1.1] 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.

1.2] Smarcad1 is essential for H3.3 deposition and Trim28 dependent silencing on retroviral sequences in mouse embryonic stem cells.

Moloney murine leukemia virus (MLV) replication is suppressed in mouse embryonic stem cells by the Trim28-ESET complex. The chromatin remodeler Smarcad1 interacts with Trim28 and is located at similar genomic sites. Moreover, Smarcad1 evicts nucleosomes from endogenous retroviruses and enables the loading of the histone variant H3.3 onto those sequences. Therefore, we wished to examine the role of Smarcad1 in MLV repression and elucidate its mechanism. To address the functional role of Smarcad1 in MLV repression, we use shRNA to deplete Smarcad1 before or after MLV infection and test the change in expression of the reporter GFP. A marked increase in expression is observed following Smarcad1 depletion, attesting to its involvement in MLV silencing. Next, we map the effect of Smarcad1 depletion on H3.3 deposition, Trim28 recruitment, and H3K9me3 heterochromatin marking on the retroviral sequences. The observed reductions suggest that the epigenetic silencing of retroviral sequences is dependent on Smarcad1. Last, we examine the interactions among H3.3, its chaperons, and the proteins that comprise retroviral silencing complex, in Smarcad1 depleted cells. This study provides evidence for the role of Smarcad1 in H3.3 and Trim28 dependent retroviral repression in embryonic stem cells.

1.3] Bovine-induced pluripotent stem cells

​Pluripotent stem cells are cells that, by definition, are capable of differentiating to all three germ layers and self-renew indefinitely. There are two methods to deriving pluripotent stem cells: 1) Harvesting from the inner cell mass of a blastocyst or 2) reprogramming differentiated cells using external factors to induce pluripotency or in other words induced pluripotent stem cells. In mice and humans,​​ the protocol to reprogram differentiated cells is already well established. However, there is no established and agreed-upon method for reprogramming cattle cells. In our research, we aim to define and establish a protocol for reprogramming bovine cells.

Derivation of bovine pluripotent cells can be applied in drug selection, human disease modeling and agriculture‐related applications. For example, the main issue in agriculture is the decreased fertility of cows due to exposure to environmental stress. Currently, the study of early developmental problems requires in vitro fertilization and maturation of blastocysts. This is both costly, time-consuming, and requires extensive training. Using pluripotent stem cells will allow us to study the effects of stress on reproduction in a more simple and controlled system. Another example is the cultured meat industry. Reprogrammed cells can self-renew indefinitely and can differentiate into any cell in the body, including muscle. Companies could then keep one line of cells that could turn into any tissue without the need to renew and re-check new cells harvested from a living cow. This would lower costs and make lab-grown meat more available, vegan and environmentally friendly. Moreover, pluripotent cells can serve as a new source for regenerative medicine in the medical and veterinary fields. With induced pluripotent cells, new avenues of stem cell therapy can be examined and tried, using the patient's own cells and the new technology of Crisper/CAS9 gene editing.