EAPP: A Modulator of E2F and p53 Dependent Transcription

E2F is a family of heterodimeric transcription factors regulating the expression of genes, whose products are essential for progression through the mammalian cell cycle. Moreover, it integrates cell-cycle progression with transcription through its cyclical interactions with important growth and cell cycle regulators, such as the retinoblastoma-tumor-suppressor-gene product (pRB), cyclins and cyclin dependent kinases. The activity of the E2F transcription factors is regulated by a multitude of mechanisms, often involving protein-protein interactions. Over-expression of E2F-1 can induce apoptosis via p53 dependent and independent pathways. E2F also links the pRB and p53 tumor suppressor networks by inducing the expression of p14ARF , which interferes with MDM2 mediated ubiquitylation of p53. In a yeast two hybrid screen we have identified a hitherto uncharacterized, E2F- binding protein. This protein is strongly phosphorylated and consequently we named it EAPP (E2F Associated PhophoProtein). Protein levels of EAPP are often elevated in transformed cells, indicating that it is growth promoting. Over-expression results in a higher fraction of S-phase cells and in apoptosis. Co-expression of EAPP with E2F-1 strongly enhanced several examined E2F-dependent promoters, but surprisingly it repressed the activity of the p14ARF promoter. Moreover, EAPP turned out to be a p53 binding protein, and this interaction might play a role in the regulation of the p14ARF promoter. In this project we will analyze the role of EAPP as a modulator of transcription in more detail. We will examine EAPP complexes by co-immunoprecipitation and chromatin immunoprecipitation experiments. With transformation assays we will investigate a possible role in the malignant transformation of a cell. To study over- expression and absence of EAPP we will create cell lines with inducible EAPP expression or inducible EAPP knock down. To characterize EAPP biochemically, we will map its nuclear localization signal and the binding sites for E2F-1 and p53, search for sequences responsible for mitotic destruction, and identify and mutate acetylation sites. Finally, with DNA microarrays we will perform a systematical analysis of gene expression of EAPP knock down cells compared to normal cells.

E2F is a family of heterodimeric transcription factors regulating the expression of genes, whose products are essential for progression through the mammalian cell cycle. Moreover, it integrates cell-cycle progression with transcription through its cyclical interactions with important growth and cell cycle regulators, such as the retinoblastoma-tumor-suppressor-gene product (pRB), cyclins and cyclin dependent kinases. The activity of the E2F transcription factors is regulated by a multitude of mechanisms, often involving protein-protein interactions. Over-expression of E2F-1 can induce apoptosis via p53 dependent and independent pathways. E2F also links the pRB and p53 tumor suppressor networks by inducing the expression of p14ARF , which interferes with MDM2 mediated ubiquitylation of p53. In a yeast two hybrid screen we have identified a hitherto uncharacterized, E2F- binding protein. This protein is strongly phosphorylated and consequently we named it EAPP (E2F Associated PhophoProtein). Protein levels of EAPP are often elevated in transformed cells, indicating that it is growth promoting. Over-expression results in a higher fraction of S-phase cells and in apoptosis. Co-expression of EAPP with E2F-1 strongly enhanced several examined E2F-dependent promoters, but surprisingly it repressed the activity of the p14ARF promoter. Moreover, EAPP turned out to be a p53 binding protein, and this interaction might play a role in the regulation of the p14ARF promoter. In this project we will analyze the role of EAPP as a modulator of transcription in more detail. We will examine EAPP complexes by co-immunoprecipitation and chromatin immunoprecipitation experiments. With transformation assays we will investigate a possible role in the malignant transformation of a cell. To study over- expression and absence of EAPP we will create cell lines with inducible EAPP expression or inducible EAPP knock down. To characterize EAPP biochemically, we will map its nuclear localization signal and the binding sites for E2F-1 and p53, search for sequences responsible for mitotic destruction, and identify and mutate acetylation sites. Finally, with DNA microarrays we will perform a systematical analysis of gene expression of EAPP knock down cells compared to normal cells.