Joseph W. Thornton, Ph.D. - US grants

DESCRIPTION (provided by applicant): The goal of this research is to develop an experimental evolution system in which hormone receptors evolve affinity for new ligands. The human estrogen receptor alpha (ERalpha and two major breast cancer drugs that are ERalpha antagonists or response modulators will be used as a test case. This system will allow the structure-function relationships that determine interactions between estrogen receptors and their ligands to be evaluated using the exponential efficiency of natural selection -- a major advance over the currently used methods of directed mutagenesis and library screening, which require the painstaking production and evaluation of mutant receptors for new functions. This system will also address fundamental questions about the dynamics and mechanistic basis of molecular evolution and applied questions about disease progression, because it will recapitulate in the laboratory the processes by which tumor cells evolve resistance to drugs and, over evolutionary time, receptor proteins in normal cells evolve new functions. He system will consist of an engineered yeast strain in which growth rate depends upon the expression of genes controlled by estrogen response elements, and expression of these genes depends in turn on the activation of a recombinant human ERalpha by ligands added to the culture medium. When cultured in the laboratory, yeast with variant ERs that are better activated by the ligand will be generated by mutation; in the presence of a growth-limiting dose of a novel ligand, these variants will increase in frequency due to natural selection. Over many generations, ERs that use the novel ligand as a high-affinity agonist will evolve. The gene sequences of evolving receptors will be obtained at frequent intervals, their functions characterized using reporter gene assays, their three-dimensional structures modeled, and the dynamics of their evolution evaluated. Repeated over numerous experimental and control replicates, this system will allow the rigorous testing of hypotheses about ER structure-function relationships and the dynamics of receptor-ligand coevolution. This system can ultimately be extended to study the coevolution of virtually any receptor-ligand interaction, including those between any hormone, growth factor, or neurotransmitter and its receptor, as well as those between host and pathogen proteins.

DISSERTATION RESEARCH: EVOLUTION OF ESTROGEN RECEPTOR FUNCTION IN THE MOLLUSK, OCTOPUS VULGARIS


Joseph W. Thornton
June Keay

University of Oregon


Virtually all the functions of living cells are regulated by specific interactions between molecules, such as those between hormones and receptors, enzymes and substrates, and transcription factors and DNA binding sites. Very little is known, however, about how these molecular interactions evolved. This project focuses on the evolution of interactions between estrogens - a group of hormones crucial for development and reproduction in vertebrates -- and the estrogen receptor (ER) protein. In vertebrates, binding of estrogens enables the ER to increase expression of hormone-responsive genes. Preliminary data indicate the ER in mollusks has lost this interaction with estradiol, and that it always activates transcription, whether or not the hormone is present. This variation presents the opportunity to study the evolution of ER's functional interaction with hormones. Specifically, this project will investigate the evolution of ER function in the common octopus (Octopus vulgaris), a mollusk in which estrogen appears to have a reproductive role. The molecular functions of the octopus ER will be determined, the ER sequence of the ancient common ancestor of vertebrates and mollusks will be reconstructed, and hypotheses about the evolution of variant ER functions will be tested by synthesizing the ancestral receptor and using site-directed mutagenesis. This research will provide important comparative data for understanding ER function and evolution in humans and other animals. It will also provide important information on the potential scope of environmental endocrine disruption, a major issue in environmental and wildlife health. This project will contribute to the next generation of scientists by supporting the research training of a talented female scientist in cutting-edge techniques of molecular evolution, gene function, and endocrinology.

Phylogenies -- evolutionary trees that represent the historical relationships among species -- provide the framework for comparative analysis in all fields of biology. Most phylogenies are now inferred from DNA or protein sequence data using methods that assume the evolutionary process is largely homogeneous. In reality, however, evolutionary dynamics often differ among sequence sites and among lineages. Our preliminary data indicate that several kinds of heterogeneity can cause current methods to infer the wrong phylogeny. The goal of this proposal is to develop, implement, and validate a new family of mixed-model phylogenetic methods that incorporate evolutionary heterogeneity in a maximum likelihood framework. The accuracy of our method versus current techniques will be evaluated with experiments using both simulated and empirical data sets. We will distribute user-friendly software to the scientific community that implements our method and provides a high-throughput platform for simulating and analyzing heterogeneous sequence data.
This project will accelerate the scientific community's success in reconstructing the Tree of Life and improve our ability to interpret genomic, developmental, and physiological data in a comparative framework. More reliable phylogenies are beneficial to society because an understanding of evolutionary relationships is crucial for characterizing biodiversity and developing strategies to preserve it. Sound phylogenetic knowledge is also central to understanding the evolutionary processes that affect agriculture, ecosystem function, and infectious disease. Our software will also provide scientists a tool for high-throughput phylogenetic experimentation and data analysis, a key goal as whole-genome sequence data become available. This project will also provide graduate and undergraduate education and research training in computer science and biology, an important need as biology becomes increasingly information-driven in the 21st Century.

