Purification and characterization of nuclear factors binding to the negative regulatory element D of human apolipoprotein A-II promoter: a negative regulatory effect is reversed by GABP, an Ets-related protein.
Purification and characterization of nuclear factors binding to the negative regulatory element D of human apolipoprotein A-II promoter: a negative regulatory effect is reversed by GABP, an Ets-related protein.
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与人载脂蛋白 A-II 启动子负调控元件 D 结合的核因子的纯化和表征：Ets 相关蛋白 GABP 逆转负调控效应。
DOI：
10.1021/bi00206a017
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Chambaz,J
中科院分区：
文献类型：
--
作者：
Cardot,P;Pastier,D;Lacorte,JM;Mangeney,M;Zannis,VI;Chambaz,J
1994年7月19日收到的修订版Mandot摘要：我们以前已经表明，人载脂蛋白A-II（apoA-II）基因的转录是由一组复杂的调控元件控制的[Cardot et al.（1993）Biochemistry 32，9080-9093]。我们还鉴定了先前描述的以及与这些元件结合并在不同程度上影响apoA-II基因转录的新活性。DNA结合和竞争分析表明，元件D结合三个新的活动，指定AUDI，AIID 2和AIID 4，以及与C/EBP相关的活动。为了进一步研究其对人apoA-II基因转录调控的作用，对活性AUDI、AIID 2和AIID 4进行了纯化和鉴定。亲和纯化的AIID 2的SDS-PAGE分析以及光亲和交联表明，它由三种蛋白质组成，分子量范围在54和63 kDa之间。从AIID 2蛋白条带中获得的胰蛋白酶肽的氨基酸序列显示，它与Ets相关蛋白GABP同源。类似的分析表明，亲和纯化的AIID 4具有130 kDa的表观分子量。AUDI活性被部分纯化;除了与apoA-II启动子结合外，AUDI还与apoCIII的调控元件C结合，并可能在两个基因的转录调控中发挥作用。G残基的甲基化干扰和T残基的高锰酸盐修饰表明，AIID 2和AIID 4的结合位点在元件D上是连续的。然而，AUDI的结合位点与AIID 2和AIID 4的结合位点重叠。这表明AUDI和AIID 2或AUDI和AIID 4的结合可能是相互排斥的，而AIID 2和AIID 4可能同时结合。当元件D缺失时，从含有apoA-II的元件AB、C和D的最小启动子的转录增加1.5至1.6倍，以及通过启动子突变消除AUDI和/或AIID 4与元件D的结合，但允许AIID 2/GABP的结合。结果表明，当D元件被蛋白AUDI和/或AIID 4占据时，对apoA-II基因的转录具有负调控作用。当元件D仅被调节因子AIID 2/GABP占据时，这种负效应被逆转。
Revised Manuscript Received July 19, 1994® abstract: We have previously shown that transcription of the human apolipoprotein A-II (apoA-II) gene is controlled by a complex set of regulatory elements [Cardot et al.(1993) Biochemistry 32, 9080-9093]. We have also identified previously described, as well as new activities which bind to these elements and influence to varying degrees the transcription of the apoA-II gene. DNA binding and competition assays indicated that element D binds three new activities, designated AUDI, AIID2, and AIID4, as well as an activity related to C/EBP. Activities AUDI, AIID2, and AIID4 were purified and characterized further in order to determinetheir function on the transcriptional regulation of human apoA-II gene. SDS-PAGE analysis as well as photoaffinity cross-linking of the affinity-purified AIID2 showed that it consists of three proteins with molecular masses ranging between 54 and 63 kDa. The amino acid sequence of tryptic peptides obtained from AIID2 protein bands revealed that it is homologous to GABP, an Ets-related protein. Similar analysis showed that affinity-purified AIID4 has an apparent molecular mass of 130 kDa. AUDI activity was purified partially; in addition to binding to the apoA-II promoter, AUDI also binds to the regulatory elementC of apoCIII and may play a role in the transcriptional regulation of both genes. Methylation interference of G residues and permanganate modification of T residues indicated that the binding sites of AIID2 and AIID4 were contiguous on element D. However, the binding site of AUDI overlaps with the binding sites of both AIID2 and AIID4. This suggests that the binding of AUDI andAIID2 or of AUDI and AIID4 may be mutually exclusive, whereas AIID2 and AIID4 may bind simultaneously. Transcription from a minimal promoter containing elements AB, C, and D of apoA-II increased 1.5-to 1.6-fold when element D is deleted, as well as by promoter mutations which eliminated the binding of both AUDI and/or AIID4 to element D, but permitted the binding of AIID2/GABP. The findings suggest that element D has a negative regulatory role on apoA-II gene transcription when it is occupied by protein AUDI and/orAIID4. This negative effect is reversedwhen element D is occupied only by the regulatory factor AIID2/GABP.