Kinetics of repression by modified p53 on the PDGF β‐receptor promoter

Abstract

Herein, we show that both exogenously transfected and endogenously activated p53 repress promoter activity and expression of PDGFRB. p53 binds the proximal promoter containing the CCAAT motif as examined by EMSA and chromatin immunoprecipitation. However, gradual induction of p53 in tet-onSAOS2 cells resulted in a transient increase of the PDGFRB-promoter activity and its expression. As binding of p53 to the promoter increased, previously bound p73, DNp73, c-Myc, HDAC1 and HDAC4 were dismissed from the repressed promoter, and p300 was recruited. The transient increase of the promoter activity was therefore induced by the release of the p73, Myc and HDACs, previously shown to act as repressors to this promoter. Along with further increase of p53, p300 was replaced by HDAC1 and HDAC4, resulting in decreased PDGFRB expression. For the repression, acetylation of the C-terminal lysines of p53 is important, and both acetyl-K373p53 and methyl-K370p53 became bound to the promoter. The acetyl-K373p53 was accumulated in the nucleus and colocalized with promyelocytic leukemia protein.Mitomycin treatment of MEF induced similar epigenetic modification of p53 and its binding to the promoter chromatin. Addition of a PDGFR tyrosine-kinase inhibitor to p53-inducing tet-onSAOS2 increased the number of apoptotic cells. These results suggest that p53 represses the PDGFRB promoter, facilitating the p53-induced apoptosis, whereas tumor cells with p53 mutation or a high level of DNp73 or Myc could become refractory to the regulation.

p19Arf Represses Platelet-Derived Growth Factor Receptor β by Transcriptional and Posttranscriptional Mechanisms

“…We demonstrated that increased binding of p53 to the proximal Pdgfr␤ promoter (but not the distal promoter or intron) was evident when Arf was expressed ectopically in 10T1/2 cells (Widau and Skapek, unpublished data). This is in agreement with a report by Yang and colleagues revealing a putative p53-binding element in the proximal Pdgfr␤ promoter in mitomycin C (MMC)-treated MEFs (63). However, we were not able to detect p53 binding to the Pdgfr␤ promoter in wild-type MEFs with or without endogenous p19…”

“…The findings generally support the concept that p53 activated by genotoxic exposure to mitomycin C represses the Pdgfr␤ promoter in MEFs by recruiting histone deacetylases 1 and 4 (63). Defining a role for p53 is further complicated by the choreographed changes in the binding of various isoforms of the p53-related p73 protein, which can also influence this promoter (13).…”

“…Defining a role for p53 is further complicated by the choreographed changes in the binding of various isoforms of the p53-related p73 protein, which can also influence this promoter (13). Despite this complexity, one consistent theme holds, i.e., both p53 and p73 impede the activity of the NF-Y transcription factor complex, which binds to the CCAAT element in the proximal Pdgfr␤ promoter (13,63). Myc represents another Pdgfr␤ repressor that acts in concert with NF-Y and the CCAAT element in the promoter (22,40), and as mentioned above, p19…”

“…This suggests that mechanisms controlling the basal Pdgfr␤ protein level in cultured MEFs likely include additional posttranscriptional events (discussed further below). Given these findings, we considered whether Arf-dependent transcriptional repression depended on p53, which has been implicated as a direct regulator of Pdgfr␤ transcription (63). As our first approach, we focused on Arf lacZ/lacZ MEFs, which were stably transduced with a retrovirus driving the expression of an shRNA that represses p53 (6) or with a control.…”

“…We designed ChIP primers to amplify regions in the distal (positions Ϫ1353 to Ϫ1011) and proximal (positions Ϫ206 to ϩ2) promoters and in the first intron (positions ϩ1054 to ϩ1188) of the mouse Pdgfr␤ locus (Fig. 6A), modeled after regions amplified in previous work (63). We demonstrated that increased binding of p53 to the proximal Pdgfr␤ promoter (but not the distal promoter or intron) was evident when Arf was expressed ectopically in 10T1/2 cells (Widau and Skapek, unpublished data).…”