Table 4

Replication of the longevity association of EXO1 promoter SNP rs1776180 in French samples (females only).

No. dbSNP ID Location Alleles MAF controls Corresponding AF P P P OR 95% CI CCA CCG APOE APOE (n=109) centenarians (n=455) 2 rs1776180 Promoter C/G 0.408 (C) 0.481 0.053 0.1567 0.0441 1.39 1.01–1.92

For abbreviations see legend to Table

the TCF3 gene (alias: E2A). Examination of TCF3 with SNPs most of the allelic variation at the locus provided no evidence an association with longevity (Table S5). In addition, regression analysis revealed no statistical interaction between EXO1 SNP rs1776180 and any of the tested TCF3 markers.

4. Discussion

We have identified exonuclease 1 (EXO1) as a candidate for a novel susceptibility gene for prolonged life expectancy in humans. Among the associated SNPs the promoter variant rs1776180 was found to be functionally relevant by influencing EXO1 gene expression. The region surrounding rs1776180 contains a binding site for transcription factor E47, which is functional only when the G allele of the SNP is present. The C allele is enriched in female centenarians and leads to a loss of transcription factor binding to the E47 consensus site. Since E47 acts as a repressor of EXO1 transcription, the substitution of G by C at rs176180 should consequently result in a higher promoter activity. Supportive evidence was obtained from the ex vivo analysis of EXO1 mRNA levels in lymphoblastoid cell lines that revealed a statistically significant increase in gene expression in female individuals. Genetic replication of rs1776180 in a French sample confirmed the initial observation. In contrast to APOE, the effect of EXO1 on longevity is moderate [OR = 1.47 in Germans, OR = 1.39 in French; compare OR = 0.42 (corresponding to 1/OR = 2.4) and 0.29 (corresponding to 1/OR = 3.4) of APOE in Germans and French in the present study, respectively]. The effect size is about the same magnitude as for FOXO3A (OR   1.4; Flachsbart et al., 2009) and conforms to the hypothesis that genes with low to moderate effect

For further analysis of functional crosstalk between E47 and EXO1 regulation, we used RNAi-mediated gene knock-down of E47 and transient over-expression of an E47-EYFP fusion protein. Knock-down of endogenous E47 in HeLa cells by RNAi (Fig. 3C) resulted in a statistically significant increase in EXO1 promoter transactivation using reporter constructs carrying the G allele of rs1776180 (Fig. 3D). Furthermore, over-expression of E47 fusion protein decreased luciferase activity, thus arguing for a role of E47 as negative regulator of EXO1 promoter activity. Using the same experimental setting, a small, but significant increase in luciferase activity was also detectable using constructs with the C allele of rs1776180 (not shown). This observation may suggest a minor contribution from other potential E47 binding sites in the proximal promoter region, which are not affected by any genetic variation influencing life expectancy. However, absolute basal luciferase activity of the C allele constructs was up to 8-fold higher than those carrying the G allele (data not shown), which is consistent with our above- mentioned results (Fig. 2A).

Since this novel regulatory mechanism is only relevant in tissues and cell types expressing both EXO1 and E47/TCF3, we analyzed the tissue distribution pattern of both genes. EXO1 is strongly expressed in thymus, spleen and testis and showed a weaker, but yet detectable, expression in brain, liver and ovary (Fig. 3E). E47/TCF3 mRNA displayed a remarkably similar expres- sion pattern with strongest signals in spleen, thymus, liver, and testis and lower expression in heart, brain and ovary. Thus, concomitant expression of EXO1 and E47/TCF3 was most pro- nounced in lymphatic tissues and the reproductive organs.

EXO1 is a 50–30 exonuclease that participates in post-replication DNA mismatch repair and other DNA metabolic pathways affecting genomic stability, including homologous recombination and DNA damage repair (Liberti and Rasmussen, 2004; Mimitou and Symington, 2008; Qiu et al., 1999). In addition, EXO1 has recently been shown to contribute to DNA damage-induced apoptosis (Bolderson et al., 2009; Klapacz et al., 2009). EXO1 physically interacts with the WRN protein that is encoded by the gene implicated in Werner Syndrome, one of the human progeria diseases (Sharma et al., 2003). WRN is crucial for the maintenance of overall genomic stability and, like EXO1, is also required for DNA repair and telomere metabolism (Lombard et al., 2005). The complexation of both proteins causes strong activation of EXO1 (Sharma et al., 2003). It has been suggested that WRN-induced stimulation of EXO1 activity may be important for the replication and stability of telomeres as well as the activation of DNA damage and signal checkpoint pathways in response to shortened telomeres (Sharma et al., 2003). An involvement of EXO1 in these pathways has been shown in both telomere-dysfunctional yeast and mouse models (Maringele and Lydall, 2002; Schaetzlein et al., 2007).

Taken together, our data indicate a major impact of variants of promoter SNP rs1776180 on EXO1 transcriptional regulation and suggest an important and hitherto undescribed role for E47 as a repressor of EXO1 expression.

3.7. EXO1-association replicated in female French centenarians