mimetic reagents, as used in the present study, have effects similar to hormesis. Therefore, resistance to stresses eventually provoked by prolonged low doses of CR mimetic reagents could promote the longevity of organisms. Thus, the results obtained in the present study are consistent with the concept of hormesis.

#### **4.4 Molecules that are involved in the regulation of the aging process**

From studies of life spans of the *C. elegans*, it has been suggested that the insulin/IGF-1 pathway influences aging (Kenyon, 2010). In this signaling system, DAF-16 (FoxO transcription factor) plays a role in activating genes that act to extend life span (van der Horst & Burgering, 2007). AMP-activated protein kinase (AMPK), which is known to extend the life span of nematodes (Apfeld et al., 2004), phosphorylates FoxO, PGC-1α, and CREB to induce various genes encoding mitochondrial and oxidative metabolism regulating factors (Cantó & Auwerx, 2010). The other biologically important function of AMPK is that it blocks the mTOR (mammalian target of rapamycin) pathway (Cantó & Auwerx, 2010). A recent study suggested that mTOR is a prime target in the genetic control of aging to determine life span and aging in yeast, worms, flies, and mice (Zoncu et al., 2011). The mTOR pathway accelerates growth by regulating signals downstream of insulin/IGF-1 receptors (Zoncu et al., 2011). Activation of mTOR is thought to speed up aging in adulthood, and reduced mTOR signaling would have the opposite effect, acting downstream of dietary restriction. Thus the antiaging effect could be expected by mTOR inhibition, such as dietary restriction, rapamycin, introduction of the AMPK expression vector, and genetic inactivation of mTOR by techniques such as RNA interference.

Characterization of 5'-Flanking Regions of Various

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#### **5. Conclusions and future perspectives**

It would be advantageous for cells to estimate the state of chromosomes just by monitoring telomeric regions. Monitoring the somatic genes, including promoter, exon, intron or other regions that harbors genetic information, would not work for that purpose, because single or multiple mutations might be lethal to the cell. Thus, microsatellite regions, including telomeres, would be suitable for a DNA damage monitoring system. Recently, it was shown that telomere length regulates mitochondrial function by activating PGC-1α (Sahin et al., 2011). This effect is the same as Rsv treatment (Lagouge et al., 2006). The CR mimetic drugs may have a common role in strengthening telomere maintenance. In the present study, we performed a multiple transfection experiment, which showed that shelterin proteinencoding gene promoters simultaneously respond to CR mimetic drugs in HeLa-S3 cells. Given that anti-aging drugs induce or activate the DNA repair system, especially by maintenance of telomeres, this multiple transfection system has demonstrable potential to contribute to the evaluation and development of such drugs.

#### **6. Acknowledgments**

The authors are grateful to Ryosuke Akiyama for outstanding technical assistance. This work was supported in part by a Research Fellowship from the Research Center for RNA Science, RIST, Tokyo University of Science, Tokyo, Japan.

#### **7. References**


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The authors are grateful to Ryosuke Akiyama for outstanding technical assistance. This work was supported in part by a Research Fellowship from the Research Center for RNA

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**5. Conclusions and future perspectives** 

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**6. Acknowledgments** 

**7. References** 


**1. Introduction** 

chromosome ends.

**30** 

Masaru Ueno *Hiroshima University* 

 *Japan* 

**Roles of DNA Repair Proteins in** 

Most eukaryots have specialized protein-DNA complexs, called telomeres at the ends of natural linear chromosomes. Telomeric DNA consists of a tandemly repeated G-rich sequence. The lengths of telomeric DNAs in *S. pombe*, *S. cerevisiae,* and human are ~300 nucleotids, ~350 nucleotides, and ~10 kb, respectively. The ends of the telomeric DNA have 3' single-stranded overhangs. The protein components of telomeres consists of doublestranded telomere-binding proteins, such as human TRF1 and TRF2, S. pombe Taz1, and single-stranded telomere-binding proteins, such as *S. cerevisiae* Cdc13, *S. pombe* Pot1, and human POT1. DNA double-strand breaks (DSBs) must be repaired to maintain genomic integrity. In contrast, natural chromosome ends should not be recognized as DSBs. The telomere is capped to protect from DNA repair activity. If this capping function is lost, this uncapped telomere is recognized as DNA damage and becomes substrate for DNA repair proteins. The first step in homologous recombination (HR) repair is processing of DNA ends by 5' to 3' degradation to create 3' single-stranded overhangs. The proteins involved in this steps include *S. cerevisiae* Mre11-Rad50-Xrs2 complex (MRX), Sae2, Sgs1, and Dna2. Recent works revealed that proteins involved in the processing of DNA DSB ends are also involved in the processing of capped or uncapped telomere. These facts raised new question of how these proteins are regulated at telomere ends. This chapter will focus on the roles of proteins involved in the processing of DBS ends at capped (functional) and uncapped (dysfunctional) telomere in *S. pombe*, *S. cerevisiae* and human. This chapter will also focus on the functional interactions between telomere-binding proteins and proteins involved in the processing of DBS ends. Resent works revealed that double-stranded and single-stranded telomerebinding proteins play critical roles to control proteins involved in DNA repair at

**2. Roles of proteins involved in DNA end-processing in telomere maintenance**  DNA DSBs are repaired by HR or non-homologous end-joining (NHEJ) [1]. *S. cerevisiae* MRX is involved in both HR and NHEJ [2]. MRX is suggested to be involved in the processng of DSB ends in HR repair. Recently, several other proteins involved the processing have been discovered. Some of the these proteins are also involved in the processing of telomere ends.

In this section, the roles of these proteins in telomere maintenance will be discussed.

**Telomere Maintenance** 

