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

Proc Natl Acad Sci U S A. 2016 May 10;113(19):5364-9. doi: 10.1073/pnas.1602886113. Epub 2016 Apr 13.


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.

Keywords: CRISPR/Cas9; MPRA; human immune gene regulation; immune tolerance; pregnancy.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Enhancer Elements, Genetic / genetics
  • Enhancer Elements, Genetic / immunology*
  • Female
  • Gene Expression Regulation, Developmental / genetics
  • Gene Expression Regulation, Developmental / immunology*
  • HLA-G Antigens / genetics
  • HLA-G Antigens / immunology*
  • Histocompatibility, Maternal-Fetal / genetics
  • Histocompatibility, Maternal-Fetal / immunology*
  • Humans
  • Immunogenetic Phenomena / genetics
  • Maternal-Fetal Exchange / genetics
  • Maternal-Fetal Exchange / immunology*
  • Placenta / immunology
  • Pregnancy / immunology*
  • Trophoblasts / immunology*


  • HLA-G Antigens