Sugimoto H, Bakovic M, Yamashita S, Vance D E
Department of Biochemistry, Gunma University School of Medicine, Maebashi 371-8511, Japan.
J Biol Chem. 2001 Apr 13;276(15):12338-44. doi: 10.1074/jbc.M100090200. Epub 2001 Jan 12.
phosphocholine cytidylyltransferase (CCT) is the rate-limiting and regulated enzyme of mammalian phosphatidylcholine biosynthesis. There are three isoforms, CCTalpha, CCTbeta1, and CCTbeta2. The mouse CCTalpha gene promoter is regulated by an enhancer element (Eb) located between -103 and -82 base pairs (5'-GTTTTCAGGAATGCGGAGGTGG-3') upstream from the transcriptional start site (Bakovic, M., Waite, K., Tang, W., Tabas, I., and Vance, D. E. (1999) Biochim. Biophys. Acta 1436, 147-165). To identify the Eb-binding protein(s), we screened a mouse embryo cDNA library by the yeast one-hybrid system and obtained 19 positive clones. Ten cDNA clones were identified as transcriptional enhancer factor-4 (TEF-4). The TEF-binding consensus sequence, 5'-(A/T)(A/G)(A/G)(A/T)ATG(C/T)(G/A)-3', was identified within the Eb binding region. Gel-shift analysis using radiolabeled Eb fragment as a probe showed that cell extracts from yeast expressing hemagglutinin-tagged TEF-4 caused a marked band retardation that could be prevented with an anti-hemagglutinin antibody. When COS-7 cells were transfected with TEF-4, CCTalpha promoter-luciferase reporter activity and CCTalpha mRNA levels increased. A TEF-4 deletion mutant containing a DNA-binding domain, mTEA(+), stimulated the CCTalpha promoter activity, whereas protein lacking the DNA binding domain, mTEA(-), did not. Unexpectedly, when the ATG core of the TEF-4 binding consensus within the Eb region was mutated, promoter activity was enhanced rather than decreased. Thus, TEF-4 might act as a dual transcriptional modulator as follows: as a suppressor via its direct binding to the Eb element and as an activator via its interactions with the basal transcriptional machinery. These results provide the first evidence that TEF-4 is an important regulator of CCTalpha gene expression.
磷酸胆碱胞苷转移酶（CCT）是哺乳动物磷脂酰胆碱生物合成中的限速酶且受其调控。CCT有三种亚型，即CCTα、CCTβ1和CCTβ2。小鼠CCTα基因启动子受位于转录起始位点上游-103至-82碱基对（5'-GTTTTCAGGAATGCGGAGGTGG-3'）之间的增强子元件（Eb）调控（巴科维奇，M.，韦特，K.，唐，W.，塔巴斯，I.，以及万斯，D.E.（1999年）《生物化学与生物物理学报》1436，147 - 165）。为鉴定与Eb结合的蛋白，我们通过酵母单杂交系统筛选了小鼠胚胎cDNA文库，获得19个阳性克隆。10个cDNA克隆被鉴定为转录增强因子-4（TEF-4）。在Eb结合区域内鉴定出TEF结合共有序列5'-(A/T)(A/G)(A/G)(A/T)ATG(C/T)(G/A)-3'。使用放射性标记的Eb片段作为探针进行凝胶迁移分析表明，表达血凝素标记的TEF-4的酵母细胞提取物导致明显的条带阻滞，而这种阻滞可被抗血凝素抗体阻止。当用TEF-4转染COS-7细胞时，CCTα启动子-荧光素酶报告基因活性和CCTα mRNA水平升高。包含DNA结合结构域的TEF-4缺失突变体mTEA(+)刺激了CCTα启动子活性，而缺乏DNA结合结构域的蛋白mTEA(-)则没有。出乎意料的是，当Eb区域内TEF-4结合共有序列的ATG核心发生突变时，启动子活性增强而非降低。因此，TEF-4可能作为一种双重转录调节因子，如下所述：通过其与Eb元件的直接结合作为抑制因子，以及通过其与基础转录机制的相互作用作为激活因子。这些结果首次证明TEF-4是CCTα基因表达的重要调节因子。
