constructed. Fig. 3C shows us that AP-2a strongly reduced the transcription of promoter from the C allele and weakly reduced the transcription of promoter from the T allele, showing consistency with the results of the protein binding assays and the expression of ATM mRNA in vivo. Together, these results indicate that AP-2a represses ATM expression in humans by pecifically binding to the SNP site.

4. Discussion

Now, it is well known that reactive oxygen species, chemical agents, UV and IR could lead to DNA double-stand breaks (DSBs), which are a critical cause for DNA lesions (Lombard et al., 2005). Once DNA lesions occur, the protein encoded by the ATM gene is activated and sequentially phosphorylates a host of downstream

 (B)AP-2a binding [7unspecific «free probe —labeled T probelabeled C probeunlabeled T probeunlabeled C probeGSTGST-AP-2a. relative ATM mRNA levelGenotype:Relative activity (%)cc CT TT120804020BB Vectori Ap-20.Callele Tallelepromoter promoter

Fig.3.AP-2arepressesATMexpression.(A)Semi-quantitativeRT-PCRwasconductedinperipheralbloodmononuclearcellsfromoldsubjectswithdifferentgenotypes.Left: images of PCR products from different concentrations of cDNA templates by a serial dilution approach. Right: PCR product was quantified as shown; b-actin expression was used as internal control. RT-PCR of each sample was performed 3 times to exclude the PCR error. (B) EMSA was performed using probes and incubated with purified GST protein (lane 1) or human recombinant GST-AP-2 protein (lanes 2–6), allele C (lane 5) and allele T (lane 6). Lanes 2–4: competition with unlabeled probes with allele C promoter (50(cid:4) or 100(cid:4)C, lanes 2–3) and allele T (100(cid:4)T, lane 4), the labeled probe as indicated. (C) Down-regulation of ATM promoter driving luciferase activity by AP-2. PGL3-basic vector and constructs with C allele promoter or T allele promoter were transfected into Hela cells. Error bars indicate SD.

Acknowledgements

effectors involved in the G1/S, intra-S and G2/M checkpoint responses and additional factors involved in DNA repair, including tumor suppressor proteins p53, BRCA1, checkpoint kinase CHK2, RAD17 and RAD9, and DNA repairs protein NBS1 (Shiloh, 2003). Therefore, ATM mutations could make individuals susceptible to cancer and are harmful for physiological activity. This suggests that the ATM gene plays an important role in lifespan.

We thank the participants and their families for their kind cooperation, generosity and patience. We also thank the following: local government officers of Dujiangyan for their great support when we visited the participants and their families, civil hospital of Dujiangyan, postgraduates from Public Health School, under- graduates of grade 2003 and postgraduates from West China Medicine School, and Sichuan University for their help in sample collection. The work was supported by the project of Science and Technology Bureau of Sichuan Province (2006Z090064), Construc- tion fund for Subjects of West China Hospital, Sichuan University (XK05001) and National Basic Research Program of China (2007CB947802), Nature Science Foundation of China to H.X. (30771228) and to X.M. (30771227).

morphisms in promoter of gene could regulate gene expression by altering binding site for transcriptional factors (Sun et al., 2007); as a result, subjects with different SNPs show distinct phenotypes. Recently, it has been noted that more and more polymorphisms in promoter or 50UTR of genes associated with certain phenotypes have been reported (Giacconi et al., 2004; Sun et al., 2007). In our results, C/T genotype in promoter of ATM is significantly associated with longevity. Bioinformatic analysis shows that there is AP-2a binding site around the SNP, which is a transcriptional factor widely involved in both physiological and pathological processes, such as morphogenesis, cell adhesion, tissue differentiation, tumorigenesis and genetic disease (Leask et al., 1991). In our study, we found that the probe containing the C allele robustly binds the AP-2a protein, and ATM transcription was repressed in the C allele further than in the T allele by AP-2a protein in luciferase assay, which is in line with mRNA expression in vivo. It is also believed that balance exists in every activity of the physiological process; extremely high or extremely low levels of activity may contribute to pathological change. For example, mutation or consistent levels of high activity of P53 causes individuals to die at an early age or senescence because of cancer or prematurity. Thus, moderate activity of genes may keep subjects from abnormal physiological function (Tyner et al., 2002).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in online version, at doi:10.1016/j.mad.2010.08.009.

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