2.6. Electrophoretic mobility shift assay (EMSA)

Synthetic double-stranded and 50 biotin-labeled and unlabeled probes were obtainedfromTakara.Inbrief,purifiedhumanrecombinantGST-AP-2aproteinand labeledprobeswereincubatedin30 ml bindingreactionmixturefor30 min at4 8C. To prevent nonspecific binding, ply (di-dC) and salmon sperm DNA were added to the mixture. Then, the samples were separated on 6% polyacrylamide gel at4 8Cfor 90 min at 100 V in 0.5(cid:4)TG buffer when the gels were pre-run for 50 min at 100 V. After electrophoresis, transfer was performed at 4 8C and 250 mA for 90 min. The products were detected by Stabilized Streptavidin-Horseradish Peroxidase Conju- gate(Roche).Unlabeledprobesat100-foldmolarexcesswereaddedtothereaction for competition.

Computational analysis showed that the third amplicon of ATM promoter region contains two Sac II restriction enzymesites, one of them is in the SNP (C/T) only when the T ! C institution at nucleotide 116 is present (Fig. 2). Therefore, we developed PCR- restriction fragment length polymorphism (RFLP) to determine genotypes. Before genotyping was carried out, we examined the RFLP reliability, and both direct sequencing and Sac II restriction digestion detected 41 DNA samples. The concurrence rate of both

(A) : promoter -125 (C/T)‘ATM —————226bp 1 +4240bpFA —__———_ 287pF2 ——F3(B) (C)a PeTeeceye sceneTecectocccacY

Fig.1.ThesinglenuclearpolymorphisminATMpromoterwasidentifiedbytheDHPLCandsequencing.(A)AsimplifiedschematicdiagramshowedthreepairsofPCRprimers tocreatethreeoverlappingfragmentsofATMpromoterregionforDHPLCanalysis.(B)SequencevariantsweredetectedbyDHPLC.PCRproductsweredenaturedbyheatingat 96 8C for 5 min and then gradually cooling down to 25 8C over a period of 30 min. DHPLC was carried out on a Transgenomic Wave DNA Fragment Analysis System (Transgenomic Inc., Omaha, NE, USA). The DNA molecules eluted from the column were detected by scanning with a UV detector at 260 nm. Profile (arrow) indicated the sample was heterozygote. (C) The SNP was identified by sequencing. Sequencing of SNP (C > T) was from genomic DNA of 23 long-lived individuals with familial history of longevity and 18 control subjects. Typical sequence results were shown. The arrow indicated the nucleotide variants.

methods was 100%, indicating that the PCR-RFLP method is reliable (data not shown).

(Supplement Tables 1 and 2), and far higher frequency in LLIs was observed (Supplement Table 3). In addition, association analysis did notshowanygenderspecificity(datanotshown).Thedistributionof the observed genotypes in long-lived individuals and control subjects conformed to the Hardy–Weinberg equilibrium.

Genotyping was conducted by PCR-RFLP in 789 long-lived individuals and 886 control subjects. Comparison of genotypic frequencies revealed that there is significant association between the genotypes and longevity (P = 0.01), but not alleles (Table 2). LLIs obviously showed higher frequency of CT genotype than controls (53.0% versus 45.9%, P = 0.004). Furthermore, the percentage of CT eterozygote increases with age in both LLIs and controls

3.3. AP-2a binds to the SNP site to repress ATM transcription

To examine whether the SNP regulates the ATM mRNA expression in vivo, we measured the ATM mRNA levels in peripheral mononuclear cells from human individuals by RT- PCR (McKinnon, 2004). The results from 42 healthy old individuals showed that TT genotype has highest level of ATM mRNA expressions than the other two genotypes (Fig. 3A), although no significant differences were found between CC and CT genotypes (CC versus TT, P = 0.001; CT versus TT, P = 0.006; CC versus CT, P = 0.818), but obvious tendency in level of ATM expression from CC to TT genotypes was observed (SupplementFig. 1). These results indicate that the SNP regulates the ATM mRNA expression in human cells in vivo.

ce:CT:TT:Sac ll Sacily_Y116 162(GCGG) (GCGG)Sacll Sacily_Y116 162(GCGG) (GCGG)SacilY16 (162(GTGG) (GCGG)SacllY116 ~—~—«162(GTGG) (GCGG)287287287Genotype: CT CT CC TT 

To figure out the mechanism in which the SNP regulates the ATM mRNA expression, we analyzed the sequences around the SNP and found that the SNP forms an activator protein 2a (AP-2a) binding site. Then, we examined the binding ability of AP-2a on the sequences of the SNP by electrophoretic mobility shift assay (EMSA). EMSA was carried out using purified human recombinant AP-2a protein and biotin-labeled probes with either DNA fragments of the C or T allele. AP-2a strongly bound to the oligonucleotide containing the C allele and weakly associated with the oligonucleotide probe with the T allele. Competition assays with unlabeled probe further confirmed a specific interaction between AP-2a and the C allele (Fig. 3B). The results of the DNA–protein interaction prompted us to study the effect of C > T on ATM transcription. The luciferase reporter vectors containing ATM promoters from the C allele or T allele were

Fig. 2. The genotypes of the ATM promoter were detected by PCR-restriction fragment lengthpolymorphism. The PCR amplified fragmentswere digested bySac II,electrophoresedonan8%polyacrylamidegelandstainedwithsilvernitrate.Left: two Sac II restriction enzyme sites in amplicon 3 of ATM promoter region: when C alleleispresent,threefragmentsof46 bp,116 bpand125 bparegeneratedandtwo fragments of 125 bp and 171 bp are generated when T allele is present. Right: examples of PCR amplified fragments digested by Sac II were shown. Genotypes were labeled as CC, CT, and TT.

Table 2

Association of rs189037 with longevity.

Genotype Allele TT CT CC Total T C LLI Number 139 418 232 789 696 882 Percentage 17.6 53.0 29.4 100 44.1 55.9 Controls Number 186 407 293 886 779 993 Percentage 21.0 45.9 33.1 100 44.0 56.0 x2 P value Genotype 8.44 0.01 Allele 0.007 0.9 CT versus othersa 8.28 0.004 TT versus othersb 3.04 0.08 CC versus othersc 2.60 0.1 a