© 2012 Nature America, Inc. All rights reserved. NATURE GENETICS ADVANCE ONLINE PUBLICATION 1 ARTICLES Precise regulation of expression is vital to most biological functions. A key challenge is to unravel how this regulation is encoded within genomes and to be able to understand, predict and design expres- sion patterns from regulatory sequences. Addressing this challenge requires knowledge of both the functional elements within regulatory sequences and the ways in which such elements combine to specify regulatory programs. Much of the research in the field has focused on the role of transcription factors and their binding sites 1 . Despite much progress, our ability to understand transcriptional regulation using only transcription factors remains limited. Recent studies suggested that nucleosome organization of regulatory regions is essential for bridging this gap. Because transcription factor–binding sites that are wrapped into nucleosomes are less accessible to binding by their cog- nate factor compared to sites located in nucleosome-free regions 2,3 , we expect that the transcriptional output directed by the same site will depend on its surrounding nucleosome organization 4–7 . A strong determinant of nucleosome organization is the presence of homopolymeric stretches of deoxyadenosine nucleotides, referred to as poly(dA:dT) tracts. These sequence elements disfavor nucleosome formation 8,9 and are strongly associated with nucleosome depletion over the tract itself and its surrounding DNA in both in vivo 10,11 and in vitro 10,12 genome-wide studies. Notably, these tracts are highly abundant in eukaryotic genomes 13 and are particularly prevalent in promoters 14 . Consistent with these observations and with a role for nucleosome organization in determining transcriptional output, a study that altered the presence and length of a native poly(dA:dT) element in one yeast promoter showed that this tract can indeed stimulate expression, most likely by conferring increased accessibility to the nearby transcription factor–binding site 15 . Thus, sequences that strongly disfavor nucleosome formation may also serve as important promoter building blocks. Despite this potentially important regulatory role of poly(dA:dT) tracts, very little is known about the extent and nature of their tran- scriptional effect. What is the magnitude of their effect compared to that of other regulatory elements? How does their effect depend on their own sequence properties and on properties of other regulatory elements such as the affinity of nearby transcription factor sites? And how does the overall arrangement of these tracts and other regulatory elements determine the transcriptional output? Here, we systematically address the above questions by measur- ing the activities of 70 different promoter variants that we designed with poly(dA:dT) tracts that differ in their length, composition and distance from several distinct transcription factor sites. We further characterize the transcriptional effect of poly(dA:dT) tracts by nucleo- some occupancy and single-cell expression measurements. Notably, we show that, by manipulating only poly(dA:dT) tracts, we can affect nucleosome organization and predictably alter the resulting tran- scriptional level to a significant extent, comparable to that attained by altering transcription factor sites. In fact, compared to binding site alterations, poly(dA:dT) manipulations can yield more gradual changes and may thus offer a genetic mechanism by which expression can be tuned with finer resolution. Manipulating nucleosome disfavoring sequences allows fine-tune regulation of gene expression in yeast Tali Raveh-Sadka 1,2,4 , Michal Levo 1,2,4 , Uri Shabi 3 , Boaz Shany 1,2 , Leeat Keren 1,2 , Maya Lotan-Pompan 1,2 , Danny Zeevi 1,2 , Eilon Sharon 1,2 , Adina Weinberger 1,2 & Eran Segal 1,2 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. 1 Department of Computer Science and Applied Mathematics, Weizmann Institute of Science, Rehovot, Israel. 2 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. 3 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel. 4 These authors contributed equally to this work. Correspondence should be addressed to E.S. (eran.segal@weizmann.ac.il) or A.W. (adina.weinberger@weizmann.ac.il). Received 26 January; accepted 3 May; published online 27 May 2012; doi:10.1038/ng.2305