CAREER: Molecular Evolution of Steroid Hormone Receptor Functions and Interactions
Joseph W. Thornton
University of Oregon

Virtually everything that living cells do is made possible by very specific interactions between molecules. For example, steroid hormones produced in the gonads or adrenal gland-such as estrogen, testosterone, and cortisol-regulate development, reproduction, behavior, and countless other processes. Each hormone produces a unique suite of effects by binding to a specific receptor protein in target cells; the hormone-bound receptor then enters the nucleus, binds to a specific set of DNA sequences, and activates the expression of nearby genes. Despite the great biological importance of specific interactions like this, there has been very little work to understand how tight molecular partnerships evolve.
This career plan integrates research on the evolution of molecular interactions with education and outreach activities that strengthen understanding of evolution, endocrinology, and the deployment of scientific knowledge in public policy. In the project's research component, steroid hormones and their receptors will be used as a model system, with the goal of reconstructing the evolutionary mechanisms by which the specific interactions between hormones and receptors evolved. First, the investigators will isolate and characterize receptors from several target species that, because of their position in the tree of life, will provide crucial information about the diversification of the receptor gene family. Phylogenetic techniques will be used to infer the dynamics by which the family diversified in number, molecular sequence, structure, and the ability to be activated by new hormones. The investigators will then test hypotheses about how receptors evolved novel functions by "resurrecting" ancestral receptor genes and studying their functions in the laboratory. Finally, experiments will be performed to determine how changes at the DNA level caused receptors to evolve new functions by re-introducing historical mutations into resurrected ancestral genes and determining their effects on the receptors' interactions with various hormones.
The education and outreach components of this proposal are focused on the interface of science with real-world policy issues. A new course will be developed, which prepares young scientists to participate in societal decision-making by teaching them to think critically about the ways that science is deployed in the policy process; environmental endocrine disrupters-pollutants that interfere with the body's steroid hormones - will be used as an extended case study. A large undergraduate course in Evolutionary Biology will be revised to better incorporate the applied implications of evolutionary knowledge. Finally, the principal investigator will serve as an occasional science advisor to two nongovernmental organizations that work directly with large constituencies on environmental endocrine disruption and human health.

DESCRIPTION (provided by applicant): The goal of this research is to elucidate the evolutionary dynamics and structural basis for the evolution of novel functions in a biomedically important gene family, the steroid hormone receptors. We will combine experimental evolution, crystallography, ancestral gene resurrection, and manipulative assays of gene function to analyze how receptors evolve tight molecular partnerships with novel hormonal ligands. The specific aims are: 1. Use an experimental evolution system in engineered yeast to select for human estrogen receptors which, like those in some mammary tumors, evolve to be stimulated by taxmoxifen, a major breast cancer drug. Using a battery of functional assays and evolutionary techniques for reconstructing the evolution of receptor sequences, we will determine the mechanistic basis for the evolution of this new ER-ligand interaction and the dynamics of receptor-ligand coevolution. 2. We will resurrect ancestral steroid receptors (using phylogenetic analysis and gene synthesis) and then evolve these receptors in this same experimental evolution system. Ancestral receptors will be selected to recapitulate the functional shifts in ligand specificity that occurred during real historical evolution over hundreds of millions of years. The mechanistic basis and evolutionary dynamics of this process will be studied in detail. 3. We will express, crystallize, and determine the three-dimensional structures of ancestral receptors, as well as those of several functionally divergent extant steroid receptors. These structures - together with the data on the evolution of new functions from Aims 1 and 2 - will provide a rich database from which to reconstruct the evolution of receptor protein structures. This is expected to reveal the structural mechanisms by which new receptor functions evolve, both in our experimental evolution system and in real historical evolution. This synthesis of manipulative, hypothesis-testing techniques will provide unprecedented detailed knowledge of the dynamics and mechanisms by which gene function evolves. These data should help resolve long-standing fundamental issues in evolutionary biology, and also improve our understanding of the structure-function relationships that determine steroid receptor function.

