Despite extensive research, our understanding of the rules according to which cis-regulatory sequences are converted into gene expression is limited. We devised a method for obtaining parallel, highly accurate gene expression measurements from thousands of designed promoters and applied it to measure the effect of systematic changes in the location, number, orientation, affinity and organization of transcription-factor binding sites and nucleosome-disfavoring sequences. Our analyses reveal a clear relationship between expression and binding-site multiplicity, as well as dependencies of expression on the distance between transcription-factor binding sites and gene starts which are transcription-factor specific, including a striking ∼10-bp periodic relationship between gene expression and binding-site location. We show how this approach can measure transcription-factor sequence specificities and the sensitivity of transcription-factor sites to the surrounding sequence context, and compare the activity of 75 yeast transcription factors. Our method can be used to study both cis and trans effects of genotype on transcriptional, post-transcriptional and translational control.
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Gene Expression Omnibus
Chiang, D.Y., Nix, D.A., Shultzaberger, R.K., Gasch, A.P. & Eisen, M.B. Flexible promoter architecture requirements for coactivator recruitment. BMC Mol. Biol. 7, 16 (2006).
Ligr, M., Siddharthan, R., Cross, F.R. & Siggia, E.D. Gene expression from random libraries of yeast promoters. Genetics 172, 2113–2122 (2006).
Kinkhabwala, A. & Guet, C.C. Uncovering cis regulatory codes using synthetic promoter shuffling. PLoS ONE 3, e2030 (2008).
Gertz, J., Siggia, E.D. & Cohen, B.A. Analysis of combinatorial cis-regulation in synthetic and genomic promoters. Nature 457, 215–218 (2009).
Cox, R.S. III., Surette, M.G. & Elowitz, M.B. Programming gene expression with combinatorial promoters. Mol. Syst. Biol. 3, 145 (2007).
Kinney, J.B., Murugan, A., Callan, C.G. Jr. & Cox, E.C. Using deep sequencing to characterize the biophysical mechanism of a transcriptional regulatory sequence. Proc. Natl. Acad. Sci. USA 107, 9158–9163 (2010).
Giniger, E. & Ptashne, M. Cooperative DNA binding of the yeast transcriptional activator GAL4. Proc. Natl. Acad. Sci. USA 85, 382–386 (1988).
Iyer, V. & Struhl, K. Poly(dA:dT), a ubiquitous promoter element that stimulates transcription via its intrinsic DNA structure. EMBO J. 14, 2570–2579 (1995).
Lam, F.H., Steger, D.J. & O'Shea, E.K. Chromatin decouples promoter threshold from dynamic range. Nature 453, 246–250 (2008).
Murphy, K.F., Balazsi, G. & Collins, J.J. Combinatorial promoter design for engineering noisy gene expression. Proc. Natl. Acad. Sci. USA 104, 12726–12731 (2007).
Patwardhan, R.P. et al. High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis. Nat. Biotechnol. 27, 1173–1175 (2009).
Patwardhan, R.P. et al. Massively parallel functional dissection of mammalian enhancers in vivo. Nat. Biotechnol. 30, 265–270 (2012).
Melnikov, A. et al. Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay. Nat. Biotechnol. 30, 271–277 (2012).
LeProust, E.M. et al. Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process. Nucleic Acids Res. 38, 2522–2540 (2010).
Kaplan, N. et al. The DNA-encoded nucleosome organization of a eukaryotic genome. Nature 458, 362–366 (2009).
Baliga, N.S. Promoter analysis by saturation mutagenesis. Biol. Proced. Online 3, 64–69 (2001).
Anderson, J.D. & Widom, J. Poly(dA-dT) promoter elements increase the equilibrium accessibility of nucleosomal DNA target sites. Mol. Cell. Biol. 21, 3830–3839 (2001).
Segal, E. & Widom, J. Poly(dA:dT) tracts: major determinants of nucleosome organization. Curr. Opin. Struct. Biol. 19, 65–71 (2009).
Zeevi, D. et al. Compensation for differences in gene copy number among yeast ribosomal proteins is encoded within their promoters. Genome Res. 21, 2114–2128 (2011).
Badis, G. et al. Diversity and complexity in DNA recognition by transcription factors. Science 324, 1720–1723 (2009).
Ghaemmaghami, S. et al. Global analysis of protein expression in yeast. Nature 425, 737–741 (2003).
Huh, W.K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003).
Zhao, Y. et al. Fine-structure analysis of ribosomal protein gene transcription. Mol. Cell. Biol. 26, 4853–4862 (2006).
