Portraits of Gene Expression in MZ Twin Pairs. Finally, we addressed Our study reveals that the pattens of epigenetic modifications in a in gene expression. We eda pigenetic modification: global change MZ twin pairs diverge as they become older. Differences in u the ultimate goal of all ined whether older twin pairs who epigenetic patterns in genetically identical individuals could be differed most with respect to DNA methylation and histone acet- explained by the influence of both external and internal factors. ylation levels in general and at specific loci also displayed the most Smoking habits, physical activity, or diet, among others, are external different gene expression profiles. To this end, we extracted RNa factors that have been proposed to have a long-term influence on from the two most distinct pairs of twins(the 3-and 50-year-old epigenetic modifications( 12, 13). However, it is possible that small pairs)and performed gene expression microarray analysis. The defects in transmitting epigenetic information through successive results obtained when each twin expression portrait was confronted cell divisions, or maintaining it in differentiated cells, accumulate in with its own sibling demonstrated that although the expression a process that could be considered as an"epigenetic drift associ- patterns of the 3-year-olds were almost identical, the 50-year-old ated with the aging process(14, 15). Identification of proteins that twins had extremely different expression profiles( Fishers exact test, mediate these effects has provided an insightful view into this P=0.029)(Fig. 4D ). From a quantitative standpoint, there were complex process and in the diseases that occur when it is perturbed four times as many differentially expressed genes in the older twin (12, 13). Accumulation of epigenetic defects would probably occur pair as in the younger twin pair. Furthermore, the older twin sibling ding to genetic mutations with the most severe DNA hypomethylation and histone H3 and H4 cause their consequences in survival are probably less dramatic hyperacetylation(the epigenetic changes usually associated with and cells have not developed a comparable amount of mechanisms transcriptional activation)was that with the highest number of to correct them. MZ twins constitute an excellent example of how genetically overexpressed genes(Fig. 4D). Most importantly, and internally identical individuals can exhibit differences and therefore provide identified by AIMS or RLGS as being differentially methylated in a unique model to study the contribution/ role of epigenetic mod- the twin pairs also showed different gene expression in our mi- does make MZ twins differ? By using whole-genome and locus- croarray analysis. specific approaches, we found that approximately one-third ofMZ 9 pigenetic Differences in MZ Twin Pairs Occur Across Different Tissue twins harbored epigenetic differences in DNA methylation an Types. Although our study had focused on the dNa methylation histone modification. These differential markers between twins distributed throughout their genomes, affecting repeat DNA se- the existence of epigenetic differences among other cell types, gene expression. We also established that these epigenetic markers especially because the described discordances between MZ twins were more distinct in MZ twins who are also present across different organs and cellular functions. To lifestyles, and had spent less of their lives together, underlining the this end, we studied the 5mC DNA content, the acetylation status nificant role of environmental factors in translating a common of histone H3 and H4, and the patterns of loci-specific dna genotype Into a difterent phenotype. Our findings also support the role of epigenetic differences in the discordant frequency/onset of methylation by the AIMS approach, methyl-specific comparative diseases in MZ twins(2, 16-18) Other evidence indicates that relatively small differences in twins couples by using epithelial mouth cells, intraabdominal fat, epigenetic patterns can have a large impact in phenotype, for and skeletal muscle biopsies. We found that in these three new instance in cloned animals(4), with MZ twins re nting natural tissues, in the same manner that we had observed in lymphocytes, human clones. Another powerful example is provided by the agouti marked epigenetic differences were present in older Mztwins with mouse( 19). In this model, diet affects the methylation status of an different lifestyles and that had spent less of their lives together inserted intracisternal A particle element that changes the animal's (Pearson test, P<0.05)(see Fig. 6, which is published as supporting coat color: an environmental factor