On the human long-arm pseudoautosomal region (XqPAR), genes that are subject to inactivation are closely linked with those that escape. Genes subject to inactivation are not only silenced on the inactive X in females, but they are also inactivated on the Y chromosome in males. One of the genes subject to this unusual inactivation pattern is the synaptobrevin-like 1 gene (SYBL1). Previously we showed that its silencing on the inactive X and the Y allele involves DNA methylation. This study explores the molecular events associated with SYBL1 silencing and investigates their relationship. Promoter DNA methylation profiles were determined by bisulfite sequencing and immunoprecipitation experiments demonstrate that chromatin on the repressed Xi and the Y alleles has underacetylated histones H3 and H4 and H3-lysine 9 methylation. In addition, the inactive X and the Y allele were found to have a condensed chromatin conformation. In contrast, the expressed allele shows H3 and H4 acetylation, H3-lysine 4 methylation and a less compacted chromatin conformation. In ICF syndrome, a human disease affecting DNA methylation, SYBL1 escapes from silencing and this correlates with altered patterns of histone methylation and acetylation. Combined, our data suggest that specific combinations of histone methylation and acetylation are involved in the somatic maintenance of permissive and repressed chromatin states at SYBL1. Although it is unclear at present how this allele-specific silencing comes about, the data also indicate that the epigenetic features of the ‘Y inactivation’ of SYBL1 are mechanistically similar to those associated with X-chromosome inactivation.
In mammals, dosage compensation of X-linked genes between males and females occurs by X inactivation (1), which silences most genes of a randomly chosen X chromosome. A number of genes, however, escape inactivation and this phenomenon, first hypothesized on the basis of the Turner phenotype in X0 individuals (2), has been shown to involve about one-fifth of the total gene number (3). Escaping genes tend to be concentrated in clusters that are mainly on the short arm of the X chromosome (4). Many of these genes appear to derive from recent autosomal additions to the sex chromosomes that show evolutionary stratification (5–7).
Among X/Y conserved regions are pseudoautosomal regions (PARs), localized at the tip of the short and long arm of both X and Y chromosomes (8). The human short-arm PAR, XpPAR, is 2.6 Mb in length and was found to ensure, through genetic recombination, the correct segregation of the sex chromosomes (9). Subsequently, the long-arm human pseudoautosomal region, XqPAR, was discovered at the end of the long arm of the sex chromosomes and is 330 kb in length. Even though recombination is six times higher in XqPAR than in autosomes, no obligate crossovers are observed during male meiosis (10). In XpPAR, all genes analysed escape inactivation, but the four genes localized in the XqPAR thus far show a strikingly distinct transcriptional and evolutionary behavior. The centromeric ones, H-SPRY3 and synaptobrevin-like 1 gene (SYBL1), are X and Y inactivated, whereas the telomeric ones escape inactivation (11–14).
Epigenetic inactivation of SYBL1 by DNA methylation is suggested by the ability to reactivate silenced alleles in somatic cell hybrids by treatment with the demethylating agent 5-deoxyazacytidine (15,16). DNA methylation-induced silencing is also supported by the observation of biallelic SYBL1 expression in two ICF syndrome cases. ICF syndrome (immunodeficiency, centromeric instability, facial abnormalities) is a human autosomal-recessive syndrome affecting DNA methylation (17) and is caused by mutations in the DNA methyltransferase 3B gene (18–20). Escape from silencing of inactive SYBL1 alleles in ICF has been correlated with advanced replication time as well as promoter hypomethylation (21).
A number of reports on different species implicate the existence of a network of interactions among the three best characterized covalent modifications: histone acetylation, histone methylation and cytosine methylation (22). In fact, DNA methylation has been associated with histone deacetylation in repressing transcription via the establishment of a repressive chromatin status. This has been supported by the in vitro finding that the methyl-CpG binding domain (MBD) of protein MeCP2 binds to histone deacetylases and represses transcription (23,24). Another class of histone modification, di-methylation of histone H3 at lysine residues 4 and 9 (H3K4 and H3K9), has been associated to the establishment of a permissive or repressed chromatin status, respectively (25,26). In particular, methylation of H3K9 has recently been linked to the establishment and maintenance of X inactivation (27–29).
