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

A Corrigendum to this article was published on 16 April 2014

A Corrigendum to this article was published on 16 April 2014

This article has been updated


There is an emerging consensus that gene regulation evolves through changes in cis-regulatory elements1,2 and transcription factors3,4,5,6. 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 regulation4,5,6,7,8,9,10. 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.

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Figure 1: CEBPB cooperatively regulates gene expression with FOXO1A in placental mammals.
Figure 2: CEBPB evolved a novel GSK-3B phosphorylation site in an internal regulatory domain.
Figure 3: Phosphorylation induces a conformation change in CEBPB.

Change history

  • 16 April 2014

    Nature 480, 383–386 (2011); doi:10.1038/nature10595 We inadvertently included a duplicate gel image in the lower right panel of Fig. 3c of this Letter. The gel image should show the loading control (‘Input’) for the image directly above it (lanes pHsa and dHsa) but instead is a duplicate of the image shown in the upper left panel of Fig.


  1. 1

    Stern, D. L. Evolutionary developmental biology and the problem of variation. Evolution 54, 1079–1091 (2000)

    CAS  Article  Google Scholar 

  2. 2

    Carroll, S. B. Evolution at two levels: on genes and form. PLoS Biol. 3, e245 (2005)

    Article  Google Scholar 

  3. 3

    Hsia, C. C. & McGinnis, W. Evolution of transcription factor function. Curr. Opin. Genet. Dev. 13, 199–206 (2003)

    CAS  Article  Google Scholar 

  4. 4

    Lynch, V. J. & Wagner, G. P. Resurrecting the role of transcription factor change in developmental evolution. Evolution 62, 2131–2154 (2008)

    CAS  Article  Google Scholar 

  5. 5

    Wagner, G. P. & Lynch, V. J. Molecular evolution of evolutionary novelties: the vagina and uterus of therian mammals. J. Exp. Zool. B 304, 580–592 (2005)

    Article  Google Scholar 

  6. 6

    Wagner, G. P. & Lynch, V. J. Evolutionary novelties. Curr. Biol. 20, R48–R52 (2010)

    CAS  Article  Google Scholar 

  7. 7

    Lynch, V. J. et al. Adaptive changes in the transcription factor HoxA-11 are essential for the evolution of pregnancy in mammals. Proc. Natl Acad. Sci. USA 105, 14928–14933 (2008)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Galant, R. & Carroll, S. B. Evolution of a transcriptional repression domain in an insect Hox protein. Nature 415, 910–913 (2002)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Ronshaugen, M., McGinnis, N. & McGinnis, W. Hox protein mutation and macroevolution of the insect body plan. Nature 415, 914–917 (2002)

    ADS  Article  Google Scholar 

  10. 10

    Löhr, U., Yussa, M. & Pick, L. Drosophila fushi tarazu: a gene on the border of homeotic function. Curr. Biol. 11, 1403–1412 (2001)

    Article  Google Scholar 

  11. 11

    Chen, L. et al. Mouse and zebrafish Hoxa3 orthologues have nonequivalent in vivo protein function. Proc. Natl Acad. Sci. USA 107, 10555–10560 (2010)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Carroll, S. B. Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution. Cell 134, 25–36 (2008)

    CAS  Article  Google Scholar 

  13. 13

    Prud’homme, B., Gompel, N. & Carroll, S. B. Emerging principles of regulatory evolution. Proc. Natl Acad. Sci. USA 104, 8605–8612 (2007)

    ADS  Article  Google Scholar 

  14. 14

    Wray, G. A. The evolutionary significance of cis-regulatory mutations. Nature Rev. Genet. 8, 206–216 (2007)

    CAS  Article  Google Scholar 

  15. 15

    Stern, D. L. & Orgogozo, V. The loci of evolution: how predictable is genetic evolution? Evolution 62, 2155–2177 (2008)

    Article  Google Scholar 

  16. 16

    Shen, F. et al. IL-17 receptor signaling inhibits C/EBPβ by sequential phosphorylation of the regulatory 2 domain. Sci. Signal. 2, ra8 (2009)

    Article  Google Scholar 

  17. 17

    Spooner, C. J., Guo, X., Johnson, P. F. & Schwartz, R. C. Differential roles of C/EBPβ regulatory domains in specifying MCP-1 and IL-6 transcription. Mol. Immunol. 44, 1384–1392 (2007)

