Tsuji Y, Akebi N, Lam T K, Nakabeppu Y, Torti S V, Torti F M
Department of Cancer Biology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157, USA.
Mol Cell Biol. 1995 Sep;15(9):5152-64. doi: 10.1128/MCB.15.9.5152.
Ferritin, the major intracellular iron storage protein of eucaryotic cells, is regulated during inflammation and malignancy. We previously reported that transcription of the H subunit of ferritin (ferritin H) is negatively regulated by the adenovirus E1A oncogene in mouse NIH 3T3 fibroblasts (Y. Tsuji, E. Kwak, T. Saika, S. V. Torti, and F. M. Torti, J. Biol. Chem. 268:7270-7275, 1993). To elucidate the mechanism of transcriptional repression of the ferritin H gene by E1A, a series of deletions in the 5' flanking region of the mouse ferritin H gene were constructed, fused to the chloramphenicol acetyltransferase (CAT) gene, and transiently cotransfected into NIH 3T3 cells with an E1A expression plasmid. The results indicate that the E1A-responsive region is located approximately 4.1 kb 5' to the transcription initiation site of the ferritin H gene. Further analyses revealed that a 37-bp region, termed FER-1, is the target of E1A-mediated repression. This region also serves as an enhancer, augmenting ferritin H transcription independently of position and orientation. FER-1 was dissected into two component elements, i.e., a 22-bp dyad symmetry element and a 7-bp AP1-like sequence. Insertion of these DNA sequences into a ferritin H-CAT chimeric gene lacking an E1A-responsive region indicated that (i) the 22-bp dyad symmetry sequence by itself has no enhancer activity, (ii) the AP1-like sequence has moderate enhancer activity which is repressed by E1A, and (iii) the combination of the dyad symmetry element and the AP1-like sequence is required for maximal enhancer activity and repression by E1A. Gel retardation assays and cotransfection experiments with c-fos and c-jun expression vectors suggested that members of the Fos and Jun families bind to the AP1-like element of FER-1 and contribute to its regulation. In addition, gel retardation assays showed that E1A reduces the ability of nuclear proteins to bind to the AP1-like sequence without affecting the levels of nuclear factors that recognize the 22-bp dyad symmetry element. Taken together, these results demonstrate that FER-1 serves as both an enhancer of ferritin H transcription and a target for E1A-mediated repression.
铁蛋白是真核细胞内主要的铁储存蛋白，在炎症和恶性肿瘤过程中受到调控。我们先前报道，在小鼠NIH 3T3成纤维细胞中，腺病毒E1A癌基因对铁蛋白H亚基（ferritin H）的转录具有负调控作用（Y. Tsuji、E. Kwak、T. Saika、S. V. Torti和F. M. Torti，《生物化学杂志》268:7270 - 7275，1993年）。为阐明E1A对铁蛋白H基因转录抑制的机制，构建了一系列小鼠铁蛋白H基因5'侧翼区的缺失片段，将其与氯霉素乙酰转移酶（CAT）基因融合，并与E1A表达质粒一起瞬时共转染到NIH 3T3细胞中。结果表明，E1A反应区位于铁蛋白H基因转录起始位点上游约4.1 kb处。进一步分析显示，一个37 bp的区域，称为FER - 1，是E1A介导的抑制作用的靶点。该区域还作为增强子，可独立于位置和方向增强铁蛋白H的转录。FER - 1被分解为两个组成元件，即一个22 bp的二元对称元件和一个7 bp的AP1样序列。将这些DNA序列插入缺乏E1A反应区的铁蛋白H - CAT嵌合基因中表明：（i）22 bp的二元对称序列本身没有增强子活性；（ii）AP1样序列具有中等增强子活性，且会被E1A抑制；（iii）二元对称元件和AP1样序列的组合是实现最大增强子活性和E1A介导的抑制作用所必需的。凝胶阻滞试验以及与c - fos和c - jun表达载体的共转染实验表明，Fos和Jun家族成员与FER - 1的AP1样元件结合并参与其调控。此外，凝胶阻滞试验表明，E1A降低了核蛋白与AP1样序列结合的能力，而不影响识别22 bp二元对称元件的核因子水平。综上所述，这些结果表明FER - 1既是铁蛋白H转录的增强子，也是E1A介导的抑制作用的靶点。
