Mapping the fine structure of a eukaryotic promoter input-output function

Journal name:
Nature Genetics
Volume:
45,
Pages:
1207–1215
Year published:
DOI:
doi:10.1038/ng.2729
Received
Accepted
Published online

Abstract

The precise tuning of gene expression levels is essential for the optimal performance of transcriptional regulatory networks. We created 209 variants of the Saccharomyces cerevisiae PHO5 promoter to quantify how different binding sites for the transcription factor Pho4 affect its output. We found that transcription-factor binding affinities determined in vitro could quantitatively predict the output of a complex yeast promoter. Promoter output was precisely tunable by subtle changes in binding-site affinity of less than 3 kcal mol−1, which are accessible by modifying 1–2 bases. Our results provide insights into how transcription-factor binding sites regulate gene expression, their possible evolution and how they can be used to precisely tune gene expression. More generally, we show that in vitro binding-energy landscapes of transcription factors can precisely predict the output of a native yeast promoter, indicating that quantitative models of transcriptional regulatory networks are feasible.

At a glance

Figures

  1. Design and assembly of the promoter library.
    Figure 1: Design and assembly of the promoter library.

    (a) Schematic of the PHO5 promoter illustrating nucleosome positioning, Pho4 and Pho2 sites. (b) Components of the synthetic promoter library. Each Pho4 site consists of an E-box (red or pink) and two flanking bases (green) on either side. The 209 promoter variants consist of Pho4 binding-site variants, ablations of Pho4 or Pho2 sites. (c) Assembly of synthetic promoters from component oligonucleotides. The promoter is broken into constant arms that flank sequences containing the modified sites, ligated, amplified and transformed into yeast at the LYS2 locus. The synthetic promoters regulate expression of mCherry (orange). The components of the synthetic promoters are shown to scale with respect to the full-size PHO5 promoter.

  2. On-chip induction, imaging and analysis.
    Figure 2: On-chip induction, imaging and analysis.

    (a) Layout of the microfluidic chemostat. (b) Promoter strains are spotted on a glass slide, which is aligned to the device. Strains are then grown and imaged in individual growth chambers. The imaged area is indicated, with a representative image shown below. The flow of the medium is from left to right (red arrows), with nutrients diffusing into each chamber. A sieve channel at the bottom prevents cells from escaping while allowing the perfusion of nutrients. The chamber image had its contrast enhanced for better visibility. (c) Single-cell fluorescence images of a promoter strain grown on the device (mCherry) and a strain with mCherry-tagged Pho4 (Pho4). Traces for the entire imaging period are shown on the right, with each curve showing the average fluorescence or fraction of nuclear mCherry of 200–300 individual cells. Scale bars, 15 μm. (d,e) Fewer cells relocate Pho4 to the nucleus as [Pi] increases (d), but localization kinetics are independent of [Pi] (e). (f) mCherry transcription and translation are modeled within the first 6 h of induction, indicated by the two dashed lines (α, basal transcription rate; σ, activated transcription rate; ϕPho4, fraction of nuclear Pho4; m, mRNA; P, nascent protein; pm, mature fluorescent protein; β, protein synthesis rate; γ, degradation rates; δt, dilution rate). (g) The model accurately fits mCherry expression for strong and weak promoters.

  3. Induction traces for each promoter in the library under Pi starvation.
    Figure 3: Induction traces for each promoter in the library under Pi starvation.

    t = 0 corresponds to the start of Pi starvation. The synthetic promoters allow expression well below and above that of the wild-type PHO5 promoter (red diamonds). Each trace is the average of 2–6 measurements.

  4. Single Pho4 site variants.
    Figure 4: Single Pho4 site variants.

    Modifications to the exposed and nucleosomal Pho4 binding sites are highlighted in red, and the site-specific sequence context is shown. (af) The effects of modifying the E-box (a,b), left-flanking bases (c,d) and both pairs of flanking bases (e,f) on promoter activity are given as transcription rates and raw induction traces. Wild-type Pho4 sites are labeled with an asterisk. Transcription rate data are presented as the mean ± s.d. of 2–6 measurements per promoter, and traces for the wild-type promoter are highlighted as in Figure 3.

