In mammals, fertilization of an oocyte by a sperm results in the generation of a totipotent embryo that can form all cell types of the embryonic and extraembryonic lineages. Before, during, and shortly after fertilization, both parental genomes are transcriptionally silent, and early embryonic events are controlled by maternal mRNAs and proteins that are stored during oocyte growth (Dai et al., 2018). Acquisition of totipotency is accompanied by the chromatin remodeling of highly differentiated parental genomes, removal of the maternal transcripts and proteins, and zygotic genome activation (ZGA) (Zhang et al., 2015; Yu et al., 2016; Sha et al., 2018b).

Before ZGA, early embryonic development is controlled exclusively by maternal products accumulated during oocyte development. After the elimination of a subset of the maternal products, transcription is re-initiated and developmental control passes to the zygotic genome (Tadros and Lipshitz, 2009). This drastic transition from a highly differentiated oocyte to a totipotent embryo is referred to as the maternal-to-zygotic transition (MZT) (Sha et al., 2019). Histone methylation is the major determinant of the formation of transcriptionally active and inactive regions of the genome and is crucial for proper chromatin remodeling during oogenesis and the MZT (Clarke and Vieux, 2015).

Gametes are highly differentiated cell types. Therefore, fertilization requires major epigenetic remodeling to reconcile the paternal and maternal genomes with the formation of a totipotent zygote (Yu et al., 2013). At the time of fertilization, the paternal genome is densely packed with protamines, and the maternal epigenome is highly specialized in terms of DNA and histone modifications. During the MZT, maternal factors unravel these specialized chromatin states to enable ZGA and embryonic development. Histone H3 lysine methylations are dynamically regulated during the MZT and have different cellular and physiological impacts, depending on the modified amino acid residue and the number of added methyl groups (mono-, di-, or tri-). However, the maternal and zygotic regulators underlying these drastic histone modifications and their impact on ZGA and embryonic development remain unknown.

Histone methylation is tightly regulated by histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs), which add and remove methyl groups, respectively. KMTs and KDMs have specificities for their target lysine residues, as well as for the number of methyl groups they can add or remove. Some of these enzymes have been genetically knocked out in mice. Deletion often leads to prenatal or perinatal lethality. To overcome early lethality, selected promoter fragments of Ddx4, Gdf9, and Zp3 were used to drive the oocyte-specific expression of CRE recombinase in the developing oocytes of transgenic mice, starting from embryonic day-16 (oocyte cyst stage) (Gallardo et al., 2007), postnatal day-3 (primordial follicle stage) (Lan et al., 2004), and day-5 (primary follicle stage) (Lewandoski et al., 1997), respectively. Using these genetic tools, the physiological functions of KMTs and KDMs during oogenesis and the MZT are being discovered.

H3K4me3 is a common histone modification at the transcription start site of actively expressing genes in eukaryotic cells (Howe et al., 2017). Because of the association between H3K4me3 and gene expression levels, H3K4me3 is generally believed to have an instructive role in gene expression and thus is an ‘activating’ histone modification. In mouse embryonic stem cells, for example, H3K4me3 is associated with gene promoters and poises them for transcriptional activation in response to developmental or environmental cues (Blackledge and Klose, 2011). However, many recent genome-wide studies have shown that under steady-state or dynamically changing conditions, transcription actually changes very little upon removal of most H3K4me3 on the chromatin (Clouaire et al., 2014). Therefore, rather than serving as instructions for transcription, the deposition of H3K4me3 onto chromatin is suggested to be a consequence of transcription and is thought to influence processes such as transcriptional consistency among cells in a population, transcriptional memory of previous differentiation states, and gene silencing. In the following sections, we will discuss the functions and regulations of H3K4 methylation associated with transcription and organization of the mammalian genome in oocytes and zygotes, some of which are similar to those observed in somatic cells, whereas others are unique to these specific cell types.

The SET1/COMPASS histone methyltransferase complex is the primary enzyme that methylates histone H3K4 (Roguev et al., 2001). While yeast contains only one SET1 protein, there are six known histone H3 methyltransferases in mammals. They are subdivided into three groups, i.e., SET domain-containing 1A/B (SETD1A/B), lysine (K) methyltransferase 2A/B (KMT2A/B), and KMT3/4 (Shilatifard, 2012). In previous publications, KMTs were also known as mixed lineage leukemia 1-4 (MLL1-4); however, these are no longer the official gene names on NCBI. These complexes are not functionally redundant, as demonstrated by the early lethality phenotypes observed upon the knockout of individual genes (Vedadi et al., 2017). These studies also showed that SETD1A/B-based enzyme complexes are the predominant H3K4 methyltransferases in most cell types (Ardehali et al., 2011; Shilatifard, 2012).

