Bosilevac J M, Gilchrist C A, Jankowski P E, Paul S, Rees A R, Hinrichs S H
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-6495, USA.
J Biol Chem. 1998 Jul 3;273(27):16874-9. doi: 10.1074/jbc.273.27.16874.
Activating transcription factor 1 (ATF1) and cAMP-responsive element (CRE)-binding protein (CREB) activate transcription through CREs located in the promoters of cellular and viral genes. We previously described a monoclonal antibody (mAb41.4) that prevents ATF1 binding to DNA and reduces CRE-driven promoter activity in vitro (Orten, D. J., Strawhecker, J. M., Sanderson, S. D., Huang, D., Prytowsky, M. B. , and Hinrichs, S. H. (1994) J. Biol. Chem. 269, 32254-32263). A single chain Fv (scFv) fragment from the mAb41.4-expressing hybridoma was generated to provide a means to investigate transcription factor function via intracellular expression of the scFv fragment. The affinity of scFv4 (subgroup: VL kappa-III, VH miscellaneous) for ATF1 was similar to that of the parental mAb and the Fab fragment, but it demonstrated greater inhibitory activity and reacted with CREB. scFv4 disrupted the binding of both ATF1 and CREB in electrophoretic mobility shift assays and reduced expression of CRE-driven expression in vitro. Transient expression of scFv had no effect on the non-CRE-containing adenovirus major late promoter. The proliferating cell nuclear antigen promoter, containing two CREs, was significantly more sensitive to inhibition by scFv than the cytomegalovirus immediate-early promoter, containing five CREs. Cotransfection of either ATF1 or CREB in the presence of scFv restored basal levels of expression. The intracellular expression of scFv provides a unique means to investigate the roles of the transcription factors ATF1 and CREB.
激活转录因子1（ATF1）和环磷酸腺苷反应元件（CRE）结合蛋白（CREB）通过位于细胞和病毒基因启动子中的CRE激活转录。我们之前描述了一种单克隆抗体（mAb41.4），它可阻止ATF1与DNA结合，并在体外降低CRE驱动的启动子活性（奥滕，D. J.，斯特劳赫克，J. M.，桑德森，S. D.，黄，D.，普里托夫斯基，M. B.和欣里克斯，S. H.（1994年）《生物化学杂志》269，32254 - 32263）。从表达mAb41.4的杂交瘤中产生了单链Fv（scFv）片段，以提供一种通过scFv片段的细胞内表达来研究转录因子功能的方法。scFv4（亚组：VL κ-III，VH杂项）对ATF1的亲和力与亲本单克隆抗体和Fab片段相似，但它表现出更强的抑制活性，并与CREB发生反应。在电泳迁移率变动分析中，scFv4破坏了ATF1和CREB的结合，并在体外降低了CRE驱动的表达。scFv的瞬时表达对不含CRE的腺病毒主要晚期启动子没有影响。含有两个CRE的增殖细胞核抗原启动子比含有五个CRE的巨细胞病毒立即早期启动子对scFv的抑制作用更敏感。在scFv存在的情况下共转染ATF1或CREB可恢复基础表达水平。scFv的细胞内表达为研究转录因子ATF1和CREB的作用提供了一种独特的方法。
