The classical DNA recognition sequence of the glucocorticoid receptor (GR) appears to be present at only a fraction of bound genomic regions. To identify sequences responsible for recruitment of this transcription factor (TF) to individual loci, we turned to the high-resolution ChIP-exo approach. We exploited this signal by determining footprint profiles of TF binding at single-base-pair resolution using ExoProfiler, a computational pipeline based on DNA binding motifs. When applied to our GR and the few available public ChIP-exo data sets, we find that ChIP-exo footprints are protein- and recognition sequence-specific signatures of genomic TF association. Furthermore, we show that ChIP-exo captures information about TFs other than the one directly targeted by the antibody in the ChIP procedure. Consequently, the shape of the ChIP-exo footprint can be used to discriminate between direct and indirect (tethering to other DNA-bound proteins) DNA association of GR. Together, our findings indicate that the absence of classical recognition sequences can be explained by direct GR binding to a broader spectrum of sequences than previously known, either as a homodimer or as a heterodimer binding together with a member of the ETS or TEAD families of TFs, or alternatively by indirect recruitment via FOX or STAT proteins. ChIP-exo footprints also bring structural insights and locate DNA:protein cross-link points that are compatible with crystal structures of the studied TFs. Overall, our generically applicable footprint-based approach uncovers new structural and functional insights into the diverse ways of genomic cooperation and association of TFs.

Significance: For proteins to be able to have context-specific activities, they can adopt context-specific conformations that enhance or restrict their activity. For transcriptional regulatory factors, such a context-specific signal is provided by the sequence of the DNA response element to which it binds. Here we show how one signal, an alternative splicing event, rewires a transcriptional regulatory protein to respond differently to a second signal, the DNA sequence to which it binds, by changing the functional interplay between protein domains. Together, our findings argue that bidirectional allosteric signaling between the DNA:protein interface and other regulatory domains fine tunes the activity of transcriptional regulatory factors toward individual target genes.

The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes.

The genomic loci bound by the glucocorticoid receptor (GR), a hormone-activated transcription factor, show little overlap between cell types. To study the role of chromatin and sequence in specifying where GR binds, we used Bayesian modeling within the universe of accessible chromatin. Taken together, our results uncovered that although GR preferentially binds accessible chromatin, its binding is biased against accessible chromatin located at promoter regions. This bias can only be explained partially by the presence of fewer GR recognition sequences, arguing for the existence of additional mechanisms that interfere with GR binding at promoters. Therefore, we tested the role of H3K9ac, the chromatin feature with the strongest negative association with GR binding, but found that this correlation does not reflect a causative link. Finally, we find a higher percentage of promoter-proximal GR binding for genes regulated by GR across cell types than for cell type-specific target genes. Given that GR almost exclusively binds accessible chromatin, we propose that cell type-specific regulation by GR preferentially occurs via distal enhancers, whose chromatin accessibility is typically cell type-specific, whereas ubiquitous target gene regulation is more likely to result from binding to promoter regions, which are often accessible regardless of cell type examined.

Although glucocorticoids (GCs) are a mainstay in the clinical management of asthma, the target cells that mediate their therapeutic effects are unknown. Contrary to our expectation, we found that GC receptor (GR) expression in immune cells was dispensable for successful therapy of allergic airway inflammation (AAI) with dexamethasone. Instead, GC treatment was compromised in mice expressing a defective GR in the nonhematopoietic compartment or selectively lacking the GR in airway epithelial cells. Further, we found that an intact GR dimerization interface was a prerequisite for the suppression of AAI and airway hyperresponsiveness by GCs. Our observation that the ability of dexamethasone to modulate gene expression in airway epithelial cells coincided with its potency to resolve AAI supports a crucial role for transcriptional regulation by the GR in this cell type. Taken together, we identified an unknown mode of GC action in the treatment of allergic asthma that might help to develop more specific therapies in the future.