Differential regulation of human hepatic flavin containing monooxygenase 3 (FMO3) by CCAAT/enhancer-binding protein β (C/EBPβ) liver inhibitory and liver activating proteins
Differential regulation of human hepatic flavin containing monooxygenase 3 (FMO3) by CCAAT/enhancer-binding protein β (C/EBPβ) liver inhibitory and liver activating proteins
复制标题
DOI：
10.1016/j.bcp.2008.05.002
复制
发表时间：
2008-07-15
影响因子：
5.8
通讯作者：
Hines, Ronald N.
中科院分区：
文献类型：
--
作者：
Klick, David E.;Shadley, Jeff D.;Hines, Ronald N.
含黄素单加氧酶 3 (FMO3) 对于氧化外源代谢很重要，但 FMO3 基因的调控仍知之甚少。 FMO3 在 HepG2 细胞中不表达，HepG2 细胞是肝基因调控研究常用的模型。转录因子瞬时表达和组蛋白脱乙酰酶或 DNA 甲基化酶抑制剂治疗发现肝核因子 (HNF) 4 α 水平降低和 DNA 高甲基化是抑制 HepG2 FMO3 表达的机制。转录机制不存在重大缺陷表明，在一定范围内，HepG2 模型适合研究 FMO3 调控。使用 HepG2 细胞和肝组织核蛋白提取物以及报告构建体瞬时表达实验进行 DNA 蛋白结合研究，以表征位置 -494 至 -439（结构域 1）的 FMO3 序列，先前已证明该序列显着影响启动子功能。尽管使用 HepG2 细胞核蛋白观察到 HNF3 β 和 CCAAT 增强子结合蛋白 (C/EBP) 与该元件特异性相互作用，但使用成人肝核蛋白仅观察到 C/EBP DNA-蛋白相互作用。使用胎儿肝核蛋白没有观察到特定的DNA/蛋白质相互作用。假定的 HNF3 β 元件的突变对 FMO3 启动子活性没有影响，而不同但重叠的 C/EBP 元件的突变导致活性降低 55%。此外，通过相同的元件，启动子活性被调节为定义的C/EBPβ肝激活蛋白：肝抑制蛋白比率的函数。染色质免疫沉淀证明 C/EBP beta 与完整细胞和成人肝组织中的 FMO3 结构域 I 元件结合。这些结果与 C/EBP beta 对于调节肝脏 FMO3 表达的重要性一致。 (C) 2008 Elsevier Inc. 保留所有权利。
Flavin-containing monooxygenase 3 (FMO3) is important for oxidative xenobiotic metabolism, but regulation of the FMO3 gene remains poorly understood. FMO3 is not expressed in HepG2 cells, a commonly employed model for hepatic gene regulation studies. Transcription factor transient expression and treatment with histone deacetylase or DNA methylase inhibitors identified decreased hepatic nuclear factor (HNF) 4 alpha levels and DNA hypermethylation as mechanisms suppressing HepG2 FMO3 expression. The absence of major deficiencies in transcriptional machinery suggested that within limits, the HepG2 model is suitable for the study of FMO3 regulation. DNA-protein binding studies with HepG2 cell and hepatic tissue nuclear protein extracts and reporter construct transient expression experiments were performed to characterize FMO3 sequences from position -494 to -439 (domain 1), previously demonstrated to significantly impact promoter function. Although both HNF3 beta and CCAAT enhancer-binding protein (C/EBP) were observed to specifically interact with this element using HepG2 cell nuclear proteins, only C/EBP DNA-protein interactions were observed using adult liver nuclear proteins. No specific DNA/protein interactions were observed using fetal liver nuclear proteins. Mutation of a putative HNF3 beta element had no effect on FMO3 promoter activity, while mutagenesis of a distinct, but overlapping C/EBP element resulted in a 55% reduction in activity. Furthermore, promoter activity was regulated as a function of defined C/EBP beta liver activating protein:liver inhibitory protein ratios through this same element. Chromatin immunoprecipitation demonstrated C/EBP beta binding to the FMO3 domain I element in intact cells and adult liver tissue. These results are consistent with C/EBP beta being important for regulating hepatic FMO3 expression. (C) 2008 Elsevier Inc. All rights reserved.