interacting with a single geno nformation on the PNAS web site). Thus, distinct profiles of DNa type, mediated by an epigenetic change, to produce a different methylation and histone acetylation patterns among many different phenotype In humans, the investigation of how assisted reproduc tissues arise during the lifetime of Mz twins that may contribute to tive technology that uses media with undisclosed concentrations of the explanation of some of their phenotypic discordances and methyl-donors associates with epigenetic errors such as imprinting underlie their differential frequency/onset of common disease defects and cancer has been proposed(20). Our comparison of Discussion twins suggests that external and/or internal factors can have an impact in the phenotype by altering the pattern of epigenetic Although genomic information is uniform among the different cells modifications and thus modulating the genetic information.Future of a complex organism, the epigenome varies from tissue to tissue, studies should now address the specific mechanisms responsible for controlling the differential expression of genes and providing the observed epigenetic drift of MZ twins. specific identity to each cell type. DNA methylation and histone We thank all our volunteer twins and Sara Casado, Lidia Lopez.Serra modifications store epigenetic information that mainly controls Miguel Alaminos, and Alicia Barroso from the Spanish National Cancer heritable states of gene expression(12, 13), and it is now well estab- Centre. M.F.F. is funded by the Foundation of the Spanish Association lished that both epigenetic layers are mechanistically linked (12, 13). Against Cancer(AECC). L. Hall. J G.& 1966)in Twins and Twinning, eds. Emery. A. E. H& 9. Paz, M. F, Wei, s Cigudosa, J C- Rodrigucz-Perales, S, Peinado. M. A Huang. T. 2. Cardno. A. isdijk, F. V- Sham, P C, Murray, R. M. McGuffin, P (2002)An 10. Costello, J F, Miraglia, D J.& Plass, C(2002)Methods 27, 144-149. iatry1s59.539-545. lachin, G. A(1996)Ant J Med. Gener. 61. 216-228. 12. Jaenisch, R& Bird, A.(2003)Nar. Genet. 33, SuppL, 245-254. Rideout, w. M. Ill, Eggan, K nisch,R.(201) Scienee293,1093-1098 5. Becker, A. Busjahn, A. Faulhaber, H. D, Bahring, S, Robertson, J. Schuster H. Luft, imada. mM.a Ukas 'im t yoki H, Masuno. M, Imaizumi K,Kojima, MWakui,K.,1过款数m.Om已Fx 17. Petronis, A Gottesman, I.I. Kan, P. Kennedy, J. L, Basile, V.S., Paterson. A. D.& Fraga. M. F Ballestar, E, Villar-Garea, A, Boix-ChorneL Bonaldi, T, Haydon, C- Ropero, S, Petrie, K, ef al.(2005)Nar. Gener. 37, 391-400 Genet Fraga et al. PNAs|y262005|vol.102|no.30|10609Portraits of Gene Expression in MZ Twin Pairs. Finally, we addressed the ultimate goal of all major epigenetic modification: global change in gene expression. We examined whether older twin pairs who differed most with respect to DNA methylation and histone acet￾ylation levels in general and at specific loci also displayed the most different gene expression profiles. To this end, we extracted RNA from the two most distinct pairs of twins (the 3- and 50-year-old pairs) and performed gene expression microarray analysis. The results obtained when each twin expression portrait was confronted with its own sibling demonstrated that although the expression patterns of the 3-year-olds were almost identical, the 50-year-old twins had extremely different expression profiles (Fishers exact test, P 0.029) (Fig. 4D). From a quantitative standpoint, there were four times as many differentially expressed genes in the older twin pair as in the younger twin pair. Furthermore, the older twin sibling with the most severe DNA hypomethylation and histone H3 and H4 hyperacetylation (the epigenetic changes usually associated with transcriptional activation) was that with the highest number of overexpressed genes (Fig. 4D). Most importantly, and internally corroborating our data, all of the aforementioned single-copy genes identified by AIMS or RLGS as being differentially methylated in the twin pairs also showed different gene expression in our mi￾croarray analysis. Epigenetic Differences in MZ Twin Pairs Occur Across Different Tissue Types. Although our study had focused on the DNA methylation and histone acetylation changes observed in the lymphocytes of MZ twins, we thought that it would be extremely interesting to analyze the existence of epigenetic differences among other cell types, especially because the described discordances between MZ twins are also present across different organs and cellular functions. To this end, we studied the 5mC DNA content, the acetylation status of histone H3 and H4, and the patterns of loci-specific DNA methylation by the AIMS approach, methyl-specific comparative genomic hybridization, and