Identification of transcriptional enhancer factor-4 as a transcriptional modulator of CTP : phosphocholine cytidylyltransferase alpha

CTP:phosphocholine cytidylyltransferase (CCT) is the rate-limiting and regu lated enzyme of mammalian phosphatidylcholine biosynthesis. There are three isoforms, CCT alpha, CCT beta1, and CCT beta2. The mouse CCT alpha gene pr omoter is regulated by an enhancer element (Eb) located between -103 and -8 2 base pairs (5'-GTTTTCAGGAAT-GCGGAGGTGG-3') upstream from the transcriptio nal 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-hybri d system and obtained 19 positive clones. Ten cDNA clones were identified a s transcriptional enhancer factor-4 (TEF-4). The TEF-binding consensus sequ ence, 5'-(A/T)(A/G)(A/G) (APT)ATG(CIT) (G/A)-3', was identified within the Eb binding region. Gel-shift analysis using radiolabeled Eb fragment as a p robe showed that cell extracts from yeast expressing hemagglutinin-tagged T EF-4 caused a marked band retardation that could be prevented with an anti- hemagglutinin antibody. When COS-7 cells were transfected with TEF-4, CCT a lpha promoter-luciferase reporter activity and CCT alpha mRNA levels increa sed. A TEF-4 deletion mutant containing a DNA-binding domain, mTEA(+), stim ulated the CCT alpha promoter activity, whereas protein lacking the DNA bin ding 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 transcripti onal modulator as follows: as a suppressor via its direct binding to the Eb element and as an activator via its interactions with the basal transcript ional machinery. These results provide the first evidence that TEF-4 is an important regulator of CCT alpha gene expression.