Evaluating the specificity spectra of DNA binding molecules is a nontrivial challenge that hinders the ability to decipher gene regulatory networks or engineer molecules that act on genomes.Here we compare the DNA sequence specificities for different classes of proteins and engineered DNA binding molecules across the entire sequence space. These high-content data are visualized and interpreted using an interactive "specificity landscape" which simultaneously displays the affinity and specificity of a million-plus DNA sequences. Contrary to expectation, specificity landscapes reveal that synthetic DNA ligands match, and often surpass, the specificities of eukaryotic DNA binding proteins. The landscapes also identify differential specificity constraints imposed by diverse structural folds of natural and synthetic DNA binders. Importantly, the sequence context of a binding site significantly influences binding energetics, and utilizing the full contextual information permits greater accuracy in annotating regulatory elements within a given genome. Assigning such context-dependent binding values to every DNA sequence across the genome yields predictive genome-wide binding landscapes (genomescapes). A genomescape of a synthetic DNA binding molecule provided insight into its differential regulatory activity in cultured cells. The approach we describe will accelerate the creation of precision-tailored DNA therapeutics and uncover principles that govern sequence-specificity of DNA binding molecules.

Interaction of estrogen receptor (ER) with DNA sequences known as estrogen response elements (ERE) is required for estrogen regulation of the expression of target genes. To characterize the affinity and specificity of ER interaction with ERE sequences in vitro under equilibrium conditions, fluorescence anisotropy assays were performed using recombinant, purified ER and a fluorescein-labeled 35-base pair oligonucleotide bearing an idealized palindromic ERE. In buffer containing 100 mM KCl, the baculovirus-expressed, purified human ER bound with similar affinity to the consensus ERE and a mutant ERE with a single base pair change per half-site. Above 225 mM KCl, ER exhibited discrimination between the consensus and mutated ERE targets. Between 225 and 275 mM KCl, binding to the consensus ERE was independent of salt concentration and occurred with an equilibrium dissociation constant (K d ) of 1.8 ؎ 0.6 nM, whereas binding to the mutant ERE was not detected at ER concentrations below 100 nM under the same conditions. At 300 mM KCl, the K d for the consensus ERE increased approximately 25-fold, suggesting complex salt concentration dependence. Both estrogen-occupied and unoccupied ER bound to the consensus ERE sequence with similar affinity, indicating that estrogen affects ER activity at a step other than DNA binding. Unlike the full-length ER, the recombinant DNA binding domain of ER did not discriminate between the consensus and mutated ERE sequences even at buffer salt concentrations greater than 200 mM NaCl, suggesting that ER sequences outside the DNA binding domain may be important in promoting specific binding.As a member of the superfamily of steroid hormone receptors, the estrogen receptor (ER) 1 is a 67-kDa nuclear protein that regulates transcription for genes involved in cellular differentiation, development of the female reproductive system, and homeostasis (1-3). ER binds to specific DNA sequences designated estrogen response elements (ERE), which are present in promoters of target genes. The consensus ERE is a 13-base pair (bp) palindromic sequence, consisting of inverted repeats of the half-sites 5Ј-GGTCA-3Ј separated by a 3-bp spacer, found in the 5Ј-flanking region of the Xenopus and chicken vitellogenin A2 genes (4 -6). ER consists of conserved structural and functional domains ( Fig. 1) for DNA-binding in region C, nuclear localization in region D, and hormone-binding in region E (7,8). Two transcriptional activation function domains, designated TAF-1 and TAF-2, are found in regions A/B and E, respectively (8 -11). The physiological ligand for ER is 17␤-estradiol (E 2 ), which diffuses into target cells and binds to ER. Binding of E 2 to ER induces conformational changes in the receptor (12-14) and dissociation of receptor-associated proteins including hsp90 and p59 (15, 16), leading to the assembly of an active transcriptional complex responsible for the positive and negative regulation of target genes. One of the principal requirements for E 2 -induced regulation of target genes is the specifi...

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The interactions of the ligand binding domain (LBD) of androgen receptor (AR) and the AR T877A mutant, found in prostate cancer, with peptides from coactivator and corepressor proteins or random phage display peptides were investigated using in vitro time-resolved fluorescence resonance energy transfer (TR-FRET). Interaction of wild-type AR LBD with the random phage display peptide D11FxxLF was observed with dihydrotestosterone (DHT), testosterone, R1881, estradiol, spironolactone, progesterone, and cortisol resulting in distinct dose dependency (EC50) values for each ligand and correlating well with the reported rank order potency of these agonists. Increasing concentrations of cyproterone acetate and mifepristone resulted in more complete disruption of the DHT-mediated AR-D11FxxLF peptide interaction, while flutamide, hydroxyflutamide, and bicalutamide caused only partial disruption of the complex. The mutant AR T877A LBD exhibited increased binding affinities for all ligands tested except for bicalutamide, mifepristone, DHT, and R1881 in a competitive binding assay as compared to wild-type AR LBD. This mutation was also characterized by increased ligand potency for agonist-induced peptide recruitment. Although usually an antagonist, hydroxyflutamide was more potent in the recruitment of D11FxxLF or an SRC3-1 LXXLL motif to AR T877A LBD than AR LBD. The antagonist cyproterone acetate behaved as a full antagonist of D11FxxLF recruitment to AR LBD and AR T877A LBD but as a more potent agonist in the recruitment of SRC3-1 to AR T877A LBD. These results suggest that the AR T877A mutation affects both ligand affinity and ligand dose dependency for peptide recruitment and may explain in part the altered responses of antagonists and increased transcriptional activation reported in androgen-independent prostate cancers.