Evolution of a derived protein–protein interaction between HoxA11 and Foxo1a in mammals caused by changes in intramolecular regulation

Abstract

Current models of developmental evolution suggest changes in gene regulation underlie the evolution of morphology. Despite the fact that protein complexes regulate gene expression, the evolution of regulatory protein complexes is rarely studied. Here, we investigate the evolution of a protein-protein interaction (PPI) between Homeobox A11 (HoxA11) and Forkhead box 01A (Foxo1a). Using extant and "resurrected" ancestral proteins, we show that the physical interaction between HoxA11 and Foxo1a originated in the mammalian stem lineage. Functional divergence tests and coimmunoprecipitation with heterologous protein pairs indicate that the evolution of interaction was attributable to changes in HoxA11, and deletion studies demonstrate that the interaction interface is located in the homeodomain region of HoxA11. However, there are no changes in amino acid sequence in the homeodomain region during this time period, indicating that the origin of the derived PPI was attributable to changes outside the binding interface. We infer that the amino acid substitutions in HoxA11 altered Foxo1a's access to the conserved binding interface at the HoxA11 homeodomain. We also found an expansion in the number of paired Hox/Fox binding sites in the genomes of mammalian lineage species suggesting the complex has a biological function. Our data indicate that the physical interaction between HoxA11 and Foxo1a evolved through noninterface changes that facilitate the PPI, which prevents inappropriate interactions, rather than through the evolution of a novel binding interface. We speculate that evolutionary changes of intramolecular regulation have limited pleiotropic effects compared with changes to interaction domains themselves.protein-protein interaction evolution | transcription factor evolution C hanges in gene regulation are the driving force in the origin and evolution of novel phenotypes. Gene expression is coordinated by the formation of multiprotein complexes that bind to cis-regulatory promoter and enhancer regions for target genes and activate or repress transcription in a signal-dependent fashion (1-3). There is strong evidence that changes in both cisregulatory elements and regulatory proteins, such as transcription factors, have led to gene regulatory evolution (4-12). However, the mechanisms by which transcription factor evolution affects gene regulation are poorly understood. For example, it has been suggested that the evolution of novel protein-protein interactions (PPIs), posttranslational modifications, and DNA-and ligandbinding specificities may all contribute to the origin of regulatory activities in transcription factors; however, to date, few studies have carefully dissected potential mechanisms.Here, we address this question by investigating the evolution of the physical interaction between two transcription factors, Homeobox A11 (HoxA11) and Forkhead box 01A (Foxo1a), which play a major role in regulating gene expression in endometrial stromal cells during pregnancy in placental mammals (8,13). By examining the a...

Functional Divergence of the Nuclear ReceptorNR2C1as a Modulator of Pluripotentiality During Hominid Evolution

“…Indeed, AGR has been used effectively to investigate the evolution of function in a wide variety of different proteins Gaucher et al 2003;Ugalde et al 2004;Gaucher et al 2008;Bridgham et al 2009; Thornton 2010, 2013;Eick and Thornton 2011;Brayer et al 2011). It is not feasible, however, to experimentally assess the functional consequences of even a subset of amino acids in each of the 48 primate NRs.…”

“…A full understanding of the phenotypic significance of primate NR evolution requires explicitly linking formal evolutionary analyses with experimental approaches focused on the functional impact of amino acid substitutions (Ugalde et al 2004;Brayer et al 2011;Kratzer et al 2014).…”

“…This is in part because sequence divergence among primates is characteristically low and it is challenging to differentiate adaptive substitutions from the background of neutral substitutions in such data. A full understanding of the phenotypic significance of primate NR evolution requires explicitly linking formal evolutionary analyses with experimental approaches focused on the functional impact of amino acid substitutions (Ugalde et al 2004;Brayer et al 2011;Kratzer et al 2014).…”

“…This is in part because sequence divergence among primates is characteristically low and it is challenging to differentiate adaptive substitutions from the background of neutral substitutions in such data. A full understanding of the phenotypic significance of primate NR evolution requires explicitly linking formal evolutionary analyses with experimental approaches focused on the functional impact of amino acid substitutions (Ugalde et al 2004;Brayer et al 2011;Kratzer et al 2014).Ancestral gene reconstruction (AGR), when combined with synthesis and assessment of the inferred ancestral protein in the laboratory, provides a powerful framework for investigating the contribution of extinct genetic systems to phenotypic change (Thornton 2001;Thornton et al 2003;Bloch et al 2015). Indeed, AGR has been used effectively to investigate the evolution of function in a wide variety of different proteins Gaucher et al 2003;Ugalde et al 2004;Gaucher et al 2008;Bridgham et al 2009; Thornton 2010, 2013;Eick and Thornton 2011;Brayer et al 2011).…”

Ancient insights into uric acid metabolism in primates

“…Although the idea of resurrecting ancient proteins using sequence information from extant organisms was first proposed half a century ago by Pauling and Zuckerkandl (4), it is only in the last decade or so that advances in phylogenetic likelihood and Bayesian models (5), combined with fast, inexpensive gene synthesis technology, have made this approach increasingly feasible. These methods have since been used with great success to investigate a variety of topics, including the evolution of protein regulatory complexes in developmental pathways (6), vertebrate steroid hormone receptors (7), paleoenvironments and the thermophilicity of early life (8), archosaur vision (9), vaccine development (10), yeast transcriptional circuits following gene duplication (11), and the evolution of complexity in coral fluorescent proteins (12). Evolutionary questions can be notoriously intractable to experimental investigation, but the recreation of ancestral proteins provides us a window to the past through which we can directly test hypotheses regarding our evolutionary history.…”