Ramos-Sánchez José M, Triozzi Paolo M, Moreno-Cortés Alicia, Conde Daniel, Perales Mariano, Allona Isabel
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM, 28223 Pozuelo de Alarcón, Madrid, Spain.
Departamento de Biotecnología-Biología Vegetal, Escuela Técnica Superior Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid (UPM), 28040 Madrid, Spain.
Plant Methods. 2017 Jun 15;13:50. doi: 10.1186/s13007-017-0199-x. eCollection 2017.
Precise control of gene expression is essential to synchronize plant development with the environment. In perennial plants, transcriptional regulation remains poorly understood, mainly due to the long time required to perform functional studies. Transcriptional reporters based on luciferase have been useful to study circadian and diurnal regulation of gene expression, both by transcription factors and chromatin remodelers. The high mobility group proteins are considered transcriptional chaperones that also modify the chromatin architecture. They have been found in several species, presenting in some cases a circadian expression of their mRNA or protein.
Transactivation experiments have been shown as a powerful and fast method to obtain information about the potential role of transcription factors upon a certain reporter. We designed and validated a luciferase transcriptional reporter using the 5' sequence upstream ATG of gene. We showed the robustness of this reporter line under long day and continuous light conditions. Moreover, we confirmed that activity reproduces the accumulation of mRNA. We performed transactivation studies by transient expression, using the reporter line as a genetic background, unraveling a new function of a high mobility group protein in poplar, which can activate the promoter in a gate-dependent manner. We also showed PtaHMGB2/3 needs darkness to produce that activation and exhibits an active degradation after dawn, mediated by the 26S proteasome.
We generated a stable luciferase reporter poplar line based on the circadian clock gene which can be used to investigate transcriptional regulation and signal transduction pathway. Using this reporter line as a genetic background, we established a methodology to rapidly assess potential regulators of diurnal and circadian rhythms. This tool allowed us to demonstrate that PtaHMGB2/3 promotes the transcriptional activation of our reporter in a gate-dependent manner. Moreover, we added new information about the PtaHMGB2/3 protein regulation along the day. This methodology can be easily adapted to other transcription factors and reporters.
精确控制基因表达对于使植物发育与环境同步至关重要。在多年生植物中，转录调控仍知之甚少，主要是因为进行功能研究所需时间较长。基于荧光素酶的转录报告基因已被用于研究转录因子和染色质重塑因子对基因表达的昼夜节律和日调节。高迁移率族蛋白被认为是转录伴侣，它们也会改变染色质结构。已在多个物种中发现它们，在某些情况下其mRNA或蛋白质呈现昼夜节律表达。
反式激活实验已被证明是一种强大且快速的方法，可用于获取有关转录因子对特定报告基因潜在作用的信息。我们使用基因ATG上游的5'序列设计并验证了一种荧光素酶转录报告基因。我们展示了该报告基因系在长日照和连续光照条件下的稳健性。此外，我们证实了活性再现了mRNA的积累。我们以该报告基因系为遗传背景，通过瞬时表达进行反式激活研究，揭示了杨树中一种高迁移率族蛋白的新功能，它可以以门控依赖的方式激活启动子。我们还表明，PtaHMGB2/3需要黑暗来产生这种激活作用，并且在黎明后由26S蛋白酶体介导表现出活性降解。
我们基于昼夜节律基因生成了一个稳定的荧光素酶报告杨树系，可用于研究转录调控和信号转导途径。以该报告基因系为遗传背景，我们建立了一种方法来快速评估昼夜节律的潜在调节因子。这个工具使我们能够证明PtaHMGB2/3以门控依赖的方式促进我们报告基因的转录激活。此外，我们补充了关于PtaHMGB2/3蛋白在一天中的调节的新信息。这种方法可以很容易地应用于其他转录因子和报告基因。
