title: Positional changes of a pluripotency marker gene during structural reorganization of fibroblast nuclei in cloned early bovine embryos.
abstract: Cloned bovine preimplantation embryos were generated by somatic cell nuclear transfer (SCNT) of bovine fetal fibroblasts with a silent copy of the pluripotency reporter gene GOF, integrated at a single site of a chromosome 13. GOF combines the regulatory Oct4/Pou5f1 sequence with the coding sequence for EGFP. EGFP expression served as a marker for pluripotency gene activation and was consistently detected in preimplantation embryos with 9 and more cells. Three-dimensional radial nuclear positions of GOF, its carrier chromosome territory and non-carrier homolog were measured in nuclei of fibroblasts, and of day 2 and day 4 embryos, carrying 2 to 9 and 15 to 22 cells, respectively. We tested, whether transcriptional activation was correlated with repositioning of GOF toward the nuclear interior either with a corresponding movement of its carrier chromosome territory 13 or via the formation of a giant chromatin loop. A significant shift of GOF away from the nuclear periphery was observed in day 2 embryos together with both carrier and non-carrier chromosome territories. At day 4, GOF, its carrier chromosome territory 13 and the non-carrier homolog had moved back toward the nuclear periphery. Similar movements of both chromosome territories ruled out a specific GOF effect. Pluripotency gene activation was preceded by a transient, radial shift of GOF toward the nuclear interior. The persistent co-localization of GOF with its carrier chromosome territory rules out the formation of a giant chromatin loop during GOF activation.
authors.affiliation: a Division of Anthropology and Human Genetics Biocenter ; LMU Munich ; Martinsried , Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Popken
authors.forename: Daniela
authors.initials: D
authors.lastname: Koehler
authors.forename: Alessandro
authors.initials: A
authors.lastname: Brero
authors.forename: Annegret
authors.initials: A
authors.lastname: Wuensch
authors.forename: Tuna
authors.initials: T
authors.lastname: Guengoer
authors.forename: Tobias
authors.initials: T
authors.lastname: Thormeyer
authors.forename: Eckhard
authors.initials: E
authors.lastname: Wolf
authors.forename: Thomas
authors.initials: T
authors.lastname: Cremer
authors.forename: Valeri
authors.initials: V
authors.lastname: Zakhartchenko
mesh_terms: D000818:Animals; D001755:Blastocyst; D002417:Cattle; D002467:Cell Nucleus / Q000235:genetics* / Q000378:metabolism; D065150:Cellular Reprogramming / Q000235:genetics; D002875:Chromosomes / Q000235:genetics*; D019976:Cloning, Organism; D004622:Embryo, Mammalian; D047108:Embryonic Development; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D053652:Nuclear Transfer Techniques; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D050814:Octamer Transcription Factor-3
keywords: (bovine) preimplantation embryos, chromosome territories, nuclear architecture, nuclear reprogramming, pluripotency gene activation, somatic cell nuclear transfer; BFF, bovine fetal fibroblasts; BTA, Bos taurus; CLSM, confocal laser scanning microscopy; CT, chromosome territory; eADS, enhanced absolute 3D distances to surfaces; IVF, in vitro fertilization; MGA, major embryonic genome activation; GOF, Oct4/Pou5f1-EGF