title: 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.
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138; leonardoferreira@fas.harvard.edu jlstrom@fas.harvard.edu.
authors.forename: Leonardo M R
authors.initials: LM
authors.lastname: Ferreira
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138;
authors.forename: Torsten B
authors.initials: TB
authors.lastname: Meissner
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138; The Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138;
authors.forename: Tarjei S
authors.initials: TS
authors.lastname: Mikkelsen
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138; The Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142;
authors.forename: William
authors.initials: W
authors.lastname: Mallard
authors.affiliation: Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138;
authors.forename: Charles W
authors.initials: CW
authors.lastname: O'Donnell
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138;
authors.forename: Tamara
authors.initials: T
authors.lastname: Tilburgs
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138;
authors.forename: Hannah A B
authors.initials: HA
authors.lastname: Gomes
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138;
authors.forename: Raymond
authors.initials: R
authors.lastname: Camahort
authors.affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115;
authors.forename: Richard I
authors.initials: RI
authors.lastname: Sherwood
authors.affiliation: Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: David K
authors.initials: DK
authors.lastname: Gifford
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138; The Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138; Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02156.
authors.forename: John L
authors.initials: JL
authors.lastname: Rinn
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138; The Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138;
authors.forename: Chad A
authors.initials: CA
authors.lastname: Cowan
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138; leonardoferreira@fas.harvard.edu jlstrom@fas.harvard.edu.
authors.forename: Jack L
authors.initials: JL
authors.lastname: Strominger
mesh_terms: D004742:Enhancer Elements, Genetic / Q000235:genetics / Q000276:immunology*; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000235:genetics / Q000276:immunology*; D059951:HLA-G Antigens / Q000235:genetics / Q000276:immunology*; D054502:Histocompatibility, Maternal-Fetal / Q000235:genetics / Q000276:immunology*; D006801:Humans; D055261:Immunogenetic Phenomena / Q000235:genetics; D008431:Maternal-Fetal Exchange / Q000235:genetics / Q000276:immunology*; D010920:Placenta / Q000276:immunology; D011247:Pregnancy / Q000276:immunology*; D014327:Trophoblasts / Q000276:immunology*
chemical_list: D059951:HLA-G Antigens
keywords: CRISPR/Cas9; MPRA; human immune gene regulation; immune tolerance; pregnancy