Cardot P, Chambaz J, Kardassis D, Cladaras C, Zannis V I
Department of Medicine, Housman Medical Research Center, Boston University Medical Center, Massachusetts 02118.
Biochemistry. 1993 Sep 7;32(35):9080-93. doi: 10.1021/bi00086a013.
We have shown previously that the hepatic and intestinal transcription of the human apolipoprotein A-II (apoA-II) gene in cell cultures is controlled by a complex set of regulatory elements A-N [Chambaz, et al. (1991) J. Biol. Chem. 266, 11676-11685; Cardot, et al. (1991) J. Biol. Chem. 266, 24460-24470]. In the present communication, we have assessed the functional importance of each of the regulatory elements. In addition, we have used DNA binding and competition assays and protein fractionation to identify the hepatic nuclear activities which are involved in the regulation of the human apoA-II gene. Such activities may be of general importance for the regulation of liver-specific genes. The DNA binding and competition analysis showed that the regulatory elements M, D, and F bind new activities which have not been identified in apolipoprotein or other liver-specific promoters. These activities have been designated AIIM1 and AIIM2 for element M, AIID1 and AIID2 for element D, and AIIF2 for element F. The activity AIIM2 is present in liver, but absent in CaCo-2 cells. A set of regulatory elements binds activities which resemble liver-enriched or ubiquitous factors previously shown to play important roles in the regulation of their target genes. Thus, element I binds to activities related to NF1, and elements L, C, D, G, AB, and F bind to C/EBP alpha as well as other heat-stable activities. The affinity of the bacterially expressed C/EBP alpha for the various apoA-II regulatory regions follows the order: AIIL approximately AIIC > AIID > AIIF > AIIG > AIIAB. Protein fractionation showed that element J binds at least three hepatic nuclear activities and is also recognized by members of the nuclear receptor family, HNF4, EAR2, EAR3, and ARP1. Another liver-enriched factor, HNF1, was shown previously to bind to element H. Despite the importance of HNF1, HNF4, NF1, and C/EBP alpha in the regulation of numerous other target genes, deletion of the HNF1, NF1, and HNF4 and several C/EBP binding sites did not drastically affect the hepatic transcription of the apoA-II gene. Rather, the hepatic and intestinal transcription is affected severely by deletion of elements A, B, K, L, and N. In addition, the intestinal transcription is affected by deletion of elements C, J, and M. The in vivo physiological importance of these elements will require analysis of their function in transgenic animals. This analysis establishes the organization of several nuclear activities on the human apoA-II promoter.(ABSTRACT TRUNCATED AT 400 WORDS)
我们之前已经表明，在细胞培养物中，人类载脂蛋白A-II（apoA-II）基因的肝脏和肠道转录受一组复杂的调控元件A-N控制[尚巴兹等人（1991年）《生物化学杂志》266卷，第11676 - 11685页；卡尔多等人（1991年）《生物化学杂志》266卷，第24460 - 24470页]。在本报告中，我们评估了每个调控元件的功能重要性。此外，我们使用了DNA结合和竞争分析以及蛋白质分级分离来鉴定参与人类apoA-II基因调控的肝细胞核活性。此类活性可能对肝脏特异性基因的调控具有普遍重要性。DNA结合和竞争分析表明，调控元件M、D和F结合了在载脂蛋白或其他肝脏特异性启动子中未鉴定出的新活性。对于元件M，这些活性被命名为AIIM1和AIIM2；对于元件D，为AIID1和AIID2；对于元件F，为AIIF2。活性AIIM2存在于肝脏中，但在CaCo - 2细胞中不存在。一组调控元件结合的活性类似于先前显示在其靶基因调控中起重要作用的肝脏富集或普遍存在的因子。因此，元件I结合与NF1相关的活性，元件L、C、D、G、AB和F结合C/EBPα以及其他热稳定活性。细菌表达的C/EBPα对各种apoA-II调控区域的亲和力顺序如下：AIIL≈AIIC＞AIID＞AIIF＞AIIG＞AIIAB。蛋白质分级分离表明，元件J结合至少三种肝细胞核活性，并且也被核受体家族的成员HNF4、EAR2、EAR3和ARP1识别。另一种肝脏富集因子HNF1先前已被证明与元件H结合。尽管HNF1、HNF4、NF1和C/EBPα在许多其他靶基因的调控中很重要，但缺失HNF1（原文有误，应为HNF1结合元件H，此处应是对原文错误理解，推测是删除结合位点）、NF1和HNF4以及几个C/EBP结合位点并没有严重影响apoA-II基因的肝脏转录。相反，肝脏和肠道转录受到元件A、B、K、L和N缺失的严重影响。此外，肠道转录受到元件C、J和M缺失的影响。这些元件在体内的生理重要性将需要在转基因动物中分析其功能。该分析确定了人类apoA-II启动子上几种核活性的组织方式。（摘要截断于400字）
