**Characterization of 5'-Flanking Regions of Various Human Telomere Maintenance Factor-Encoding Genes**

Fumiaki Uchiumi1,4, Takahiro Oyama1, Kensuke Ozaki1 and Sei-ichi Tanuma2,3,4 *1Department of Gene Regulation, Faculty of Pharmaceutical Sciences 2Department of Biochemistry, Faculty of Pharmaceutical Sciences 3Genome and Drug Research Center 4Research Center for RNA Science, RIST, Tokyo University of Science Japan* 

#### **1. Introduction**

Telomeres are the unique nucleoprotein complex structures located at the end of linear eukaryotic chromosomes (Blackburn, 2000; de Lange, 2006). They are composed of TTAGGG repeats that are typically 10 kb at birth and gradually shorten with cell divisions (de Lange, 2006). Telomerase is composed of the protein subunit TERT and the RNA subunit TERC (TR). It elongates the telomere by adding telomeric repeats (Greider & Blackburn, 1987). The 50 to 300 nucleotides from the terminal end of the telomeres are single stranded 3'-protluded Goverhang structures which make the t-loop configuration (de Lange, 2006; Griffith et al., 1999). Mammalian telomeres are included in heterochoromatin and attached to the nuclear matrix (Oberdoerffer & Sinclair, 2007; Gonzalez-Suarez & Gonzalo, 2008). Telomere shortening causes instability of the ends of chromosomes to lead to replicative senescence (O'Sullivan & Karlseder, 2010; Lundblad & Szostak, 1989). Therefore, the ends of telomeres should be protected from damaging or cellular activities. The t-loop structures are regulated by shelterin protein factors, TRF1, TRF2, Rap1, TIN2, TPP1, POT1 (Gilson & Geli, 2007; O'Sullivan & Karlseder, 2010), and Rec Q DNA helicases, WRN and BLM (Chu & Hickson, 2009). TRF1 and TRF2, which bind to duplex telomeric DNA and retain shelterin on the telomere repeats, were shown to interact with various functional proteins (Giannone et al., 2010). Molecular structural analysis of Rap1 revealed that its mechanism of action involves interaction with TRF2 and Taz1 proteins (Chen et al., 2011). A recent study showed that depletion of TPP1 and its partner TIN2 causes a loss of telomerase recruitment to telomeres (Abreu et al., 2010). POT1 is an important regulator of telomerase length, in stimulating the RecQ helicases WRN and BLM (Opresko et al., 2005). Tankyrase-1 (TANK1), which is classified as a poly(ADP-ribose) polymerase family protein, is also known to regulate telomere homeostasis by modifying TRF1 (Smith et al., 1998; Schreiber et al., 2006). Dyskerin, which is encoded by the *DKC1* gene, is a key auxiliary protein that is contained in a Cajal body with TERT (Cohen et al., 2007). Defects in the shelterin components and telomerase are thought to down-regulate telomere structure

Characterization of 5'-Flanking Regions of Various

Name Sequence

associated genes

**2.4 Transient transfection assay** 

Human Telomere Maintenance Factor-Encoding Genes 587

into the *Kpn*I-*Xho*I site of the pGL4-basic vector (pGL4[luc 2.10]) (Promega). The resultant cloned plasmids were designated pGL4-DKC1, pGL4-POT1, pGL4-RAP1, pGL4-TANK1, pGL4-TANK2, pGL4-TIN2, pGL4-TPP1, pGL4-TRF1, and pGL4-TRF2. Clone sequences were confirmed using a DNA Sequencing System (Applied Biosystems, Foster City, CA) with Rv (5'-TAGCAAAATAGGCTGTCCCC-3' and GL (5'-CTTTATGTTTTTGGCGTCTT-

hDysk-7065 5'-TCGGTACCGTGAGCCCAGGCGCAGGCGC-3' AhDysk-7414 5'-ATCTCGAGGGAACGACCGCAGACTCCC-3' hPOT-1509 5'-TCGGTACCTGAGAACTGAATATTGCTGTG-3' AhPOT-1164 5'-ATCTCGAGAATATCATCTTACCAAAGAC-3' hRAP1-5667 5'-TCGGTACCTCGCGGCGCTTCCCAGCCC-3' AhRAP1-5970 5'-ATCTCGAGCTGTCACCGCAGACGCCTC-3' hTANK1-8541 5'-TCGGTACCGACTGAAAGTGAGAAATGC-3' AhTANK1-8860 5'-ATCTCGAGAGCGACGCGACGCCGCCATC-3' hTANK2-4227 5'-TCGGTACCAGGAGAAAGGGATGTGGAAG-3' AhTANK2-4519 5'-ATCTCGAGGCGGCGCGAAGGGTTTGTGG-3' hTIN2-8835 5'-TCGGTACCGCAGGCTCCGCGAAGAAAGC-3' AhTIN2-8508 5'-ATCTCGAGTGGAGAAGCTGACCGTCTC-3' hTPP1-8283 5'-TCGGTACCTCGACGATGCTATCGGGAC-3' AhTPP1-7995 5'-ATCTCGAGCGTGATGACGCAAGAGCGGA-3' hTRF1-1070 5'-TCGGTACCTCCTCCTATCCTAATCTCGC-3' AhTRF1-1371 5'-ATCTCGAGGAAACATCCTCCGCCATGTT-3' hTRF2-9454 5'-TCGGTACCGATCCCGGCCTGTTTTTCAG-3' AhTRF2-9170 5'-ATCTCGAGCGGGGCCCGCCGTCCCGGC-3'

