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A global proteomics approach by 2-D gel electrophoresis and mass spectrometry was used to analyze the differential protein patterns of three placentae obtained after postnatal death of fetuses from SCNT of Korean Native cattle and three normal placentae obtained after birth of AI fetuses, as previously reported (Kim et al. 2004 Reprod. Fertil. Develop. 16, 145). One differentially up-regulated proteins in SCNT placenta was identified as TIMP-2 protein that is related to extracellular matrix degradation and tissue remodeling processes. To ensure that the identified protein was truly up-regulated in SCNT placenta, a small portion of the protein lysates were subjected to Western blotting with the antibodies against TIMP-2 and β-actin protein. Band images were scanned with a GT-6000 Scanner (Epson-Seiko, Suwa, Nagano, Japan) and densitometric analyses were performed using NIH Image (version 1.56). Proteins were normalized against the levels of β-actin protein. Indeed, Western blot analysis revealed a significant increase in TIMP-2 protein level (about 5-fold) in SCNT placenta compared with normal. Epigenetic programming of the genome by DNA methylation and histone modification ensures proper gene expression during development. To examine how the TIMP-2 gene was programmed to be active in SCNT placenta, we first investigated the status of DNA methylation in the promoter region of the TIMP-2 gene. Sodium bisulfite mapping revealed that most CpG sites in SCNT placenta cells were unmethylated in agreement with expression of TIMP-2 compared with those in normal placenta cells, suggesting that TIMP-2 gene programming by hypomethylation of CpG sites in the promoter region was responsible for the expression in SCNT placenta. We then examined histone modification of the nucleosome in the coding region of the TIMP-2 gene of SCNT placenta cells and normal placenta cells. Chromatin immunoprecipitation (ChIP) assay revealed that H3-K9/K14 acetylation of the TIMP-2 locus in SCNT placenta cells was about 2-fold higher than in normal placenta cells. Therefore, it is postulated that the SCNT placenta at the end of gestation seems to be unusual in tissue remodeling via improper expression of TIMP-2 by abnormal epigenetic modification, and that may affect placental abnormality such as enlarged and dysfunctional placentae as reported in cloned bovine and mouse.

223 ABERRENT TIMP-2 GENE EXPRESSION IN CLONED BOVINE NEONATAL PLACENTA IS DUE TO ABNORMAL EPIGENETIC MODIFICATIONS