To address the involvement of these epigenetic modifications and their interplay in SYBL1 regulation, detailed methylation patterns at the SYBL1 X/Y promoter region have been obtained by genomic bisulfite sequencing on cell lines derived from normal males and females. In addition, we carried out chromatin immunoprecipitation (30) and DNaseI experiments on the same cell lines and on a cell line derived from a male ICF patient.
Combined, our findings support the hypothesis that cytosine methylation and specific histone modifications act together to repress transcription on the inactive X and on the Y allele of SYBL1, a process which involves the establishment of a condensed chromatin conformation. Importantly, it appears that the maintenance of SYBL1 repression is identical on the inactive X in females as compared with the Y chromosome in males. Although these two different mechanisms might be distinct in their establishment, the same epigenetic features seem to be involved in their somatic maintenance.
Monoallelic SYBL1 expression in males and skewed females
The SYBL1 gene structure has been reported recently (16). SYBL1 regulatory regions include a canonical CpG island, spanning its minimal promoter, including the first untranslated exon. A schematic map of the region is reported in Figure 1.
To better analyse allele-specific modifications of the SYBL1 gene, we looked for RFLPs in the promoter proximal transcribed region. Exon flanking primers were developed and a shifted fragment was identified in the 5′UTR of exon 1. Sequencing of this fragment revealed a C–T transition, creating an XhoI site at position +65 (Fig. 1). The RFLP heterozygosity was 42% in 200 chromosomes analysed (not shown). XhoI restriction analysis of the heterozygous samples shows three fragments, the undigested 189 bp and the digested 112+77 bp fragments (Fig. 1 and data not shown).
To analyse the expression of the SYBL1 gene, we used primers spanning the XhoI RFLP, located in exons 1 and 2, in all RT–PCR experiments (237 and 190+47 bp fragments; see Fig. 1). Y allele-specific expression was been analysed in heterozygous males, whereas to assess inactive X expression we took advantage of two highly skewed female fibroblast cell lines, GM07693 and GM03884, heterozygous for the described RFLP. Monoallelic expression of the heterozygous males and females was confirmed using an RT–PCR assay, followed by XhoI digestion (data not shown). For the following experiments, we used one male lymphoblastoid cell line (AB) and one female fibroblast cell line with highly skewed X inactivation (GM07693).
Bisulfite analysis in somatic cell hybrids, heterozygous male and female individuals with skewed X inactivation
SYBL1 monoallelic expression has been linked to hypermethylation of its associated CpG island using a methylation-sensitive restriction enzyme-based approach (15). Only a few methylation sensitive sites can be checked for their status with this method. Our previous studies determined that the minimal SYBL1 promoter (15,16) was restricted to roughly 170 bp near the main transcription initiation site (nt −163 to +3). Using bisulfite analysis (31) we analysed the methylation status of the CpG island, which spans nucleotides −252 to +227 bp and includes 37 CpG dinucleotides (Fig. 2A).
Following bisulfite treatment of somatic cell hybrid DNAs (11), HY136C (active X), THX88 (inactive X) or GM06317 (Y chromosome), the lower DNA strand was analysed, using conditions described elsewhere (32,33). Assessment of cytosine deamination was determined by noting the state of cytosines not belonging to CpG doublets. Absence of cytosine in such instances implies full DNA conversion. Treated DNA was cloned and 20 clones were sequenced for each hybrid analysed.
Taking advantage of the XhoI heterozygous individuals showing SYBL1 monoallelic expression, we assessed the methylation profile of the gene in the male (AB) and female (GM07693) cell lines, whose expression patterns had been previously determined. These patterns can be correlated with allele-specific methylation status because the polymorphic nucleotide is preserved in the antisense DNA strand following bisulfite treatment.
The methylation profiles of the three alleles in the human cell lines are shown in Figure 2B–D. Each clone represents the methylation pattern of a single chromosome. The active X chromosome, which expresses SYBL1, shows complete absence of methylation, whereas both the repressed alleles are methylated throughout the region. The correlation between CpG density and SYBL1 repression, however, cannot exclude the importance of specific CpG dinucleotide methylation.
Similar results were obtained when hybrid cell lines, retaining the active or inactive X chromosomes and the Y chromosome, were analysed (not shown). The active X allele is similarly completely unmethylated. The inactive alleles are similarly methylated when compared with the human cell lines.
