Tsuji Y, Moran E, Torti S V, Torti F M
Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.
J Biol Chem. 1999 Mar 12;274(11):7501-7. doi: 10.1074/jbc.274.11.7501.
We previously identified a major enhancer of the mouse ferritin H gene (FER-1) that is central to repression of the ferritin H gene by the adenovirus E1A oncogene (Tsuji, Y., Akebi, N., Lam, T. K., Nakabeppu, Y., Torti, S. V., and Torti, F. M. (1995) Mol. Cell. Biol. 15, 5152-5164). To dissect the molecular mechanism of transcriptional regulation of ferritin H, E1A mutants were tested for their ability to repress FER-1 enhancer activity using cotransfection with ferritin H-chloramphenicol acetyltransferase (CAT) reporter constructs. Here we report that p300/CBP transcriptional adaptor proteins are involved in the regulation of ferritin H transcription through the FER-1 enhancer element. Thus, E1A mutants that failed to bind p300/CBP lost the ability to repress FER-1, whereas mutants of E1A that abrogated its interaction with Rb, p107, or p130 were fully functional in transcriptional repression. Transfection with E1A did not affect endogenous p300/CBP levels, suggesting that repression of FER-1 by E1A is not due to repression of p300/CBP synthesis, but to E1A and p300/CBP interaction. In addition, we have demonstrated that transfection of a p300 expression plasmid significantly activated ferritin H-CAT containing the FER-1 enhancer, but had a marginal effect on ferritin H-CAT with FER-1 deleted. Furthermore, both wild-type p300 and a p300 mutant that failed to bind E1A but retained an adaptor function restored FER-1 enhancer activity repressed by E1A. Sodium butyrate, an inhibitor of histone deacetylase, mimicked p300/CBP function in activation of ferritin H-CAT and elevation of endogenous ferritin H mRNA, suggesting that the histone acetyltransferase activity of p300/CBP or its associated proteins may contribute to the activation of ferritin H transcription. Recruitment of these broadly active transcriptional adaptor proteins for ferritin H synthesis may represent an important mechanism by which changes in iron metabolism are coordinated with other cellular responses mediated by p300/CBP.
我们先前鉴定出小鼠铁蛋白H基因（FER-1）的一个主要增强子，它在腺病毒E1A癌基因对铁蛋白H基因的抑制中起核心作用（Tsuji, Y., Akebi, N., Lam, T. K., Nakabeppu, Y., Torti, S. V., and Torti, F. M. (1995) Mol. Cell. Biol. 15, 5152 - 5164）。为了剖析铁蛋白H转录调控的分子机制，我们利用与铁蛋白H-氯霉素乙酰转移酶（CAT）报告基因构建体共转染，测试了E1A突变体抑制FER-1增强子活性的能力。在此我们报告，p300/CBP转录衔接蛋白通过FER-1增强子元件参与铁蛋白H转录的调控。因此，无法结合p300/CBP的E1A突变体失去了抑制FER-1的能力，而消除其与Rb、p107或p130相互作用的E1A突变体在转录抑制方面功能完全正常。用E1A转染不影响内源性p300/CBP水平，这表明E1A对FER-1的抑制不是由于p300/CBP合成的抑制，而是由于E1A与p300/CBP的相互作用。此外，我们已经证明，转染p300表达质粒可显著激活含有FER-1增强子的铁蛋白H-CAT，但对缺失FER-1的铁蛋白H-CAT影响很小。此外，野生型p300和一个无法结合E1A但保留衔接蛋白功能的p300突变体都恢复了被E1A抑制的FER-1增强子活性。组蛋白脱乙酰酶抑制剂丁酸钠在激活铁蛋白H-CAT和提高内源性铁蛋白H mRNA水平方面模拟了p300/CBP的功能，这表明p300/CBP或其相关蛋白的组蛋白乙酰转移酶活性可能有助于铁蛋白H转录的激活。募集这些对铁蛋白H合成具有广泛活性的转录衔接蛋白可能代表了一种重要机制，通过该机制铁代谢的变化与由p300/CBP介导的其他细胞反应得以协调。
