Unraveling the Structural, Dynamic, Thermodynamic, and Kinetic Heterogeneity in DNA Site Recognition by Structurally Homologous ETS Transcription Factors

Despite increasing awareness of their biological importance, mechanisms of DNA site discrimination by structurally homologous and functionally non-redundant transcription factors remain poorly defined concepts. Members of the ETS family of transcription factors, which regulate the differentiation of hematopoietic stem cells to the gamut of blood cell lineages, exemplify this conundrum. While the ETS-family members PU.1 and Ets-1 share structurally superimposable DNA-binding domains, the two homologs direct distinct cohorts of genes and induce mutually exclusive outcomes in hematopoietic cell-fate decisions. Using an array of biochemical, spectroscopic, and calorimetric techniques, we have compared DNA site recognition by PU.1 and Ets-1. The data indicate a startling level of structural, dynamic, thermodynamic, and kinetic heterogeneity associated with DNA site recognition by these proteins, which may be traced to their differential coupling of molecular hydration and counter-ion release with site-specific binding. Whereas site recognition by Ets-1 appears homogeneous with respect to DNA sequence identity, high- and low-affinity sites unmask major heterogeneity in PU.1 binding by all experimental observations. These differences are intrinsic to the two homologous DNA-binding domains; additional intramolecular interactions in adjacent domains exert no effect on the apparent homogeneity in Ets-1/DNA binding. The emerging evidence suggests a strong coupling between molecular hydration, DNA curvature, conformational dynamics, electrostatics as a mechanism for PU.1 to enforce different sequence preferences and kinetic behavior relative to Ets-1, and offer new insight into the functional differences between the two homologs. [This investigation is supported by NIH R15GM112002-01 to GMKP, NIH R01AI083803 to JKB, and NSF MCB1411502 to GMKP and WDW.]