Histone modifications mark allele-specific repression of SYBL1 gene
Many reports describe the link between cytosine methylation, histone acetylation and chromatin conformation in the repression of target genes (34,35). More generally, changes in histone modifications, often mutually exclusive, mark an expressed versus a repressed status of a certain gene and/or genomic region (25–29). This has been recently postulated as ‘the histone code hypothesis’ (36). To determine if histone modifications play a role in SYBL1 allele-specific silencing, chromatin immunoprecipitations of the AB (male) and GM07693 (female) cell lines were performed using commercial antibodies to acetyl-histone H3 and H4, acetylated and di-methylated H3K9 and di-methylated H3K4.
To perform chromatin immuno precipitation (IP) assays, cells were fixed with formaldehyde followed by sonication (30). This procedure produced chromatin fragments containing 200–1000 bp of DNA, which corresponded to one or several nucleosomes (data not shown). Chromatin IPs were performed with antisera that recognized histone H4 acetylated on any of its four N-terminal lysine residues (anti-AcH4) and antisera that recognized hyperacetylated isoforms of histone H4 but did not detect mono- and di-acetyl histone H4 (anti-HyAcH4). For histone H3, antisera that recognized acetylated histone H3 on lysines 9 and 14 (anti-AcK9/14H3), di-methylated lysines 4 (anti-mK4H3) and acetylated (anti-AcK9H3) and di-methylated lysine 9 (anti-mK9H3) were used.
Following chromatin immunoprecipitation, the antibody-enriched fractions of genomic DNA were purified and isolated. The enriched DNA was analysed by PCR for the presence of the SYBL1 CpG island fragment using primers spanning from −49 to +141 bp. A protocol of hot-stop PCR (37) was developed to circumvent the problem of heteroduplex formation.
To distinguish between the X and Y alleles for the male cell line we followed the strategy for allele-specific analysis outlined in Figure 1. XhoI digestion of the PCR product produces three fragments, the undigested 189 bp and the digested 112+77 bp fragments corresponding to the Y and X alleles, respectively (Fig. 1). Genomic DNA from AB and his father (GB) share the undigested allele, which is therefore the Y chromosome (Fig. 3A, far left panel). The undigested allele represents the inactive allele also for the female cell line, GM07693.
Figure 3A and B shows the results of the outlined chromatin immunoprecipitations. Density profiles are normalized to the no antibody lane. The unmethylated and expressed X alleles in male (AB) and female (GM07693) reveal a strong enrichment for acetylated and hyperacetylated histone H4 isoforms as well as acetylated histone H3 isoforms. The active X allele is also specifically precipitated by the antibody that is specific for acetylated H3K9. The methylated lysine, in the same residue (K9), marks only the repressed, inactive X and Y alleles. As expected from other studies, methylation of H3K4 is only associated with the active allele.
SYBL1 PCR product was amplified in the ‘no antibody’ negative control almost exclusively in the unbound fraction, as expected (non-specific binding of chromatin to the protein A-Sepharose beads without antibody was very low). Both fractions (bound+unbound) represent the starting chromatin.
CpG methylation and specific histone modifications are associated with condensed chromatin at the SYBL1 promoter
Chromatin accessibility assays were performed to determine if the cytosine methylation and histone H3 and H4 modifications of the SYBL1 promoter were associated with the remodeling of chromatin to a transcriptionally repressive state. Chromatin conformation can be studied by determining its sensitivity to DNaseI in specific chromosomal regions. Since DNaseI normally digests all sequences at about the same efficiency, it constitutes an excellent general marker of chromatin structure. Generally the differences detected in generalized nuclease sensitivity between the two chromosomes may be indicative of specific intranucleosomal modifications (such as core-histone acetylation) (38).
Nuclei purified from AB and GM07693 cell lines were incubated at increasing concentrations of DNaseI after which genomic DNA was extracted and analysed for sensitivity of the alleles to this enzyme. We found that the expressed SYBL1 X allele was more sensitive to DNaseI than the repressed inactive X and Y chromosomes (Fig. 4). The uncut fragment (189 bp), corresponding to SYBL1 inactive X or Y repressed alleles, were highly resistant to the digestion, whereas the digested, double fragment (112+77 bp) corresponding to the active X allele was more readily digested. We also checked the DNaseI accessibility in a larger region with similar results (data not shown).
