Tzameli I, Zannis V I
Section of Molecular Genetics, Center for Advanced Biomedical Research, Cardiovascular Institute, Boston University Medical Center, Boston, Massachusetts 02118-2394, USA.
J Biol Chem. 1996 Apr 5;271(14):8402-15. doi: 10.1074/jbc.271.14.8402.
Three proximal regulatory elements, AIB, AIC, and AID, of the apoA-I gene are necessary and sufficient for its hepatic expression in vivo and in vitro. DNA binding and competition assays showed that elements AIB and AID contain hormone response elements composed of imperfect direct repeats that support the binding of the hepatic nuclear factor-4, other nuclear orphan receptors, and the ligand-dependent nuclear receptors retinoic X receptor (RXRalpha), RXRalpha/RARalpha, and RXRalpha/T3Rbeta. Substitution mutations on repeats 1 and 2 in the hormone response sites of elements AIB and AID, respectively, abolished the binding of all nuclear receptors and reduced promoter activity to background levels, indicating the importance of both hormone response elements for the hepatic expression of the apoA-I gene. Cotransfection experiments in HepG2 cells with normal and mutated promoter constructs and plasmids expressing nuclear hormone receptors showed that RXRalpha homodimers transactivated the wild type promoter 150% of control, in the presence of 9-cis-retinoic acid (RA), whereas RXR alpha/T3R beta heterodimers repressed transcription to 60% of control, in the presence of T3. RXR alpha/RAR alpha and hepatic nuclear factor-4 did not affect the transcription, driven by the proximal apoA-I promoter. Potassium permanganate and dimethyl sulfate interference experiments showed that RXRalpha homodimers, RXRalpha/RARalpha, and RXRalpha/T3Rbeta heterodimers participate in protein-DNA interactions with 12, 13, and 11 out of the 14 nucleotides, respectively, that span repeats 1 and 2 and the spacer region separating them on the hormone response element of element AID. The binding of RXRalpha homodimers and RXRalpha/T3Rbeta heterodimers is associated with ligand-dependent activation by 9-cis-RA or repression by T3. Upon deletion or mutation of repeat 1, homodimeric binding of RXRalpha is lost whereas heterodimeric binding is retained. This heterodimeric binding to the mutated element AID is mediated solely by interactions with repeat 2 and one adjacent nucleotide and is confined to a heptameric core recognition motif. The interactions of the RXRalpha heterodimers with repeat 2 are associated with low levels of ligand-independent transcriptional activity. The findings suggest that the specific types of homo- and heterodimers of nuclear hormone receptors occupying the hormone response elements of apoA-I and the availability of the ligand may play an important role in the transcriptional regulation of the human apoA-I gene.
载脂蛋白A-I基因的三个近端调控元件AIB、AIC和AID对于其在体内和体外的肝脏表达是必需且充分的。DNA结合和竞争试验表明，元件AIB和AID含有由不完全直接重复序列组成的激素反应元件，这些元件支持肝细胞核因子-4、其他核孤儿受体以及配体依赖性核受体视黄酸X受体（RXRα）、RXRα/RARα和RXRα/T3Rβ的结合。分别在元件AIB和AID的激素反应位点的重复序列1和2上进行取代突变，消除了所有核受体的结合，并将启动子活性降低到背景水平，表明这两个激素反应元件对于载脂蛋白A-I基因的肝脏表达都很重要。在HepG2细胞中用正常和突变的启动子构建体以及表达核激素受体的质粒进行共转染实验表明，在9-顺式视黄酸（RA）存在的情况下，RXRα同二聚体使野生型启动子的转录激活至对照的150%，而在T3存在的情况下，RXRα/T3Rβ异二聚体将转录抑制至对照的60%。RXRα/RARα和肝细胞核因子-4不影响由近端载脂蛋白A-I启动子驱动的转录。高锰酸钾和硫酸二甲酯干扰实验表明，RXRα同二聚体、RXRα/RARα和RXRα/T3Rβ异二聚体分别与跨越重复序列1和2以及元件AID的激素反应元件上分隔它们的间隔区的14个核苷酸中的12、13和11个核苷酸参与蛋白质-DNA相互作用。RXRα同二聚体和RXRα/T3Rβ异二聚体的结合分别与9-顺式视黄酸的配体依赖性激活或T3的抑制相关。在重复序列1缺失或突变后，RXRα的同二聚体结合丧失，而异二聚体结合得以保留。这种与突变的元件AID的异二聚体结合仅由与重复序列2和一个相邻核苷酸的相互作用介导，并局限于一个七聚体核心识别基序。RXRα异二聚体与重复序列2的相互作用与低水平的非配体依赖性转录活性相关。这些发现表明，占据载脂蛋白A-I激素反应元件的核激素受体的同二聚体和异二聚体的特定类型以及配体的可用性可能在人类载脂蛋白A-I基因的转录调控中起重要作用。
