Manipulating nucleosome disfavoring sequences allows fine-tune regulation of gene expression in yeast

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Abstract

Understanding how precise control of gene expression is specified within regulatory DNA sequences is a key challenge with far-reaching implications. Many studies have focused on the regulatory role of transcription factor–binding sites. Here, we explore the transcriptional effects of different elements, nucleosome-disfavoring sequences and, specifically, poly(dA:dT) tracts that are highly prevalent in eukaryotic promoters. By measuring promoter activity for a large-scale promoter library, designed with systematic manipulations to the properties and spatial arrangement of poly(dA:dT) tracts, we show that these tracts significantly and causally affect transcription. We show that manipulating these elements offers a general genetic mechanism, applicable to promoters regulated by different transcription factors, for tuning expression in a predictable manner, with resolution that can be even finer than that attained by altering transcription factor sites. Overall, our results advance the understanding of the regulatory code and suggest a potential mechanism by which promoters yielding prespecified expression patterns can be designed.

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Figure 1: Schematic of library design, strain construction and promoter activity measurements.
Figure 2: Poly(dA:dT) tracts significantly affect the transcriptional outcome, likely by altering nucleosome organization.
Figure 3: The transcriptional effects of poly(dA:dT) tracts are evident in promoters regulated by different transcription factors, and its magnitude is inversely proportional to the affinity of the transcription factor site.
Figure 4: The transcriptional effects of poly(dA:dT) tracts depend on their distance from other promoter elements.
Figure 5: Changes to poly(dA:dT) tracts may allow tuning of expression levels with finer resolution than that allowed by changes to the transcription factor site.
Figure 6: A mechanistic model for transcription accounts for many of the transcriptional effects of poly(dA:dT) tracts.
Figure 7: Poly(dA:dT) tracts affect cell-to-cell expression variability.

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Acknowledgements

We wish to dedicate this paper to Jon Widom who inspired and assisted us greatly throughout this project. This work was supported by grants from the European Research Council (ERC) and the US National Institutes of Health (NIH) to E. Segal. E. Segal is the incumbent of the Soretta and Henry Shapiro career development chair. T.R.-S. and M.L. thank the Azrieli Foundation for the award of an Azrieli Fellowship. We thank G. Hornung for his help with the analysis of flow cytometry measurements and E. Mochly for his help with the preparation of Supplementary Figure 9.

Author information

T.R.-S., M.L. and E. Segal conceived the project, designed promoter variants and analyzed the data. T.R.-S., M.L., A.W. and E. Segal planned all experiments. D.Z. and A.W. developed protocols for robotic strain assembly and activity measurements. D.Z., U.S. and M.L.-P. constructed the master strain. U.S. participated in the design of the variants, constructed the majority of variants and, together with M.L.-P., constructed strains. T.R.-S. and M.L. performed expression measurements. B.S. performed nucleosome occupancy measurements. L.K. participated in expression measurements and in their initial analysis. E. Sharon, together with T.R.-S., M.L. and L.K., developed the analysis pipeline. T.R.-S., M.L. and E. Segal wrote the manuscript. A.W. and E. Segal supervised and guided the research.

Correspondence to Adina Weinberger or Eran Segal.

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Supplementary Figures 1–11, Supplementary Tables 1–5 and Supplementary Note (PDF 810 kb)

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Raveh-Sadka, T., Levo, M., Shabi, U. et al. Manipulating nucleosome disfavoring sequences allows fine-tune regulation of gene expression in yeast. Nat Genet 44, 743–750 (2012) doi:10.1038/ng.2305

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