Palmer C N, Hsu M H, Griffin H J, Johnson E F
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037, USA.
J Biol Chem. 1995 Jul 7;270(27):16114-21. doi: 10.1074/jbc.270.27.16114.
The gene encoding cytochrome P-450 4A6 (CYP4A6) is transcriptionally activated by peroxisome proliferators. This response is dependent on a strong enhancer element (Z) and weaker elements (X and -27). The peroxisome proliferator response is mediated by the binding of heterodimers containing the peroxisome proliferator-activated receptor alpha (PPAR alpha) and the retinoid X receptor alpha (RXR alpha) to these elements. These peroxisome proliferator response elements (PPREs) contain imperfect direct repeats of the nuclear receptor consensus recognition sequence with a spacing of one nucleotide (DR1) (AGGTCA N AGGTCA). This DR1 motif is seen in the binding sites for other nuclear receptor complexes, such as ARP-1, HNF-4, and RXR alpha homodimers. Mutational analysis of the Z element reveals that the DR1 motif is required for the transcriptional activation of the CYP4A6 gene by peroxisome proliferators; however, deletion of sequences immediately upstream of this motif also abolishes this response. Oligonucleotides corresponding to truncated and mutated Z elements were assayed by gel retardation for binding to RXR alpha, PPAR alpha, and ARP-1. Deletions or mutations within six nucleotides 5' of the DR1 motif dramatically diminish PPAR alpha.RXR alpha binding without reducing the binding of either RXR alpha or ARP-1 homodimers, whereas mutation or deletion of the core DR1 sequences abolishes the binding of PPAR alpha.RXR alpha heterodimers and of RXR alpha or ARP-1 homodimers. Thus, the DR1 motif in the Z element is not sufficient to constitute a PPRE. Moreover, the binding of PPAR alpha.RXR alpha to the Z element requires sequences immediately 5' of the DR1. These sequences are conserved in natural PPREs and promote binding of PPAR alpha.RXR alpha heterodimers in preference to potential competitors such as ARP-1 and RXR alpha.
编码细胞色素P-450 4A6（CYP4A6）的基因可被过氧化物酶体增殖剂转录激活。这种反应依赖于一个强增强子元件（Z）和较弱的元件（X和-27）。过氧化物酶体增殖剂反应是由包含过氧化物酶体增殖物激活受体α（PPARα）和视黄酸X受体α（RXRα）的异二聚体与这些元件的结合介导的。这些过氧化物酶体增殖剂反应元件（PPREs）包含核受体共有识别序列的不完全直接重复，间隔一个核苷酸（DR1）（AGGTCA N AGGTCA）。这种DR1基序存在于其他核受体复合物的结合位点中，如ARP-1、肝细胞核因子-4（HNF-4）和RXRα同二聚体。对Z元件的突变分析表明，DR1基序是过氧化物酶体增殖剂对CYP4A6基因进行转录激活所必需的；然而，删除该基序上游紧邻的序列也会消除这种反应。通过凝胶阻滞试验检测了与截短和突变的Z元件相对应的寡核苷酸与RXRα、PPARα和ARP-1的结合情况。DR1基序5'端六个核苷酸内的缺失或突变会显著减少PPARα.RXRα的结合，而不会降低RXRα或ARP-1同二聚体的结合，而核心DR1序列的突变或缺失则会消除PPARα.RXRα异二聚体以及RXRα或ARP-1同二聚体的结合。因此，Z元件中的DR1基序不足以构成一个PPRE。此外，PPARα.RXRα与Z元件的结合需要DR1 5'端紧邻的序列。这些序列在天然PPREs中是保守的，并且优先促进PPARα.RXRα异二聚体的结合，而不是像ARP-1和RXRα这样的潜在竞争者。
