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. Threedimensional 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.

Three-dimensional analysis of nuclear heterochromatin distribution during early development in the rabbit

“…Indeed, only a few studies have been carried out in the context of early development on bovine or rabbit embryos (Koehler et al 2009; Pichugin et al 2010; Yang et al 2013; Popken et al 2014a, b; review in Borsos and Torres-Padilla 2016). …”

Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Development and Its Functional Implications

“…Recently, we observed a massive reorganization of nuclear architecture in bovine preimplantation embryos generated either by in vitro fertilization (IVF) or by somatic cell nuclear transfer (SCNT) [ 1 , 2 ]. These changes were most prominent during the transit through minor and major genome activation (mGA and MGA) which occur at the 2-cell stage [ 3 ] and at the 8-cell stage [ 4 ], respectively.…”

Transcriptome activity of isochores during preimplantation process in human and mouse

“…2). Moreover, the weaker coefficients obtained for the early stages suggest a weaker activity of the GC-rich and gene-rich isochores, this can be explained by the minor gene activation and silencing of the maternal genes during early stages [32], and a subsequent increased activity at the EGA and PGA stages [41]. Both, EGA and PGA, can explain the differences we detected in the patterns of the compositional correlations between the two species, for example, mouse EGA initiates at the two-cell stage, human four-to eight-cell stages [33,34].…”