Laboratory of Biochemistry and Cell Biology (URBC), NAmur Research Institute for LIfe Sciences, University of Namur, 61 rue de Bruxelles, 5000 Namur, Belgium.
Nucleic Acids Res. 2012 Nov;40(21):e168. doi: 10.1093/nar/gks733. Epub 2012 Aug 16.
To depict the largest picture of a core promoter interactome, we developed a one-step DNA-affinity capture method coupled with an improved mass spectrometry analysis process focused on the identification of low abundance proteins. As a proof of concept, this method was developed through the analysis of 230 bp contained in the 5'long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1). Beside many expected interactions, many new transcriptional regulators were identified, either transcription factors (TFs) or co-regulators, which interact directly or indirectly with the HIV-1 5'LTR. Among them, the homeodomain-containing TF myeloid ectopic viral integration site was confirmed to functionally interact with a specific binding site in the HIV-1 5'LTR and to act as a transcriptional repressor, probably through recruitment of the repressive Sin3A complex. This powerful and validated DNA-affinity approach could also be used as an efficient screening tool to identify a large set of proteins that physically interact, directly or indirectly, with a DNA sequence of interest. Combined with an in silico analysis of the DNA sequence of interest, this approach provides a powerful approach to select the interacting candidates to validate functionally by classical approaches.
为了描绘核心启动子相互作用组的最大图谱，我们开发了一种一步 DNA 亲和捕获方法，结合了改进的质谱分析过程，重点是鉴定低丰度蛋白质。作为概念验证，该方法通过分析人类免疫缺陷病毒 1 (HIV-1) 的 5'长末端重复 (LTR) 中包含的 230 个碱基对来开发。除了许多预期的相互作用外，还鉴定了许多新的转录调节剂，无论是转录因子 (TF) 还是共调节剂，它们直接或间接与 HIV-1 5'LTR 相互作用。其中，含同源域的 TF 髓系异位病毒整合位点被证实与 HIV-1 5'LTR 中的特定结合位点具有功能相互作用，并作为转录抑制剂起作用，可能通过募集抑制性 Sin3A 复合物。这种强大且经过验证的 DNA 亲和方法也可以用作有效的筛选工具，以鉴定与感兴趣的 DNA 序列直接或间接物理相互作用的大量蛋白质。与对感兴趣的 DNA 序列进行计算机分析相结合，这种方法提供了一种强大的方法来选择相互作用的候选者，以通过经典方法验证功能。
