Cohen L, Lacoste J, Parniak M, Daigneault L, Skup D, Hiscott J
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Canada.
Cell Growth Differ. 1991 Jul;2(7):323-33.
The human interferon beta (IFN-beta) regulatory element consists of multiple enhanson domains which are targets for transcription factors involved in inducible expression of the promoter. To further characterize the protein-DNA interactions mediating IFN-beta induction, positive regulatory domain (PRD) II binding proteins were purified from phorbol ester induced Jurkat T-cells and from IFN primed, cycloheximide/polyinosinic-polycytidylic acid treated HeLa S3 cells. From HeLa cells, two major proteins of 52 and 45 kilodaltons (kD) copurified with DNA binding activity, whereas from T-cells, four proteins--a major protein of 52 kD and three minor proteins of 82, 67, and 43-47 kD--were purified. Also, an induction specific DNA binding protein was purified from HeLa cells that interacted with the (AAGTGA)4 tetrahexamer sequence and the PRDI domain. This protein is immunologically distinct from IRF-1/ISGF2. Uninduced or Sendai virus induced HeLa extracts were used to examine transcription in vitro using a series of IFN beta promoter deletions. Deletions upstream of the PRDII element increased transcription in the uninduced extract, indicating predominantly negative regulation of the promoter. A 2-4-fold increase in IFN-beta promoter transcription was observed in Sendai virus induced extracts, and deletion of PRDI and PRDII elements decreased this induced level of transcription. When purified PRDII and tetrahexamer binding proteins were added to the induced extract, a 4-fold increase in transcription was observed. These experiments demonstrate that it is possible to modulate IFN-beta transcription in vitro but indicate that additional proteins may be required to fully activate IFN-beta transcription.
人β干扰素（IFN-β）调控元件由多个增强子结构域组成，这些结构域是参与启动子诱导性表达的转录因子的作用靶点。为了进一步表征介导IFN-β诱导的蛋白质 - DNA相互作用，从佛波酯诱导的Jurkat T细胞以及IFN预刺激、环己酰亚胺/聚肌苷酸 - 聚胞苷酸处理的HeLa S3细胞中纯化了正调控结构域（PRD）II结合蛋白。从HeLa细胞中，两种主要的蛋白质，分子量分别为52和45千道尔顿（kD），与DNA结合活性一起共纯化，而从T细胞中纯化出了四种蛋白质——一种主要的52 kD蛋白质和三种分子量分别为82、67和43 - 47 kD的次要蛋白质。此外，从HeLa细胞中纯化出一种诱导特异性DNA结合蛋白，它与（AAGTGA）4四聚体序列和PRDI结构域相互作用。这种蛋白质在免疫学上与IRF-1/ISGF2不同。未诱导的或仙台病毒诱导的HeLa提取物用于使用一系列IFN-β启动子缺失来检测体外转录。PRDII元件上游的缺失增加了未诱导提取物中的转录，表明对启动子主要起负调控作用。在仙台病毒诱导的提取物中观察到IFN-β启动子转录增加了2 - 4倍，并且PRDI和PRDII元件的缺失降低了这种诱导的转录水平。当将纯化的PRDII和四聚体结合蛋白添加到诱导提取物中时，观察到转录增加了4倍。这些实验表明在体外调节IFN-β转录是可能的，但表明可能需要其他蛋白质来完全激活IFN-β转录。
