Cohesin protein SMC1 represses the nuclear receptor CAR-mediated synergistic activation of a human P450 gene by xenobiotics
Cohesin protein SMC1 represses the nuclear receptor CAR-mediated synergistic activation of a human P450 gene by xenobiotics
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DOI：
10.1042/bj20060109
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发表时间：
2006-08-15
影响因子：
4.1
通讯作者：
Negishi, Masahiko
中科院分区：
文献类型：
--
作者：
Inoue, Kaoru;Borchers, Christoph H.;Negishi, Masahiko
CAR（组成型活性/雄烷受体）调节远端增强子PBREM（苯巴比妥响应增强子模块）和近端元件OARE [OA（冈田酸）响应元件]，以协同上调HepG 2细胞中的内源性CYP 2B 6（其中OARE是细胞色素P450）基因。在这种上调中，CAR充当转录因子和共调节因子，在被外源性物质如TCPOBOP（1，4-双-[2 -（4-甲基-1，4-二氧戊环-1，4-氧戊环-（3，5-二氯吡啶氧基）]苯}并响应于OA而与OARE间接缔合[Swales，Kakizaki，Yamamoto，Inoue，小林和Negishi（2005）J.Biol.Chem.280，34583466]。我们现在已经确定了粘附蛋白SMC 1（染色体1的结构维持）作为CAR结合蛋白，并将其表征为OARE活性的负调节因子，从而抑制协同作用。SMC 1小干扰RNA处理增强了HepG 2细胞中CYP 2B 6表达的协同上调20倍，而SMC 1剪接形式的瞬时共表达废除了1.8 kb CYP 2B 6启动子的协同激活。SMC 1间接结合CYP 2B 6启动子中OARE下游紧邻的19 bp序列（-236/-217）。DNA亲和性和染色质免疫沉淀分析均显示OA处理使SMC 1与CYP 2B 6启动子解离，使CAR与OARE的间接结合往复。这些结果与以下结论一致：SMC 1结合抑制OARE活性，其解离允许CAR募集至OARE，协同PBREM活性和CYP 2B 6基因表达。
CAR (constitutive active/androstane receptor) regulates both the distal enhancer PBREM (phenobarbital-responsive enhancer module) and the proximal element OARE [OA (okadaic acid) response element] to synergistically up-regulate the endogenous CYP2B6 (where CYP is cytochrome P450) gene in HepG2 cells. In this up-regulation, CAR acts as both a transcription factor and a co-regulator, directly binding to and enhancing PBREM upon activation by xenobiotics such as TCPOBOP {1,4-bis-[2 -(3,5-dichloropyridyloxy)]benzene} and indirectly associating with the OARE in response to OA [Swales, Kakizaki, Yamamoto, Inoue, Kobayashi and Negishi (2005) J. Biol. Chem. 280, 34583466]. We have now identified the cohesin protein SMC1 (structural maintenance of chromosomes 1) as a CAR-binding protein and characterized it as a negative regulator of OARE activity, thus repressing synergy. Treatment with SMC1 small interfering RNA augmented the synergistic up-regulation of CYP2B6 expression 20-fold in HepG2 cells, while transient co-expression of spliced form of SMC1 abrogated the synergistic activation of a 1.8 kb CYP2B6 promoter. SMC1 indirectly binds to a 19 bp sequence (-236/-217) immediately downstream from the OARE in the CYP2B6 promoter. Both DNA affinity and chromatin immunoprecipitation assays showed that OA treatment dissociates SMC1 from the CYP2B6 promoter, reciprocating the indirect binding of CAR to OARE. These results are consistent with the conclusion that SMC1 binding represses OARE activity and its dissociation allows the recruitment of CAR to the OARE, synergizing PBREM activity and the expression of the CYP2B6 gene.