No abstract

The expression of the PDGF β-receptor is tightly regulated during a normal cell cycle. c-Myc and p73α repress transcription of the receptor through interaction with NF-Y. In ST15A cells which stably express the temperature-sensitive SV40 large T antigen (LT) the receptor expression and ligand binding decreased under the permissive condition. Transient expression of the LT, but not small t, decreased the endogenous receptor expression at mRNA and protein levels in NIH3T3 cells but not in the myc-null HO15.19 cells. The wild-type LT, but not the various pRb or p53 binding defective LT mutants, represses the PDGF β-receptor promoter activity. Moreover, the inability of the LT-mediated repression in the myc-null cells, the Rb-null 3T3 cells, and the Saos-2 cells lacking pRb and p53, indicates that Myc, pRb and p53 are all necessary elements. PDGF β-receptor promoter-luciferase assays revealed that the CCAAT motif is important for the repression. Furthermore, p53 was found to increase the promoter activity mainly via the upstream Sp1 binding sites together with the CCAAT motif in the NIH 3T3 cells. This was confirmed by Schneider's Drosophila line (SL2) cells deficient in both endogenous NF-Y and Sp1. Chromatin immunoprecipitation using ST15A cells revealed that both LT and p53 bound the PDGF β-receptor promoter and the binding of p53 diminished when LT was expressed in the permissive condition. However, LT binds the promoter in the absence of pRb and p53 in Saos-2 cells stably expressing LT. These results suggest that LT binds the promoter and interferes with NF-Y and Sp1 to repress it in the presence of Myc, pRb and p53.

We investigated mechanisms of the p73α-mediated repression of the platelet-derived growth factor β-receptor (PDGFRB) promoter caused by its interaction with NF-Y. Treatment of cells with the histone deacetylase (HDAC) inhibitor, Trichostatin A, increases PDGFRB promoter activity through the CCAAT motif and counteracts the repression caused by p73α. Activation of the PDGFRB promoter by the co-activator p300 also occurs through the CCAAT motif. Expression of p73α counteracts both p300- and P/CAF-mediated activation of the PDGFRB promoter, and expression of p300 or P/CAF attenuates the p73α-mediated repression of the promoter activity. In concordance, p73α decreases the p300-mediated acetylation of NF-YC, p300 competes with p73α for binding NF-YB, and P/CAF competes with p73α for binding NF-YB and NF-YC. Furthermore, p73α, but not the oncogenic ΔNp73α, binds directly to HDAC1. We performed chromatin immunoprecipitation with antibodies against p73, ΔNp73, NFYB, p300 and HDAC1 at different periods after serum stimulation in serum-starved NIH3T3 cells. A marked decrease of ΔNp73, NF-YB and p300 was detected 6 hours after serum stimulation when the expression of PDGFRB decreased. Conversely, HDAC1 was found bound at its maximum and the anti-p73 detecting both TAp73 and ΔNp73 was found at all time points, indicating that p73, but not ΔNp73, remains bound at this time. Double immunofluorescence staining of TAp73 and HDAC1 revealed that both of these molecules exist in the nucleus at this time point, supporting the presence of endogenous interaction. These results suggest that p73 and ΔNp73 behave as physiological regulators for the transcription of the PDGFRB promoter.

The mouse PDGF beta-receptor promoter is tightly controlled by NF-Y that binds to a CCAAT box located upstream of the initiation site [1, 2]. In this report, we show that Sp1 plays an essential role in the PDGF beta-receptor transcription. Within the upstream GC rich area there are two Sp1 binding sites located in close proximity to the CCAAT box. Deletion of the GC rich region resulted in a 50% decrease of the transcriptional activity of the promoter, and a complete loss of its responsiveness to over-expression of Sp1. There was an additive effect between NF-Y and Sp I in reporter activity when they were co-transfected together with the promoter-reporter construct. Furthermore, transfection of NF-Y failed to enhance transcriptional activity when the Sp1 binding sites were deleted from the promoter, suggesting an important role for Sp1 in this NF-Y controlled transcription. We have recently reported that c-Myc represses PDGF beta-receptor transcription through its interference with the transactivation activity of NF-Y [3]. In the case of p21(wafl/cip1) transcription, c-Myc was shown to repress its transcription by sequestering Sp1 [4]. However, we could not find any effect of Sp1 in the c-Myc-mediated repression on the PFDGF beta-receptor promoter, since the deletion of SpI binding sites could not attenuate the repression by c-Myc on the promoter activity.