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

There is an emerging consensus that gene regulation evolves through changes in cis-regulatory elements and transcription factors. 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. 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.

[1]  M. Baer,et al.  CRP2 (C/EBP beta) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity. , 1995, The EMBO journal.

[2]  G. Wagner,et al.  Molecular evolution of evolutionary novelties: the vagina and uterus of therian mammals. , 2005, Journal of experimental zoology. Part B, Molecular and developmental evolution.

[3]  S. Carroll,et al.  Evolution at Two Levels: On Genes and Form , 2005, PLoS biology.

[4]  Vincent J. Lynch,et al.  Resurrecting the Role of Transcription Factor Change in Developmental Evolution , 2008, Evolution; international journal of organic evolution.

[5]  S. Carroll,et al.  Emerging principles of regulatory evolution , 2007, Proceedings of the National Academy of Sciences.

[6]  C. Amemiya,et al.  Mouse and zebrafish Hoxa3 orthologues have nonequivalent in vivo protein function , 2010, Proceedings of the National Academy of Sciences.

[7]  D. Stern.  PERSPECTIVE: EVOLUTIONARY DEVELOPMENTAL BIOLOGY AND THE PROBLEM OF VARIATION , 2000, Evolution; international journal of organic evolution.

[8]  G. Wray.  The evolutionary significance of cis-regulatory mutations , 2007, Nature Reviews Genetics.

[9]  J. Brosens,et al.  Cyclic AMP-induced Forkhead Transcription Factor, FKHR, Cooperates with CCAAT/Enhancer-binding Protein β in Differentiating Human Endometrial Stromal Cells* , 2002, The Journal of Biological Chemistry.

[10]  Joel L Sussman,et al.  Operational definition of intrinsically unstructured protein sequences based on susceptibility to the 20S proteasome , 2007, Proteins.

[11]  M. Manns,et al.  Transactivation of LAP/NF-IL6 Is Mediated by an Acidic Domain in the N-terminal Part of the Protein (*) , 1995, The Journal of Biological Chemistry.

[12]  J. Brosens,et al.  Functional association of PR and CCAAT/enhancer-binding protein beta isoforms: promoter-dependent cooperation between PR-B and liver-enriched inhibitory protein, or liver-enriched activatory protein and PR-A in human endometrial stromal cells. , 2002, Molecular endocrinology.

[13]  L. Pick,et al.  Drosophila fushi tarazu a gene on the border of homeotic function , 2001, Current Biology.

[14]  S. Carroll.  Evo-Devo and an Expanding Evolutionary Synthesis: A Genetic Theory of Morphological Evolution , 2008, Cell.

[15]  Phillip P. Le,et al.  Orthogonal analysis of C/EBPbeta targets in vivo during liver proliferation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Sean B. Carroll,et al.  Evolution of a transcriptional repression domain in an insect Hox protein , 2002, Nature.

[17]  Yongchang Chen,et al.  Cyclic GMP-dependent Protein Kinase Regulates CCAAT Enhancer-binding Protein β Functions through Inhibition of Glycogen Synthase Kinase-3* , 2005, Journal of Biological Chemistry.

[18]  Vincent J. Lynch,et al.  Adaptive changes in the transcription factor HoxA-11 are essential for the evolution of pregnancy in mammals , 2008, Proceedings of the National Academy of Sciences.

[19]  Günter P. Wagner,et al.  Evolutionary novelties , 2010, Current Biology.

[20]  Peter F. Johnson,et al.  Differential roles of C/EBP beta regulatory domains in specifying MCP-1 and IL-6 transcription. , 2007, Molecular immunology.

[21]  William McGinnis,et al.  Evolution of transcription factor function. , 2003, Current opinion in genetics & development.

[22]  William McGinnis,et al.  Hox protein mutation and macroevolution of the insect body plan , 2002, Nature.

[23]  A. Leutz,et al.  Novel mechanism of C/EBP beta (NF-M) transcriptional control: activation through derepression. , 1994, Genes & development.

[24]  Maria Miller,et al.  CCAAT/Enhancer-binding Protein β DNA Binding Is Auto-inhibited by Multiple Elements That Also Mediate Association with p300/CREB-binding Protein (CBP)*♦ , 2010, The Journal of Biological Chemistry.

[25]  B. Gellersen,et al.  CCAAT/Enhancer-binding Proteins Are Mediators in the Protein Kinase A-dependent Activation of the Decidual Prolactin Promoter* , 1999, The Journal of Biological Chemistry.

[26]  David Baker,et al.  Protein structure prediction and analysis using the Robetta server , 2004, Nucleic Acids Res..

[27]  David L Stern,et al.  The Loci of Evolution: How Predictable is Genetic Evolution? , 2008, Evolution; international journal of organic evolution.

[28]  E. Ziff,et al.  cAMP stimulates the C/EBP-related transcription factor rNFIL-6 to trans-locate to the nucleus and induce c-fos transcription. , 1991, Genes & development.

[29]  B. Doble,et al.  IL-17 Receptor Signaling Inhibits C/EBPβ by Sequential Phosphorylation of the Regulatory 2 Domain , 2009, Science Signaling.

[30]  G. Dittmar,et al.  Crosstalk between C/EBPb phosphorylation, arginine methylation, and SWI/SNF/Mediator implies an indexing transcription factor code This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution,andreproductioninanymedium,providedthe , 2010 .