Ingrassia R, Savoldi G F, Caraffini A, Tironi M, Poiesi C, Williams P, Albertini A, Di Lorenzo D
Institute of Chemistry, School of Medicine, University of Brescia, Italy.
DNA Cell Biol. 1994 Jun;13(6):615-27. doi: 10.1089/dna.1994.13.615.
The liver alpha-1-acid glycoprotein (AGP) gene promoter contains several positive cis-acting sequences that are involved in the hormone regulation of its expression. We have characterized a new functionally important sequence located at -155 to -143 upstream from the glucocorticoid regulatory element (GRE, -120 to -105). At least three nuclear proteins bind to this sequence (CTGTGGGAACAG), called the upstream regulatory element (URE). One of these proteins, AGP nuclear factor 4 (ANF-4), is the major component of the DNA-protein complex we detected in footprint and electrophoresis mobility shift assay (EMSA) experiments using rat liver, HTC(JZ-1) rat hepatoma cell extracts and affinity-purified proteins. Another is C/EBP beta, which also binds to three elements downstream from the GRE. The third protein is shown to have a molecular weight of 102 kD. Deletions and site-directed mutagenesis demonstrated that this complex of proteins is involved in the positive hormonal regulation of AGP gene transcription. Binding experiments revealed that ANF-4 and C/EBP beta binding sites are partially overlapping and require the palindromic structure of the URE for high-affinity binding. Southwestern (DNA-protein blot analysis) and cross-linking experiments with nuclear extracts from rat liver and HTC(JZ-1) rat hepatoma cells, revealed two identical constitutive binding activities with molecular masses of 66 and 102 kD. We concluded that this transcription complex is composed of three distinct proteins, ANF-4, C/EBP beta, and a 102-kD protein, and that they play an important role for the hormone regulation of AGP.
肝脏α-1-酸性糖蛋白（AGP）基因启动子包含几个正向顺式作用序列，这些序列参与其表达的激素调节。我们已经鉴定出一个新的功能重要序列，位于糖皮质激素调节元件（GRE，-120至-105）上游-155至-143处。至少有三种核蛋白与这个序列（CTGTGGGAACAG）结合，该序列称为上游调节元件（URE）。这些蛋白之一，AGP核因子4（ANF-4），是我们在足迹和电泳迁移率变动分析（EMSA）实验中使用大鼠肝脏、HTC（JZ-1）大鼠肝癌细胞提取物和亲和纯化蛋白检测到的DNA-蛋白质复合物的主要成分。另一种是C/EBPβ，它也与GRE下游的三个元件结合。第三种蛋白的分子量为102 kD。缺失和定点诱变表明，这种蛋白复合物参与AGP基因转录的正向激素调节。结合实验表明，ANF-4和C/EBPβ结合位点部分重叠，并且需要URE的回文结构进行高亲和力结合。用大鼠肝脏和HTC（JZ-1）大鼠肝癌细胞核提取物进行的蛋白质免疫印迹（DNA-蛋白质印迹分析）和交联实验，揭示了两种分子量分别为66和102 kD的相同组成性结合活性。我们得出结论，这种转录复合物由三种不同的蛋白ANF-4、C/EBPβ和一种102-kD蛋白组成，并且它们在AGP的激素调节中起重要作用。
