Transcriptional activation of the murine CTP : phosphocholine cytidylyltransferase gene (Ctpct): combined action of upstream stimulatory and inhibitory cis-acting elements

CTP:phosphocholine cytidylyltransferase plays a key role in regulating the rate of phosphatidylcholine biosynthesis. However, the proximal regulatory elements for the gene (Ctpct) that encode this enzyme and the cognate trans cription factors involved have not been characterized. Ctpct promoter activ ities were deduced from promoter deletion constructs linked to a luciferase reporter and transiently transfected into C3H10T1/2 and McArdle RH7777 cel ls. Positive regulatory elements were located between -130 and -52 bp from the transcription start site. Basal expression resided downstream between - 52 and +38 bp. DNase I protection and electromobility-shift assays indicate d that Sp1-related nuclear factors bind to a stimulatory, a possible inhibi tory and minimal promoter element. Gel-shift assays confirmed that all thre e regulatory regions bound Sp1. Sp1 was further implicated when Sp1-deficie nt Drosophila cells were co-transfected with promoter-reporter constructs a nd an Sp1 construct. DNase I assays also indicated that the Ap1 binding ele ments could be occupied in the proximal activator and minimal promoter regi ons. Gel-shift assays demonstrated that the distal activator region could b ind Ap1 and an unknown transcription factor. We conclude that Sp1, Ap1 and an unknown transcription factor have important roles in regulating expressi on of the Ctpct gene. (C) 1999 Elsevier Science B.V. All rights reserved.