THE HISTONE METHYLTRANSFERASE G9A ADOPTS A NUCLEAR LOCALIZATION IN CLEAVED PORCINE EMBRYOS, BUT RARELY IN PRONUCLEAR STAGE EMBRYOS

Abstract

Epigenetic modifications to chromatin play key roles in regulating transcription. Dimethylation of the lysine 9 residue of histone protein H3 (H3/K9) plays a central role in transcriptional repression and heterochromatin formation. Porcine, unlike murine embryos, posess dimethylated H3/K9 in both maternal and paternal pronuclei, although some paternal pronuclei appear to lack this modification. We hypothesized that some pronuclei lacked detectable dimethylated H3/K9 due to an asymmetric localization of histone methyltransferases that mediate dimethylation of H3/K9. The aim of this study was to characterize the localization of G9a, a histone methyltransferase that mediates H3/K9 dimethylation, during early embryonic development in the pig. An experiment was carried out in which mRNA encoding the fusion protein of human G9a and GFP (gift from Yoichi Shinkai) was injected into porcine oocytes, and the localization of the translated protein detected in pronuclear and cleavage stage embryos. Porcine oocytes were matured in vitro for 40 hours in a chemically defined maturation medium (TCM 199 supplemented with 0.1% PVA, 0.069mg/mL cysteine, 10 ng/mL EGF, 0.5 IU/mL LH, and 0.5 IU/mL FSH) at 39°C in 5% CO2. The oocytes were denuded of cumulus cells and placed into Hepes-buffered medium with 0.3% BSA. Two separate treatment groups were microinjected intracytoplasmically with 1000ng/μL mRNA of GFP or G9a-GFP, respectively. Microinjection was performed using a FemtoJet microinjector by Eppendorf. The treatment groups and non-injected controls were then separately fertilized in vitro and cultured in PZM supplemented with 4mg/mL BSA for 12 or 48 hours at 39°C in 5% CO2. Embryos were examined under UV light; embryos expressing GFP were selected for processing. After removal of the zona pellucida using 0.1% PVA in acid Tyrode solution, embryos were fixed in 3.7% paraformaldehyde for 2 hours and washed in PBS and 0.1% Tween20. Indirect immunocytochemical staining was then performed using a mouse anti-GFP antibody from Chemicon (MAB3580), and a FITC conjugated goat anti-mouse antibody from Sigma (F5262). Embryos were stained with Hoechst and mounted on slides. Embryos were examined using epifluorescence and confocal microscopy. We found that embryos injected with G9a-GFP mRNA did not possess nuclear localized GFP in most pronuclear stage embryos (n=19/22) while all cleaved embryos displayed nuclear localization (n=7/7). Embryos injected with GFP mRNA did not display nuclear localization of GFP at any stage of development. These results indicate that G9a is localized in cleaved porcine embryos but does not display localization in most pronuclear stage embryos. These data suggest that histone methyltransferases other than G9a may be responsible for the asymmetric dimethylation pattern observed in porcine pronuclei, and that G9a may play a role in dimethylation in cleaved embryos. (poster)