Measuring cis-regulatory energetics in living cells using allelic manifolds
Abstract
Gene expression in all organisms is controlled by cooperative interactions between DNA-bound transcription factors (TFs), but quantitatively measuring TF-DNA and TF-TF interactions remains difficult. Here we introduce a strategy for precisely measuring the Gibbs free energy of such interactions in living cells. This strategy centers on the measurement and modeling of 'allelic manifolds', a multidimensional generalization of the classical genetics concept of allelic series. Allelic manifolds are measured using reporter assays performed on strategically designed cis-regulatory sequences. Quantitative biophysical models are then fit to the resulting data. We used this strategy to study regulation by two Escherichia coliTFs, CRP and σ^(70) RNA polymerase. Doing so, we consistently obtained energetic measurements precise to ∼0.1 kcal/mol. We also obtained multiple results that deviate from the prior literature. Our strategy is compatible with massively parallel reporter assays in both prokaryotes and eukaryotes, and should therefore be highly scalable and broadly applicable.
Additional Information
© 2018 Forcier et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Received: 31 July 2018; Accepted: 27 November 2018; Published: 20 December 2018. We thank Stirling Churchman, Barak Cohen, David McCandlish, Bryce Nickels, and Saurabh Sinha for helpful discussions. We also thank Naama Barkai, Ulrich Gerland, Richard Neher, and one anonymous referee for reviewing this manuscript and providing helpful feedback. This work was supported by a CSHL/Northwell Health Alliance grant to JBK and by NIH Cancer Center Support Grant 5P30CA045508. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Author contributions: Talitha L Forcier, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing-review and editing; Andalus Ayaz, Data curation, Validation, Investigation, Methodology, Writing-review and editing; Manraj S Gill, Data curation, Validation, Investigation, Methodology; Daniel Jones, Conceptualization, Investigation, Methodology; Rob Phillips, Supervision, Funding acquisition, Writing—review and editing; Justin B Kinney, Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing. Data availability: All data used to make the Figures is available in Supplementary file 1. The PSAM for RNAP, previously published by Kinney et al. (2010), is also provided in Supplementary file 1 (with permission). Raw data, processed data, and analysis scripts are also available at https://github.com/jbkinney/17_inducibility (copy archived at https://github.com/elifesciences-publications/17_inducibility). No datasets have been deposited in public databases as part of this work.Attached Files
Published - elife-40618-v1.pdf
Submitted - 380972.full.pdf
Supplemental Material - elife-40618-supp1-v1.xlsx
Supplemental Material - elife-40618-transrepform-v1.docx
Files
Additional details
- Eprint ID
- 91275
- Resolver ID
- CaltechAUTHORS:20181128-093526476
- Cold Spring Harbor Laboratory/Northwell Health Alliance
- 5P30CA045508
- NIH
- Created
-
2018-11-28Created from EPrint's datestamp field
- Updated
-
2023-06-02Created from EPrint's last_modified field