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．     

Identification of novel splice variants of ARNT and ARNT2 in the rat

Most of the biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated by the bHLH/PAS protein AH receptor (AHR). For regulation of gene activities, AHR dimerizes with another member of the bHLH/PAS protein family, AHR nuclear translocator (ARNT). A substrain of Wistar rats, Han/Wistar (Kuopio) (H/W), is about 1000-fold more resistant to the acute lethality of TCDD than other strains, exemplified by Long-Evans (Turku/AB) (L-E); the LD50 values for these two strains are >9600 and 10-20 microg/kg, respectively. Previous studies have demonstrated that the major reason for the exceptional TCDD resistance of H/W rats lies in their AHR, which is remodeled at its C-terminal transactivation domain, but there appears to be another contributing gene product. The present study set out to compare the primary structure of ARNT and the closely related ARNT2 proteins in H/W and L-E rats by cDNA cloning. To our surprise, we found several isoforms of these proteins only one of which has previously been reported in rats. All of the isoforms appeared to arise from alternative splicing. For ARNT, isoforms with deletions at exon 5, 3(') end of exon 6 or 5(') end of exon 11, or with an insertion at 5(') end of exon 20 were discovered. There was also interindividual variation in the number of glutamine-encoding codons at 5(') end of exon 16. The most exciting new variant was revealed for ARNT2, because the insertion found at 5(') end of exon 19 disrupts the functionally critical transactivation domain in the protein, implying a dominant negative role for this isoform. The relative expression levels of the variants did not differ in the two rat strains, nor did TCDD modify the ratios, suggesting that the variants do not contribute to TCDD resistance. However, the regulation of ARNT and ARNT2 activities may be more intricate than previously assumed.