Despite extensive studies on H3K4me3 in the context of transcription-related cellular events, the direct function of H3K4me3 with respect to chromatin behavior during cell division has been elusive because of the absence of H3K4me3 in most cell types affects the transcription of a broad spectrum of house-keeping genes and causes cell cycle arrest in the G1 or S phase. Considering these technical difficulties, a fully grown mammalian oocyte is an ideal model to study transcription-independent functions of epigenetic modifications because de novo gene transcription is neither required nor active during the two sequential meiotic divisions.

In the ovaries of female mammals, all oocytes within the pre-antral follicles have a non-surrounded nucleolus (NSN) type of chromatin configuration. The chromatin of fully grown germinal vesicle oocytes undergoes a transformation from the NSN to the surrounding nucleolus (SN) type when the follicles grow to the antral stage (Tan et al., 2009). SN type oocytes have better developmental competence after fertilization than NSN oocytes (Ma et al., 2013; Zhang et al., 2019). In developing mouse oocytes, H3K4me3 levels on chromatin increase during the transition of chromatin configuration from the NSN to SN type (Yu et al., 2017). Following meiosis resumption, H3K4me1 and H3K4me2 levels increase, but H3K4me3 levels decrease after GV breakdown (GVBD) and reach the lowest point in anaphase I (Sha et al., 2018a). The meiotic maturation-coupled fluctuation of H3K4 methylation levels suggested that these histone modifications perform previously unrecognized transcription-independent functions with respect to regulating the meiotic divisions of oocytes.

When Gdf9-Cre was employed to knock out Cxxc1 in oocytes as early as the primordial follicle stage, the Cxxc1-null oocytes exhibited reduced GVBD and polar body 1 (PB1) emission rates during meiotic maturation than wild type oocytes (Sha et al., 2018a). They also fail to assemble bipolar spindles because chromosomes are not able to align at the equatorial plates of the meiotic spindles. CXXC1 is likely to be directly involved in these processes because the expression of a dominant-negative CXXC1 mutant using mRNA microinjection in fully grown oocytes leads to defects similar to those observed in Cxxc1 null oocytes. Because the genome of the fully grown mammalian oocytes does not have transcriptional activities, these results suggest that CXXC1-mediated H3K4 trimethylation might have a transcription-independent role during mouse oocyte meiotic maturation.

The phosphorylation of histone H3 at threonine-3 (H3T3ph) during the G2-M transition is required for both mitotic and meiotic divisions (Wang et al., 2012, 2016). CXXC1-dependent H3K4 trimethylation is a permissive signal for the subsequent H3T3 phosphorylation (Sha et al., 2018a), but the exact mechanism is unclear. Hypothetically, a decreased H3K4me3 level in oocytes results in chromatin tightening. As a result, haspin, a kinase that triggers H3T3 phosphorylation, cannot easily access the chromatin during meiosis resumption. The interplay between K4 methylation and T3 phosphorylation in histone H3 has also been investigated using biochemical studies. Analysis of the haspin crystal structure revealed that the bulkiness of methylated K4 prevents the interaction of the H3 tail with the narrow substrate-binding groove of haspin (Eswaran et al., 2009). In vitro kinase assay results indicate that K4 methylation impairs the T3 phosphorylation of histone H3 by haspin (Eswaran et al., 2009). Furthermore, H3T3ph is located adjacent to—while not overlapping with—H3K4me3 on the chromosomes of mouse oocytes (Sha et al., 2018a). All these studies indicate that H3K4me3 regulates T3 phosphorylation in an intermolecular manner. These hierarchical histone modifications on the maternal genome are involved in the precise regulation of meiotic cell cycle progression.

CXXC1 proteins are rapidly degraded in oocytes after meiotic resumption, remain undetectable at the MII stage, and reaccumulate after fertilization (Sha et al., 2018a). The same study has shown that CXXC1 degradation is mediated by 65 amino acids present at the C-terminal. As a cell cycle-coupled regulator of CXXC1, CDK1 directly interacts with CXXC1 and phosphorylates it at two conserved consensus CDK1 target sites (Ser-138 and Ser-143) during the G2–M transition and triggers CXXC1 degradation. The ubiquitin E3 ligase that mediates CXXC1 degradation has not yet been identified. CDK1 may trigger CXXC1 degradation by modulating the activity of this E3 ligase or its accessibility to CXXC1 during the G2-M transition. In addition, phosphorylation of CXXC1 at Ser-138 and Ser-143 impairs its binding to histone H3, thereby inhibiting the ability of CXXC1 to mediate H3K4 trimethylation (Sha et al., 2018a) (Figure 2A).

