Epigenetic regulation of the imprinted U2af1-rs1 gene during retinoic acid-induced differentiation of embryonic stem cells.
Epigenetic modifications such as DNA methylation and changes in chromatin structure are changes in the chemical composition or structure of DNA that work by regulating gene expression. Their mechanisms of action have been generally studied in imprinted genes. The present work analyzes the involvement of these mechanisms in the expression of the U2af1-rs1 imprinted gene during the differentiation process of embryonic stem (ES) cells induced by retinoic acid. By DNA digestion with methylation-dependent or independent restriction enzymes and consecutive Southern blot, we have found that methylation of the U2af1-rs1 gene increases in differentiated ES cells and in embryoid bodies. However, northern blot and real-time reverse transcription-polymerase chain reaction analysis showed a higher expression of the U2af1-rs1 gene in differentiated ES cells and in embryoid bodies than in undifferentiated ones. On the other hand, the sensitivity to DNase-I assay demonstrated an open chromatin conformation for differentiated cells with regard to undifferentiated ES cells. Our results suggest that the expression of the U2af1-rs1 gene would be regulated by changes in chromatin structure rather than by DNA methylation during the RA-induced process of differentiation of ES cells.
Andollo
Noelia
N
Department of Cell Biology and Histology, Faculty of Medicine and Dentistry, University of the Basque Country, Leioa, Vizcaya, Spain.
Boyano
María D
MD
Andrade
Ricardo
R
Aréchaga
Juan M
JM
D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D002843:Chromatin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D019003:Deoxyribonuclease HpaII / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D005786:Gene Expression Regulation / Q000187:drug effects; D018392:Genomic Imprinting; D051379:Mice; D046529:Microscopy, Electron, Transmission; D008957:Models, Genetic; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism; D000072668:Splicing Factor U2AF; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D014212:Tretinoin / Q000494:pharmacology*
D002843:Chromatin; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607495:Zrsr1 protein, mouse; D014212:Tretinoin; D004247:DNA; D019003:Deoxyribonuclease HpaII; D003850:Deoxyribonuclease I; D000469:Alkaline Phosphatase