Epigenetic changes at the SYBL1 promoter in a hypomethylated DNA background
We recently demonstrated that, in cell lines derived from ICF patients who are characterized by marked DNA hypomethylation at certain heterochromatic loci, SYBL1 is biallelically expressed. This abnormal escape from silencing is also correlated with advanced replication time (21). To investigate the interaction between CpG methylation, histone modifications and chromatin condensation status of the SYBL1 regulatory region in an ICF cell, we analysed an ICF male cell line derived from patient PT5 that is heterozygous for the XhoI RFLP of the SYBL1 promoter (21).
The active X chromosome is the uncut allele in PT5, whereas the Y allele is digested using XhoI (not shown). We performed essentially the same experiments as described for the normal male and female cell lines. The chromatin features of the SYBL1 gene in ICF are, however, quite different: both alleles of this gene show hyperacetylation of histones H3 and H4 and methylation of K4 on H3 as predicted from the evidence that both alleles are expressed. Methylation of H3K9 which marks repressed alleles is, as expected, not detected at significant levels on either allele (Fig. 5).
Marked chromatin sensitivity to DNaseI digestion for both alleles is seen in the PT5 ICF cell line (Fig. 6). The Y-linked SYBL1 promoter region, therefore, is in an abnormally open chromatin state in this line.
The SYBL1 gene is unusual in that it is subject to inactivation not only on the inactive X in females, but also on the Y chromosome in males. The fact that the Y-linked allele can be reactivated by treatment with the demethylating agent (5AC) suggested that its silencing involves promoter methylation (15). However, the establishment and/or maintenance of transcriptional states is generally thought to not only involve DNA methylation, but also covalent modifications of the histone proteins by which DNA is packaged in chromatin. Such alterations include the acetylation and methylation of histone tail residues such as lysines 4 and 9 of histone H3 (27–29). In this report, we analyse histone modifications at silent and expressed SYBL1 alleles and show that their allelic differences are comparable to those at other differentially expressed X-linked (39–41) or autosomal mammalian loci (42). Consistently, the active X allele of SYBL1 is found to be associated with hyperacetylated histones H3 and H4 and H3 lysine 4 methylation. In contrast, chromatin on the silenced Y and inactive X alleles has hypo-acetylated histones H3 and H4 and is methylated at lysine 9 of H3. These data support the idea that specific combinations of histone methylation and histone acetylation are involved in the somatic maintenance of the opposite epigenetic states at SYBL1.
Several reports link binding of MBD proteins, such as MeCP2, to methylated DNA, which in turn recruits multimeric complexes including HDACs as silencing factors (23,24). The ICF syndrome provides a model to study the interplay between these epigenetic features as these patients have mutations in DNMT3B that lead to hypomethylation in heterochromatic regions. We recently found that SYBL1 is deregulated in ICF cell lines and has biallelic expression (21). When we analysed the level of histone acetylation and methylation for the SYBL1 promoter in a male ICF cell line, both the Y and the X allele exhibited H3 and H4 acetylation and H3K4 methylation. In contrast, no H3K9 was detected in the ICF cell line. The mechanistic link between DNA and histone methylation, already proposed in N. crassa (43) and A. thaliana (44), seems therefore evident also for SYBL1 in this hypomethylated background.
These results support the idea of MBD proteins recruiting histone deacetylases (HDACs) to deacetylate histones, which then become targets for histone methylation (26). Another possibility is that DNA methylation may recruit methyl–CpG binding complexes containing histone methyltransferases that directly methylate histone tails (26). It has been suggested that K9 methylation is involved in the repression of DNA methylated promoters by MeCP2 and that K9 HMTase directly associates with MeCP2 and is delivered to specific methylated promoters (T. Kouzarides, manuscript in preparation). Intriguingly, preliminary observations suggest that the allelic SYBL1 repression is also mediated by MeCP2 binding to the methylated gene promoter (M.R. Matarazzo et al., manuscript in preparation).