CXXC1 depletion during meiotic division is of physiological importance. Chromatin undergoes extensive reorganization during oocyte meiosis and fertilization, transitioning from a relatively relaxed configuration during maturation to a highly condensed state in mature eggs and returning to the interphase state after fertilization (Zhang et al., 2015). The degradation of CXXC1 is a robust means of ensuring the absence of SETD1-CXXC1 complex from the chromatin during this transition, thereby facilitating chromosome condensation. If the chromosome-bound CXXC1 proteins are not degraded in maturing oocytes, they can hinder chromosomal condensation during meiotic spindle assembly. Overexpression of stabilized CXXC1 (C-terminus-deleted CXXC1) in fully grown GV oocytes results in decreased GVBD and PB1 emission rates, as well as defects in spindle assembly. Furthermore, these phenotypes become more prominent upon additional deletions of CXXC1 phosphorylation sites (Sha et al., 2018a) (Figure 2B).

There are three variants of histone H3 in mammals, namely H3.1, H3.2, and H3.3. H3.1 and H3.2 variants are deposited on chromatin during the S phase of the cell cycle because their deposition is DNA replication-dependent. In contrast, the H3.3 variant is expressed and deposited in chromatin throughout the cell cycle in a DNA replication-independent manner. Dynamic histone exchanges in the chromatin of the oocytic and zygotic genomes are essential for maintaining normal transcriptional activity. Particularly, histone modifications associated with active gene expression, such as H3K4me3, are enriched in H3.3. Accordingly, the deposition of H3.3 is correlated with transcriptionally active genes.

Akiyama et al. (2011) examined the global deposition of histone H3 variants after fertilization in mice. H3.1 and H3.3 occupy unusual heterochromatic and euchromatin locations, whereas H3.2 is incorporated into the transcriptionally silent heterochromatin. Maternal H3.3 protein is incorporated into the paternal genome as early as 2 h post-fertilization, and is detectable in the paternal genome until the morula stage (Kong et al., 2018). The knockdown of maternal H3.3 results in compromised transcription from the paternal genome after fertilization as well as embryonic development (Wen et al., 2014). Collectively, these findings indicate that active changes in the deposition of histone H3 variants are critical for chromatin reorganization during MZT.

Related to the focus of this review, the replacement of H3 variants is also regulated by post-translational modifications of H3. H3.3 is enriched in H3K4me3, whereas H3.1 is enriched in dimethylated H3 lysine-9 (H3K9me2). In a histone replacement experiment—involving microinjection of mRNAs encoding Flag-tagged histone H3 variants into GV stage-arrested Cxxc1-null oocytes—the H3.3 incorporation rates were significantly decreased when compared with those in the control oocytes. The newly translated H3.3 proteins underwent remarkable de novo lysine-4 trimethylation in wild type oocytes in a CXXC1-dependent manner, even when DNA replication and transcription activities were absent. Furthermore, DNase I digestion assay for assessing chromatin accessibility in oocytes revealed that the genomic DNA of CXXC1-deleted oocytes was more resistant to DNase I digestion than the DNA from wild type oocytes. These results indicate that SETD1-CXXC1-mediated H3K4 trimethylation is essential for maintaining proper chromatin configurations, which makes the genome accessible to protein factors that facilitate transcription during oocyte development.

In mammals, the maternal and paternal genomes are not functionally equivalent; both the maternal and paternal genomes are required for embryonic development. The most distinguishing feature that differentiates the sperm genome from the oocyte genome is that it is globally compacted with protamine proteins rather than with histone proteins (Feil, 2009). However, the protamines are promptly removed after fertilization and are replaced by maternal histones stored in the oocytes. Unlike the maternal chromatin, which maintains all types of histone H3 methylations throughout embryonic development, paternal chromosomes acquire new and unmodified histones during or after the formation of the male pronucleus.

Shortly after the formation of the male and female pronuclei, H3K4me1 exhibits comparable levels in both pronuclei; however, H3K4me2 and H3K4me3 are initially present at lower levels in male pronuclei than in the female pronuclei. The acquisition of H3K4me2/me3 in the male pronucleus only occurs at the latest pronuclear stages (Lepikhov and Walter, 2004). Researchers have logically assumed that the H3K4me3 modifications in the female pronuclei are inherited from those that were deposited on the maternal chromosomes during oogenesis. However, a recent study indicated that this might not be so simple (Yu et al., 2017).