DESCRIPTION (provided by applicant): 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.

How organisms adapt to environments is a fundamental question in biology, and exploring this process can enrich our understanding of factors that shape the evolution of forms, functions, and species. A precise way to study adaptive traits is by experimentally characterizing the mechanisms through which genes drive evolution. This approach can reveal general principles about how changes at the DNA level affect function over time. Here, this framework is used to rigorously characterize alcohol tolerance in fruit flies, a classic model of adaptive evolution. The researchers will investigate a long-standing hypothesis about adaptation to ethanol and the relationship of variation at genetic, biochemical, and physiological levels. This work will demonstrate how biochemical methods can be deployed in evolutionary contexts to yield novel insights. Broader impacts of this project include training of a graduate student in the emerging discipline of evolutionary biochemistry as well as collaboration with local high school teachers to design, present, and publicly distribute an educational module emphasizing the interface of biochemistry and evolutionary genetics.

The evolution of alcohol dehydrogenase (ADH) in Drosophila melanogaster is an iconic example of adaptation: inter- and intraspecific variations in ADH harbor signatures of positive and balancing selection, respectively, and past works have hypothesized the responsible selective forces. The signature of positive selection on ADH is thought to reflect protein evolution in response to expansion and adaptation to ethanol-rich environments by D. melanogaster. This research utilizes ancestral sequence reconstruction of proteins, biochemical characterization, and physiological assays of transgenic organisms to test if functional changes occurred in ADH as predicted. The signature of balancing selection - supported by recurrent and dynamic allele frequency clines across continents - is thought to be due to allele-by-temperature interactions. This project investigates the role of temperature interactions as a mechanism. It will test a hypothesis about how temperature might shape patterns of genetic variance by characterizing the propensity of natural alleles to misfold at environmentally realistic temperatures. These experiments will have broad significance as methodological exemplars of utilizing detailed, mechanistic studies in evolutionary contexts to elucidate general insights pertaining to the functional determinants of adaptation.

This project will combine evolutionary genetics and biochemistry to dissect how a fundamental system of gene regulation in animals has evolved. It will explore how specific control molecules that can turn particular genes on or off have evolved. This will be done by tracing the ancient evolutionary changes in the structure of critical control molecules and their corresponding genes. It will integrate laboratory tests of protein to DNA binding with computer analysis of molecular evolution. Broader impacts include training of a graduate student in the integration of computer and experimental approaches to molecular evolution. Dissemination of techniques in statistical programming through software boot camps and science outreach presentations will also increase the representation of women in inter-disciplinary science.

Transcription is the first step in the expression of any gene and hence is thought by many to be the fundamental step in regulation of the genome. Proper regulation of an organism's genes depends on the correct pairing of thousands of different proteins, called transcription factors, with their respective genes. Understanding how new control pairing occurs without loss of original functions is central to regulation in complex genomes. Steroid receptors are biologically important transcription factors. They are also an excellent model system for the study of transcriptional control since they consist of two related but functionally divergent groups that differ in the way they bind to specific genes. One group predominantly binds to a major groove of the DNA molecule; the other group binds to both major and minor grooves of DNA. To understand the evolution of DNA binding and recognition in these two groups this research utilizes ancestral sequence reconstruction, along with functional laboratory assays. Computational analysis of receptor function and evolution will be combined with laboratory protein binding assays to explore the hypothesis that the shape of the DNA minor groove as well as the DNA sequence was important in the evolution of novel specificity. The proposed experiments will delineate the genetic and physical mechanisms by which different modes of DNA recognition diversified in this group of molecules.

