2.8. Cell culture and transfection
Human embryonic kidney HEK293 (ACCregion containing all three SNPs upstream of the luciferase gene in the reporter gene construct. We generated four haplotypes based on the allele combinations of these three SNPs (AGC, GGA, AGT and GGT). We observed no significant difference in the reporter gene activity between any of the haplotypes (Fig. S2A).  To investigate whether the 50-UTR haplotypes may affect EXO1 expression levels, we quantified EXO1 mRNA expression in lymphoblastoid cell lines of homozygous carriers of each haplotype (Fig. S2B). We observed no significant difference in EXO1 mRNA expression levels between any of the haplotypes.  4. Discussion  In this study, we have investigated the association of cSNPs in DNA repair genes with human longevity and provide evidence for a functional role of two promoter SNPs (rs1776181 and rs1776180) located in the DNA repair gene EXO1. Our finding that the C allele of rs1776180 is associated with increased promoter activity and higher EXO1 mRNA expression levels provides a possible explanation for our observation that this allele is overrepresented among centenarians compared to younger controls. However, we found no evidence that any of the analyzed cSNPs affect the ability of EXO1 to repair UV-induced DNA damage or that any of the other analyzed SNPs in EXO1 or other DNA repair genes are significantly associated with human longevity.  The lack of association between cSNPs in most DNA repair genes and human longevity is consistent with previous studies (Mockett et al., 2010; Tanaka et al., 2003). Our study has focused on non-synonymous coding SNPs in DNA repair genes, whereas previous studies have investigated a broader range of genetic variants, including regulatory SNPs and copy number variations (CNVs). The latter studies have identified several genetic loci that are associated with human longevity, including the APOE gene (Nebel et al., 2011; Schachter et al., 1994), which we also confirmed in our study.  The functional relevance of the two promoter SNPs in EXO1 is supported by our observation that the C allele of rs1776180 is associated with increased promoter activity and higher EXO1 mRNA expression levels. This finding is consistent with a previous study reporting that the C allele of rs1776180 is associated with increased EXO1 mRNA expression levels in human lymphoblastoid cell lines (Yamamoto et al., 2009). However, the functional consequence of this increased expression on DNA repair capacity or other cellular functions remains to be investigated.Fragment in front of luciferase mRNA was used to create a heterologous reporter gene system. Using site-directed mutagenesis, we established constructs representing the common UTR haplotypes that show a frequency >1% in the German control individuals (i.e. AGC, GGC, AGT, GAC and GAT, see Table S4). Comparison of the five 50-UTR haplotypes by dual luciferase assay in HeLa cells did not reveal any differential reporter gene levels (Fig. S2), indicating that none of the analyzed 50-UTR haplotypes influences post-transcriptional processing or translation efficiency of EXO1. Fig. 3 shows that transcription factor E47 acts as a transcriptional repressor of EXO1. 

We observed a remarkable influence of the rs1776180 alleles on EXO1 promoter activity (Fig. 2A). Both haplotypes containing the C allele of rs1776180 showed a 69-fold higher activity than their G bearing counterpart (P < 0.01 for both comparisons). No significant difference in promoter activity was observed between the constructs carrying either allele of rs1776181. The C allele of rs1776180 is therefore most likely to enhance EXO1 promoter activity regardless of the rs1776181 allele. Interestingly, the C allele of rs1776180 was statistically significantly over-represented in centenarians (Table 2). 

The above-mentioned results prompted us to investigate promoter SNP-dependent EXO1 mRNA expression levels in human lymphoblastoid cell lines in relation to their rs1776180 genotype. No significant genotype-dependent EXO1 mRNA regulation was observed in cell lines originating from male individuals (P = 0.50; 17 CC vs. 15 GG genotypes; (cid:3)1.06-fold induction). In contrast, EXO1 transcript levels were significantly up-regulated (P = 0.03; 1.34-fold induction) in cell lines originating from females with the CC genotype (n = 10) when compared to women with the GG genotype (n = 23) and significantly up-regulated (P = 0.02; 1.45-fold induction) when compared to women with the genotype CG (n = 36; see real-time PCR data in Fig. 2B). These data support the notion of a female-specific functional effect, as already observed in the genetic analysis (Table 3).