title: Aberrant Expression of TIMP-2 and PBEF Genes in the Placentae of Cloned Mice Due to Epigenetic Reprogramming Error.
abstract: Cloned mice derived from somatic or ES cells show placental overgrowth (placentomegaly) at term. We had previously analyzed cloned and normal mouse placentae by using two-dimensional gel electrophoresis and mass spectrometry to identify differential protein expression patterns. Cloned placentae showed upregulation of tissue inhibitor of metalloproteinase-2 (TIMP-2), which is involved in extracellular matrix degradation and tissue remodeling, and downregulation of pre-B cell colony enhancing factor 1 (PBEF), which inhibits apoptosis and induces spontaneous labor. Here, we used Western blotting to further analyze the protein expression levels of TIMP-2 and PBEF in cloned placentae derived from cumulus cells, TSA-treated cumulus cells, intracytoplasmic sperm injection (ICSI), and natural mating (NM control). Cloned and TSA-treated cloned placentae had higher expression levels of TIMP-2 compared with NM control and ICSI-derived placentae, and there was a positive association between TIMP-2 expression and the placental weight of cloned mouse concepti. Conversely, PBEF protein expression was significantly lower in cloned and ICSI placentae compared to NM controls. To examine whether the observed differences were due to abnormal gene expression caused by faulty epigenetic reprogramming in clones, we investigated DNA methylation and histone modification in the promoter regions of the genes encoding TIMP-2 and PBEF. Sodium bisulfite sequencing did not reveal any difference in DNA methylation between cloned and NM control placentae. However, ChIP assays revealed that the level of H3-K9/K14 acetylation at the TIMP-2 locus was higher in cloned placentae than in NM controls, whereas acetylation of the PBEF promoter was lower in cloned and ICSI placenta versus NM controls. These results suggest that cloned placentae appear to suffer from failure of histone modification-based reprogramming in these (and potentially other) developmentally important genes, leading to aberrant expression of their protein products. These changes are likely to be involved in generating the abnormalities seen in cloned mouse placentae, including enlargement and/or a lack of proper placental function.
authors.affiliation: Department of Animal Science & Biotechnology, Chungnam National University, Daejeon, Republic of Korea.
authors.forename: Hong Rye
authors.initials: HR
authors.lastname: Kim
authors.affiliation: Department of Animal Science & Biotechnology, Chungnam National University, Daejeon, Republic of Korea.
authors.forename: Jae Eun
authors.initials: JE
authors.lastname: Lee
authors.affiliation: Department of Animal Science & Biotechnology, Chungnam National University, Daejeon, Republic of Korea.
authors.forename: Reza Kheirkhahi
authors.initials: RK
authors.lastname: Oqani
authors.affiliation: Department of Animal Science & Biotechnology, Chungnam National University, Daejeon, Republic of Korea.
authors.forename: So Yeon
authors.initials: SY
authors.lastname: Kim
authors.affiliation: Faculty of Life and Environmental Science, University of Yamanashi, Yamanashi, Japan.
authors.forename: Teruhiko
authors.initials: T
authors.lastname: Wakayama
authors.affiliation: School of Medicine, Tongi University, Shanghai, China.
authors.forename: Chong
authors.initials: C
authors.lastname: Li
authors.affiliation: Department of Animal Resource Development, National Institute of Animal Science, Cheonan, Republic of Korea.
authors.forename: Su Jin
authors.initials: SJ
authors.lastname: Sa
authors.affiliation: Department of Animal Resource Development, National Institute of Animal Science, Cheonan, Republic of Korea.
authors.forename: Je Seok
authors.initials: JS
authors.lastname: Woo
authors.affiliation: Department of Animal Science & Biotechnology, Chungnam National University, Daejeon, Republic of Korea.
authors.forename: Dong Il
authors.initials: DI
authors.lastname: Jin
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D065150:Cellular Reprogramming / Q000187:drug effects / Q000235:genetics*; D002585:Cesarean Section; D019976:Cloning, Organism; D016207:Cytokines / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects / Q000235:genetics; D044127:Epigenesis, Genetic / Q000187:drug effects; D005260:Female; D006657:Histones / Q000378:metabolism; D006877:Hydroxamic Acids / Q000494:pharmacology; D008297:Male; D051379:Mice; D054409:Nicotinamide Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D009929:Organ Size / Q000187:drug effects; D010920:Placenta / Q000187:drug effects / Q000378:metabolism*; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020554:Sperm Injections, Intracytoplasmic; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics* / Q000378:metabolism
chemical_list: D016207:Cytokines; D006657:Histones; D006877:Hydroxamic Acids; D012333:RNA, Messenger; D019716:Tissue Inhibitor of Metalloproteinase-2; C012589:trichostatin A; D054409:Nicotinamide Phosphoribosyltransferase; C495867:nicotinamide phosphoribosyltransferase, mouse