We propose the first comprehensive characterization of sequence space around an ancestral protein. This work will 1) characterize the effects on function of all possible mutations and pairs of mutations across the protein's entire length and of all possible combinations of mutations at a key subset of sites, 2) illuminate how the distribution of function through this multidimensional sequence space would have affected the processes of protein evolution (a key goal in molecular evolution), and 3) quantify the complete set of main-effect and epistatic genetic determinants of DNA specificity in a transcription factor and elucidate their biochemical causes ? an important goal for protein biochemistry and molecular gene regulation. We use the steroid hormone receptor DNA-binding domain as an ideal model system, because it is of great biomedical importance; it is experimentally and phylogenetically tractable; and its specificity for DNA targets diversified through a well-understood evolutionary process, with a known set of historical mutations and biophysical mechanisms. The proposed work will reveal why this history occurred relative to the many other mutational trajectories the protein could have taken as it evolved its new specificity. With the map of sequence space in hand, we will then apply locus-specific, replicated experimental evolution to the ancestral protein, placing it under strong selection to explore sequence space and evolve the same novel specificity that it acquired during historical evolution. By identifying commonalities and differences among the historical trajectory, experimental evolution trajectories, and the many other possible pathways through sequence space, we will gain fundamental insight into the roles of contingency and determinism in evolution and illuminate underlying mechanistic factors that caused those phenomena. Specific questions include: how many ways were there to evolve the derived DNA specificity, and how many were accessible under selection and drift? Did the historical outcome evolve because it was the optimal genotype, because it was the best or only accessible genotype, or simply due to chance? If more optimal genotypes exist, what prevented the evolving protein from reaching them? To what extent must new specificities evolve through promiscuous intermediates, and how many mutations does it take to evolve a new specificity? We will also characterize sequence space and experimental evolutionary trajectories around ancient receptors that existed at different times during history; this will reveal how the protein's evolvability and robustness fluctuated over evolutionary time due to epistatically acting mutations. Finally, by fully characterizing the main and epistatic genetic determinants of the protein's DNA specificity, we will identify common biophysical mechanisms that underlie DNA recognition, contributing to an important goal in molecular biology, biochemistry, cell biology, and development. The methods and conceptual tools we develop will be applicable to studying other transcription factors and the evolution of many other protein families.

We propose the first experimental studies of the historical origin and elaboration of molecular complexes. Virtually all proteins assemble with specific molecular partners into precise geometric arrangements, but we know little about the genetic and structural mechanisms by which these complexes evolved or the evolutionary forces that explain their origin, elaboration, and long-term persistence. We will combine ancestral protein reconstruction with biochemical, structural, and functional experiments to reconstruct the evolution of molecular complexes in three model protein families, enabling us to formulate and test general hypotheses about the evolutionary causes and consequences of changes in stoichiometry, allostery, and specificity. All three protein families are biologically essential, experimentally tractable, and exemplify distinct questions. The models are: 1) hemoglobin, the major oxygen carrier in vertebrates, a heterotetramer that is biochemistry's iconic case of an allosterically regulated molecular complex; 2) citrate synthase, an essential metabolic enzyme that is a dimer in some lineages and an allosterically regulated hexamer in others, which provides a rich case-study of the evolutionary relationship and long-term persistence of allostery and complexity; and 3) steroid hormone receptors, a family of transcription factors that regulate vertebrate reproduction and development and which evolved after gene duplication to specifically assemble as homodimers, each with distinct regulatory functions. The project will address these questions: 1) How and why do complexes originate and increase in stoichiometry from simpler forms? 2) What genetic and biophysical mechanisms mediate evolution of new and specific interfaces? 3) By what mechanisms did allostery evolve? 4) Does selection for oligomer-associated functions account entirely for the emergence and long-term persistence of molecular complexes? Or did substitutions compatible only with the assembled form occur neutrally and entrench the complex, creating an evolutionary ratchet towards greater complexity? And 5) After duplication, how did homomers evolve to selectively assemble with copies of themselves (excluding their sister paralogs), and was evolution of new functions constrained until this selectivity was achieved? By combining advanced techniques from protein biochemistry and evolutionary biology, the project will articulate and test at unprecedented resolution hypotheses about the evolutionary forces and biochemical mechanisms that underlie the physical and functional properties of molecular complexes. It will also help to explain why multimers are so widespread and, by revealing how evolution achieved specificity and allosteric regulation, enhance engineering efforts to design complexes with these properties. The project will also provide a methdological and conceptual template for future studies of other molecular complexes.

