157 DYNAMIC CHANGES IN LOCALIZATION OF HP1α DURING BOVINE EMBRYOGENESIS

Abstract

The Cbx gene family, consisting of Polycomb and Heterochromatin Protein 1 genes, is involved in transcriptional repression, cell cycle regulation, and chromatin remodeling. In Drosophila embryos, the single family member HP1 localizes to the nuclei from the blastoderm stage, nuclear division cycle 10, suggesting that heterochromatin formation occurs around the time of embryonic transcriptional activation (James et al. 1989 Eur. J. Cell Biol. 50, 170–180). In mice, HP1� is absent in the first cell cycle in both parental nuclei following fertilization (van der Heijden et al. 2005 Mech. Dev. 122, 1008–1022). We report the first study of gene expression, mRNA and protein localization, of the Cbx gene HP1� in in vitro-produced bovine embryos. Expression of the HP1� gene was assessed non-quantitatively in amplified cDNA from 3 individual oocytes or embryos for each stage. HP1� (Cbx5) transcripts were detected in all samples except one oocyte, three 4-cell embryos, and one Day 7 blastocyst. The lack of transcripts at the 4-cell stage may reflect an early degradation of maternal transcripts, or indicate that transcript levels were too low to be detected with this assay. Immuno-localization of HP1� was then performed by using an anti-HP1� monoclonal antibody (Upstate, Auspep Pty., Ltd., Parkville, Victoria, Australia), revealing HP1� to be localized evenly in the cytoplasm of oocytes, and then progressing to both cytoplasmic and nuclear staining at the 2-cell stage. This was followed by nuclear staining from the 4-cell stage onward. Higher power investigation of the subnuclear localization of HP1� showed a diffuse type staining pattern within the nuclei of 2- and 4-cell embryos, followed by punctate staining within the euchromatin in the 8-cell embryos, and within the heterochromatin by the 16-cell stage. At the blastocyst stage, staining appeared more diffuse, but localized to the heterochromatin. This suggests that HP1� is localized to the euchromatin early, followed by subsequent localization to the heterochromatin following the MET. HP1� localization to the euchromatin was recently described (Grigoryev et al. 2004 J. Cell Sci. 117, 6153–6162) during a differentiation event in murine lymphocytes. It is evenly distributed throughout the nucleus in quiescent lymphocytes, but localizes to the vicinity of centromeric chromatin during lymphocyte reactivation. HP1-TIF1α interaction was also recently shown to be required during a short period of time within primitive endoderm-like cells for terminal differentiation (Cammas et al. 2004 Genes Dev. 18, 2147–2160). The results thus indicate that HP1� behaves similarly in embryonic differentiation and lymphocyte reactivation, implying a common mechanism of chromatin remodeling in the 2 cellular systems. Collectively, the results suggest that dynamic changes of the nuclear–cytoplasmic and subnuclear distribution of HP1� may play a key role in the bovine MET.