  5. Effects of split sites, dual site modifications and ablations.
    Figure 5: Effects of split sites, dual site modifications and ablations.

    (a) Split-exposed Pho4 binding sites. Promoters with secondary low-affinity binding sites are indicated with asterisks. The trace of the wild-type promoter is plotted as in Figure 3. (b) Fifteen promoters with pairs of simultaneously modified nucleosomal and exposed sites. Promoters marked with a hash contain reconstituted but shifted wild-type Pho4 sites. (c) The promoter library included a subset of promoters whose Pho4 (pink or red) and Pho2 (blue) sites were systematically ablated, and their induction was measured. Transcription rates (middle) and fluorescence (right) decreased considerably with the loss of all Pho2 and Pho4 sites. Nucl, nucleosomal; exp, exposed.

  6. Correlation between in vitro binding-site affinity and in vivo promoter output.
    Figure 6: Correlation between in vitro binding-site affinity and in vivo promoter output.

    (a) Measured transcription rates correlate well with the affinity of the modified site calculated directly from the in vitro binding energy (R2 = 0.826; Supplementary Fig. 9). (b) Calculating promoter occupancy (Pbound) using binding-site affinity, Cbf1 competition, nucleosome remodeling and transcription-factor competition quantitatively predicts in vivo promoter output. The wild-type promoter is marked with an asterisk. (c) One or two mutations to either site, or a single mutation to both sites, is sufficient to access the entire dynamic range of the promoter. Occupancies based on every single- and double-base mutation to the nucleosomal (nuc) and/or exposed (exp) sites were calculated, and the transcription rate was predicted. WT, wild type.

  7. Phosphate-dependent activation of the PHO5 promoter.
    Figure 7: Phosphate-dependent activation of the PHO5 promoter.

    (a) Histograms of single-cell fluorescence over time reveal its broadening and bifurcation as [Pi] is increased. (b) Side-by-side comparison of microscopic images of the corresponding chamber used in a and a representative Pho4 localization at the end of induction. Scale bars, 15 μm. The red dashed lines indicate the measured position of the fluorescent front. The lower edge of the images corresponds to the location of the sieve channels at the bottom of each chamber. (c) mCherry profiles across the chamber for the same promoter and [Pi] as in a and b. The boxes indicate the mean fluorescence for 5-μm intervals ± 1 s.d. The dashed lines indicate the position of the front, as in b.

  8. Promoter dynamic range and induction threshold.
    Figure 8: Promoter dynamic range and induction threshold.

    (a) Plotting promoter output for a subset of the library at various [Pi] shows that the synthetic promoters decouple activation threshold from dynamic range for a certain range of output levels. No output is observed at intermediate [Pi] for promoters with a sufficiently low output under full induction, whereas promoters with high-affinity binding sites and correspondingly high output under full induction induced appreciably at intermediate [Pi]. The dotted lines indicate the wild-type promoter, and the dashed line is set at 200 AU. (b) Induction ratio (full induction output/output at intermediate [Pi]) as a function of full induction. Induction ratio plots are colored by exposed (E) or nucleosomal (N) binding-site affinity. The dotted and dashed lines are the same as those in a.

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Author information

Affiliations

  1. Institute of Bioengineering, School of Engineering, Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland.

    • Arun S Rajkumar,
    • Nicolas Dénervaud &
    • Sebastian J Maerkl

Contributions

S.J.M. and A.S.R. designed the library constructs. A.S.R. synthesized the promoter libraries, performed nucleosome occupancy measurements and characterized the promoter library in bulk. N.D. carried out the on-chip experiments. N.D. and S.J.M. designed and implemented the transcription model. A.S.R. and S.J.M. analyzed the data. A.S.R. and S.J.M. wrote the manuscript.

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The authors declare no competing financial interests.

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