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 proteinand 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 crosslink points that are compatible with crystal structures of the studied TFs. Overall, our generically applicable footprintbased approach uncovers new structural and functional insights into the diverse ways of genomic cooperation and association of TFs.

Antigen uptake and processing by innate immune cells is crucial to initiate the immune response. Therein, the endocytic C-type lectin receptors serve as pattern recognition receptors, detecting pathogens by their glycan structures. Herein, we studied the carbohydrate recognition domain of Langerin, a C-type lectin receptor involved in the host defense against viruses such as HIV and influenza as well as bacteria and fungi. Using a combination of nuclear magnetic resonance and molecular dynamics simulations, we unraveled the molecular determinants underlying cargo capture and release encoded in the receptor architecture. Our findings revealed receptor dynamics over several time scales associated with binding and release of the essential cofactor Ca(2+) controlled by the coupled motions of two loops. Applying mutual information theory and site-directed mutagenesis, we identified an allosteric intradomain network that modulates the Ca(2+) affinity depending on the pH, thereby promoting fast ligand release.

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.

No abstract