Lamb B T, Satyamoorthy K, Solter D, Basu A, Xu M Q, Weinmann R, Howe C C
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Mol Cell Biol. 1992 Nov;12(11):4824-33. doi: 10.1128/mcb.12.11.4824-4833.1992.
The retinoic acid-induced differentiation of F9 cells into parietal endoderm-like cells activates transcription of the endogenous mouse retrovirus, the intracisternal A-particle (IAP). To investigate the elements that control IAP gene differentiation-specific expression, we used methylation interference, Southwestern (DNA-protein), and transient-transfection assays and identified the IAP-proximal enhancer (IPE) element that directs differentiation-specific expression. We find that the IPE is inactive in undifferentiated F9 cells and active in differentiated parietal endoderm-like PYS-2 cells. Three proteins of 40, 60, and 68 kDa bind to the sequence GAGTAGAC located between nucleotides -53 and -47 within the IPE. The 40- and 68-kDa proteins from both the undifferentiated and differentiated cells exhibit similar DNA-binding activities. However, the 60-kDa protein from differentiated cells has greater binding activity than that from undifferentiated cells, suggesting a role for this protein in F9 differentiation-specific expression of the IAP gene. The IAP gene is negatively regulated by the adenovirus E1A proteins, and the E1A sequence responsible for repression is located at the N terminus, between amino acids 2 and 67. The DNA sequence that is the target of E1A repression also maps to the IPE element. Colocalization of the differentiation-specific and E1A-sensitive elements to the same protein-binding site within the IPE suggests that the E1A-like activity functions in F9 cells to repress IAP gene expression. Activation of the IAP gene may result when the E1A-like activity is lost or inactivated during F9 cell differentiation, followed by binding of the 60-kDa positive regulatory protein to the enhancer element.
维甲酸诱导F9细胞分化为壁内胚层样细胞可激活内源性小鼠逆转录病毒即胞内A颗粒（IAP）的转录。为了研究控制IAP基因分化特异性表达的元件，我们使用了甲基化干扰、西南杂交（DNA-蛋白质）和瞬时转染分析，并鉴定了指导分化特异性表达的IAP近端增强子（IPE）元件。我们发现IPE在未分化的F9细胞中无活性，而在分化的壁内胚层样PYS-2细胞中具有活性。三种分子量分别为40、60和68 kDa的蛋白质与IPE内核苷酸-53至-47之间的GAGTAGAC序列结合。未分化和分化细胞中的40 kDa和68 kDa蛋白质表现出相似的DNA结合活性。然而，分化细胞中的60 kDa蛋白质比未分化细胞中的具有更强的结合活性，表明该蛋白质在IAP基因的F9分化特异性表达中起作用。IAP基因受到腺病毒E1A蛋白的负调控，负责抑制的E1A序列位于N端，氨基酸2至67之间。E1A抑制的靶DNA序列也定位于IPE元件。分化特异性和E1A敏感元件在IPE内的同一蛋白质结合位点共定位，表明E1A样活性在F9细胞中发挥作用以抑制IAP基因表达。当F9细胞分化过程中E1A样活性丧失或失活，随后60 kDa阳性调节蛋白与增强子元件结合时，可能导致IAP基因的激活。
