Plant Physiology, Center for Plant Molecular Biology, University of Tuebingen, Tuebingen, Germany.
PLoS One. 2013 Oct 11;8(10):e75177. doi: 10.1371/journal.pone.0075177. eCollection 2013.
DNA-binding proteins (DBPs), such as transcription factors, constitute about 10% of the protein-coding genes in eukaryotic genomes and play pivotal roles in the regulation of chromatin structure and gene expression by binding to short stretches of DNA. Despite their number and importance, only for a minor portion of DBPs the binding sequence had been disclosed. Methods that allow the de novo identification of DNA-binding motifs of known DBPs, such as protein binding microarray technology or SELEX, are not yet suited for high-throughput and automation. To close this gap, we report an automatable DNA-protein-interaction (DPI)-ELISA screen of an optimized double-stranded DNA (dsDNA) probe library that allows the high-throughput identification of hexanucleotide DNA-binding motifs. In contrast to other methods, this DPI-ELISA screen can be performed manually or with standard laboratory automation. Furthermore, output evaluation does not require extensive computational analysis to derive a binding consensus. We could show that the DPI-ELISA screen disclosed the full spectrum of binding preferences for a given DBP. As an example, AtWRKY11 was used to demonstrate that the automated DPI-ELISA screen revealed the entire range of in vitro binding preferences. In addition, protein extracts of AtbZIP63 and the DNA-binding domain of AtWRKY33 were analyzed, which led to a refinement of their known DNA-binding consensi. Finally, we performed a DPI-ELISA screen to disclose the DNA-binding consensus of a yet uncharacterized putative DBP, AtTIFY1. A palindromic TGATCA-consensus was uncovered and we could show that the GATC-core is compulsory for AtTIFY1 binding. This specific interaction between AtTIFY1 and its DNA-binding motif was confirmed by in vivo plant one-hybrid assays in protoplasts. Thus, the value and applicability of the DPI-ELISA screen for de novo binding site identification of DBPs, also under automatized conditions, is a promising approach for a deeper understanding of gene regulation in any organism of choice.
DNA 结合蛋白（DBP），如转录因子，构成真核基因组中约 10%的蛋白质编码基因，通过与短链 DNA 结合，在调节染色质结构和基因表达方面发挥着关键作用。尽管它们的数量和重要性很大，但只有一小部分 DBP 的结合序列被揭示出来。允许鉴定已知 DBP 的 DNA 结合基序的方法，如蛋白质结合微阵列技术或 SELEX，还不适合高通量和自动化。为了弥补这一差距，我们报告了一种自动化的 DNA-蛋白质相互作用（DPI）-ELISA 筛选优化的双链 DNA（dsDNA）探针文库的方法，该方法允许高通量鉴定六核苷酸 DNA 结合基序。与其他方法相比，这种 DPI-ELISA 筛选可以手动或使用标准实验室自动化进行。此外，输出评估不需要广泛的计算分析来推导出结合共识。我们证明，DPI-ELISA 筛选可以揭示给定 DBP 的全部结合偏好谱。作为一个例子，AtWRKY11 被用于证明自动化的 DPI-ELISA 筛选揭示了体外结合偏好的全部范围。此外，还分析了 AtbZIP63 的蛋白质提取物和 AtWRKY33 的 DNA 结合结构域，这导致对其已知 DNA 结合共识的改进。最后，我们进行了 DPI-ELISA 筛选，以揭示尚未表征的推定 DBP AtTIFY1 的 DNA 结合共识。发现了一个回文 TGATCA 共识，并且我们证明 GATC 核心对于 AtTIFY1 结合是必需的。AtTIFY1 与其 DNA 结合基序之间的这种特定相互作用通过质体中的体内植物单杂交测定得到了证实。因此，DPI-ELISA 筛选用于鉴定 DBP 的新结合位点，即使在自动化条件下，也是深入了解任何所选生物体基因调控的一种很有前途的方法。
