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

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.

These 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.

CRISPR/Cas9 Genome Editing:

JEG3 cells were transfected with Cas9-2A-GFP and gRNAs targeting Enhancer L. GFP⁺ cells were sorted 48 h posttransfection and plated at clonal density in 10-cm dishes. Approximately 10 d after plating, single-cell–derived colonies were picked into 96-well plates and cultured for an additional 10 d. For PCR analysis, cells were harvested and genomic DNA extracted using prepGEM. Selected WT and KO clones were then expanded and further characterized.