Smith Alexander E F, Ford Kevin G
King's College London, Department of Haematological and Molecular Medicine, King's College School of Medicine, The Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK.
Nucleic Acids Res. 2005 Oct 20;33(18):6011-23. doi: 10.1093/nar/gki907. Print 2005.
Oct-4 is a POU domain transcription factor that is critical for maintaining pluripotency and for stem cell renewal. Previous studies suggest that transcription regulation by Oct-4 at particular enhancers requires the input of a postulated E1A-like cofactor that is specific to pluripotent cells. However, such studies have been limited to the use of enhancer elements that bind other POU-protein family members in addition to Oct-4, thus preventing a 'clean' assessment of any Oct-4:cofactor relationships. Other attempts to study Oct-4 functionality in a more 'stand-alone' situation target Oct-4 transactivation domains to DNA using heterologous binding domains, a methodology which is known to generate artificial data. To circumvent these issues, an altered-specificity binding Oct-4 (Oct-4RR) and accompanying binding site, which binds Oct-4RR only, were generated. This strategy has previously been shown to maintain Oct-1:cofactor interactions that are highly binding-site and protein/binding conformation specific. This system therefore allows a stand-alone study of Oct-4 function in pluripotent versus differentiated cells, without interference from endogenous POU factors and with minimal deviation from bound wild-type protein characteristics. Subsequently, it was demonstrated that Oct-4RR and the highly transactive regions of its N-terminus determined here, and its C-terminus, have the same transactivation profile in pluripotent and differentiated cells, thus providing strong evidence against the existence of such a pluripotent cell-specific Oct-4 cofactor.
八聚体结合转录因子4（Oct-4）是一种POU结构域转录因子，对维持多能性和干细胞更新至关重要。先前的研究表明，Oct-4在特定增强子处的转录调控需要一种假定的类似E1A的辅因子的参与，该辅因子对多能细胞具有特异性。然而，这些研究仅限于使用除Oct-4之外还能结合其他POU蛋白家族成员的增强子元件，因此无法对任何Oct-4与辅因子的关系进行“纯粹”的评估。其他在更“独立”情况下研究Oct-4功能的尝试，是利用异源结合结构域将Oct-4反式激活结构域靶向到DNA上，这种方法已知会产生人为数据。为了规避这些问题，我们构建了一种特异性改变的结合型Oct-4（Oct-4RR）及其伴随的结合位点，该结合位点仅与Oct-4RR结合。此前已证明这种策略能够维持Oct-1与辅因子的相互作用，这种相互作用具有高度的结合位点特异性以及蛋白质/结合构象特异性。因此，该系统能够在不受到内源性POU因子干扰且与结合的野生型蛋白质特征偏差最小的情况下，对Oct-4在多能细胞与分化细胞中的功能进行独立研究。随后，研究表明Oct-4RR及其在此确定的N端高反式激活区域以及C端，在多能细胞和分化细胞中具有相同的反式激活谱，从而为不存在这种多能细胞特异性Oct-4辅因子提供了有力证据。
