Dynamic changes in localization of chromobox (CBX) family members during the maternal to embryonic transition

Abstract

The Chromobox domain (Cbx) gene family, consisting of Polycomb and Heterochromatin Protein 1 genes, is involved in transcriptional repression, cell cycle regulation and chromatin remodeling. We report the first study of gene expression and protein localization of the Cbx genes in in vitro produced bovine embryos. All but one gene (Cbx6) were expressed. This was confirmed by immunolocalization for HP1alpha, beta, gamma, and Pc2, 3. HP1beta was found in the nuclei of embryos from the two-cell stage onwards, whereas HP1gamma showed diffuse cytoplasmic/nuclear localization at the two- and eight-cell stages, and predominantly nuclear localization at the four-cell stage and the 16-cell stage onwards. Leptomycin B (LMB), a specific inhibitor of the nuclear export protein CRM-1 (chromosomal regional maintenance-1), was found to increase nuclear localization of HP1gamma at the eight-cell stage, and to prevent progression past this stage of embryogenesis. This indicates that HP1gamma possesses a CRM-1-dependent nuclear export pathway which may represent part of the basis of HP1gamma's ability to shuttle between the nucleus and the cytoplasm in dynamic fashion. HP1alpha was expressed in embryonic nuclei at all stages, but was found to relocalise from euchromatin to heterochromatin during the maternal to embryonic transition (MET). In contrast, Pc2 and Pc3 were evenly distributed between cytoplasm and nucleus until the eight- and sixteen-cell stages or the morula stage, respectively, before relocating preferentially to the cytoplasm. Collectively, the results suggest that dynamic changes of the nuclear-cytoplasmic and subnuclear distribution of members of the Cbx family may be central to the MET.

Chromobox homolog 2 protein: A novel biomarker for predicting prognosis and Taxol sensitivity in patients with breast cancer

“…However, certain points require further study. Firstly, dynamic localization of the nuclear-cytoplasmic and/or sub-nuclear distribution of members of the Cbx family occurs during the maternal-to-embryonic transition (42). The present study revealed that Cbx2 was predominantly expressed in the cytoplasm.…”

Lineage-specific expression of heterochromatin protein 1γ in post-compaction, in vitro-produced bovine embryos

“…Fluorescence in situ hybridisation probe generation and digoxigenin labelling A partial polymerase chain reaction (PCR) product (288 bp) of HP1g (Ruddock-D'Cruz et al 2008) was ligated into pGEM-T Easy plasmid and grown on agar according to the manufacturer's instructions (pGEM-T Easy Vector System; Promega). Clones containing the HP1g insert were cultured and cDNA produced from the whole plasmid by PCR.…”

“…HP1a, HP1b and HP1g) share conserved N-terminal (chromodomain) and C-terminal (chromoshadow domain) domains, coupled by a less conserved hinge domain. Despite considerable primary sequence homology, mammalian HP1 variants differentially bind mono-and olignucleosomes and linker histones in vitro (Nielsen et al 2001) and can localise to euchromatic and/or heterochromatic regions (Minc et al 2000;De Lucia et al 2005;Ruddock-D'Cruz et al 2008). The localisation and activity of the HP1 protein is regulated by ubiquitous and isoform-specific acetylation, methylation, sumoylation and ubiquitination (Lomberk et al 2006).…”

“…The HP1g transcript is expressed throughout human folliculogenesis and preimplantation embryogenesis, although it is undetectable in mature oocytes (Hinkins et al 2005). In contrast, bovine HP1g protein is expressed in mature oocytes and throughout early embryogenesis (Ruddock-D'Cruz et al 2008). Interestingly, Exportin-1-dependent evacuation of HP1g from the nuclear compartment has been shown to occur before the MET during bovine embryonic development (Ruddock-D'Cruz et al 2008).…”

“…In contrast, bovine HP1g protein is expressed in mature oocytes and throughout early embryogenesis (Ruddock-D'Cruz et al 2008). Interestingly, Exportin-1-dependent evacuation of HP1g from the nuclear compartment has been shown to occur before the MET during bovine embryonic development (Ruddock-D'Cruz et al 2008). In embryonic cells in Drosophila, RNA interference (RNAi) knockdown of HP1 induces a marked increase in apoptosis and transcriptional aberrations in euchromatic genes (De Lucia et al 2005).…”