This study reveals that epistasis, or interactions between genetic variants, facilitates the evolution of new functions in proteins, particularly in an ancient transcription factor. Using combinatorial deep mutational scanning (DMS), the researchers characterized the genetic architecture of DNA recognition in a reconstructed ancestral steroid hormone receptor. They found that the genetic architecture of DNA recognition involves a dense set of main and pairwise effects, with higher-order epistasis playing a minor role. Pairwise interactions significantly expand the number of functional sequences and are the primary determinants of specificity for different DNA elements. These interactions also enable single-residue mutations to switch specificity between DNA targets, facilitating the evolution of new functions. The study shows that epistasis enhances rather than constrains evolutionary paths by bringing functionally distinct variants close together in sequence space. This allows for more efficient transitions between different functions. The genetic architecture of the protein's DNA recognition is complex, with many amino acid states and interactions contributing to its function. The results suggest that pairwise interactions are more important than higher-order interactions in determining functional specificity. The study also highlights that the genetic architecture of the protein's DNA recognition has evolved over time, with historical changes in amino acid states leading to the transition from ERE-specific to SRE-specific DNA binding. These changes were made possible by pairwise epistasis, which allowed for the functional activation of previously non-functional variants. The findings indicate that epistasis plays a crucial role in shaping the genetic architecture of proteins and facilitating the evolution of new functions. The study provides a comprehensive understanding of the genetic architecture of protein function and evolution, emphasizing the importance of pairwise interactions in determining functional specificity.This study reveals that epistasis, or interactions between genetic variants, facilitates the evolution of new functions in proteins, particularly in an ancient transcription factor. Using combinatorial deep mutational scanning (DMS), the researchers characterized the genetic architecture of DNA recognition in a reconstructed ancestral steroid hormone receptor. They found that the genetic architecture of DNA recognition involves a dense set of main and pairwise effects, with higher-order epistasis playing a minor role. Pairwise interactions significantly expand the number of functional sequences and are the primary determinants of specificity for different DNA elements. These interactions also enable single-residue mutations to switch specificity between DNA targets, facilitating the evolution of new functions. The study shows that epistasis enhances rather than constrains evolutionary paths by bringing functionally distinct variants close together in sequence space. This allows for more efficient transitions between different functions. The genetic architecture of the protein's DNA recognition is complex, with many amino acid states and interactions contributing to its function. The results suggest that pairwise interactions are more important than higher-order interactions in determining functional specificity. The study also highlights that the genetic architecture of the protein's DNA recognition has evolved over time, with historical changes in amino acid states leading to the transition from ERE-specific to SRE-specific DNA binding. These changes were made possible by pairwise epistasis, which allowed for the functional activation of previously non-functional variants. The findings indicate that epistasis plays a crucial role in shaping the genetic architecture of proteins and facilitating the evolution of new functions. The study provides a comprehensive understanding of the genetic architecture of protein function and evolution, emphasizing the importance of pairwise interactions in determining functional specificity.