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

CTP:phosphocholine cytidylyltransferase (CCT) is the rate-limiting and regulated enzyme of mammalian phosphatidylcholine biosynthesis. There are three isoforms, CCT alpha, CCT beta1, and CCT beta2. The mouse CCT alpha gene promoter is regulated by an enhancer element (Eb) located between -103 and -82 base pairs (5'-GTTTTCAGGAAT-GCGGAGGTGG-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) (APT)ATG(CIT) (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, CCT alpha promoter-luciferase reporter activity and CCT alpha mRNA levels increased. A TEF-4 deletion mutant containing a DNA-binding domain, mTEA(+), stimulated the CCT alpha 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 CCT alpha gene expression.