To shed more light on the functional mechanisms underlying the allele-dependent promoter activity of EXO1, we performed electrophoretic mobility shift assays (EMSA) with oligonucleotides representing the genomic region surrounding rs1776180 and a panel of nuclear extracts derived from different human cell lines. With all extracts tested, we detected a dramatic reduction in specific band shifts using the C allele of rs1776180 compared to the G allele (Fig. 3A, left panel). Specificity of protein binding was demonstrated by omitting nuclear extract in the binding reaction and by competition experiments (Fig. 3A, right panel). Using the JASPAR binding site prediction tool, we found that the region directly surrounding rs1776180 contains a potential binding site for the basic helix-loop-helix (bHLH) E-Box-binding transcription factor E47 that is depleted by presence of the C allele of rs1776180. To demonstrate direct binding of E47 to the promoter region surrounding rs1776180, DNA-binding complexes were analyzed for the presence of E47 protein by EMSA and subsequent immunoblotting (Shift-Western). As shown in Fig. 3B, E47 protein was detectable in two individual protein complexes that bound to the G allele of rs1776180 while no E47 was found binding to the C allele. E47, which is capable of forming homo- and heterodimers like other members of the E-Box protein subfamily, is encoded by the TCF3 gene (alias: E2A). 

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, overexpression of E47 fusion protein decreased luciferase activity, thus suggesting a role of E47 as a 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, the 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 weaker but yet detectable expression in the brain, liver, and ovary (Fig. 3E). E47/TCF3 mRNA displayed a remarkably similar expression pattern with strongest signals in spleen, thymus, liver, and testis and lower expression in the heart, brain, and ovary. Thus, concomitant expression of EXO1 and E47/TCF3 was most pronounced in lymphatic tissues and reproductive organs. EXO1 is a 5030 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. The functional results prompted us to focus on the genetic replication of the association between EXO1 and longevity on the promoter SNP rs1776180. Since the effect of this functionally relevant polymorphism was apparent only in German women (Table 3 and Fig. 2B), we considered females only for validation. We analyzed rs1776180 in a collection of 455 French female centenarians and 109 younger individuals whose age range was comparable to that of the German controls. In this French sample, the APOE-adjusted analysis confirmed the association signal (P = 0.044) (Table 4). The French allele and genotype APOE frequencies were similar to the original findings in the Germans, and an enrichment of the C allele in the centenarians compared to the controls was apparent in both populations. Therefore, the combined genetic and functional analyses point to a pivotal role of rs1776180 in the allele-specific regulation of EXO1, which in turn influences the ability to survive to exceptional old age.