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Intersecting transcription networks constrain gene regulatory evolution

Nature volume 523, pages 361–365 (2015)Cite this article

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Abstract

Epistasis—the non-additive interactions between different genetic loci—constrains evolutionary pathways, blocking some and permitting others1,2,3,4,5,6,7,8. For biological networks such as transcription circuits, the nature of these constraints and their consequences are largely unknown. Here we describe the evolutionary pathways of a transcription network that controls the response to mating pheromone in yeast9. A component of this network, the transcription regulator Ste12, has evolved two different modes of binding to a set of its target genes. In one group of species, Ste12 binds to specific DNA binding sites, while in another lineage it occupies DNA indirectly, relying on a second transcription regulator to recognize DNA. We show, through the construction of various possible evolutionary intermediates, that evolution of the direct mode of DNA binding was not directly accessible to the ancestor. Instead, it was contingent on a lineage-specific change to an overlapping transcription network with a different function, the specification of cell type. These results show that analysing and predicting the evolution of cis-regulatory regions requires an understanding of their positions in overlapping networks, as this placement constrains the available evolutionary pathways.

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Figure 1: Evolution of the pheromone response in budding yeast.
Figure 2: Ste12 is recruited to asg promoters in K. lactis without its binding site.
Figure 3: a2 mediates the a-specific gene pheromone response in K. lactis.
Figure 4: Repression by α2 is necessary for the gain of Ste12 sites.
Figure 5: Summary of key events in the evolution of asg regulation.

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Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

ChIP-Seq data has been deposited at the Gene Expression Omnibus (GEO) repository under accession number GSE65792.

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Acknowledgements

We thank S. Coyle, I. Nocedal, C. Dalal, C. Britton, C. Baker, and V. Hanson-Smith for valuable comments on the manuscript. We also thank K. Pollard, E. Mancera, C. Nobile, and Q. Mitrovich for advice and assistance with experiments and analysis. We thank K. Boundy-Mills of the Phaff Yeast Culture Collection, University of California, Davis for the K. lactis isolates. The work was supported by grant R01 GM037049 from the National Institutes of Health. T.R.S. was supported by a Graduate Research Fellowship from the National Science Foundation.

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Author notes

  1. Lauren N. Booth & Brian B. Tuch

    Present address: †Present addresses: Department of Genetics, Stanford University, Stanford, California 94305, USA (L.N.B.); Loxo Oncology, South San Francisco, California 94080, USA (B.B.T.),

Authors and Affiliations

  1. Department of Biochemistry & Biophysics, Department of Microbiology & Immunology, University of California, San Francisco, 94158, California, USA

    Trevor R. Sorrells, Lauren N. Booth, Brian B. Tuch & Alexander D. Johnson

  2. Tetrad Graduate Program, University of California, San Francisco, 94158, California, USA

    Trevor R. Sorrells, Lauren N. Booth & Alexander D. Johnson

  3. Biological and Medical Informatics Graduate Program, University of California, San Francisco, 94158, California, USA

    Brian B. Tuch

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Contributions

T.R.S. performed gene expression experiments, reporter assays, ChIP-Seq, and bioinformatics analyses. L.N.B. obtained and sequenced the asg promoters of the K. lactis isolates. B.B.T. conceived of, designed, and performed a preliminary analysis of the phylogenetic distribution of Ste12 sites across the asgs. T.R.S., L.N.B., and A.D.J. designed and interpreted experiments and edited the manuscript. T.R.S. and A.D.J. wrote the manuscript.

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Correspondence to Alexander D. Johnson.

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Extended data figures and tables

Extended Data Figure 1 Conservation of Ste12 function.

Expression of the asg STE2 and the general pheromone-activated gene FUS3 in the presence of K. lactis Ste12, in the absence of Ste12, and in the presence of S. cerevisiae Ste12. Expression under uninduced and pheromone-induced conditions are shown as mean fluorescence ±s.d. of three independent genetic isolates, grown and analysed separately.

Extended Data Figure 2 Distribution of Ste12 cis-regulatory sites.

Individual a-specific genes and general pheromone-activated genes were scored for the presence of Ste12 cis-regulatory motifs in their upstream regulatory regions. Shown is the number of consensus Ste12 sites within 600 bp of the translation start site. Orthologues that could not be identified in a given species are shown in grey. The enrichment for the Ste12 motif in each set of genes in each species is shown in Fig. 1c.

Extended Data Figure 3 Distribution of mismatched Ste12 cis-regulatory sites.

asgs were scored for the presence of mismatched Ste12 binding sites in their upstream regulatory regions. The enrichment of the Ste12 site in the asgs in each species is shown in the bottom row.

Extended Data Figure 4 Ste12 sites are required in S. cerevisiae asgs.

The S. cerevisiae STE2 promoter was fused to GFP and the role of Ste12 cis-regulatory sites in expression. Expression under uninduced and pheromone-induced conditions are shown as mean fluorescence ±s.d. of three independent genetic isolates, grown and analysed separately. Symbols are described in Fig. 2d.

Extended Data Figure 5 Mcm1 sites are required in K. lactis asgs.

The K. lactis STE2 GFP reporter was tested with a mutated Mcm1 cis-regulatory site. Shown is mean fluorescence ±s.d. of three independent genetic isolates, grown and analysed separately. Symbols are described in Fig. 3d.

Extended Data Figure 6 a2 sites are sufficient for pheromone activation.

The K. lactis STE2 GFP reporter and heterologous reporter containing the a2-Mcm1 binding site were transformed into wild type and ste12Δ cells. Shown is mean fluorescence ±s.d. of three independent genetic isolates, grown and analysed separately.

Extended Data Figure 7 Mcm1 sites do not compensate for the loss of a2.

a, The presence of the Mcm1 binding site is unchanged across the Candida, Kluyveromyces, and Saccharomyces clades22,24. asgs were scored for the strength of the Mcm1 binding site in their upstream regulatory regions. The enrichment of the strength of the Mcm1 site in the asgs in each species is shown in the bottom row. b, A construct with increased Mcm1 binding site strength, marked with an ‘S’, was tested for its ability to compensate for the deletion of a2. Shown is the mean fluorescence ±s.d. of three independent genetic isolates, grown and analysed separately.

Extended Data Figure 8 Loss of Ste12 sites decreased in the Saccharomyces clade.

The yeast phylogeny along with the number of Ste12 binding sites in extant species was used to estimate the gain and loss rate of the sites over evolutionary time. The Saccharomyces clade was allowed to have different rates (orange) (LRT, X2 = 49.33, df = 2, P = 1.94 × 10−11). The gain and loss rate units are cis-regulatory sites per amino acid substitution in the species phylogeny.

Extended Data Figure 9 Saccharomyces Ste12 binding sites evolve under purifying selection.

The evolutionary rate of nucleotides in Ste12 binding sites was compared to rates in the rest of the upstream regulatory regions of the a-specific genes, shown as violin plots. Promoters between closely related species were aligned and the evolutionary rate of each base pair in the alignment was determined after model selection.

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Sorrells, T., Booth, L., Tuch, B. et al. Intersecting transcription networks constrain gene regulatory evolution. Nature 523, 361–365 (2015). https://doi.org/10.1038/nature14613

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