Smarcad1 is responsible for H3.3 deposition on retroviral sequences in embryonic stem cells

Histone 3 variant, H3.3, obtains different modifications according to its position in the chromatin. It occupies gene bodies and active transcription zones as well as repeats and retroviral sequences, which are mostly repressed. Hence, the contribution of H3.3 deposition to gene regulation is not yet clear.
Here we show that H3.3 depletion disrupts retroviral silencing in mouse embryonic stem cells (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 MLV-like virus, we demonstrate a distinct upregulation of incoming retroviral expression. Conversely, H3.3b knockout did not show a similar effect.
The upregulation of retroviral expression was associated with the exclusion of Trim28 and H3K9me3 chromatin mark from the retroviral sequences. Deletion of DAXX, a specific H3.3 chaperon, did not affect H3.3 loading on retroviral sequences or the viral expression. Conversely, depletion of Smarcad1, which evicts nucleosomes and enables the loading of H3.3, leads to a marked reduction of H3.3 deposition, Trim28 recruitment, and H3K9me3 heterochromatin marking from the retroviral sequences. Moreover, a significant increase in expression is observed following Smarcad1 depletion, attesting to its involvement in retroviral silencing via H3.3 and Trim28. In conclusion, our results show a distinct function for the two H3.3 genes and demonstrate its importance in retroviral regulation in ESC. We propose a model whereby Smarcad1 safeguards heterochromatin structure by binding to Trim28 and enabling H3.3a deposition on the repressed retroviral sequence.