Down Regulation of DHFR Promoter Activity by PML Transcription Repressor

The PML gene encodes a nuclear protein with transcriptional regulatory activity. It directly interacts with the Sp1 transcription factor and abrogates its activation of the epidermal growth factor receptor (EGFR) gene promoter. Here, the effects of PML on the activity of dihydrofolate reductase (DHFR) promoter, mainly regulated by Sp1, were investigated. On functional analysis, transient transfection of PML into mammalian cells, U2Os and HeLa, resulted in a significant repression of the DHFR promoter. The DHFR promoter also contains binding sites for the E2F transcription factor. When the Sp1 DNA-binding site, but not the E2F-binding site of the promoter, was replaced with an unrelated binding site (GAL4), the repressive effects of PML were lost. Moreover, electrophoretic mobility shift assay (EMSA) using Sp1-containing oligonucleotide probes showed significant reduction in Sp1 binding in the presence of PML, confirming the necessity of the Sp1 DNA-binding sites for PML’s repressive effects. Analysis of DNA synthesis using [3H]thymidine incorporation assay showed a significant reduction in DNA synthesis in HeLa cells overexpressing PML. Together, the data demonstrated that PML could function as a negative regulator of the DHFR promoter, which may represent a novel mechanism for the known repressive effects of PML on cellular growth.