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

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.

Regulatory activities of transposable elements: from conflicts to benefits

“…2b). In a third example, a screen for cis -elements regulating the pregnancy-specific histocompatibility gene HLA-G combining massively parallel reporter assays and CRISPR genome editing identified a LTR71-derived distal enhancer required for HLA-G expression in the placenta 76 .…”

Transcription factor–DNA binding: beyond binding site motifs

“…CRISPR/Cas9-mediated genome editing was used to characterize an obesity-associated variant that disrupted a conserved ARID binding site motif; this variant was associated with altered gene expression and cellular metabolism [78]. These advances in genome editing are beginning to allow in situ validation of candidate causal variants identified by MPRAs [79-81] or other approaches. Saturating mutagenesis of a BCL11A erythroid enhancer using ZF nucleases, TALENs, and CRISPR/Cas9 identified a key functional GATA1 binding site [82,83].…”

Co-option of endogenous viral sequences for host cell function

“…The trophoblast layer of the placenta exhibits globally elevated EVE expression, which is potentiated by a seemingly general hypomethylation of repetitive DNA [66–68]. In addition, the LTRs of several ERV families exhibit placenta-specific enhancer activity [69,70] (Figure 3A). Together these properties open the door for the cooption of certain LTRs to drive novel adaptive pattern of host gene expression.…”