Blaiseau, P.L., Lesuisse, E. & Camadro, J.M. Aft2p, a novel iron-regulated transcription activator that modulates, with Aft1p, intracellular iron use and resistance to oxidative stress in yeast. J. Biol. Chem. 276, 34221–34226 (2001).
Lamb, T.M. & Mitchell, A.P. The transcription factor Rim101p governs ion tolerance and cell differentiation by direct repression of the regulatory genes NRG1 and SMP1 in Saccharomyces cerevisiae. Mol. Cell. Biol. 23, 677–686 (2003).
Hanlon, S.E., Rizzo, J.M., Tatomer, D.C., Lieb, J.D. & Buck, M.J. The stress response factors Yap6, Cin5, Phd1, and Skn7 direct targeting of the conserved co-repressor Tup1-Ssn6 in S. cerevisiae. PLoS ONE 6, e19060 (2011).
Canizares, J.V., Pallotti, C., Sainz-Pardo, I., Iranzo, M. & Mormeneo, S. The SRD2 gene is involved in Saccharomyces cerevisiae morphogenesis. Arch. Microbiol. 177, 352–357 (2002).
Akache, B., Wu, K. & Turcotte, B. Phenotypic analysis of genes encoding yeast zinc cluster proteins. Nucleic Acids Res. 29, 2181–2190 (2001).
Woudt, L.P., Smit, A.B., Mager, W.H. & Planta, R.J. Conserved sequence elements upstream of the gene encoding yeast ribosomal protein L25 are involved in transcription activation. EMBO J. 5, 1037–1040 (1986).
Lieb, J.D., Liu, X., Botstein, D. & Brown, P.O. Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association. Nat. Genet. 28, 327–334 (2001).
Nutiu, R. et al. Direct measurement of DNA affinity landscapes on a high-throughput sequencing instrument. Nat. Biotechnol. 29, 659–664 (2011).
Maerkl, S.J. & Quake, S.R. A systems approach to measuring the binding energy landscapes of transcription factors. Science 315, 233–237 (2007).
Bulyk, M.L., Gentalen, E., Lockhart, D.J. & Church, G.M. Quantifying DNA-protein interactions by double-stranded DNA arrays. Nat. Biotechnol. 17, 573–577 (1999).
Raveh-Sadka, T. et al. Manipulating nucleosome disfavoring sequences allows fine-tune regulation of gene expression in yeast. Nat. Genet. (in the press).
Kim, J.H., Polish, J. & Johnston, M. Specificity and regulation of DNA binding by the yeast glucose transporter gene repressor Rgt1. Mol. Cell. Biol. 23, 5208–5216 (2003).
Karolchik, D. et al. The UCSC Genome Browser Database. Nucleic Acids Res. 31, 51–54 (2003).
Zhu, C. et al. High-resolution DNA binding specificity analysis of yeast transcription factors. Genome Res. 19, 556–566 (2009).
Cleary, M.A. et al. Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis. Nat. Methods 1, 241–248 (2004).
Fazekas, A., Steeves, R. & Newmaster, S. Improving sequencing quality from PCR products containing long mononucleotide repeats. Biotechniques 48, 277–285 (2010).
Sheff, M.A. & Thorn, K.S. Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. Yeast 21, 661–670 (2004).
Breslow, D.K. et al. A comprehensive strategy enabling high-resolution functional analysis of the yeast genome. Nat. Methods 5, 711–718 (2008).
Otsuka, C. et al. Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo. Mutat. Res. 502, 53–60 (2002).
Hoaglin, D.C., Mosteller, F. & Tukey, J.W. Understanding Robust and Exploratory Data Anlysis (Wiley, 1983).
We thank J. Widom for assistance and inspiration throughout this project. This work was supported by grants from the European Research Council and the US National Institutes of Health to E. Segal. E. Segal is the incumbent of the Soretta and Henry Shapiro career development chair. We thank S. Lubliner for help with computational analyses. We thank C. Boone (University of Toronto) for kindly giving us the Y8205 strain.
The authors declare no competing financial interests.
About this article
Cite this article
Sharon, E., Kalma, Y., Sharp, A. et al. Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters. Nat Biotechnol 30, 521–530 (2012). https://doi.org/10.1038/nbt.2205
BMC Genomics (2021)
Nature Communications (2021)
Nature Genetics (2021)
Nature Reviews Genetics (2021)
Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems
Nature Communications (2021)