Functional role of 5-hydroxymethylcytosine in early lineage gene expression during human development
Figure 1 – Dynamics and epigenetic role of 5hmC in the early embryonic stages.
Abstract
This study focused on the dynamics of 5-hydroxymethylcytosine (5hmC) during the different early stages of embryogenesis, starting from gametogenesis. It provides a landscape of 5hmC distribution across the genome over time, unraveling that it may not only work as an intermediate in DNA demethylation, but also have a direct regulatory role in the transcription of certain lineage-specific genes. Further analyses confirmed that 5hmC is enriched in transcription factor binding sites, whose knockdown compromises the zygotic genome activation (ZGA) and, for these reasons, it is actively involved in the early gene expression. This study represents an important breakthrough, as it lays the foundation for future analyses in which other regulatory mechanisms involving 5hmC can be further investigated.
Review
At the first stages of fertilization and embryogenesis, the zygotic genome undergoes a drastic reprogramming phase, which consists in a widespread DNA active demethylation and histone modifications resetting. The demethylation process is operated by dyoxygenases of the TET family, which oxidize 5-methylcytosine (5mC) to 5hmC and other subsequent intermediates, that will be excised and substituted with unmodified cytosine by the TDG enzyme (1). Notably, 5hmC is much more prevalent than the other oxidized forms, suggesting functions beyond being a demethylation intermediate. In contrast to somatic cells, mammalian zygotes undergo rapid epigenetic reprogramming, with TET3-mediated 5hmC formation playing a key role in mice, where it is enriched mainly on the paternal genome and has limited impact on development (2). These observations motivate the investigation of 5hmC dynamics and function in humans, which this study addresses by analyzing 5hmC in human gametes, preimplantation embryos and embryonic stem cells.
Pre-fertilization 5hmC dynamics
APOBEC coupled epigenomic sequencing (ACE-seq) technique (2) has been used to generate genome-wide maps of 5hmC in samples from different developmental stages, revealing that 5hmC is globally low but highly enriched in early stages, particularly in oocytes and early cleavage embryos (3). Human oocytes showed exceptionally high 5hmC levels in CpG sites, which persist in the zygote and early divisions before progressively declining toward the blastocyst, indicating a transient but functional role. 5hmC distribution is uniform across chromosomes, enriched in gene bodies, repetitive elements and maternal imprinted regions (3), suggesting it could be involved in genomic imprinting, while it is largely absent from CH sites (H = A, C, or T), highlighting a hydroxymethylation program that is coordinated, CpG-specific and specific for human during early development.
The origin and fate of 5hmC during human oocyte maturation and embryogenesis have been investigated. Human oocytes generate 5hmC de novo during maturation, producing far more hydroxymethylated regions than in mouse (2). Single-cell multi-omics analyses have been performed on the same stages. It has been observed that these regions are linked to open chromatin, transcription and de novo methylation, suggesting that TET activity could be coordinated. While the majority of 5hmC sites are maintained after fertilization in the maternal genome, in the paternal one it is accumulated with demethylation. The fact that inherited maternal 5hmC is correlated with higher expression of associated genes, leads to the conclusion that it could be a transient but functional intermediate (3).
Post-fertilization 5hmC dynamics
It has been observed that after fertilization, the human paternal genome rapidly acquires 5hmC, peaking at the two-cell stage and then declining. Unlike in mouse, immunostaining detection confirmed that 5hmC is present in both parental pronuclei, indicating less asymmetric reprogramming in humans (2). This process is mediated by maternal TET3 acting on conserved loci in both genomes, with paternal 5hmC linked to active demethylation and maternal 5hmC remaining stable, reflecting complementary rather than symmetric epigenetic reprogramming (4).
The relationship between 5hmC and DNA methylation in human zygotes is more complex than in mouse. In humans, 5hmC not only accompanies demethylation but is also stably retained at maintained and newly methylated regions, particularly in the paternal genome. Unlike mouse, de novo methylated loci in humans unexpectedly gain 5hmC, indicating that 5mC and 5hmC can coexist as regulatory signals (2). The targets of TET3 could be coordinated, as demethylated and maintained regions are mostly overlapped, while de novo methylation regions are specific for parental pronuclei.
It has been observed that, although 5hmC is not enriched at promoters of ZGA genes, it is consistently enriched within their gene bodies across all preimplantation stages, and this is correlated with active transcription during zygotic genome activation (ZGA), concluding that 5hmC is an epigenetic marker for the transcriptional activity specific of gene bodies and not a “switch” located on promoters.
