Large-scale mapping of gene regulatory logic reveals context-dependent repression by transcriptional activators

Transcription factors (TFs) are key mediators that propagate extracellular and intracellular signals through to changes in gene expression profiles. However, the rules by which promoters decode the amount of active TF into target gene expression are not well understood. To determine the mapping between promoter DNA sequence, TF concentration, and gene expression output, we have conducted in budding yeast a large-scale measurement of the activity of thousands of designed promoters at six different levels of TF. We observe that maximum promoter activity is determined by TF concentration and not by the number of binding sites. Surprisingly, the addition of an activator site often reduces expression. A thermodynamic model that incorporates competition between neighboring binding sites for a local pool of TF molecules explains this behavior and accurately predicts both absolute expression and the amount by which addition of a site increases or reduces expression. Taken together, our findings support a model in which neighboring binding sites interact competitively when TF is limiting but otherwise act additively.This work was supported by the Spanish Ministerio de Economía y Competitividad and FEDER through project BFU2015-68351-P to L.B.C. and by grant 2014SGR0974 from the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) to L.B.C. This work was supported by grants from the European Research Council (ERC) and the US National Institutes of Health (NIH) to E.S. D.vD. was supported by Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) Rubicon fellowship 825.14.016

Large-scale mapping of gene regulatory logic reveals context-dependent repression by transcriptional activators

Transcription factors (TFs) are key mediators that propagate extracellular and intracellular signals through to changes in gene expression profiles. However, the rules by which promoters decode the amount of active TF into target gene expression are not well understood. To determine the mapping between promoter DNA sequence, TF concentration, and gene expression output, we have conducted in budding yeast a large-scale measurement of the activity of thousands of designed promoters at six different levels of TF. We observe that maximum promoter activity is determined by TF concentration and not by the number of binding sites. Surprisingly, the addition of an activator site often reduces expression. A thermodynamic model that incorporates competition between neighboring binding sites for a local pool of TF molecules explains this behavior and accurately predicts both absolute expression and the amount by which addition of a site increases or reduces expression. Taken together, our findings support a model in which neighboring binding sites interact competitively when TF is limiting but otherwise act additively.This work was supported by the Spanish Ministerio de Economía y Competitividad and FEDER through project BFU2015-68351-P to L.B.C. and by grant 2014SGR0974 from the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) to L.B.C. This work was supported by grants from the European Research Council (ERC) and the US National Institutes of Health (NIH) to E.S. D.vD. was supported by Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) Rubicon fellowship 825.14.016

Measurements of the impact of 3' end sequences on gene expression reveal wide range and sequence dependent effects

A full understanding of gene regulation requires an understanding of the contributions that the various regulatory regions have on gene expression. Although it is well established that sequences downstream of the main promoter can affect expression, our understanding of the scale of this effect and how it is encoded in the DNA is limited. Here, to measure the effect of native S. cerevisiae 3′ end sequences on expression, we constructed a library of 85 fluorescent reporter strains that differ only in their 3′ end region. Notably, despite being driven by the same strong promoter, our library spans a continuous twelve-fold range of expression values. These measurements correlate with endogenous mRNA levels, suggesting that the 3′ end contributes to constitutive differences in mRNA levels. We used deep sequencing to map the 3′UTR ends of our strains and show that determination of polyadenylation sites is intrinsic to the local 3′ end sequence. Polyadenylation mapping was followed by sequence analysis, we found that increased A/T content upstream of the main polyadenylation site correlates with higher expression, both in the library and genome-wide, suggesting that native genes differ by the encoded efficiency of 3′ end processing. Finally, we use single cells fluorescence measurements, in different promoter activation levels, to show that 3′ end sequences modulate protein expression dynamics differently than promoters, by predominantly affecting the size of protein production bursts as opposed to the frequency at which these bursts occur. Altogether, our results lead to a more complete understanding of gene regulation by demonstrating that 3′ end regions have a unique and sequence dependent effect on gene expressionThis work was supported by the ‘Ideas’ program of the European Research Council and the Ben May Charitable Trust