转录因子DEC1和DEC2通过Ebox元件下调大鼠Gclc基因的表达

DEC1 and DEC2 Down-regulate the Expression of Rat Gclc through Ebox Element

为研究Ebox元件在谷氨酸-半胱氨酸连接酶催化亚基(glutamate-cysteine ligase catalyticsubunit,GCLC)基因表达中的地位,构建含Gclc上游5.9 kb调控序列,及突变Ebox(-3 853～-3 848)的萤火虫荧光素酶报道载体.转染大鼠Ⅱ型肺泡上皮细胞(L2),比较野生与突变报道载体的转录活性.共转染野生型载体与转录因子分化型胚胎软骨发育基因1/2(differentiated embryochondrocyte expressed gene1/2,DEC1/2)真核表达载体,检测DEC1/2对Gclc转录活性的影响;电泳迁移率(electrophoretic mobility shift assays,EMSA)和超级迁移率实验(supershift assay)检测Ebox元件是否与DEC1/2特异结合.蛋白免疫印迹技术检测Dec1/2过表达对Gclc表达的影响.结果显示,载体构建符合预期;突变Ebox元件可显著上调Gclc荧光素酶活性(P<0.01);共转染DEC1/2表达载体显著下调Gclc荧光素酶活性(P<0.01);EMSA证实Ebox元件(-3 853～-3 848)与核蛋白结合,且特异性强;超级迁移率显示,结合的核蛋白有转录因子DEC1、DEC2;Western印迹结果显示,DEC1/2的表达明显下调Gclc的内源性表达.结果提示,转录因子DEC1与DEC2具有Gclc表达抑制活性,可能与Ebox(-3 853～-3 848)有关.

To investigate the role of Ebox element in the expression of rat glutamate-cysteine ligase catalytic subunit （GCLC） , a luciferase reporter vector containing rat GCLC 5.9 kb proximal regulatory sequence and a reporter vector mutated the Ebox （ -3 853 - -3 848） element were constructed. Both vectors were transfected into rat lung alveolar type Ⅱ ceils （ L2 ） to determine the relative contribution of Ebox on Gclc transcriptional activity. The GCLC-LUC plasmids and plasmids expressing differentiated embryo-chondrocyte expressed genel/2 （DEC1/2）, which supposed to bind Ebox element, were transiently co-transfected into L2 cells along with a calibration reporter pRL-SV40 to observe whether DEC1 or DEC2 effects its transcription. Eleetrophoretic mobility shift assays （EMSA） and supershift assays were used to confirm the specific binding of the transcription factor DEC1/2 to the Ebox element. The expression level of GCLC after DEC1 and DEC2 expression was analyzed by Western blotting. Theresults showed that plasmids were successfully constructed. The mutation of Ebox （ -3853 - -3848） significantly increased the lueiferase activity compared with the wild-type Ebox （P 〈 0.01 ）. The luciferase activity was significantly decreased after co-transfected with the DEC1 or DEC2 expression plasmid （P 〈 0.01 ）. EMSA showed that the Ebox element （ - 3 853 - 3 848） binds nuclear proteins in L2 cells. Supershift assay confirmed that the transcription factor DEC1 and DEC2 were the proteins binding to the Ebox. Western blotting showed that both DEC1 and DEC2 expression reduce the expression level of GCLC. We concluded that the transcription factor DEC1 and DEC2 down-regulate the expression of rat GCLC, and that was possibly related to Ebox element （ - 3 853 - - 3 848）.