Table 1. Primers used for amplifying 5'-upstream region of various human telomere-

Luc sample (2.5 μL) and Protein Assay Reagent (Bio-Rad Lab., Hercules, CA, USA).

Transient transfection of Luc-reporter plasmids was performed using multi-well culture plates that had been prepared and treated with DNA/DEAE-dextran (Uchiumi et al., 2010a). After 4 h of transfection, 2DG or Rsv was added to the culture medium (Zhou et al., 2009; Uchiumi et al., 2011). After a further incubation (19 to 24 h), cells were collected and lysed with 40 μL of 1 x Cell culture lysis reagent, mixed, and stored at -80°C. Luc assays were performed according to the manufacturer's instructions (Promega). In brief, Luc assay reagent (40 μL) was added to 10 μL of protein sample and mixed briefly. Immediately after mixing, chemiluminescence was measured for 7.5 sec with a Minilumat LB9506 luminometer (Berthold, Bad Wildbad, Germany). Protein assays were performed with the

CC-3') primers purchased from Operon Biotechnologies (Tokyo, Japan).

and length (O'Sullivan & Karlseder, 2010). The shelterin proteins also play important roles in protecting chromosomal ends from being recognized by DNA damage response (DDR) machinery (O'Sullivan & Karlseder, 2010). Although the biological significance of the shelterin complex proteins has been studied, the molecular mechanisms that regulate expression of those genes encoding telomere associated proteins is less well-characterized. We hypothesized that expressions of those telomere-associated protein-encoding genes are regulated by a similar mechanism. In order to analyze these promoter activities promptly, we isolated 200 to 300-bp of the 5'-upstream regions of these telomere regulatory protein-encoding genes and applied them to a multiple transfection assay system (Uchiumi et al., 2010a). Previously, we have observed that *WRN* and *TERT* promoter activities were up-regulated by 2-deoxy-Dglucose (2DG) and *trans*-resveratrol (Rsv) in accordance with the activation of telomerase (Zhou et al., 2009; Uchiumi et al., 2011). A potent inhibitor of glucose metabolism, 2DG is thought to mimic glucose deprivation *in vivo* such that it is mimetic of caloric restriction (CR) (Roth et al., 2001). Resveratrol (Rsv), which is a polyphenol contained in grape skins and red wine, activates sirtuin-mediated deacetylation (Stefani et al., 2007; Knutson & Leeuwenburgh, 2008). We report here that most of the promoters of the shelterin protein-encoding genes positively responded to the CR mimetic agents, 2DG and Rsv. These results suggest that telomerase and telomere maintenance factors are simultaneously regulated at the initiation of the transcription.

#### **2. Materials and methods**

#### **2.1 Chemicals**

The reagents 2-deoxy-D-glucose (2DG) and *trans*-resveratrol (Rsv) were purchased from Wako Chemicals (Tokyo, Japan) and Cayman Chemicals (Ann Arbor, MI), respectively.

#### **2.2 Cells and cell culture**

HeLa-S3 cells (Zhou et al., 2009) were cultured in Dulbecco's modified eagle (DME) medium supplemented with heat-inactivated 10% fetal calf serum (FCS) (Sanko-Pure Chemical, Tokyo, Japan), 2 mM L-glutamine (Invitrogen, CA, USA), penicillin (100 IU/mL) (MEIJI SEIKA, Tokyo, Japan), and streptomycin (100 μg/mL) (MEIJI SEIKA).

#### **2.3 Construction of Luc-reporter plasmids**

Luc reporter plasmids carrying promoter regions for the human *TERT* and *TERC* genes have been constructed and designated as pGL4-TERT, and pGL4-TERC, respectively (Zhou et al., 2009; Uchiumi et al., 2010a). Extraction of DNA from HeLa-S3 cells, and subsequent PCR for the promoter regions of interest were performed as described previously (Uchiumi et al., 2010a; Zhou et al., 2009). Primer-sets were designed against human genomic sequences from the Cross-Ref NCBI-data base (http://www.ncbi.nlm.nih.gov/sites/gquery/) for the 5' flanking regions of the genes of interest (Table. 1). PrimeStar Taq polymerase (Takara, Kyoto, Japan) was used for all amplifications.

