GENOMICS

Genomics 85 (2005) 258–263

www.elsevier.com/locate/ygeno

A novel VNTR enhancer within the SIRT3 gene, a human homologue of SIR2, is associated with survival at oldest ages

Dina Bellizzi , Giuseppina Rose , Paola Cavalcante , Giuseppina Covello , Serena Dato , a a b c c De Rango , Valentina Greco , Marcello Maggiolini , Emidio Feraco , Vincenzo Mari , Claudio Franceschid , Giuseppe Passarinoa , Giovanna De Benedictisa,*

aDepartment of Cell Biology, University of Calabria, 87036 Rende, Italy bDepartment of Pharmaco-Biology, University of Calabria, 87036 Rende, Italy cIstituto Nazionale Riposo e Cura dellTAnziano, Cosenza, Italy dDepartment of Experimental Pathology, University of Bologna, Bologna, Italy

Abstract

SIR2 genes control life span in model organisms, playing a central role in evolutionarily conserved pathways of aging and longevity. We wanted to verify whether similar effects may act in humans too. First, we searched for variability in the human sirtuin 3 gene (SIRT3) and discovered a VNTR polymorphism (72-bp repeat core) in intron 5. The alleles differed both for the number of repeats and for presence/ absence of potential regulatory sites. Second, by transient transfection experiments, we demonstrated that the VNTR region has an allele- specific enhancer activity. Third, by analyzing allele frequencies as a function of age in a sample of 945 individuals (20–106 years), we found that the allele completely lacking enhancer activity is virtually absent in males older than 90 years. Thus the underexpression of a human sirtuin gene seems to be detrimental for longevity as it occurs in model organisms. D 2004 Elsevier Inc. All rights reserved.

Keywords: SIRT3; Human longevity; VNTR enhancer

FOXO-family transcription factor that affects the rate of aging in response to the insulin/IGF1 pathway [5]. SIR2 proteins constitute an evolutionarily conserved family of NAD-dependent deacetylases called sirtuins [6–8]. In model organisms the expression levels of SIR2 modulate life span [9–11]. Since sirtuins are NAD+ dependent these proteins through different routes may link energy metabolism, genome maintenance, and aging [11,12]. Thus SIR2 genes may play a crucial role in conserved pathways of aging and longevity.

Aging can be viewed as a lethal by-product of activities, such as reproduction and food intake, that are controlled by genes [1]. Since most of these genes are evolutionarily conserved, distant species may share common pathways of aging [2]. The insulin/insulin-like growth factor 1 (IGF1) signaling pathway could be one such common pathway, as it modulates aging in many species, including Caenorhabditis elegans, Drosophila, mice [3], and possibly humans [4]. An elegant study carried out in C. elegans by applying microarray techniques showed that a member of the SIR2- like protein family is regulated downstream of DAF-16, a

A human homologue of the SIR2 genes, SIRT3, lies at the telomeric terminal on chromosome 11p15.5 (www.ncbi. nlm.nih.gov/omim; MIM 604481; contig NT_035113). The gene is ubiquitously expressed, particularly in metabolically active tissues, and the SIRT3 protein is targeted to mitochondria through an N-terminus signal necessary for

* Corresponding author. Fax: +

E-mail address: g.debenedictis@unical.it (G. De Benedictis).

These authors contributed equally to this work.

G477T and VNTR. The disequilibrium was highly signifi- b cant ( p 0.001), with 10 observed haplotypes of the 12 expected (estimated by MLE from both homozygous and heterozygous genotypes).

mitochondrial localization [13,14]. By analyzing a large sample of individuals including people older than 100 years of age, we found an association between longevity and the silent marker G477T of SIRT3 [15]. Owing to the silent nature of the SNP, we hypothesized a linkage disequilibrium between the G477T marker and an unknown functional variant of SIRT3. The aim of the present study was to identify the functional variant that could account for the association we observed between the silent marker of SIRT3 and survival. Thus, first we searched for variability in the SIRT3 gene in a large population sample; then we characterized the functional activity, and its association with longevity, of a VNTR polymorphism we identified in intron 5.

occur within the VNTR alleles. By sequencing the PCR- DNA fragments of all the subjects homozygous for the alleles having 1 to 4 repeats (see Table 1) we identified a T/C variation located in the second repeat, 63 nt from its starting point. MatInspector V2.2 analysis carried out on the 72-bp core fragment revealed that the T/C variation trans- formed a GATA3 site into a DeltaEF1 site, potentially having different regulatory activities [17,18]. On the occurrence of the GATA3/DeltaEF1 site in the second repeat, the length-alleles having one to four repeats were resolved into seven alleles. In fact, with the exception of allele 1 (which lacked a second repeat and thus had the GATA3 core sequence in all cases), each of the remaining common length-alleles was resolved into two categories, one having the GATA3 site (alleles 2a, 3a, and 4a) and the other the DeltaEF1 site (alleles 2b, 3b, 4b). Fig. 1 shows the position of the VNTR with respect to the G477T silent marker we previously identified in exon 3 [15], the seven length-alleles spanning one to four repeats, and the GATA3/

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