转录因子DEC1和DEC2通过Ebox元件下调大鼠Gclc基因的表达

为研究Ebox元件在谷氨酸 半胱氨酸连接酶催化亚基(glutamate cysteine ligase catalytic subunit,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 embryo chondrocyte 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.     

Molecular cloning and characterization of DEC2, a new member of basic helix-loop-helix proteins

DEC1 is a basic helix-loop-helix (bHLH) protein related to Drosophila Hairy, Enhancer of split and HES, and involved in the control of proliferation and/or differentiation of chondrocytes, neurons, etc. We report here the identification and characterization of human, mouse and rat DEC2, a novel member of the DEC subfamily. DEC2 had high (97%) and moderate (52%) similarities in the bHLH region and the Orange domain with DEC1, respectively. However, DEC2, but not DEC1, had alanine and glycine-rich regions in the C-terminal half. Unlike Hairy, Enhancer of split and HES, DEC2 lacked the WRPW motif for interaction with the corepressor Groucho. The DEC2 gene was mapped to human chromosome 12p11.23-p12.1, mouse chromosome 6 G2-G3 and rat chromosome 4q43 distal-q4, where the conserved linkage homology has been identified among these species. Unlike DEC1, which was broadly expressed in many tissues, DEC2 showed a more restricted pattern of mRNA expression. The DEC subfamily proteins may play an important role in tissue development.     

Characterization of adjacent E-box and nuclear factor 1-like DNA binding sequence in the human CYP1A2 promoter

To better understand the molecular mechanisms of cytochrome P450 1A2 (CYP1A2) regulation, we have characterized a region of the promoter (+3 to -176) that contains a single E-box and an adjacent nuclear factor 1 (NF1)-like DNA binding site. The E-box was shown to specifically bind nuclear proteins that were recognized by antibodies against upstream stimulatory factor (USF) 1 and 2. Comparison of NF1 binding proteins in HepG2 cells and primary cultures of rat hepatocytes revealed different patterns of DNA-protein complexes, all of which were recognized by a general NF1 antibody. Mutations of the E-box resulted in substantial reduction of promoter activity in either primary hepatocytes or HepG2 cells regardless of the presence in the reporter constructs of other CYP1A2 regulatory elements, such as the hepatic nuclear factor 1 (HNF-1) binding site. In contrast, reporter gene activity of the promoter construct harboring the mutated NF1-like binding site was affected by upstream sequences when transfected into HepG2 cells, but not in primary hepatocytes. We conclude that both USF proteins and different isoforms of NF1 contribute to the constitutive expression of CYP1A2.