Unraveling determinants of transcription factor binding outside the core binding site

Michal Levo*,1,2, Einat Zalckvar*,1,2, Eilon Sharon1, Ana Carolina Dantas Machado3, Yael Kalma2, Maya Lotam-Pompan2, Adina Weinberger1,2, Zohar Yakhini4,5, Remo Rohs3 and Eran Segal1,2

Binding of transcription factors (TFs) to regulatory sequences is a pivotal step in the control of gene expression. Despite many advances in the characterization of sequence motifs recognized by TFs, our ability to quantitatively predict TF binding to different regulatory sequences is still limited. Here, we present a novel experimental assay termed BunDLE-seq that provides quantitative measurements of TF binding to thousands of fully designed sequences of 200 bp in length within a single experiment. Applying this binding assay to two yeast TFs we demonstrate that sequences outside the core TF binding site profoundly affect TF binding. We show that TF-specific models based on the sequence or DNA shape of the regions flanking the core binding site are highly predictive of the measured differential TF binding. We further characterize the dependence of TF binding, accounting for measurements of single and co-occurring binding events, on the number and location of binding sites and on the TF concentration. Finally, by coupling our in vitro TF binding measurements, and another application of our method probing nucleosome formation, to in vivo expression measurements carried out with the same template sequences now serving and promoters, we offer insights into mechanisms that may determine the different expression outcomes observed. Our assay thus paves the way to a more comprehensive understanding of TF binding to regulatory sequences, and allows the characterization of TF binding determinants within and outside ofcore binding sites.

Correspondence to: Eran Segal1,2 eran.segal@weizmann.ac.il
* These authors contributed equally to this work, and are listed alphabetically.
1 Department of Computer Science and Applied Mathematics, Weizmann Institute of Science, Rehovot 76100, Israel.
2 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel
3 Molecular and Computational Biology Program, Departments of Biological Sciences, Chemistry, Physics, and Computer Science, University of Southern California, Los Angeles, CA 90089, USA.
4 Computer Science Department, Technion - Israel Institute of Technology, Haifa 32000, Israel.
5 Agilent Laboratories, Santa Clara, CA 95051, USA.
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