title: Systematic dissection of genomic features determining transcription factor binding and enhancer function.
abstract: Enhancers regulate gene expression through the binding of sequence-specific transcription factors (TFs) to cognate motifs. Various features influence TF binding and enhancer function-including the chromatin state of the genomic locus, the affinities of the binding site, the activity of the bound TFs, and interactions among TFs. However, the precise nature and relative contributions of these features remain unclear. Here, we used massively parallel reporter assays (MPRAs) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo binding assays, to systematically dissect the contribution of each of these features to the binding and activity of genomic regulatory elements that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that distinct sets of features govern PPARγ binding vs. enhancer activity. PPARγ binding is largely governed by the affinity of the specific motif site and higher-order features of the larger genomic locus, such as chromatin accessibility. In contrast, the enhancer activity of PPARγ binding sites depends on varying contributions from dozens of TFs in the immediate vicinity, including interactions between combinations of these TFs. Different pairs of motifs follow different interaction rules, including subadditive, additive, and superadditive interactions among specific classes of TFs, with both spatially constrained and flexible grammars. Our results provide a paradigm for the systematic characterization of the genomic features underlying regulatory elements, applicable to the design of synthetic regulatory elements or the interpretation of human genetic variation.
authors.affiliation: Broad Institute, Cambridge, MA 02142.|Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.|Health Sciences and Technology, Harvard Medical School, Boston, MA 02215.
authors.forename: Sharon R
authors.initials: SR
authors.lastname: Grossman
authors.affiliation: Broad Institute, Cambridge, MA 02142.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Broad Institute, Cambridge, MA 02142.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: Broad Institute, Cambridge, MA 02142.|Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139.
authors.forename: Jesse
authors.initials: J
authors.lastname: Engreitz
authors.affiliation: Broad Institute, Cambridge, MA 02142.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Melnikov
authors.affiliation: Broad Institute, Cambridge, MA 02142.
authors.forename: Peter
authors.initials: P
authors.lastname: Rogov
authors.affiliation: Broad Institute, Cambridge, MA 02142.|Faculty of Arts and Sciences Center for Systems Biology, Harvard University, Cambridge, MA 02138.|Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138.
authors.forename: Ryan
authors.initials: R
authors.lastname: Tewhey
authors.affiliation: Institute of Bioengineering, CH-1015 Lausanne, Switzerland.|Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland.
authors.forename: Alina
authors.initials: A
authors.lastname: Isakova
authors.affiliation: Institute of Bioengineering, CH-1015 Lausanne, Switzerland.|Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland.
authors.forename: Bart
authors.initials: B
authors.lastname: Deplancke
authors.affiliation: Broad Institute, Cambridge, MA 02142.|Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114.|Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114.
authors.forename: Bradley E
authors.initials: BE
authors.lastname: Bernstein
authors.affiliation: Broad Institute, Cambridge, MA 02142.|Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138.|Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138.
authors.forename: Tarjei S
authors.initials: TS
authors.lastname: Mikkelsen
authors.affiliation: Broad Institute, Cambridge, MA 02142; lander@broadinstitute.org.|Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.|Department of Systems Biology, Harvard Medical School, Boston, MA 02215.
authors.forename: Eric S
authors.initials: ES
authors.lastname: Lander
mesh_terms: D041721:3T3-L1 Cells; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005786:Gene Expression Regulation; D023281:Genomics / Q000379:methods*; D051379:Mice; D009154:Mutation; D059372:Nucleotide Motifs / Q000235:genetics; D047495:PPAR gamma / Q000378:metabolism; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D047495:PPAR gamma; D014157:Transcription Factors
keywords: gene regulation; systems biology; transcription factor binding