The data reported here support a causal relationship between the effect of methylation on the acetylation level of histones H3 and H4 which, in turn, would affect SYBL1 expression. Recently, it has been demonstrated using a somatic cell hybrid system that X inactivation of specific genes is dependent on promoter specific hypo-acetylation (40). Our data establish that the X-linked, active, SYBL1 allele displays loss of methylation and hyperacetylation of histones H3 and H4 coupled to an open chromatin structure, characterized by generalized DNaseI sensitivity. In contrast, inactive X and Y alleles are characterized by the presence of DNA methylation, histone hypoacetylation and by a conformation that is remarkably resistant to DNaseI. Because the data we have presented suggest a similarity in the silencing mechanisms used for the Y and inactive X allele, we suggest introducing the term ‘Y inactivation’. This phenomenon has not been described before the identification of SYBL1 (11) and would also include the silencing of the Y allele of the H-SPRY3 gene (14).
What could be the primary cause of Y inactivation, given that this process is most likely XIST independent? One possibility would be that Y inactivation is somehow related to the presence of a heterochromatin block close to the YqPAR in Yq12. It should be relevant to test whether, upon transmission through the male germ line, chromatin at this repeat block becomes inactivated and maintains and spreads its repressive state in cis during embryonic development. Since DNMT3B localizes at pericentromeric regions and methylates them in addition to satellite DNA (45), a local hypomethylation of this heterochromatin block could be the primary cause of escape from silencing of the Y copy of SYBL1 in ICF syndrome. Analysis of other pathologies that bring about changes in DNA methylation may shed further light on the mechanisms of Y inactivation. These include the ATRX (α-talassemia, mental retardation X linked syndrome; OMIM 301040), in which satellite sequences on the Y chromosome were recently found to have substantially reduced levels of DNA methylation (46). A complementary approach to test the proposed involvement of satellite sequences in the initiation and maintenance of Y inactivation would be the analysis of individuals lacking, at least in part, the Y-specific heterochromatic block close to the XqPAR region (47). In case of specific sequence elements present on the Y chromosome, but not on the X and indeed found to be involved in triggering Y inactivation, it should be relevant to determine the precise extent of spreading of repressive chromatin features in cis and why it only affects some of the genes on XqPAR.
MATERIALS AND METHODS
Epstein–Barr virus-transformed lymphoblastoid cell line AB was derived from a normal male individual. GM07693 and GM03884 (NIGMS) are fibroblast cell lines from two females with highly skewed X inactivation (>95:5) (48).
PCR and RT–PCR
Exon 1 flanking primers spanning the 5′UTR XhoI RFLP are as follows: EX1telf, 5′-CGTGTCGCCTGCTGCCATTG-3′ (nt +89 to −69); EX1telr, 5′-CGACACCTCCGGCCTCCCTGG-3′ (nt +163 to +142). PCR amplification was performed on genomic DNA at 60°C for 33 cycles. Restriction analysis of the heterozygous samples, after XhoI digestion shows three fragments: the undigested 251 bp and the digested 150+101 bp fragments (data not shown).
Total RNA was collected from the cells with Trizol (Life Technologies, Rockville, MD, USA) following the manufacturer's instructions. RT–PCR was carried out using Super Script II (Life Technologies) following the manufacturer's protocol. For SYBL1 expression analysis, cDNA was amplified with primers: EX1RT, 5′-TGGGAGCGGGCAGTTGGCGA-3′ (nt +17 to +37); EX2RT, 5′-TTGCCATGTGAGTACGTTAGTTT-3′ (nt +8340 to +8317). PCR amplification was done at 62°C; the PCR product was digested with XhoI, which cleaves the T allele into 190 and 47 bp fragments (data not shown).
Bisulfite sequencing of the SYBL1 promoter
Five micrograms of the XbaI digested genomic DNA were modified with bisulfite sodium under the conditions previously described (32) with some modifications. Briefly, DNA was denatured with 0.3 m NaOH and incubated with a urea/bisulfite solution and hydroquinone to final concentration of 5.36 m, 3.44 m and 0.5 mm, respectively (33). The reaction was performed in a PCR machine (480 Thermocycler PE Biosystem, Foster City, CA, USA): 20 cycles of 55°C for 15 min followed by denaturation at 95°C for 30 s. The PCR products were obtained through two rounds of PCR using a nested primers approach. The primers were designed to be fully complementary to the deaminated DNA lower strand.
