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Dalton JC, Bätz U, Liu J, et al. A Modified Reverse One-Hybrid Screen Identifies Transcriptional Activation Domains in PHYTOCHROME-INTERACTING FACTOR 3. Front Plant Sci. 2016;7:881doi: 10.3389/fpls.2016.00881.
Dalton, J. C., Bätz, U., Liu, J., Curie, G. L., & Quail, P. H. (2016). A Modified Reverse One-Hybrid Screen Identifies Transcriptional Activation Domains in PHYTOCHROME-INTERACTING FACTOR 3. Frontiers in plant science, 7881. https://doi.org/10.3389/fpls.2016.00881
Dalton, Jutta C, et al. "A Modified Reverse One-Hybrid Screen Identifies Transcriptional Activation Domains in PHYTOCHROME-INTERACTING FACTOR 3." Frontiers in plant science vol. 7 (2016): 881. doi: https://doi.org/10.3389/fpls.2016.00881
Dalton JC, Bätz U, Liu J, Curie GL, Quail PH. A Modified Reverse One-Hybrid Screen Identifies Transcriptional Activation Domains in PHYTOCHROME-INTERACTING FACTOR 3. Front Plant Sci. 2016 Jun 17;7:881. doi: 10.3389/fpls.2016.00881. eCollection 2016. PMID: 27379152; PMCID: PMC4911399.
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Front Plant Sci. 2016 Jun 17;7:881. doi: 10.3389/fpls.2016.00881. eCollection 2016.

A Modified Reverse One-Hybrid Screen Identifies Transcriptional Activation Domains in PHYTOCHROME-INTERACTING FACTOR 3.

Frontiers in plant science

Jutta C Dalton, Ulrike Bätz, Jason Liu, Gemma L Curie, Peter H Quail

Affiliations

  1. Peter H. Quail Lab, Plant Gene and Expression Center, Department of Plant and Microbial Biology, University of California Berkeley Albany, CA, USA.

PMID: 27379152 PMCID: PMC4911399 DOI: 10.3389/fpls.2016.00881

Abstract

Transcriptional activation domains (TADs) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput manner. A plant transcriptional activator, PIF3 (phytochrome interacting factor 3), was fused to the yeast GAL4-DNA-binding Domain (BD), driving expression of the URA3 (Orotidine 5'-phosphate decarboxylase) reporter, and used for negative selection on 5-fluroorotic acid (5FOA). Randomly mutagenized variants of PIF3 were then selected for a loss or reduction in transcriptional activation activity by survival on FOA. In the process, we developed a strategy to eliminate false positives from negative selection that can be used for both reverse-1- and 2-hybrid screens. With this method we were able to identify two distinct regions in PIF3 with transcriptional activation activity, both of which are functionally conserved in PIF1, PIF4, and PIF5. Both are collectively necessary for full PIF3 transcriptional activity, but neither is sufficient to induce transcription autonomously. We also found that the TAD appear to overlap physically with other PIF3 functions, such as phyB binding activity and consequent phosphorylation. Our protocol should provide a valuable tool for identifying, analyzing and characterizing novel TADs in eukaryotic transcription factors, and thus potentially contribute to the unraveling of the mechanism underlying transcriptional activation.

Keywords: PHYTOCHROME INTERACTING FACTOR; light signaling; loss of function screens; phytochrome; transcriptional activation; yeast-one hybrid

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