Sp-1 Binds Promoter Elements That Are Regulated by Retinoblastoma and Regulate CTP:Phosphocholine Cytidylyltransferase-α Transcription

The retinoblastoma (Rb) protein is implicated in transcriptional regulation of at least five cellular genes. Co-transfection of Rb and truncated promoter constructs has defined a discrete element (retinoblastoma control element (RCE)) within the promoters of each of these genes as being necessary for Rb-mediated transcriptional control. In the present report we demonstrate that two RCEs identified within the CTP:phosphocholine cytidylyltransferase-α (CTα) proximal promoter are essential to promote transcription. Mutations that abolished each RCE markedly decreased CTα transcription. Co-transfection of Rb and truncated promoter constructs demonstrated that Rb regulates CTα expression by different mechanisms depending on the phase of the cell cycle. The regulation of CTα expression by Rb required both the Sp1 and the RCEs. Maximal expression occurred when both Rb and Sp1 were overexpressed. Moreover, RCEs were required for Rb association with the DNA. This regulatory mechanism alters CTα activity and thereafter changes PC availability and cell physiology. This is the first report demonstrating not only that surrounding Sp1 binding sites alter regulation mediated by Rb, but also that the expression of a gene involved in PC biosynthesis shares a common regulatory pathway with genes responsible for cell growth and differentiation. The retinoblastoma (Rb) protein is implicated in transcriptional regulation of at least five cellular genes. Co-transfection of Rb and truncated promoter constructs has defined a discrete element (retinoblastoma control element (RCE)) within the promoters of each of these genes as being necessary for Rb-mediated transcriptional control. In the present report we demonstrate that two RCEs identified within the CTP:phosphocholine cytidylyltransferase-α (CTα) proximal promoter are essential to promote transcription. Mutations that abolished each RCE markedly decreased CTα transcription. Co-transfection of Rb and truncated promoter constructs demonstrated that Rb regulates CTα expression by different mechanisms depending on the phase of the cell cycle. The regulation of CTα expression by Rb required both the Sp1 and the RCEs. Maximal expression occurred when both Rb and Sp1 were overexpressed. Moreover, RCEs were required for Rb association with the DNA. This regulatory mechanism alters CTα activity and thereafter changes PC availability and cell physiology. This is the first report demonstrating not only that surrounding Sp1 binding sites alter regulation mediated by Rb, but also that the expression of a gene involved in PC biosynthesis shares a common regulatory pathway with genes responsible for cell growth and differentiation. Phosphatidylcholine (PC) 3The abbreviations used are: PC, phosphatidylcholine; CT; CTP:phosphocholine cytidylyltransferase; FBS, fetal bovine serum; LUC, luciferase; Rb, retinoblastoma protein; RCE, retinoblastoma control element; RCP, retinoblastoma control protein; qPCR, quantitative PCR; CMV, cytomegalovirus; ChIP, chromatin immunoprecipitation. is the major phospholipid in mammalian cells and is a precursor for the synthesis of sphingomyelin and phosphatidylserine. PC biosynthesis occurs in all nucleated mammalian cells via the Kennedy (CDP-choline) pathway in which CTP:phosphocholine cytidylyltransferase (CT) catalyzes the regulated and rate-limiting step (1Vance D.E. Biochem. Cell Biol. 1990; 68: 1151-1165Crossref PubMed Scopus (147) Google Scholar, 2Johnson J.E. Cornell R.B. Mol. Membr. Biol. 1999; 16: 217-235Crossref PubMed Scopus (240) Google Scholar, 3Lykidis A. Jackson P. Jackowski S. Biochemistry. 2001; 40: 494-503Crossref PubMed Scopus (41) Google Scholar). 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We explored the nature of these putative RCEs and their possible role in CTα regulation in C3H10T1/2 mouse embryonic fibroblasts. Materials—The luciferase vector, pGL3-basic, which contains the cDNA for Photinus pyraris luciferase, and the dual-luciferase Reporter Assay System were obtained from Promega (Madison, WI). Lipofectamine and PLUS reagent, Dulbecco's modified Eagle's medium, Schneider's medium, fetal bovine serum, and fetal calf serum were from Invitrogen. Anti-Rb and anti-Sp1 antibodies were from Santa Cruz Bio

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