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

Susanne Lingrell,Claudia Banchio,Dennis E. Vance

doi:10.1074/jbc.m700527200

Abstract

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-alpha (CTalpha) proximal promoter are essential to promote transcription. Mutations that abolished each RCE markedly decreased CTalpha transcription. Co-transfection of Rb and truncated promoter constructs demonstrated that Rb regulates CTalpha expression by different mechanisms depending on the phase of the cell cycle. The regulation of CTalpha 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 CTalpha 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.

Highlights

Pathway in which CTP:phosphocholine cytidylyltransferase (CT) catalyzes the regulated and rate-limiting step [1,2,3]
At the level of gene expression, CT␣ mRNA has been shown to increase after growth factor stimulation [15], during liver development [16], in proliferating liver tissue following partial hepatectomy [17], and during the S phase of the cell cycle [18]
retinoblastoma gene product (Rb) regulates the expression of several genes through cis-acting elements in a cell typedependent manner through the retinoblastoma control element (RCE), which is present in some genes responsible for cell growth and differentiation [22]

Summary

EXPERIMENTAL PROCEDURES

Materials—The luciferase vector, pGL3-basic, which contains the cDNA for Photinus pyraris luciferase, and the dual-. Cell Culture—C3H10T1/2 mouse embryo fibroblasts and human osteosarcoma (SaOs-2) cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with penicillin G (100 units/ml), streptomycin (100 ␮g/ml), and 10 –15% fetal bovine serum in a 5% CO2 humidified incubator at 37 °C. Schneider SL2 cells were cultured in Schneider’s medium supplemented with 10% heat-inactivated fetal calf serum at room temperature. Transient transfections with CT␣ promoter-luciferase reporter plasmids containing mutations that alter transcription factor binding, LUC.C7 (Ϫ1268/ϩ38), LUC.C7delRCE1, LUC.C7delRCE2, LUC.C7delSp1B, and LUC.C7delSp1C (1 ␮g), were performed using a cationic liposome method [30]. For each binding reaction (40 ␮l), 1 ␮g of poly(dI-dC)-poly(dI-dC), 20 ␮l of 2ϫ binding buffer (100 mM Tris-HCl, pH 7.4), 10 mM MgCl2, 250 mM NaCl, 5 mM EDTA, 50% glycerol, 0.5% Nonidet P-40, 5 mM dithiothreitol), 1 ␮g of nuclear extract, and labeled probe (20,000 cpm) were incubated for 30 min at room temperature.

Mutations That Alter the RCEs

Findings

RESULTS