Resf1 is required for proper placental development and configuration of trophoblast cell-specific heterochromatin.

Retroelement silencing factor 1 (Resf1) is involved in retroelement silencing in cooperation with H3K9 methyltransferase SETDB1 by regulating H3K9 methylation in mouse embryonic stem cells (mESCs). However, it remains unknown whether Resf1 functions in retroelement silencing in vivo and has a role in development. Here, we established Resf1-deficient mice, which exhibit developmental delay, partial embryonic lethality, and placental defects. Notably, retroelements are also upregulated in the Resf1-deficient placenta, correlating with increased expression of nearby genes. To further assess whether Resf1 functions within the trophoblast lineage, we generate Resf1-deficient trophoblast stem cell (TSC) lines. Both undifferentiated TSCs and differentiated TSCs (D-TSCs) display increased retroelement expression along with elevated levels of genes associated with placental development. Moreover, Resf1-deficient TSCs exhibit compromised maintenance of H3K9me3 domains in a manner independent of SETDB1. Collectively, our findings reveal that Resf1 plays multifaceted roles beyond retroelement silencing, underscoring its importance in development and its critical function in trophoblast lineage regulation.