Sequence-Specific Control of ETS Transcription Factors

Abstract

The ETS family of transcription factors participate in a broad array of developmental and disease processes. ETS proteins are united by a conserved DNA-binding domain that targets 10-bp sequences harboring a central 5’-GGAA/T-3’ consensus. A primary recognition helix confers specificity for the consensus by direct readout of the major groove while adjoining mobile elements contact backbone contacts of flanking 5' and 3' sequences. Although flanking contacts play a secondary role in specifying the ETS binding site, they profoundly influence its affinity and transactivational activity. Elucidating the biophysical basis for the intrinsic selectivity of the ETS domain is therefore essential to understanding other interactions within the transcription regulatory complex. We have characterized the structure and thermodynamics of the ETS complexes with a high-, low-affinity, and nonspecific sequence. Calorimetric measurements reveal that the unbound ETS domain is a noncovalent, entropically-driven dimer. DNA binding induces ETS dissociation with sequence-dependent thermodynamics, suggesting distinct conformational arrangements accompanying the binding of each sequence variant. Footprinting titrations show that hypersensitivity to DNase I at the consensus is an experimental hallmark of sequence-specific ETS binding, regardless of affinity, indicating the importance of DNA distortion triggered by indirect readout. High- and low-affinity ETS cognates can be readily differentiated in terms of sensitivity to methylation in the latter. Based on this data, we present a model for ETS-DNA interactions in which indirect and direct readout are structurally linked to achieve sequence-specific binding. Complex formation in turn induces rheostatic rearrangements of ETS conformation (as manifest in terms of complex stability) that represent specific permissive or inhibitory states for protein-protein interactions (as manifest in ETS dimerization). Thus, the sequence identity of an ETS binding site may serve a functional role in initiating regulatory functions as well as an address for localization in the genome.