Antibodies are routinely used to study the activity of transcription factors, using various in vitro and in vivo approaches such as electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, genome-wide method analysis coupled with next generation sequencing, or mass spectrometry. More recently, a new application for antibodies has emerged as crystallisation scaffolds for difficult to crystallise proteins, such as transcription factors. Only in a few rare cases, antibodies have been used to modulate the activity of transcription factors, and there is a real gap in our knowledge on how to efficiently design antibodies to interfere with transcription. The molecular function of transcription factors is underpinned by complex networks of protein-protein interaction and in theory, setting aside intra-cellular delivery challenges, developing antibody-based approaches to modulate transcription factor activity appears a viable option. Here, we demonstrate that antibodies or an antibody single-chain variable region fragments are powerful molecular tools to unravel complex protein-DNA and protein-protein binding mechanisms. In this study, we focus on the molecular mode of action of the transcription factor SOX18, a key modulator of endothelial cell fate during development, as well as an attractive target in certain pathophysiological conditions such as solid cancer metastasis. The engineered antibody we designed inhibits SOX18 transcriptional activity, by interfering specifically with an 8-amino-acid motif in the C-terminal region directly adjacent to α-Helix 3 of SOX18 HMG domain, thereby disrupting protein-protein interaction. This new approach establishes a framework to guide the study of transcription factors interactomes using antibodies as molecular handles.

通常使用抗体在体内和体外进行多种抗体研究转录因子的活性电泳迁移率测定，酶联免疫吸附测定，全基因组方法分析和下一代测序或质谱分析等方法。最近，针对难以结晶的蛋白质，例如转录因子的结晶支架，出现了抗体的新应用。仅在极少数情况下，抗体才被用于调节转录因子的活性，在我们如何有效设计抗体来干扰转录方面，我们的知识确实存在空白。蛋白质与蛋白质相互作用的复杂网络为转录因子的分子功能提供了支撑，理论上，撇开细胞内传递挑战，开发基于抗体的方法来调节转录因子活性似乎是一个可行的选择。这里，我们证明抗体或抗体单链可变区片段是强大的分子工具，可揭示复杂的蛋白质-DNA和蛋白质-蛋白质结合机制。在这项研究中，我们专注于转录因子SOX18的分子作用模式，该因子是发育过程中内皮细胞命运的关键调节剂，并且在某些病理生理条件下（例如实体癌转移）也具有吸引力。我们设计的工程抗体可通过特异性干扰SOX18 HMG结构域的α-Helix3紧邻的C端区域中的8个氨基酸基序来抑制SOX18转录活性。这种新方法建立了一个框架，以使用抗体作为分子柄来指导转录因子相互作用组的研究。