    CAS  Article  Google Scholar 

  18. 18

    Friedman, J. R. et al. Orthogonal analysis of C/EBPβ targets in vivo during liver proliferation. Proc. Natl Acad. Sci. USA 101, 12986–12991 (2004)

    ADS  CAS  Article  Google Scholar 

  19. 19

    Christian, M., Pohnke, Y., Kempf, R., Gellersen, B. & Brosens, J. J. Functional association of PR and CCAAT/enhancer-binding proteinβ 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. Mol. Endocrinol. 16, 141–154 (2002)

    CAS  PubMed  Google Scholar 

  20. 20

    Christian, M. et al. Cyclic AMP-induced forkhead transcription factor, FKHR, cooperates with CCAAT/enhancer-binding protein β in differentiating human endometrial stromal cells. J. Biol. Chem. 277, 20825–20832 (2002)

    CAS  Article  Google Scholar 

  21. 21

    Pohnke, Y., Kempf, R. & Gellersen, B. CCAAT/enhancer-binding proteins are mediators in the protein kinase A-dependent activation of the decidual prolactin promoter. J. Biol. Chem. 274, 24808–24818 (1999)

    CAS  Article  Google Scholar 

  22. 22

    Williams, S. C., Baer, M., Dilliner, A. J. & Johnson, P. F. CRP2 (C/EBPβ) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity. EMBO J. 14, 3170–3183 (1995)

    CAS  Article  Google Scholar 

  23. 23

    Kowenz-Leutz, E., Twamley, G., Ansieau, S. & Leutz, A. Novel mechanism of C/EBPβ (NF-M) transcriptional control: activation through derepression. Genes Dev. 8, 2781–2791 (1994)

    CAS  Article  Google Scholar 

  24. 24

    Lee, S., Miller, M., Shuman, J. D. & Johnson, P. F. CCAAT/enhancer-binding protein β DNA binding is auto-inhibited by multiple elements that also mediate association with p300/CREB-binding protein (CBP). J. Biol. Chem. 285, 21399–21410 (2010)

    CAS  Article  Google Scholar 

  25. 25

    Kowenz-Leutz, E., Pless, O., Dittmar, G., Knoblich, M. & Leutz, A. Crosstalk between C/EBPβ phosphorylation, arginine methylation, and SWI/SNF/mediator implies an indexing transcription factor code. EMBO J. 29, 1105–1115 (2010)

    CAS  Article  Google Scholar 

  26. 26

    Metz, R. & Ziff, E. cAMP stimulates the C/EBP-related transcription factor rNFIL-6 to trans-locate to the nucleus and induce c-fos transcription. Genes Dev. 5, 1754–1766 (1991)

    CAS  Article  Google Scholar 

  27. 27

    Zhao, X., Zhuang, S., Chen, Y., Boss, G. R. & Pilz, R. B. Cyclic GMP-dependent protein kinase regulates CCAAT enhancer-binding protein β functions through inhibition of glycogen synthase kinase-3. J. Biol. Chem. 280, 32683–32692 (2005)

    CAS  Article  Google Scholar 

  28. 28

    Trautwein, C., Walker, D. L., Plümpe, J. & Manns, M. P. Transactivation of LAP/NF-IL6 is mediated by an acidic domain in the N-terminal part of the protein. J. Biol. Chem. 270, 15130–15136 (1995)

    CAS  Article  Google Scholar 

  29. 29

    Kim, D. E., Chivian, D. & Baker, D. Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res. 32, W526–W531 (2004)

    CAS  Article  Google Scholar 

  30. 30

    Tsvetkov, P. et al. Operational definition of intrinsically unstructured protein sequences based on susceptibility to the 20S proteasome. Proteins Struct. Funct. Bioinf. 70, 1357–1366 (2008)

    CAS  Article  Google Scholar 

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This work was funded by a grant from the John Templeton Foundation, number 12793 Genetics and the Origin of Organismal Complexity; results presented here do not necessarily reflect the views of the John Templeton Foundation. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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V.J.L. and G.P.W. designed experiments and wrote the manuscript. V.J.L. performed experiments and analysed data. G.M. cloned and sequenced CEBPB genes from non-mammalian species.

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Correspondence to Vincent J. Lynch.

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The authors declare no competing financial interests.

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Lynch, V., May, G. & Wagner, G. Regulatory evolution through divergence of a phosphoswitch in the transcription factor CEBPB. Nature 480, 383–386 (2011).

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