One of the most striking features of the organization of chromatin following completion of the first mitosis is the active and rapid removal of a number of histone modifications, including the methylation of two lysine residues in the globular domain of histone H3, K64 and K79 (). H3K64me3, which is strongly enriched in the maternal chromatin throughout the pronuclear phases, is dramatically removed by the two-cell stage independently of DNA replication and remains undetectable until implantation [29
]. H3K79me3 however, is gradually removed from the maternal chromatin during pronuclear stages before DNA replication begins and is also undetectable by the two-cell stage until post-implantation [30
]. Both of these, (presumably) demethylation events are also observed upon parthenogenetic activation demonstrating that the mechanism for this active removal is sufficiently provided by the oocyte itself (). The removal of these hydrophobic marks in the globular domain of histone H3 may unlock the chromatin structure allowing a higher level of plasticity for developmental transitions and transcription to occur during the two-cell and later stages.
Figure 2: Distribution of heterochromatin marks in two-cell stage embryos from normal fertilization or parthenogenetic activation of oocytes. In two-cell embryos, the maternal and paternal chromosomes remain compartmentalized and therefore the maternally derived (more ...)
During the first mitosis, the chromosomes move together but they are compartmentalized and remain separated even in two-cell stage embryos, indicating that the epigenetic programmes remain distinct. Concordantly, the differences between the maternal and paternal chromosomes’ epigenetic marks can still be visualized at syngamy [8
]. In particular H3K9me2/3, H4K20me3 and H3K64me3 are strikingly enriched on only half the metaphase plate, presumably the maternal genome, and H4K20me3 and H3K64me3 are then lost rapidly by the two-cell stage () [23
]. On the other hand, H3K9me2/3 asymmetry can still be observed in two-cell embryos and decreases passively through the first replication cycles due to the absence of de novo
]. It is likely therefore that this is due to the continued absence of Suv39h activity. Although the genomes remain compartmentalized in the four-cell stage, H3K9me2/3 asymmetry is lost and this is likely to be explained by the observation that methylation levels begin to increase at this stage, possibly due to the expression of the genes responsible from the zygotic genome or the repression of a potential inhibitor(s) [23
]. It is also possible that only after this stage, the global chromatin configuration of the embryonic chromatin becomes ‘receptive’ for Suv39h activity. Notably, levels of H3K9 methylation during these early cleavage stages have been correlated with successful development of cloned bovine embryos, where developmental success is low and defects in chromatin structure are often observed [61
In addition to H3K9me2/3 passive dilution, DNA methylation is lost from the maternal genome across replication cycles due to the lack of maintenance DNA methyltransferase activity, except for certain imprinted genes and repetitive sequences [11
]. However, maternal and zygotic Dnmt1 are both required for the maintenance of most DNA methylation imprints in the pre-implantation embryo [62
] in contrast to previous reports suggesting that the maternal Dnmt1o is excluded to the cytoplasm up to the 8-cell stage and the zygotic Dnmt1s is not expressed until post-implantation [63–65
]. Therefore, the zygotic Dnmt1s must be present at low levels but specifically targeted to the particular imprinted regions during mitoses, by an unknown mechanism that could be via distinct epigenetic modifications. The maintenance of the methylation of particular genes above a certain threshold is essential for development as knockout embryos for the Dnmt1 methyltransferase arrest at the late gastrulation stage, with hypomethylated repetitive elements and imprinted genes [66–68
]. Lineage-specific de novo
DNA methylation does not begin thereafter until the late morula stage [13
] and is also essential for development as knockouts of both the Dnmt3a and b enzymes also results in embryonic lethality at E11.5 [13
The first differentiation event occurs before implantation, with the allocation of the trophectoderm and inner cell mass lineages commencing at the 8- to 16-cell stage [70
]. It is likely that epigenetic dynamics during the cleavage divisions of the zygote are important for this lineage specification, through their potential to regulate gene expression in a heritable manner [72
]. On a global level the ICM displays higher levels of DNA methylation and H3K9me3 and H3K27me3 as well as lower levels of H2A/H4 phosphorylation [13
]. Furthermore, embryos lacking methyltransferases responsible for these DNA and histone methylations result in more severely affected embryonic than trophectoderm tissues and often fail in the transition from pre- to post-implantation development, suggesting that these asymmetries are likely to be functionally important [26
Specific evidence for the hypothesis that epigenetic modifications may play instructive rather than merely consequential roles, derives from the observation that, in a proportion of embryos, the vegetal blastomeres of 4-cell embryos have lower levels of H3R26me2 and are more likely to develop into trophectoderm tissues [75
]. Significantly, increasing the levels of H3R26me2 resulted in re-allocation into blastomeres of the ICM, providing evidence for a ‘driver’ role for an epigenetic modification during development [76
]. This also supports a degree of flexibility in the early embryo, as the expression of an epigenetic modifier at the four-cell stage is able to change the allocation of the blastomere. With this in mind it would be interesting to determine whether at later stages this flexibility is lost. It is also important to note that it is likely to be the relationship with the surrounding cells and the relative levels of histone modifications and transcripts between cells rather than absolute levels that governs their eventual fate such that feedback and/or feedforward loops can then be set up to stabilize these effects.
Histone acetylation and other arginine methylations show cell-cycle regulated dynamics in cleavage-stage embryos. Hyperacetylated histone H4 is not observed on metaphase chromatin of blastomeres at the four-cell stage but becomes apparent at later stages suggesting that the appropriate acetyltransferase and deacetylase activities are present at this time. Dimethylation of arginines H3R17 and H4/H2AR3 are not associated with metaphase stage chromatin, although H4/H2AR3 becomes apparent at metaphase of mitotic blastomeres in the blastocyst [39
]. H3R2me2 is also abundant during cleavage stages, at least between the two- and the eight-cell stages, although for this mark there is no known relationship to mitosis [76
The structure and organization of chromatin regions differs fundamentally in zygotes and early embryos compared to somatic cells and this is likely to reflect the distinct plasticities and potencies of the genomes. On a general level chromatin progressively accumulates epigenetic marks during development that are likely to establish a heritable state of gene expression determining lineage allocation. Thus by extension, reprogramming requires the removal of the marks for the specification of the highly specialized sperm and oocyte. However the situation is clearly more complex, highlighted by the significant asymmetry in the temporal and spatial dynamics of epigenetic modifications in paternal and maternal pronuclei, which is likely coupled to differences in transcription timing and the regulation of chromatin architecture in the pronuclei. The distinction of parental origin of the genomes at the epigenetic level is thus preserved despite the equalization of overall chromatin structure in the mature totipotent zygote and it remains to be determined whether this has functional consequences, for example, in imprinting or early development and differentiation events. It seems likely that histone lysine and arginine methylation may play a particularly crucial role during reprogramming and development due perhaps to their more stable nature than phosphorylation or acetylation and therefore greater potential for heritability, although these marks also clearly undergo dramatic dynamic regulation during reprogramming. Importantly, how all these epigenetic dynamics are achieved mechanistically remains obscure. Therefore the role and regulation of specific chromatin modifications in controlling gene expression during reprogramming and early development and differentiation needs to be addressed by modulation of the enzymatic activities responsible in temporally and spatially higher resolution studies.
- There is a fundamentally distinct structure and organization of chromatin in mouse zygotes and early embryos, of heterochromatin in particular.
- The paternal and maternal genomes undergo very different epigenetic programmes in the zygote.
- We propose that histone lysine and arginine methylation may play a particularly important role during reprogramming and early differentiation events.
- Higher resolution studies, both temporally and spatially need to be performed to address the role of specific chromatin modifications during pre-implantation development.