Epistatic interactions within proteins can, in principle, make the paths and outcomes of evolution contingent on chance events; they can also entrench proteins with residues that appear to be optimal but are accidents of history. The extent to which epistasis actually affected the trajectory and outcomes of molecular evolution depends on the fitness effects of substitutions when they occurred in history compared to their potential effects earlier or later in time and on the temporal order in which interacting substitutions occurred. Deep mutational scanning studies have revealed pervasive epistasis among the huge number of possible mutations, but no studies have directly assessed how the fitness effects of substitutions that happened during history changed over time as the protein evolved. We will perform the first comprehensive experimental analysis of the fitness effects of all amino acid states that evolved in a protein during a long-term phylogenetic trajectory, both at the time they occurred and if they had occurred at other points in history. These data will be analyzed in the ordered temporal context of the protein's phylogeny and supplemented with biochemical experiments, enabling a deep characterization of the causes and consequences of epistasis, contingency and entrenchment across the billion-year history of an essential protein. Our model system is ideal for this purpose. Hsp90, the essential molecular chaperone in all eukary- otic cells, plays key roles in protein folding and maturation, cell signaling, and a wide range of diseases. Strong phylogenetic signal allows confident reconstruction of the billion-year evolutionary history of Hsp90's protein sequence from the last common ancestor of animals, fungi and related protists to present-day Saccharomyces cerevisiae. We will generate targeted protein libraries containing every ancestral and derived state that occurred during this phylogenetic trajectory, singly and in every possible pair, in the background of all 30 reconstructed ancestral proteins along the trajectory. Using a high- resolution bulk competition assay in yeast, we will precisely measure selection coefficients and epistatic interactions and quantify how these properties changed over time. This will reveal the fitness effects and interactions of every substitution at the approximate time it occurred, as well as the effects and interactions it would have had if it happened (or reverted to the ancestral state) at any point earlier or later during the trajectory. We will also apply biophysical and structural techniques to elucidate the underlying biochemical mechanisms that drove these genetic and evolutionary phenomena. This work will provide deep new insight into the ways in which proteins' genetic and physical architecture influences, and is influenced by, the processes by which they evolve; it will also strengthen our understanding of sequence-structure-function relationships in a biologically essential protein.

Recent technological advances have transformed genetics research, and social changes have caused major shifts in best practices for graduate education, research training, and mentoring. We propose an innovative interdisciplinary predoctoral T32 program, Genetic Mechanisms and Evolution (GME), which is specifically crafted to meet the challenges and opportunities presented by these changes. The GME program will train a diverse group of world-class Ph.D. scientists in molecular, statistical, and evolutionary genetics research who will serve as the next generation of innovative scientific leaders in genetics. Training will ensure development of multidisciplinary competence across these fields, with a strong foundation in quantitative and computational analysis for every student. The GME training program leverages the world-class strength of the University of Chicago in genetics. Mentors include 56 faculty with extraordinary records of research and graduate training, drawn from 14 departments across the fields of evolutionary, statistical, and molecular genetics. Further, the University?s unique organizational structure brings all areas of genetics into a single division and makes possible the interdisciplinary program we propose. Trainees for 18 funded positions will be selectively drawn from 9 graduate programs across disciplinary areas. The pool of potential trainees is extraordinarily well-qualified and diverse (49% women and 26% URM over the last 5 years). Trainees will be funded in years 2-3 of their studies, but they will participate in training and advising activities from matriculation through graduation. A new interdisciplinary core course and breadth requirements will develop student foundations in molecular, statistical, and evolutionary genetics and build strong skills in programming and statistics. Specialized workshops and an annual hackathon will provide further rigorous training in computational and quantitative analysis of modern genetic data. Formal writing instruction along with workshops in grant-writing and oral presentation skills will train scientists for effective communication and help ameliorate disparities in preparation among students from diverse backgrounds. Individual development plans, mentor-mentee contracts, faculty mentor training, and peer mentoring will facilitate trainee success and allow growth of a mutually supportive community of faculty and students. Participation in a pioneering career development program will support trainees in finding and preparing for a variety of post-PhD career paths. Recruitment and retention of an increasingly diverse group of students will be further strengthened by participating in pipeline and outreach programs, bridge activities for new students, and faculty training to enhance the inclusivity of the training environment and admissions process. All these activities -- building on the strengths of an exceptional cadre of trainees, trainers and institutional support ? will allow us to recruit and train the future leaders of 21st century genetics research.