External primer 1, 5′-ATGATTTTGATTTGTTTTTTTT-3′ (nt −339 to −317); external primer 2, 5′-ATTTAACTCTCTTTCAATTATA-3′ (nt +334 to +356); nested primer 3, 5′-GTAGGAGAGTAAAAGGATTTAG-3′ (nt −252 to −233); nested primer 4, 5′-TTAACCTTAACAACTATAATCTG-3′ (nt +206 to +227). Nucleotide positions are based on the published SYBL1 promoter region (16). First round amplification was performed in 25 ml reaction under following conditions: 95°C for 1 min followed by 30 cycles of 95°C/30 s, 47°C/45 s, 72°C/45 s. Two milliliters of the primary PCR product was used for nested PCR as follows: 95°C for 1 min followed by 30 cycles of 95°C/30 s, 59°C/45 s, 72°C/45 s. All the PCR products were cloned into TOPO TA cloning vector (Invitrogen, Carlsbad, CA, USA). About 20 clones for each allele were isolated from each somatic cell hybrids/individuals and sequenced using ABI automated sequencer (PE Biosystem, Foster City, CA, USA).
Chromatin immunoprecipitations and PCR
A sample of 1×108 cells was rinsed in 1× PBS and treated with 1% formaldehyde for 10 min at 37°C (30). Chromatin IPs were then performed using various polyclonal antisera according to the manifacturer's instructions (Upstate Biotech, Lake Placid, NY, USA). Briefly, sonication of crosslinked nuclei was performed under conditions that gave a range in DNA fragments from 200–1000 bp. Anti-acetyl-histone H3, H4 and hyperAcH4 and anti di-methylated K9 and K4 of histone H3 antisera were incubated overnight with precleared nuclear lysates. The chromatin–antibody complexes were collected using Protein A Sepharose beads. After purification, 50 ng of each DNA sample was used for PCR (36 cycles with annealing temperature of 60°C). Based on hot stop PCR protocol only the last cycle of PCR was performed in the presence of α32P dCTP.
Selected SYBL1 primers amplified a 189 bp fragment from the promoter region: forward, syb17, 5′-CCAAGAGGCCACGCGTAG-3′ (nt −49 to −31); reverse: syb26, 5′-GTTCTCTCCCCGCCTCCC-3′ (nt +141 to +123). Five milliliters of the PCR product were digested with polymorphic XhoI enzyme at 37°C. The products of digestion were resolved by MDE acrylamide gel (Biowitthaker Molecular Applications Corp., Walkersville, MD, USA) electrophoresis. After being dried the gels were exposed to X-ray films, after which the relative band intensities were calculated using Quantity-One imaging software (Bio-Rad Laboratories, Hercules, CA, USA). Density profiles were normalized to the no antibody lane. Then, the Y/X and/or Xi/Xa ratio relative to the experiments on the normal cell lines (AB and GM07693) and X/Y ratio relative to the experiments on ICF cell line (PT5) were measured.
Nuclease sensitivity assays
Nuclei were isolated from cultured cells and nuclease sensitivity assays were performed as described elsewhere (35). Briefly, purified nuclei were suspended in DNaseI digestion buffer at about 107 nuclei/ml. For DNaseI assay, aliquots of nuclei suspension were incubated for 10 min, 25°C with increasing amount of enzyme (Roche Diagnostics Gmbh, Germany): 100, 200, 400, 600, 800 and 1000 U/ml. After DNA purification, for all the samples the hot-stop PCR protocol was performed with the same primers used for the ChIP assay (see above) as well as the following XhoI digestion and electrophoresis.
In addition, PCR amplification of a larger fragment of the SYBL1 5′ region including all four MspI sites (shown in Fig. 1) was performed with following primers (data not shown): A, 5′-CTTGGTTTTGGTGGGTTTTG-3′ (nt +1010 to −990); B, 5′-CACCCCACTCCATTTACCTG-3′ (nt +115 to +94).
The authors gratefully acknowledge Drs S. Gartler and V. Orlando for helpful suggestions and critical reading of the manuscript and Dr L. Carrel for the gift of cell lines GM07693 and GM03884. This work received financial support from Telethon grant GGP02308 to M.D'E., from P.F. Biotecnologia CNR to M.D'U., from the Human Frontier Science Program, the CNRS and the Fondation pour la Recherche Médicale to R.F. and from the National Institute of Health to R.S.H. (grant HD16659).
To whom correspondence should be addressed at: Institute of Genetics and Biophysics, ‘A. Buzzati Traverso’, CNR, via Marconi 10, 80125, Naples, Italy. Tel: +39 0817257250; Fax: +39 0815936123; Email: email@example.com