Defining the DNA binding energetics of the glucocorticoid receptor

DNA-binding proteins bind to specific sequences to direct their activity to defined loci in the genome. Regulation of gene expression, for example, is dependent on the recognition of specific DNA sequences by transcription factors (TFs). These TFs receive input from cellular signals to control panels of genes to meet the needs of the cells. Critical to this function is the recognition and binding of TFs to the correct DNA sequence. The main focus of this thesis is to quantitatively determine how proteins, including TFs, distinguish DNA sequences, and to understand how DNA sequence affect their function. Primarily using the Glucocorticoid receptor (GR) as the model TF, I developed novel methods to measure the DNA binding specificity over long binding sites. These methods: 1) Distinguished the sequence specificity of GR and closely related androgen receptor (AR), which helped to both account for differential genomic localization between the two factors, and explained how GR can functionally substitute for AR in castration-resistant prostate cancer (Chapter II); 2) Explored the effect of DNA sequence on GR-regulated transcription through the specification of monomeric versus dimeric binding. Sequence motifs that bias GR binding toward the monomeric state were discovered (Chapter III); 3) Demonstrated a conserved role of intrinsic specificity in directing the degree of GR genomic occupancy in vivo in a fixed chromatin context (Chapter V); 4) Quantitatively modeled and decoupled the DNA binding and cleavage specificities of CRISPR-Cas9 system, providing a rapid pipeline to characterize the genome-editing reagents (Chapter IV). In summary, we showed here that DNA binding specificity is only the initial step in directing the activity of the bound protein. Beyond the affinity-based recruitment, DNA sequences can regulate the protein activity through alternative mechanisms, such as modulating the binding cooperativity, or directly serving as an allosteric ligand for protein function that is independent of DNA binding affinity.