ZSCAN4 AND REPLICATION STRESS: SAFEGUARDING GENOME STABILITY IN MOUSE EMBRYONIC STEM CELLS

The accuracy of DNA replication is essential for maintaining genome stability. However, replication can be disrupted by both endogenous and exogenous factors, leading to a condition known as replication stress (RS). RS slows down and eventually stalls replication forks. If stalled forks are not resolved, they can give rise to DNA double-strand breaks, thereby promoting genomic instability—a hallmark of cancer. Our laboratory discovered that exposure of mouse embryonic stem cells (mESC) to RS induces the expression of Zscan4, a marker of the 2C stage in mouse embryo development. Depletion of Zscan4 in mouse zygotes reduces the number of blastocysts formed, which eventually fail to grow and implant. ZSCAN4 also plays a crucial role in mESC by contributing to telomere maintenance and genomic stability. Its prolonged knockdown leads to cell death, accompanied by karyotype abnormalities and telomere shortening. Moreover, deregulation of Zscan4 has been implicated in cancer development and maintenance. Despite this, the mechanisms through which Zscan4 promotes genome maintenance in mESC, particularly within the context of RS, remain poorly understood. We analyzed the effect of Zscan4 expression modulation on DNA replication in mESC using DNA fiber assays and electron microscopy. We found that ZSCAN4 accelerates fork progression while concomitantly reducing single-stranded DNA gaps at fork junction. In addition, whole genome bisulfite sequencing revealed that Zscan4 overexpression induces global DNA demethylation in mESC, whereas RS increases global DNA methylation. Finally, short nascent strand sequencing analysis showed that RS leads to increased origin usage, while Zscan4 overexpression decreases origin usage. Together, these findings reveal a novel function of ZSCAN4 in promoting replication fork progression and remodeling in mESC. Furthermore, Zscan4-induced genome-wide demethylation appears to facilitate DNA accessibility, for instance to DNA repair factors, while RS-induced hypermethylation has the opposite effect. Strikingly, the DNA methylation status correlates with the profile of origin usage: Zscan4 overexpression decreases origin usage, whereas RS increases it. These results suggest the existence of an underexplored connection between DNA methylation and DNA replication.