bisulfite genomic sequencing in MZ twins couples by using epithelial mouth cells, intraabdominal fat, and skeletal muscle biopsies. We found that in these three new tissues, in the same manner that we had observed in lymphocytes, marked epigenetic differences were present in older MZ twins with different lifestyles and that had spent less of their lives together (Pearson test, P  0.05) (see Fig. 6, which is published as supporting information on the PNAS web site). Thus, distinct profiles of DNA methylation and histone acetylation patterns among many different tissues arise during the lifetime of MZ twins that may contribute to the explanation of some of their phenotypic discordances and underlie their differential frequencyonset of common diseases. Discussion Although genomic information is uniform among the different cells of a complex organism, the epigenome varies from tissue to tissue, controlling the differential expression of genes and providing specific identity to each cell type. DNA methylation and histone modifications store epigenetic information that mainly controls heritable states of gene expression (12, 13), and it is now well estab￾lished that both epigenetic layers are mechanistically linked (12, 13). Our study reveals that the patterns of epigenetic modifications in MZ twin pairs diverge as they become older. Differences in epigenetic patterns in genetically identical individuals could be explained by the influence of both external and internal factors. Smoking habits, physical activity, or diet, among others, are external factors that have been proposed to have a long-term influence on epigenetic modifications (12, 13). However, it is possible that small defects in transmitting epigenetic information through successive cell divisions, or maintaining it in differentiated cells, accumulate in a process that could be considered as an ‘‘epigenetic drift’’ associ￾ated with the aging process (14, 15). Identification of proteins that mediate these effects has provided an insightful view into this complex process and in the diseases that occur when it is perturbed (12, 13). Accumulation of epigenetic defects would probably occur at a faster rate than that corresponding to genetic mutations because their consequences in survival are probably less dramatic and cells have not developed a comparable amount of mechanisms to correct them. MZ twins constitute an excellent example of how genetically identical individuals can exhibit differences and therefore provide a unique model to study the contributionrole of epigenetic mod￾ifications in the establishment of the phenotype (2, 16–18). What does make MZ twins differ? By using whole-genome and locus￾specific approaches, we found that approximately one-third of MZ twins harbored epigenetic differences in DNA methylation and histone modification. These differential markers between twins are distributed throughout their genomes, affecting repeat DNA se￾quences and single-copy genes, and have an important impact on gene expression. We also established that these epigenetic markers were more distinct in MZ twins who were older, had different lifestyles, and had spent less of their lives together, underlining the significant role of environmental factors in translating a common genotype into a different phenotype. Our findings also support the role of epigenetic differences in the discordant frequencyonset of diseases in MZ twins (2, 16–18). Other evidence indicates that relatively small differences in epigenetic patterns can have a large impact in phenotype, for instance in cloned animals (4), with MZ twins representing natural human clones. Another powerful example is provided by the agouti mouse (19). In this model, diet affects the methylation status of an inserted intracisternal A particle element that changes the animal’s coat color: an environmental factor interacting with a single geno￾type, mediated by an epigenetic change, to produce a different phenotype. In humans, the investigation of how assisted reproduc￾tive technology that uses media with undisclosed concentrations of methyl-donors associates with epigenetic errors such as imprinting defects and cancer has been proposed (20). Our comparison of MZ twins suggests that external andor internal factors can have an impact in the phenotype by altering the pattern of epigenetic modifications and thus modulating the genetic information. Future studies should now address the specific mechanisms responsible for the observed epigenetic drift of MZ twins. We thank all our volunteer twins and Sara Casado, Lidia Lopez-Serra, Miguel Alaminos, and Alicia Barroso from the Spanish National Cancer Centre. 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