RESUMO
DNA methylation patterns are important for establishing cell, tissue, and organism phenotypes, but little is known about their contribution to natural human variation. To determine their contribution to variability, we have generated genome-scale DNA methylation profiles of three human populations (Caucasian-American, African-American, and Han Chinese-American) and examined the differentially methylated CpG sites. The distinctly methylated genes identified suggest an influence of DNA methylation on phenotype differences, such as susceptibility to certain diseases and pathogens, and response to drugs and environmental agents. DNA methylation differences can be partially traced back to genetic variation, suggesting that differentially methylated CpG sites serve as evolutionarily established mediators between the genetic code and phenotypic variability. Notably, one-third of the DNA methylation differences were not associated with any genetic variation, suggesting that variation in population-specific sites takes place at the genetic and epigenetic levels, highlighting the contribution of epigenetic modification to natural human variation.
Assuntos
Metilação de DNA , Variação Genética , População/genética , Adulto , Negro ou Afro-Americano/genética , Asiático/genética , Epigênese Genética , Feminino , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Masculino , Locos de Características Quantitativas , População Branca/genéticaRESUMO
Epigenetic modifications have been shown to be important in developmental tumors as Ewing sarcoma. We profiled the DNA methylation status of 15 primary tumors, 7 cell lines, 10 healthy tissues and 4 human mesenchymal stem cells lines samples using the Infinium Human Methylation 450K. Differential methylation analysis between Ewing sarcoma and reference samples revealed 1166 hypermethylated and 864 hypomethylated CpG sites (Bonferroni p < 0.05, δ-ß-value with absolute difference of >0.20) corresponding to 392 and 470 genes respectively. Gene Ontology analysis of genes differentially methylated in Ewing sarcoma samples showed a significant enrichment of developmental genes. Membrane and cell signal genes were also enriched, among those, 11 were related to caveola formation. We identified differential hypermethylation of CpGs located in the body and S-Shore of the PTRF gene in Ewing sarcoma that correlated with its repressed transcriptional state. Reintroduction of PTRF/Cavin-1 in Ewing sarcoma cells revealed a role of this protein as a tumor suppressor. Restoration of caveolae in the membrane of Ewing sarcoma cells, by exogenously reintroducing PTRF, disrupts the MDM2/p53 complex, which consequently results in the activation of p53 and the induction of apoptosis.
Assuntos
Neoplasias Ósseas/genética , Caveolina 1/genética , Metilação de DNA , Epigênese Genética , Perfilação da Expressão Gênica/métodos , Genes Supressores de Tumor , Proteínas de Ligação a RNA/genética , Sarcoma de Ewing/genética , Animais , Apoptose , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Camundongos Nus , Fosforilação , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Transdução de Sinais , Espanha , Transfecção , Carga Tumoral , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Human embryonic stem (hES) cells and fetal mesenchymal stem cells (fMSC) offer great potential for regenerative therapy strategies. It is therefore important to characterise the properties of these cells in vitro. One major way the environment impacts on cellular physiology is through changes to epigenetic mechanisms. Genes subject to epigenetic regulation via genomic imprinting have been characterised extensively. The integrity of imprinted gene expression therefore provides a measurable index for epigenetic stability. Allelic expression of 26 imprinted genes and DNA methylation at associated differentially methylated regions (DMRs) was measured in fMSC and hES cell lines. Both cell types exhibited monoallelic expression of 13 imprinted genes, biallelic expression of six imprinted genes, and there were seven genes that differed in allelic expression between cell lines. fMSCs exhibited the differential DNA methylation patterns associated with imprinted expression. This was unexpected given that gene expression of several imprinted genes was biallelic. However, in hES cells, differential methylation was perturbed. These atypical methylation patterns did not correlate with allelic expression. Our results suggest that regardless of stem cell origin, in vitro culture affects the integrity of imprinted gene expression in human cells. We identify biallelic and variably expressed genes that may inform on overall epigenetic stability. As differential methylation did not correlate with imprinted expression changes we propose that other epigenetic effectors are adversely influenced by the in vitro environment. Since DMR integrity was maintained in fMSC but not hES cells, we postulate that specific hES cell derivation and culturing practices result in changes in methylation at DMRs.
Assuntos
Metilação de DNA/fisiologia , Células-Tronco Embrionárias/metabolismo , Impressão Genômica/fisiologia , Células-Tronco Mesenquimais/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Feto , Perfilação da Expressão Gênica/métodos , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Medicina Regenerativa/métodosRESUMO
BACKGROUND: Imprinted genes are expressed from only one allele in a parent-of-origin dependent manner. Loss of imprinted (LOI) expression can result in a variety of human disorders and is frequently reported in cancer. Biallelic expression of imprinted genes in adult blood has been suggested as a useful biomarker and is currently being investigated in colorectal cancer. In general, the expression profiles of imprinted genes are well characterised during human and mouse fetal development, but not in human adults. METHODOLOGY/PRINCIPAL FINDINGS: We investigated quantitative expression of 36 imprinted genes in adult human peripheral blood leukocytes obtained from healthy individuals. Allelic expression was also investigated in B and T lymphocytes and myeloid cells. We found that 21 genes were essentially undetectable in adult blood. Only six genes were demonstrably monoallelic, and most importantly, we found that nine genes were either biallelic or showed variable expression in different individuals. Separated leukocyte populations showed the same expression patterns as whole blood. Differential methylation at each of the imprinting control loci analysed was maintained, including regions that contained biallelically expressed genes. This suggests in some cases methylation has become uncoupled from its role in regulating gene expression. CONCLUSIONS/SIGNIFICANCE: We conclude that only a limited set of imprinted genes, including IGF2 and SNRPN, may be useful for LOI cancer biomarker studies. In addition, blood is not a good tissue to use for the discovery of new imprinted genes. Finally, lymphocyte DNA methylation status in the adult may not always be a reliable indicator of monoallelic gene expression.