Regulatory evolution through divergence of a phosphoswitch in the transcription factor CEBPB

Evolution of gene regulation Changes in cis -regulatory regions of the genome play an important part in the evolution of gene regulation. Lynch et al . explore the influence of amino acid substitutions in transcription factors on gene regulation. They show how, over evolutionary time, amino acid substitutions in the transcription factor CEBPB in the stem-lineage of placental mammals reorganize the location of key phosphorylation sites, thereby changing the way it responds to cAMP/PKA signalling. There is an emerging consensus that gene regulation evolves through changes in cis -regulatory elements 1 , 2 and transcription factors 3 , 4 , 5 , 6 . Although it is clear how nucleotide substitutions in cis -regulatory elements affect gene expression, it is not clear how amino-acid substitutions in transcription factors influence gene regulation 4 , 5 , 6 , 7 , 8 , 9 , 10 . Here we show that amino-acid changes in the transcription factor CCAAT/enhancer binding protein-β (CEBPB, also known as C/EBP-β) in the stem-lineage of placental mammals changed the way it responds to cyclic AMP/protein kinase A (cAMP/PKA) signalling. By functionally analysing resurrected ancestral proteins, we identify three amino-acid substitutions in an internal regulatory domain of CEBPB that are responsible for the novel function. These amino-acid substitutions reorganize the location of key phosphorylation sites, introducing a new site and removing two ancestral sites, reversing the response of CEBPB to GSK-3β-mediated phosphorylation from repression to activation. We conclude that changing the response of transcription factors to signalling pathways can be an important mechanism of gene regulatory evolution.