An RLTR11B element regulates gene expression at the imprinted Sfmbt2/Gm13261 locus

Abstract

Endogenous retrovirus K (ERVK)-derived long terminal repeats (LTRs) have previously been implicated as regulatory elements at placenta-specific non-canonically imprinted (NCI) loci. In common with other NCI genes, the monoallelic H3K4me3 peak at the paternally expressed Sfmbt2/Gm13261 locus coincides with an ERVK LTR (RLTR11B), leading to the question of whether this LTR is instrumental in the persistent, placenta-specific imprinting and if it contributes to transcriptional regulation at this locus. To address this, the Sfmbt2/Gm13261 locus was used as a model to investigate the regulatory role of RLTR11B in vitro and in vivo. In trophoblast stem cells (TSCs), RLTR11B was found to drive expression of the lncRNA Gm13261, which in turn promotes Sfmbt2 expression in cis, consistent with a transcription-dependent, promoter-like enhancer function. Deletion of RLTR11B resulted in reduced Sfmbt2 expression, transcriptional mis-regulation cell-cycle progression and proliferation associated genes, and increased evidence of trophoblast giant cell differentiation, indicative of Sfmbt2 haploinsufficiency during trophoblast development. In vivo, paternal deletion of RLTR11B led to placental hypoplasia, foetal growth restriction, and a trend towards increased pre-weaning mortality. Despite this, Sfmbt2 and Gm13261 expression were maintained at variable levels across conceptuses, suggesting the presence of compensatory regulatory mechanisms. Notably, homozygous deletion of RLTR11B resulted in partial embryonic lethality, indicating a previously undefined contribution from both parental alleles. However, allele-specific analysis in hybrid placentas demonstrated that Sfmbt2 expression remained robustly paternal at midgestation, indicating that RLTR11B is not the primary element controlling imprinting at this locus. Additionally, these findings suggest that the Sfmbt2 locus displays regulatory plasticity, consistent with multiple regulatory inputs contributing to the maintenance of gene expression during placental development. This work demonstrates a previously undefined role for the ERVK-derived RLTR11B element in trophoblast development, where it functions as a context dependent cis-regulatory element that supports robust Sfmbt2 expression required for normal placental growth and function.