Amplification conditions consisted of: 30 cycles of 98°C for 10 sec, 55°C for 5 sec, and 72°C for 30 sec. PCR products were digested with *Kpn*I and *Xho*I and then separated on 0.9% agarose gels.

After electrophoresis, DNA bands of the correct length were recovered from the gel with Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and subcloned

and length (O'Sullivan & Karlseder, 2010). The shelterin proteins also play important roles in protecting chromosomal ends from being recognized by DNA damage response (DDR) machinery (O'Sullivan & Karlseder, 2010). Although the biological significance of the shelterin complex proteins has been studied, the molecular mechanisms that regulate expression of those genes encoding telomere associated proteins is less well-characterized. We hypothesized that expressions of those telomere-associated protein-encoding genes are regulated by a similar mechanism. In order to analyze these promoter activities promptly, we isolated 200 to 300-bp of the 5'-upstream regions of these telomere regulatory protein-encoding genes and applied them to a multiple transfection assay system (Uchiumi et al., 2010a). Previously, we have observed that *WRN* and *TERT* promoter activities were up-regulated by 2-deoxy-Dglucose (2DG) and *trans*-resveratrol (Rsv) in accordance with the activation of telomerase (Zhou et al., 2009; Uchiumi et al., 2011). A potent inhibitor of glucose metabolism, 2DG is thought to mimic glucose deprivation *in vivo* such that it is mimetic of caloric restriction (CR) (Roth et al., 2001). Resveratrol (Rsv), which is a polyphenol contained in grape skins and red wine, activates sirtuin-mediated deacetylation (Stefani et al., 2007; Knutson & Leeuwenburgh, 2008). We report here that most of the promoters of the shelterin protein-encoding genes positively responded to the CR mimetic agents, 2DG and Rsv. These results suggest that telomerase and telomere maintenance factors are simultaneously regulated at the initiation of

The reagents 2-deoxy-D-glucose (2DG) and *trans*-resveratrol (Rsv) were purchased from Wako Chemicals (Tokyo, Japan) and Cayman Chemicals (Ann Arbor, MI), respectively.

HeLa-S3 cells (Zhou et al., 2009) were cultured in Dulbecco's modified eagle (DME) medium supplemented with heat-inactivated 10% fetal calf serum (FCS) (Sanko-Pure Chemical, Tokyo, Japan), 2 mM L-glutamine (Invitrogen, CA, USA), penicillin (100 IU/mL) (MEIJI

Luc reporter plasmids carrying promoter regions for the human *TERT* and *TERC* genes have been constructed and designated as pGL4-TERT, and pGL4-TERC, respectively (Zhou et al., 2009; Uchiumi et al., 2010a). Extraction of DNA from HeLa-S3 cells, and subsequent PCR for the promoter regions of interest were performed as described previously (Uchiumi et al., 2010a; Zhou et al., 2009). Primer-sets were designed against human genomic sequences from the Cross-Ref NCBI-data base (http://www.ncbi.nlm.nih.gov/sites/gquery/) for the 5' flanking regions of the genes of interest (Table. 1). PrimeStar Taq polymerase (Takara,

Amplification conditions consisted of: 30 cycles of 98°C for 10 sec, 55°C for 5 sec, and 72°C for 30 sec. PCR products were digested with *Kpn*I and *Xho*I and then separated on 0.9%

After electrophoresis, DNA bands of the correct length were recovered from the gel with Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and subcloned

SEIKA, Tokyo, Japan), and streptomycin (100 μg/mL) (MEIJI SEIKA).

the transcription.

**2.1 Chemicals** 

agarose gels.

**2. Materials and methods** 

**2.2 Cells and cell culture** 

**2.3 Construction of Luc-reporter plasmids** 

Kyoto, Japan) was used for all amplifications.

into the *Kpn*I-*Xho*I site of the pGL4-basic vector (pGL4[luc 2.10]) (Promega). The resultant cloned plasmids were designated pGL4-DKC1, pGL4-POT1, pGL4-RAP1, pGL4-TANK1, pGL4-TANK2, pGL4-TIN2, pGL4-TPP1, pGL4-TRF1, and pGL4-TRF2. Clone sequences were confirmed using a DNA Sequencing System (Applied Biosystems, Foster City, CA) with Rv (5'-TAGCAAAATAGGCTGTCCCC-3' and GL (5'-CTTTATGTTTTTGGCGTCTT-CC-3') primers purchased from Operon Biotechnologies (Tokyo, Japan).


Table 1. Primers used for amplifying 5'-upstream region of various human telomereassociated genes
