GCLC基因上游AHR/ARNT元件负性调控GCLC基因在大鼠支气管上皮细胞的表达

研究谷氨酰半胱氨酸合成酶催化亚单位(GCLC)基因上游调控序列中2个AHR/ARNT元件的功能,从而了解γ-谷氨酰半胱氨酸合成酶(γ-GCS)基因转录调节特征．分别构建缺失2个位点AHR/ARNT元件的GCLC基因上游近端序列的萤光素酶报道基因载体以及含有2个AHR/ARNT元件核心序列的萤光素酶报道基因载体．转染大鼠支气管上皮细胞(RTE),比较检测野生与缺失报道载体的基因转录调控效率;利用电泳迁移率变动实验（EMSA）和超级迁移率变动实验检测AHR/ARNT元件与AHR以及ARNT因子的特异性结合;通过转染AHR因子真核表达质粒进一步确定AHR/ARNT元件与AHR结合在GCLC基因表达中的最终作用．结果显示,相比其野生序列,缺失AHR/ARNT元件（-1 090～-1 085）和双缺失AHR/ARNT元件（-1 090～-1 085,-215～-210）的GCLC上游调控序列报道载体在RTE显著提高萤光素酶表达（均P＜0.05）,而缺失AHR/ARNT元件（-215～-210）则未见显著影响（P＞0.05）; 独立AHR/ARNT元件（-1 090～-1 085）具有转录促进作用（P＜0.05）而独立AHR/ARNT元件（-215～-210）无明显影响（P＞0.05）．转染CMV2-AHR能够抑制野生型和缺失型报道载体的萤光素酶表达（P＜0.05）．EMSA证实GCLC基因上游调控区域的2个AHR/ARNT元件均有核蛋白结合,并且超级迁移率变动实验显示结合的蛋白主要含有转录因子AHR以及ARNT．因此,2个AHR/ARNT元件均可以与异源二聚体AHR/ARNT结合,AHR/ARNT元件（-1 090～-1 085）是GCLC基因中重要的抑制元件．     

Characterization of the aryl hydrocarbon receptor repressor gene and association of its Pro185Ala polymorphism with micropenis

BACKGROUND: Genetic background of a fetus contributes to the abnormal development after teratogen exposure. In rodents, in utero exposure to dioxins affects male external genital development. The effects of dioxins are mediated via the aryl hydrocarbon receptor (AHR) and its binding protein, aryl hydrocarbon receptor nuclear translocator (ARNT). In mice, aryl hydrocarbon receptor repressor (AHRR), which binds to ARNT in competition with AHR, plays a critical negative regulatory role in AHR signaling. We attempt to characterize the human AHRR gene and investigate the relationship between AHRR polymorphisms and the incidence of micropenis, a phenotype of undermasculinization. METHODS: We identified and characterized the human homolog of mouse AHRR, taking advantage of the publicly available draft version of the human genome sequence. After detecting an AHRR protein polymorphism by the direct sequencing of pooled human genomic DNA, we evaluated the association between the polymorphism and the presence or absence of micropenis (< -2.5 SD) in patients with micropenis and control subjects. RESULTS: The deduced sequence for human AHRR (715 residues) and the mouse AHRR protein exhibited 81% sequence homology to each other. The Pro185Ala polymorphism was identified between the PAS-A region and the highly conserved arginine/cysteine-rich RCFRCRL/VRC region. Forty-six percent (27/59) of patients with micropenis and 27% (22/80) of the controls were homozygous for 185Pro; this difference in frequencies was significant (P = 0.03). CONCLUSIONS: Homozygosity for the 185Pro allele of AHRR may increase the susceptibility of a fetus to the undermasculinizing effects of dioxin exposure in utero, presumably through the diminished inhibition of AHR-mediated signaling.     

Conditional disruption of the aryl hydrocarbon receptor nuclear translocator (Arnt) gene leads to loss of target gene induction by the aryl hydrocarbon receptor and hypoxia-inducible factor 1alpha

To determine the function of the aryl hydrocarbon receptor nuclear translocator (ARNT), a conditional gene knockout mouse was made using the Cre-loxP system. Exon 6, encoding the conserved basic-helix-loop-helix domain of the protein, was flanked by loxP sites and introduced into the Arnt gene by standard gene disruption techniques using embryonic stem cells. Mice homozygous for the floxed allele were viable and had no readily observable phenotype. The Mx1-Cre transgene, in which Cre is under control of the interferon-gamma promoter, was introduced into the Arnt-floxed mouse line. Treatment with polyinosinic-polycytidylic acid to induce expression of Cre resulted in complete disruption of the Arnt gene and loss of ARNT messenger RNA (mRNA) expression in liver. To determine the role of ARNT in gene control in the intact animal mouse liver, expression of target genes under control of an ARNT dimerization partner, the aryl hydrocarbon receptor (AHR), was monitored. Induction of CYP1A1, CYP1A2, and UGT1*06 mRNAs by the AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin was absent in livers of Arnt-floxed/Mx1-Cre mice treated with polyinosinic-polycytidylic. These data demonstrate that ARNT is required for AHR function in the intact animal. Partial deletion of the Arnt allele was found in kidney, heart, intestine, and lung. Despite more than 80% loss of the ARNT expression in lung, maximal induction of CYP1A1 was found, indicating that the expression level of ARNT is not limiting to AHR signaling. Cobalt chloride induction of the glucose transporter-1 and heme oxygenase-1 mRNAs was also markedly abrogated in mice lacking ARNT expression, suggesting an inhibition of HIF-1alpha activity. These studies establish a critical role for ARNT in AHR and HIF-1alpha signal transduction in the intact mouse.