Binding Does Not Equal Function: Linking Glucocorticoid Receptor Binding to the Regulation of Genes in the Endogenous Genomic Context

The glucocorticoid receptor (GR) binds to tens of thousands of genomic binding sites, yet seems to regulate a much smaller number of genes. In a simple scenario, binding of GR would influence the expression of associated genes whereas in the absence of binding, nearby genes would not be affected. In practice however, the link between GR binding and gene regulation is fuzzy, with the majority of binding events apparently not resulting in the regulation of nearby genes. This raises the question: What distinguishes GR binding events that result in the regulation of gene expression from those that do not? We try to answer this question using a variety of approaches, which advanced our understanding in several ways. First, computational approaches indicate that productive GR binding events are more likely when the three-dimensional organization of the genome brings such regions proximal to the promoter of genes. Second, using massively parallel reporter assays, we determined the regulatory potential of tens of thousands of GR-bound regions to test if only a subset has an intrinsic regulatory potential. Finally, using genome-editing approaches, we identify single GR-bound recognition sequences that contribute to the GR-dependent regulation of genes. By dissecting an enhancer element with multiple GR recognition sequences and by mutating these, alone or in combination, we uncover that the regulatory potential of the enhancer depends on cooperative interactions between multiple GR binding sites. Furthermore, by building synthetic endogenouscis-regulatory elements we investigated the minimal sequence requirements of a functional response element and were able to expand the repertoire of GR target genes. Together, these studies yield several insights into the molecular mechanisms that discriminate “productive” GR interactions with the genome resulting in gene regulation from “non-productive” binding events.