A Modified Reverse One-Hybrid Screen Identifies Transcriptional Activation Domains in PHYTOCHROME-INTERACTING FACTOR 3.
A Modified Reverse One-Hybrid Screen Identifies Transcriptional Activation Domains in PHYTOCHROME-INTERACTING FACTOR 3.
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DOI：
10.3389/fpls.2016.00881
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发表时间：
2016
影响因子：
5.6
通讯作者：
Quail PH
中科院分区：
文献类型：
--
作者：
Dalton JC;Bätz U;Liu J;Curie GL;Quail PH
转录激活域（TAD）很难预测和识别，因为它们不保守并且几乎没有共识。在这里，我们描述了一种基于酵母的筛选方法，该方法能够以高通量方式识别参与转录激活的单个氨基酸残基。植物转录激活因子 PIF3（光敏色素相互作用因子 3）与酵母 GAL4-DNA 结合域 (BD) 融合，驱动 URA3（乳清苷 5'-磷酸脱羧酶）报告基因的表达，并用于 5-氟乳清酸 (5FOA) 的负选择。然后选择 PIF3 的随机诱变变体，以通过在 FOA 上存活而导致转录激活活性的损失或减少。在此过程中，我们开发了一种策略来消除阴性选择中的假阳性，该策略可用于反向 1 和 2 杂交筛选。通过这种方法，我们能够识别 PIF3 中具有转录激活活性的两个不同区域，这两个区域在 PIF1、PIF4 和 PIF5 中都是功能保守的。两者对于 PIF3 的完整转录活性都是必需的，但都不足以诱导自主转录。我们还发现 TAD 似乎与其他 PIF3 功能在物理上重叠，例如 phyB 结合活性和随后的磷酸化。我们的协议应该提供一个有价值的工具来识别、分析和表征真核转录因子中的新型 TAD，从而可能有助于揭示转录激活的机制。
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.