1.1] The dynamic control of retroviral suppression during the transition into pluripotency

Pluripotency undergoes dynamic transitions starting from the inner cell mass (ICM) of the blastocyst through the epiblast stage, encompassing both naïve and primed states in mouse embryonic stem cells (ESCs). These states exhibit distinct gene expressions, DNA methylation status and chromatin structures. Recently, our research demonstrated the essential role of H3.3 deposition in silencing repressed retroviral sequences in naïve mouse ESCs. However, understanding of how H3.3 dynamics and epigenetic differences regulate retroviral silencing during the transition from naïve to primed pluripotency remains limited.

Sharon Schlesinger

Here, we focus on evaluating how H3.3 impacts the transcription of retroviral sequences during this pluripotency transition in mouse ESCs. We compared the transcriptional statuses of newly integrated Moloney leukemia virus (MLV) provirus between naïve and formative pluripotent states using wild-type (wt) and H3.3 null ESCs. Our findings reveal that MLV provirus repression is compromised in naïve (2i/LIF) ESCs upon H3.3 depletion, whereas wt ESCs effectively suppress viral expression. In contrast, epiblast-like cells (EpiLC- FGF2/Activin) infected with MLV show reduced silencing in wt and H3.3 null cells.

1.2] The H3.3 coding genes have distinct roles in retroviral silencing in ESC

Histone 3 variant, H3.3, obtains different modifications according to its position in the chromatin. It was shown to occupy gene body and active transcription zones, as well as centromeres and highly conserved repeat elements, which are mostly repressed. Although H3.3 was shown to occupy endogenous retroviruses sequences, the contribution of this variant to retroviral silencing in embryonic stem cells (ESC) is not yet clear.

Here we show that H3.3 depletion disrupts retroviral sequences' silencing in mouse ESC. Interestingly, our results show a differential impact on the depletion of the two genes coding for H3.3, H3f3a, and H3f3b, on retroviral expression. By infecting H3.3A-depleted cells with a retroviral vector, we demonstrate a transient upregulation of incoming retroviral expression, as well as that of ERVs. Conversely, H3.3B- knock-out did not show a similar effect, and retroviral repression was maintained. Notably, the depletion of both genes activated retroviral expression in a stable manner.
The upregulation of retroviral expression was associated with the depletion of the silencing mediator Trim28 and the H3K9me3 chromatin mark from the retroviral sequences. Deletion of DAXX, a specific H3.3 chaperon, did not affect either the expression or H3.3 loading on retroviral sequences, whereas depletion of Trim28 did affect both. Without Trim28, retroviral expression is upregulated and the H3.3 accumulation on the retroviral promoter is abolished.  Thus, our results show for the first time a distinct function for the two H3.3 genes in retroviral regulation in ESC and suggest a functional interplay between Trim28 recruitment and H3.3 loading.

1.3] Bovine pluripotent stem cells

Mammalian development follows fundamental principles, yet species-specific variations critically influence early embryogenesis. By utilizing blastoids—lab-derived structures mimicking early, pluripotent embryos—we aim to investigate interspecies differences in pluripotency, early cell lineage commitment, and molecular mechanisms underlying embryogenesis. A key focus of our research is the successful generation of bovine blastoids to (1) Examine critical developmental events, such as early lineage specification, and better understand the molecular control of lineage specification in bovine embryos compared to other mammals. (2) Study epigenetic modifications during early development. For instance, bovine blastoids provide a controlled model to explore how metabolic states influence epigenetic reprogramming, offering novel insights into these dynamic processes. (3) Analyze the impact of environmental conditions on early developmental stages, which were previously inaccessible for detailed investigation.
Bovine blastoids offer significant ethical and practical advantages by reducing dependency on natural embryos, addressing ethical concerns, and enabling high-throughput research. They facilitate systematic cross-species comparisons under controlled conditions, revealing species-specific adaptations and evolutionary conservation of gene regulatory networks and implantation mechanisms.
Potential applications in livestock breeding include overcoming the bottleneck of limited embryo availability for artificial reproductive technologies (ART). Bovine blastoids could accelerate genetic improvement programs by enabling scalable embryo production without reliance on IVF or somatic cell nuclear transfer (SCNT), transforming animal breeding and genetics.
Future directions involve further optimization and validation of bovine blastoid models through comparisons with in vivo embryos to ensure fidelity and applicability. These advancements promise to enhance both fundamental biological understanding and practical applications in agriculture and biomedicine.

Sharon Schlesinger