We propose the first experimental analysis of the mechanisms by which transcription factors (TFs) evolved specificity for new DNA binding sites. The diversity and specificity of TFs allows organisms to precisely regulate cellular processes in development and physiology;modulation of TF action is also a critical means by which organisms evolve. But little is known about the mechanisms and dynamics by which TF's evolved their DNA specificities. Comparative studies of extant proteins have had limited success, because the causes of protein diversity occurred in the deep past, so historical approaches are required to distinguish them from the many other changes that have accrued since that time. Here we combine a powerful strategy for analyzing evolutionary mechanisms and processes-ancestral protein reconstruction-with advanced biophysical analysis and high-throughput screening of variant protein libraries to analyze the evolution of DNA specificity in the steroid hormone receptor (SR) protein family, a superb model of TF diversification. SRs play key roles in development, reproduction, homeostasis, cancer, and many diseases. The two classes of SRs-estrogen receptors on one hand and the receptors for androgens, progestagens, glucocorticoids, and mineralocorticoids (APGMRs) on the other-recognize different DNA binding sites. Preliminary data indicate that this diversity evolved via a sharp shift in DNA recognition that occurred between the ancestor of all steroid receptors (AncSR1) and the ancestor of the APGMRs (AncSR2). Our goals are to: 1) Dissect this evolutionary shift by combining phylogenetic inference with functional and biochemical/ biophysical techniques to "resurrect" AncSR1 and AncSR2 and experimentally characterize them;2) Identify the historical mutations that switched DNA specificity and characterize the mechanisms by which they did so, using targeted genetic manipulations and experimental analysis in ancestral backgrounds;3) Identify permissive mutations that were required for AncSR1 to tolerate the mutations that shifted its DNA recognition and determine the mechanisms for their effects;and 4) Develop a new high-throughput method to identify the functional effects and interactions of all historical mutations between AncSR1 and AncSR2. Our experiments will establish a complete mechanistic account for the evolution of novel TF specificity, linking historical genetic changes to shifts in protein function and biochemistry that generated a new gene regulatory system. This complete causal chain will elucidate how the biophysical architecture of extant proteins evolved and how that architecture structured the evolutionary genetic process. As the first-of-its-kind case study of the mechanistic evolution of TF function, this project will establish a methodological exemplar for future studies. Because the architecture of SR binding to DNA is classical, our work will establish baseline knowledge of evolutionary processes that is likely to apply to other TF families. The resulting structure-function knowledge will facilitate efforts to engineer TFs with new DNA-binding specificities in synthetic biology and biomedicine.

Public Health Relevance

Steroid hormone receptors bind specific DNA sequences and regulate the expression of genes that play key roles in a wide variety of diseases, including cancer, reproductive and immune dysfunction, cardiovascular disease, and disruptions of metabolic and osmotic homeostasis. Understanding how steroid receptors evolved to recognize their DNA targets can help explain how and why these important proteins have the underlying architecture that determines their functions, thus providing fundamental knowledge that will aid in predicting and understanding the effects of steroid receptor genetic variation on changes in transcriptional regulation leading to disease. This project uses computational techniques to infer the history of gene sequences in the receptor family followed by manipulative biochemical techniques to resurrect ancient receptor proteins, experimentally determine their structures and functions, and reconstruct the specific genetic and biochemical mechanisms by which their DNA-binding functions evolved.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM104397-01A1S1
Application #
8795931
Study Section
Genetic Variation and Evolution Study Section (GVE)
Program Officer
Eckstrand, Irene A
Project Start
2013-09-17
Project End
2017-05-31
Budget Start
2013-09-17
Budget End
2014-05-31
Support Year
1
Fiscal Year
2014
Total Cost
$64,592
Indirect Cost
Name
University of Chicago
Department
Biology
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Hart, Kathryn M; Harms, Michael J; Schmidt, Bryan H et al. (2014) Thermodynamic system drift in protein evolution. PLoS Biol 12:e1001994
Bridgham, Jamie T; Keay, June; Ortlund, Eric A et al. (2014) Vestigialization of an allosteric switch: genetic and structural mechanisms for the evolution of constitutive activity in a steroid hormone receptor. PLoS Genet 10:e1004058
McKeown, Alesia N; Bridgham, Jamie T; Anderson, Dave W et al. (2014) Evolution of DNA specificity in a transcription factor family produced a new gene regulatory module. Cell 159:58-68
Harms, Michael J; Thornton, Joseph W (2014) Historical contingency and its biophysical basis in glucocorticoid receptor evolution. Nature 512:203-7
Harms, Michael J; Thornton, Joseph W (2013) Evolutionary biochemistry: revealing the historical and physical causes of protein properties. Nat Rev Genet 14:559-71