HNF-3β, C/EBPβ, and HNF-4 act in synergy to enhance transcription of the human apolipoprotein B gene in intestinal cells
HNF-3β, C/EBPβ, and HNF-4 act in synergy to enhance transcription of the human apolipoprotein B gene in intestinal cells
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DOI：
10.1089/104454901750070265
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发表时间：
2001-02-01
影响因子：
3.1
通讯作者：
Levy-Wilson, B
中科院分区：
文献类型：
--
作者：
Antes, TJ;Levy-Wilson, B
最近，我们鉴定了一个 315 bp 的肠道增强子 (IE)，位于人类载脂蛋白 B (apoB) 基因转录起始上游超过 55 kb 处，可在小鼠肠道中表达人类 apoB 转基因。肠道富集转录因子肝细胞核因子 (HNF)-3 beta、CAAT 增强子的四个功能结合位点 结合蛋白 (C/EBP)beta 和 HNF-4 在 315 bp IE 中得到证实。在本报告中，我们扩展了这些早期研究，并检查了这三种转录因子对增强子活性的相对贡献以及它们彼此相互作用的机制。使用 HNF-3 β、C/EBP β 和 HNF-4 表达载体的共转染实验表明，HNF-3 β 与位点 1 结合，C/EBP β 与位点 2 结合，HNF-4 与 315 bp IE 内的位点 3 结合，并且这些位点协同作用以增强 apoB 的肠道表达。这四个结合位点中的每一个都发生了突变，并且 将突变构建体转染到肠源性 CaCo-2 细胞中，以评估每个结合位点在增强子活性中的作用。诱变实验的结果证实，HNF-3 β 和 HNF-4 位点对于增强子活性最重要，其次是 C/EBP β 位点 2。与位点 1、2 和 3 结合的所有三个因子必须协同作用，以获得 apoB IE 的最佳活性。
Recently, we identified a 315-bp intestinal enhancer (IE), localized over 55 kb upstream from the transcriptional start of the human apolipoprotein B (apoB) gene, that confers expression of human apoB transgenes in the intestines of mice, Four functional binding sites for the intestine-enriched transcription factors hepatocyte nuclear factor (HNF)-3 beta, CAAT enhancer binding protein (C/EBP)beta, and HNF-4 were demonstrated within the 315-bp IE, In this report, we extend these earlier studies and examine the relative contributions of these three transcription factors to the activity of the enhancer as well as their mechanism of interaction with one another. Cotransfection experiments with the expression vectors for HNF-3 beta, C/EBP beta, and HNF-4 revealed that HNF-3 beta bound to Site 1, C/EBP beta bound to Site 2, and HNF-4 bound to Site 3 within the 315-bp IE and that the sites act synergistically to enhance intestinal expression of apoB, Each one of these four binding sites was mutated, and mutant constructs were transfected into intestine-derived CaCo-2 cells to evaluate the role of each of these binding sites in enhancer activity. The results of the mutagenesis experiments confirmed that the HNF-3 beta and HNF-4 sites are most important for the enhancer activity, followed by C/EBP beta Site 2, All three factors bound to Sites 1, 2, and 3 must act synergistically for optimal activity of the apoB IE.