Epigenetic marks and nuclear reprogramming in early embryos: short and long term effects

ln mammals, the embryonic genome is first transcriptionnally inactive alter fertilization. The onset of embryonic gene expression is initiated later during the preimplantation developmental period, during the so-called "embryonic genome activation (EGA)". EGA is dependent on the presence of the basal transcriptional machinery components but also on parental genomes reprogramming. Among the mechanisms of this reprogramming, it appears that modifications of epigenetic marks are essential. One of the main reprogrammed elements is pericentric heterochromatin and the related epigenetic mark H3K9me3 (Martin et al., 2006). Remarkably, it has been shown that interference with pericentric heterochromatin reprogramming allers development (Probst et al., 2010; Santenard et al., 2010). To further investigate pericentric heterochromatin reorganization, we decided to study three other epigenetic markers, H3S10P 1 H3K9me3S10P and 5-hydroxymethylcytosine (5-hMeC) in early mouse embryos. Our results indicate that H3K9me3S1OP followed similar dynamics but only labeled the maternai pericentric heterochromatin, even more specifically than H3K9me3. On the other hand, we found that H3S1OP overlaps with the pericentric sequences at the 1-cell and 2-cell stages on both parental genomes. We then looked at 5-hMeC that is believed to complement 5-MeC during paternal genome demethylation at 1-cell. We demonstrate thal 5-MeC is mainly enriched on the paternal pericentric heterochromatin whil5-hMeC overlaps only with a subset of the maternai pericentric sequences. Moreover, their kinetics do not exactly complement each other; in particular, a peak of 5-hMeC is observed at the end of the 1-cell stage in bath pronuclei. Based on these findings, we used these epigenetic modifications as markers of pericentric heterochromatin reprogramming in embryos derived by nuclear transfer (NT). NT refers to the injection of a donor nucleus into an enucleated egg. However only a very small proportion of NT embryos can develop to term alter transfer into recipient uteri and it is believed that one of the causes for this is incomplete genome reprogramming. ln mouse, we indeed showed that remodeling into an embryonic-like nuclear organization occurs quite rapidly alter NT, within two-cell cycles. However, abnormal nuclear remodeling is frequently observed: NT embryos olten present irregular chromatin reorganization with pericentromeric heterochromatin clumps like in the donor nuclei, suggesting thal the somalie nuclear configuration is not completely lost alter NT. lnterestingly, the percentage of such abnormalities is correlated with developmental efficiency and seems to depend on the differentiation state of the starting material. When we studied epigenetic markers in these embryos, we observed thal two of these markers, H3S10P and H3K9me3S10P, are the ones found on the part of the pericentromeric heterochromatin which is correctly remodeled, resembling exactly the embryonic heterochromatin configuration of naturally fertilized embryos. Conversely, H3K9me3 and HP1associated protein were also detected in the perinuclear clumps of heterochromatin, making obvious the maintenance of the somalie epigenetic signature within these nuclear regions. Finally, we also observed that transient treatment with histone deacetylase inhibitors (trichostatin A or scriptaid) improves nuclear remodeling in NT embryos as weil as embryonic development at subsequent stages. Together, the results suggest that proper organization of constitutive heterochromatin in embryos is involved in the initial reprogramming steps and might have long term consequences. We also