Neddylation plays a crucial part in the early embryonic development in mice, according to this study. Embryonic development arrested at 2-cell stage after specific inhibition with MLN4924. ZGA marker detection revealed that the Zygote genome was not activated and that the large nucleoli in the MLN4924 treatment group were increased, indicating a change in nuclear outcome. Transcriptome profiling showed that gene expression of key pathways related to “cell fate determination”, “cell differentiation”, and “cytoskeletal proteins” has changed significantly. We discovered that histone methylation H3K4me3 were decreased, and MLN4924 treatment reduced histone methylation modifications in 2-cell embryos by reducing methyltransferases. We also found that the activity of YAP component in the Hippo pathway was reduced. This agrees with existing reports that maternal YAP1 gene knockout led to ZGA initiation disorder, prolonged 2-cell stage and difficulty in entering the 4-cell stage[27].
In many organisms, mice included the transition from zygote to embryo occurs in the silence of transcription [8]. Thus, maternal mRNA and proteins synthesized during oogenesis are essential for early embryonic development. Studies have shown that the degradation of maternal proteins by the ubiquitin proteasome pathway is crucial for the transformation of oocytes into embryos and depends on normal zygotic genome initiation[6]. Ubiquitin is a micromolecular polypeptide that can covalently bind with and modify a variety of proteins in cells. Most of the modified proteins will be identified and degraded by proteasome[28]. Compared with other ubiquitin-like molecules, Nedd8 has the structure closest to that of ubiquitin[9]. Although neddylation modification is similar to ubiquitination, the difference is that neddylation-modified proteins cannot be degraded by protein enzymes like ubiquitinated proteins and thus affect the abundance of substrates, but instead regulates the activity of proteins of substrates[29]. This implies that the mechanism of neddylation regulating embryonic development may be different from that of ubiquitin, which agrees with our experimental results. There have only been two reports showing the phenotypes of neddylation inhibition in the oocyte maturation and early embryonic development of mammals. Inhibition of neddylation caused mouse oocyte maturation failure with MI (meiotic metaphase I) arrest phenotype and spindle damages with overactivated spindle assembly checkpoints[11]. After interdiction of neddylation, there was no cumulus cell expansion, an elevated number of polyspermy occurrences, and a statistically significant deterioration of embryonic development. In this study, it was found that inhibition of the neddylation pathway resulted in 2-cell arrest while inhibition of the ubiquitination pathway resulted in cleavage failure. Our study is a powerful supplement to the function of this ubiquitination-like modification, neddylation in ZGA initiation. We speculate that the activation or function of key proteins might be specifically finetuned by neddylation, whose inhibition thus triggers the downstream cascade reaction leading to the final failure of zygotic genome activation. More detailed targetome of neddylation in early embryos should be profiled and these key proteins remain to be unraveled.
To study how the neddylation pathway affects early embryonic development, we treated fertilized zygotes with MLN4924, which can form covalent adducts with Nedd8 and selectively lock NAE1 in an inactive state[30]. As previously stated, the primary substrates of Nedd8 are cullins[9]. We detected the expression of cullins in treated and untreated embryos by quantitative PCR. The results showed that Cul1, 2, 3, 4b and 5 were down-regulated, which indicated that these cullins were likely to be the main substrates of neddylation in mouse embryonic cells. The expression of Nedd8-modified cullins had changed when the neddylation pathway was inhibited. In this way, neddylation modification can further regulate cullins-mediated ubiquitination in mouse embryonic cells. Interestingly, we found that Cul9 expression was up-regulated after MLN4924 treatment. In U2OS cells, Cul9 and E3 ligases cooperate and promote ubiquitination and degradation of survivin, expressing only in tumor and embryonic tissues[31], which is an essential anti-apoptotic factor in preimplantation embryos[32]. This may partly explain the embryonic development arrest phenotype in the present study. In addition, substrates of cullins were detected and the expression of transcription factor Elob was down-regulated. This may partially explain the altered transcriptome found in the MLN4924-treated group.
Transcriptome analysis showed down-regulated transcripts involved in “cell fate determination”, “cell differentiation” and “cytoskeleton protein” in MLN4924-treated embryos. It is known that genes related to cell fate determination endow the embryo with polarity to differentiate into endoderm and ectoderm[33]. Cytoskeletal proteins are crucial for correct chromosome arrangement and separation, during both oocyte maturation and polar body extrusion[34, 35]. Furthermore, our findings agree with the notion that the actin network is necessary for the freshly formed zygote's initial mitotic division[36]. Figure 2e shows the expression changes during these crucial processes and the critical changes in embryonic cell division and development. Nucleolus is the centre of RNA metabolism and the movement of chromatin towards or away from nucleolus triggers the activation and silencing of gene transcription[37]. The nucleolus of early embryo undergoes a violent transformation during the transition from oocyte to embryo[38]. While using immunofluorescence to detect the transcriptional activity of embryos, we accidentally found that inhibition of neddylation reduced the expression of RNA polymerase II and the number of nucleolus and increased the proportion of large nucleolus in embryonic cells. Previous studies have shown that neddylation inhibition with MLN4924 can remodel the morphology of nucleolus, increase the nucleolar size and change the composition of nuclear proteome in human and nematode germline cells[39]. MLN4924 activates the tumor suppressor p53 through RPL11/RPL5-MDM2 pathway and causes nuclear stress[40]. Evidence suggests that zygotic genome activation is closely associated with histone modifications[41]. Our results also showed that after neddylation was blocked in 2-cell embryos, methyltransferases were reduced in the embryos, resulting in a reduction of H3K4me3 in the embryonic nucleus. Data from previous studies suggests that in lymphoma B, FBXW7 targets the degradation of the methyltransferase KMT2D, a process that is antagonistically regulated by neddylation modification[42].
Taken together, neddylation modification negatively regulates the degradation of methyltransferase (KMT2D) by ubiquitin system. After the neddylation modification is blocked, the dose of KMT2D in the embryo is insufficient and subsequent histone H3K4 trimethylation cannot be completed at the appropriate time. Subsequently, histone modification determines chromosome accessibility and may also explain the reduction of RNA polymerase signal in the nuclei of MLN4924-treated 2-cell embryos.
It is reported that Hippo-Yap pathway is involved in mouse embryonic development[27]. It has been shown that Hippo activation leads to the phosphorylation of transcription factor Yap1, a substrate of cullin[43], thus preventing its translocation to the nucleus. In the absence of nuclear Yap1, its downstream partner Tead4 could not activate target gene transcription[44]. Therefore, the inactivation or deletion of Yap1 would result in ZGA initiation failure[27]. We detected the enhanced expression of inactivated YAP1 in MLN4924-treated embryos. This indicates that neddylation inhibition would reduce the activity of YAP1 and cause embryonic development arrest, which agrees with existing reports in the literature. The hypothesis that suppression of neddylation pathway could cause embryonic development arrest by suppressing YAP1 has been reinforced by our findings.