Leonardo M. R. Ferreira, Torsten B. Meissner, Tarjei S. Mikkelsen, William Mallard, Charles W. O’Donnell et al.
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.
Leonardo M. R. Ferreira, Torsten B. Meissner, Tarjei S. Mikkelsen, William Mallard, Charles W. O’Donnell, Tamara Tilburgs, Hannah A. B. Gomes, Raymond Camahort, Richard I. Sherwood, David K. Gifford, John L. Rinn, Chad A. Cowan, Jack L. Strominger. A distant trophoblast-specific enhancer controls HLA-G expression at the maternal–fetal interface. 2016-04-13. https://doi.org/10.1073/pnas.1602886113
A 12,000-oligonucleotide library of partially overlapping 121-bp tiles spanning a 27-kb HLA-G locus was cloned into pGL4 reporter plasmids and tested in JEG3 cells with minimal TATA-box (minP) and strong SV40 (SV40P) promoters. The table contains the published Fig. 1A marker observations, recovered from the source PDF as approximate figure values.
A second MPRA tested approximately 12,000 Enhancer L variants, including all single substitutions plus series of consecutive substitutions and small insertions/deletions, with an average of 16 tags per variant and approximately 200,000 variant–tag combinations. The table is a 121-position summary of the SV40P mutual-information bars and significance colors published in Fig. 4A.
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