| Description |
Families of regulatory transcription factors share highly conserved DNA binding domains and overlapping DNA target specificity, creating a paradox in transcriptional control. How do proteins with closely related DNA binding domains assume unique roles in gene regulation? Potential solutions to this problem include tissue-specific expression, binding site selectivity, combinatorial control and functional regulation by autoinhibition and phosphorylation. This thesis explores specificity mechanisms within the ets family of transcriptional regulatory proteins. The closely related ets proteins Ets-1 and Ets-2 were studied to identify specificity mechanisms acting at the level of DNA binding regulation. Quantitative DNA binding studies revealed that the DNA binding affinity of Ets-2 was 10 fold lower than that of Ets-1 for the same site. The DNA binding domains of Ets-1 and Ets-2 are 96% identical, suggesting that differences in autoinhibition of DNA binding might cause the lower affinity of Ets-2 for DNA. Partial proteolysis coupled with DNA binding studies indicated that Ets-2, like Ets-1, is regulated by autoinhibition, supporting the hypothesis that the basis for the 10-fold difference in affinity could be enhanced autoinhibition of Ets-2. Distinct responses to calcium signaling also may promote specificity between Ets-1 and Ets-2. While nuclear extract phosphorylated Ets-1 on multiple sites and dramatically inhibited its DNA binding, Ets-2 was phosphorylated less efficiently, producing only modest effects on DNA binding. Phosphorylation-dependent regulation of Ets-1 DNA binding was studied in greater detail to determine the molecular mechanism of the regulation. Phosphorylation of four serines by either nuclear extract or CaMKII inhibited Ets-1 DNA binding 50-fold. Autoinhibition was required for this effect, and partial proteolysis studies showed that phosphorylation stabilized an autoinhibitory conformation of Ets-1. These results suggest that the inhibitory module of Ets-1 provides a switch that inhibits DNA binding in response to phosphorylation. Together, the studies presented in this thesis suggest that differential regulation of DNA binding by both autoinhibition and phosphorylation may contribute to specificity among closely related transcription factors. These studies also indicate that autoinhibitory mechanisms can mediate inhibitory signaling events, highlighting the importance of autoinhibition as a transcriptional regulatory strategy. |