Experimental validation of 5hmC role
High 5hmC levels have been identified at nucleosome-depleted regulatory regions (NDRs) and in active enhancers. In these distal regions, there are high levels of H3K27ac, which coincide with active chromatin and high transcriptional activity (5). It has been discovered that 5hmC is present on enhancers before their activation and it promotes the erasure of repressive histone modifications, like H3K9me3, indicating it could be a priming signal for gene expression. Moreover, in order to verify its importance, some different approaches have been applied. At first, the reduction of 5hmC through two different chemical compounds resulted in a downregulation of key ZGA genes.
Then, to test the opposite effect, a gain-of-function experiment has been performed, through the ectopic expression of mouse enhanced dyoxygenasic activity Tet3 mutant. This approach has revealed a genome-wide increase in 5hmC and a loss of 5mC, confirming the link between hydroxymethylation and methylation. Most affected regions overlapped with natural TET targets in human oocytes and zygotes, indicating conserved targeting between human and mouse. Artificial increase of 5hmC activated hundreds of normally silent genes in early embryos, demonstrating that 5hmC is an active epigenetic effector which can directly promote gene expression during early human development.
Subsequently, a motif enrichment analysis of 5hmC-enriched enhancers in eight-cell embryos was performed. Several transcription factors that could cooperate with 5hmC during early development have been identified. It has been found that, among the genes that were conserved between human and mouse, the OTX2 transcription factor, more expressed in human, showed high score of motif enrichment at enhancers that were marked with 5hmC, suggesting that 5hmC could prime regulatory elements for the transcription factor binding and the later gene activation.
Then, the role of OTX2 in human embryos has been investigated. It is expressed from the oocyte stage and reaches its peak at the four and at the eight-cell stages, where it plays a key regulatory role in early human embryos. The siRNA-mediated knockdown of OTX2 led to a strong downregulation of embryonic genes and the integration with overexpression results revealed that there are 999 OTX2 target genes, many of which have OTX2 motifs in distal regulatory elements. CUT&Tag analysis revealed that OTX2 tends to bind enhancers and intragenic regions with high 5hmC levels. Moreover, most of the genes targeted by OTX2 are activated during early embryogenesis, suggesting that OTX2 is crucial during early lineage gene expression and 5hmC could improve its recruitment and activity.
Finally, the cooperation between 5hmC and OTX2 in the embryonic gene expression regulation has been examined, using DUSP4 locus as a model, a developmentally regulated gene. DUSP4 enhancers are enriched in 5hmC from the first stages and the chromatin in these loci becomes more accessible after fertilization, coinciding with the accumulation of activating H3K27ac modification. This discovery confirms that 5hmC could prime enhancers bound to OTX2 for transcriptional activation, linking hydroxymethylation to lineage-specific gene regulation in early human embryos.
Limits and strengths
Among the limitations of this study, the fact that the comparison of such evolutionarily distinct species, like mouse and human, could lead to limited conclusions, must be considered. Moreover, although the gain and loss-of-function resulted in significant outcomes, these experimental systems do not fully represent physiological conditions, so there could be many other factors that may bias the correct interpretation of the data.
On the other hand, the use of advanced techniques, like ACE-seq and CUT&Tag, provided a widespread view of the 5hmC dynamics and its functional role in early gene regulation.
Conclusions
This study provides further insight into the role of 5hmC in early human embryonic development, starting from the marked differences between human and mouse. They have seen that while during human oogenesis there is an accumulation of high levels of 5hmC, that is also associated to de novo DNA methylation and activating histone modifications, in mouse oocytes there are lower and less stable levels of 5hmC, leading to the conclusion that it has a different influence on the two species (6). Thus, it has been demonstrated that 5hmC is not only a resulting intermediate of the active demethylation process, but an active effector involved in gene expression, particularly during ZGA. Another important fact that supports the thesis is that TDG, the enzyme involved in active demethylation which excides the oxidized intermediates of 5mC, is expressed only from the eight-cell stage, highlighting the importance of 5hmC levels stability in this temporal window.
Moreover, since OTX2 is expressed even in mouse (7), but after the implantation, and the epiblasts (2, 3) are enriched with 5hmC, the relationship between 5hmC and OTX2 should be investigated in this species too.
Finally, this work opens new perspectives to the study of epigenetic diseases, even cancer, but also to the improvement of in vitro fertilization techniques and of induced pluripotent stem cells, as 5hmC unique distribution could be used as a molecular benchmark for evaluating in vitro-derived human embryonic models in the future.
References
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