The Cxxc1-deleted oocytes can be fertilized, but none of the embryos developed beyond the 2-cell stage, indicating that the absence of maternal CXXC1 in oocytes affects the developmental potential of the resulting zygotes after fertilization (Yu et al., 2017); these zygotes exhibited severe defects in ZGA. In a rescue experiment—involving the microinjection of mRNAs encoding CXXC1 into the maternal Cxxc1-deleted zygotes—CXXC1 reaccumulated in both male as well as female pronuclei. Surprisingly, H3K4me3 levels were restored only in the female but not in male pronuclei (Yu et al., 2017). This asymmetrical H3K4 trimethylation in rescued zygotes that mimic wild type zygotes, but reveals two previously unknown facts, i.e., (Dai et al., 2018) the H3K4me3 modification in female pronuclei are not only inherited from the maternal chromosome but are also generated de novo after fertilization, and (Sha et al., 2018b) H3K4me3 modification is incapable of being generated in male pronuclei, even in the presence of CXXC1. Further investigations, such as the localization of SETD1A/B and chromosome accessibility to CXXC1 in male pronuclei, are needed to understand the mechanisms underlying this phenomenon.

Consistent with the essential role of the H3K4me3 modification in ZGA, maternal HIRA—a chaperone for the histone variant H3.3 mediating its chromosomal deposition—is required for zygote development in mice (Lin et al., 2014; Nashun et al., 2015). The formation of the male pronucleus is inhibited upon the knockout of maternal Hira owing to the absence of the nucleosome assembly in the paternal genome after fertilization (Lin et al., 2014). Because H3K4 trimethylation occurs predominantly on H3.3, the absence of maternal HIRA may result in zygotic developmental arrest by impairing H3K4me3 generation in the pronuclei. Furthermore, Hira-null oocytes fail to develop parthenogenetically, indicating a role for HIRA in the female pronuclei (Lin et al., 2014).

After fertilization, the maternal and paternal genomes form haploid female and male pronuclei, respectively. These two separate nuclei coexist in the zygote. How these two epigenetically distinct genomes are temporally and spatially reorganized following the initiation of embryo development is poorly understood. Recently, several groups have developed single-nucleus high-resolution chromosome conformation capture methods that provide greater sensitivities than the previous methods to investigate three-dimensional chromatin organization in rare cell types, including the oocytes, zygotes, and blastomeres of early embryos (Du et al., 2017; Ke et al., 2017). They showed that the chromatin organization of germ cells and zygotes is fundamentally different from that of somatic cells. Oocytes display distinct features of genomic organization, but these chromatin architectures are uniquely reorganized during the MZT. Understanding the role of H3K4 modification in these processes could potentially provide insights into reprogramming differentiated cells to a totipotent state.

Accumulating evidence indicates that H3K4me3 is essential for ZGA, but the underlying mechanisms are not adequately addressed. The timing of the transcription initiation after fertilization (known as minor ZGA) is species-specific and occurs at the mid-1-cell stage in mice (Abe et al., 2018). A low level of enhancer-independent transcription occurs promiscuously in a large proportion of genomic regions during minor ZGA. The expression pattern of these genes is very different from that at later stages (Abe et al., 2015). Generally, the chromatin structure is repressive for transcription, and enhancers are required to help transcription factors access the gene promoters. In zygotes, however, transcriptional activity is not stimulated by enhancers. A plausible hypothesis suggests that in zygotes with an extremely loose chromatin structure, transcription factors can easily access the DNA and cause promiscuous gene expression (Yamamoto and Aoki, 2017). The role of H3K4me3 in this working model remains to be elucidated.

Lastly, the findings described above indicate that the spatiotemporal-specific establishment and erasure of H3K4me3 during oocyte development enables the oocyte genome to establish the competence to generate a healthy embryo in a cell-autonomous manner. Nevertheless, the extent to which this epigenetic modification forms a cell non-autonomous instructive component of ovarian follicle development remains unclear. Recent studies have revealed that appropriate levels of H3K4me3 accumulation in growing oocytes are necessary to maintain the expression of genes encoding oocyte-derived paracrine factors, including growth and differentiation factor 9 (GDF9), bone morphogenic protein 15 (BMP15), and fibroblast growth factor 8 (FGF8) (Sha et al., 2020). CXXC1-dependent expression of these genes facilitates communication between an oocyte and the surrounding ovarian somatic cells and is required for the establishment of distinct gene expression patterns in granulosa cells and cumulus cells. Oocytes that have high levels of H3K4me3 have greater potential to support follicle development to the ovulation stage, whereas follicles containing oocytes with low H3K4me3 levels are prone to undergo atresia before ovulation. Future investigations are required to elucidate the cell non-autonomous role of H3K4me3 in germ cells and blastomeres of preimplantation embryos.

H-YF and Q-QS conceived and wrote the manuscript. JZ prepared the figures. All the authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.