Fig. 3. Transcription factor E47 acts as transcriptional repressor of EXO1.    PghTOPPog Pro ge eee> ¢ 8 sf rPESEE ME FSE 7 SEL<ER Pol181776180_C 1s1776180_G + + + + - nuclear extractsB( ) Input (C) ,. Ss= ow? aw?WB: E47 ce FS FY>Rd oF7 7oe oewo& &  ©(E)¢e SF SS % @ e aXS S& S S g #(bp) PF PF HPF MHF FF WK oO

remarkable influence of the rs1776180 alleles on EXO1 promoter activity (Fig. 2A). Both haplotypes containing the C allele of rs1776180 showed a 6–9-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).

3.5. EXO1 mRNA level is up-regulated in females carrying the C allele of SNP rs1776180

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).

3.6. Allele-specific binding of E47 contributes to transcriptional repression of EXO1

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 bandshifts 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

Fig. 3. Transcription factor E47 acts as transcriptional repressor of EXO1. (A) Presence of the C allele of rs1776180 results in decreased binding of nuclear proteins.BandshiftassayswereperformedwitholigonucleotidesrepresentingtheC or G allele of rs1776180, respectively, and nuclear extracts of HeLa, HEK293, THP1, Jurkat and pancreatic carcinoma (PC) cells. For all nuclear extracts analyzed, decreased protein binding is observed for the C allele (left panel). Detection of two specific bandshifts reflects the presence of more than one DNA-binding protein complex. Specificity of binding was shown for the G allele by omitting nuclear extracts in the binding reaction and by competition experiments, using unlabeled specific oligonucleotide or unspecific oligonucleotide at 100- or 200-fold molar excess (right panel). FP: free probe, arrowheads: specific bandshifts. (B) Since computational analyses revealed an effect of rs1776180 alleles on binding of transcription factor E47 to the promoter region, DNA-binding complexes of HeLa cells were analyzed for the presence of E47 protein by EMSA and subsequent immunoblotting(‘Shift-Western’).Twoindividualproteincomplexesthatboundto the G allele of rs1776180 contained E47 protein while no E47 was detected bound to the C allele. Protein extracts used for the binding reaction were analyzed in parallelforendogenouslevelsofE47(upperpanel).(C)RNAi-mediatedknock-down of the E47 protein. HeLa cells were transiently transfected with pSUPER constructs carrying independent target sequences directed against E47 (si1, s2) or unspecific

control sequence (ctrl-si). Proteins were extracted 48 h later and subjected to Western blot analysis using anti-E47 antibody. Both constructs resulted in significant reduction in E47 protein expression and were used in dual luciferase assay. (D) Dual luciferase assay with pGL3Enhancer constructs representing promoter haplotypes carrying rs1776180_G were performed as described. Gene silencingofE47byRNAivectorconstructsincreasedEXO1promotertransactivation significantly while unspecific control small hairpin (sh-)RNA (ctrl-si) showed no effect incomparisontovectorcontrol(vc;emptypGL3Enhancer).Inaddition, over- expressionofE47-EYFPfusionproteinimpairedreportergeneexpression.*P < 0.05; **P < 0.01 experiment versus ctrl-si. (E) Overlapping tissue expression patterns of EXO1 and E47/TCF3. Commercially available human cDNA tissue panels were used toanalyzetissuedistributionbyRT-PCR.Overlappingexpressioncanbedetectedin spleen, thymus, liver, testis and ovary. Expression of GAPDH is shown as control.

Table 4