A distant trophoblast-specific enhancer controls HLA-G expression at the maternal–fetal interface

Significance

Successful pregnancy poses an immunological paradox, as the mother’s immune system does not reject a fetus, even though it is a partially foreign tissue. Fetal extravillous trophoblasts (EVTs) deeply invade the uterus and interact with maternal immune cells without facing rejection. The nonclassical major histocompatibility complex (MHC) molecule HLA-G is essential for immune tolerance induction in pregnancy, yet the mechanism by which EVTs uniquely express HLA-G remains unknown. Using high-throughput cis-regulatory element dissection and genome editing tools, we discovered a remote enhancer essential for HLA-G expression in human EVTs, describing the basis for its selective expression at the maternal–fetal interface. These findings provide insight into immune tolerance induction during pregnancy and may yield new therapeutic targets for pregnancy-related disorders.

Abstract

HLA-G, a nonclassical HLA molecule uniquely expressed in the placenta, is a central component of fetus-induced immune tolerance during pregnancy. The tissue-specific expression of HLA-G, however, remains poorly understood. Here, systematic interrogation of the HLA-G locus using massively parallel reporter assay (MPRA) uncovered a previously unidentified cis-regulatory element 12 kb upstream of HLA-G with enhancer activity, Enhancer L. Strikingly, clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of this enhancer resulted in ablation of HLA-G expression in JEG3 cells and in primary human trophoblasts isolated from placenta. RNA-seq analysis demonstrated that Enhancer L specifically controls HLA-G expression. Moreover, DNase-seq and chromatin conformation capture (3C) defined Enhancer L as a cell type-specific enhancer that loops into the HLA-G promoter. Interestingly, MPRA-based saturation mutagenesis of Enhancer L identified motifs for transcription factors of the CEBP and GATA families essential for placentation. These factors associate with Enhancer L and regulate HLA-G expression. Our findings identify long-range chromatin looping mediated by core trophoblast transcription factors as the mechanism controlling tissue-specific HLA-G expression at the maternal–fetal interface. More broadly, these results establish the combination of MPRA and CRISPR/Cas9 deletion as a powerful strategy to investigate human immune gene regulation.