Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Departamento de Inmunología y Virología, San Nicolás de los Garza, Nuevo León, México.
Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Departamento de Biología Celular y Genética, San Nicolás de los Garza, Nuevo León, México.
Hereditas. 2022 May 30;159(1):23. doi: 10.1186/s41065-022-00239-8.
Hox proteins finely coordinate antero-posterior axis during embryonic development and through their action specific target genes are expressed at the right time and space to determine the embryo body plan. As master transcriptional regulators, Hox proteins recognize DNA through the homeodomain (HD) and interact with a multitude of proteins, including general transcription factors and other cofactors. HD binding specificity increases by protein-protein interactions with a diversity of cofactors that outline the Hox interactome and determine the transcriptional landscape of the selected target genes. All these interactions clearly demonstrate Hox-driven transcriptional regulation, but its precise mechanism remains to be elucidated.
Here we report Antennapedia (Antp) Hox protein-protein interaction with the TATA-binding protein (TBP) and the formation of novel trimeric complexes with TFIIEβ and Extradenticle (Exd), as well as its participation in transcriptional regulation. Using Bimolecular Fluorescence Complementation (BiFC), we detected the interaction of Antp-TBP and, in combination with Förster Resonance Energy Transfer (BiFC-FRET), the formation of the trimeric complex with TFIIEβ and Exd in living cells. Mutational analysis showed that Antp interacts with TBP through their N-terminal polyglutamine-stretches. The trimeric complexes of Antp-TBP with TFIIEβ and Exd were validated using different Antp mutations to disrupt the trimeric complexes. Interestingly, the trimeric complex Antp-TBP-TFIIEβ significantly increased the transcriptional activity of Antp, whereas Exd diminished its transactivation.
Our findings provide important insights into the Antp interactome with the direct interaction of Antp with TBP and the two new trimeric complexes with TFIIEβ and Exd. These novel interactions open the possibility to analyze promoter function and gene expression to measure transcription factor binding dynamics at target sites throughout the genome.
Hox 蛋白在胚胎发育过程中精细地协调前后轴，通过其作用，特定的靶基因在正确的时间和空间表达，从而决定胚胎的体节模式。作为主转录调控因子，Hox 蛋白通过同源域（HD）识别 DNA，并与多种蛋白质相互作用，包括一般转录因子和其他辅助因子。HD 结合特异性通过与多种辅助因子的蛋白-蛋白相互作用增加，这些辅助因子勾勒出 Hox 相互作用组，并决定所选靶基因的转录景观。所有这些相互作用都清楚地表明了 Hox 驱动的转录调控，但确切的机制仍有待阐明。
在这里，我们报告了触角（Antp）Hox 蛋白与 TATA 结合蛋白（TBP）的相互作用以及与 TFIIEβ 和 Extradenticle（Exd）形成新型三聚体复合物的情况，以及其参与转录调控的情况。我们使用双分子荧光互补（BiFC）检测到 Antp-TBP 的相互作用，并结合Förster 共振能量转移（BiFC-FRET），在活细胞中形成与 TFIIEβ 和 Exd 的三聚体复合物。突变分析表明，Antp 通过其 N 端多谷氨酰胺延伸与 TBP 相互作用。使用不同的 Antp 突变来破坏三聚体复合物，验证了 Antp-TBP 与 TFIIEβ 和 Exd 的三聚体复合物的形成。有趣的是，三聚体复合物 Antp-TBP-TFIIEβ 显著增加了 Antp 的转录活性，而 Exd 则减弱了其转录激活。
我们的发现为 Antp 相互作用组提供了重要的见解，即 Antp 与 TBP 的直接相互作用以及与 TFIIEβ 和 Exd 的两个新的三聚体复合物。这些新的相互作用为分析启动子功能和基因表达提供了可能性，以测量整个基因组中靶位点的转录因子结合动力学。
