Lynch, V., May, G., & Wagner, G. (2011). Regulatory evolution through divergence of a phosphoswitch in the transcription factor CEBPB Nature, 480 (7377), 383-386 DOI: 10.1038/nature10595
This paper demonstrates that change in the response of transcription factors to signalling pathways is an important mechanism in the evolution of novel developmental functionalities.
During mammalian pregnancy, the expression of Prolactin (PRL) in endometrial stromal cells (ESCs) is activated by an interaction of the transcription factors CEBPB (CCAAT/ enhancer binding protein b) and FOXO1A. Both of these transcription factors are present in non-mammalian animals, so to test whether this function evolved coincidentally with the origin of pregnancy, the investigators designed a biochemical assay in which activation of an enhancer for ESC Prolactin expression could be measured by expression of a luciferase reporter gene. Expressing human CEBPB and FOXO1A together transactivated the expression of luciferase. However, Coexpression of the two proteins from chicken or from opossum (a marsupial) failed to cooperatively transactivate expression from the ESC PRL enhancer. To test the hypothesis that this cooperative interaction evolved in placental mammals (Eutheria) they phylogenetically reconstructed the putative ancestral sequences of CEBPB and FOXO1A from the common ancestor of both Theria (all mammals excluding the egg bearing monotremes) (AncTheria) and placental mammals (AncEutheria), synthesised them and tested their ability to activate ESC PRL expression in the assay. As predicted the AncEutherian CEBPB/FOXO1A complex strongly transactivated tuciferase expression, whilst the AncTherian complex did not.
Having ruled out change in FOXO1A being responsible for this derived cooperative function, the researchers set out to determine which domains of CEBPB are responsible by making truncated forms of the gene and assaying their transactivation ability (in the presence of FOXO1A). The deletion of an internal regulatory domain (RD2) reduced the AncEutherian protein’s transactivation ability to that of the AncTherian CEBPB. Having shown that RD2 plays a critical role in PRL regulation, they then tested the roles of five eutherian specific amino acid substitutions in this region. Back mutations from the AncEutherian CEBPB and forward mutations from the AncTherian CEBPB showed amino acid substitutions at sites 3 and 4 contributed to the derived regulatory ability but that they are dependent on the substitution at site 5. Upon mapping of predicted sites for phosphorylation of CEBPB it was clear that the three amino acid substitutions corresponded to AncEutherian CEBPB having lost serine phosphosites at positions 3 and 4 and having gained one at site 5. To test the importance of phosphorylation (by the kinase GSK3b) in potentiating transactivation by CEBPB they repeated their experiments in the presence of a phosphorylation inhibitor. In accordance with their predictions, inhibition of phosphorylation prevented AncEutherian CEBPB mediated transactivation. Interestingly however, it potentiated transactivation by AncTherian CEBPB. This shows that AncEutherian CEBPB is activated by GSK3b whereas AncTherian CEBPB is repressed by it. These different responses to GSK3b are mediated by the differences in phosphorylation of the three amino acid positions in RD2.
The researchers went on to use protein structure modelling simulations to infer the consequences of repositioning phosphorylation sites in RD2. Unphosphorylated AncEutherian CEBPB is predicted to be collapsed into a knot-like bundle in which the various domains are in contact with each other – an intrinsically repressed conformation. Phosphorylation of the AncEutherian protein is predicted to give rise to an open conformation in which the domains don’t contact each other, freeing the DNA binding domain from an intramolecular masking effect. The AncTherian CEBPB model however shows the transcriptionally inactive conformation.
The importance of this paper is that it shows changing the response of transcription factors to signalling pathways can be an important mechanism of genetic regulatory evolution. It is known that the interactions of cis-regulatory elements and transcription factors are the main substrate on which evolution acts to change developmental processes. However, more emphasis has been put on changes to cis-regulatory elements. This is partly due to their amenability, but also probably partly due to the pleiotropic expression of transcription factors and other developmentally important molecules, ie they are expressed in lots of different places but have different developmental roles. The thinking would be that it is easier to evolve novel enhancers, that in combination with other cis-elements integrate a transcription factor code to drive differential gene expression, whereas evolution of transcription factors themselves would be more likely to affect a large amount of developmental processes. This work shows how evolution can affect transcription factor function in a cell type specific way without potentially deleterious pleiotropic effects. The other important aspect of this paper is that it shows how experimental evolutionary developmental biology can be done. Reconstruction of ancestral genes and testing their function yielded insights into CEBPB regulation and the evolution of mammalian pregnancy. This type of reconstructive biology is open to criticism as it will always have a speculative and ultimately unknowable aspect to it. However if evolutionary developmental biology is to be anything more than molecular comparative anatomy this more experimental approach is the way to go.