Acute loss of DP1, but not DP2, induces p53 mRNA and augments p21Waf1/Cip1 and senescence
Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan.Cell Biochemistry and Function (Impact Factor: 2.01). 01/2012; 30(1). DOI: 10.1002/cbf.1818
The transcription factors DP1 and DP2 have been implicated in crucial gene regulation as heterodimer partners of E2F; however, the functional differences between DP1 and DP2 remain poorly understood. To gain insight into DPs in human somatic cells, we first suppressed endogenous DP1 and DP2 using RNA interference and examined the effect of their loss on gene expression changes in HeLa cervical cancer cells. A DNA microarray and gene pathway analysis revealed that the suppression of well-known E2F/DP-regulated pathways, including the G1 to S phase transition of the cell cycle and DNA replication, was manifested in accordance with the acute loss of DP1 and DP2. On the other hand, the acute loss of DP1 and DP2 increased the p21Waf1/Cip1 mRNA level compared with the control RNA treatment. We further showed that the inactivation of DP1, but not DP2, resulted in mRNA induction for p53, an upstream regulator of p21Waf1/Cip1. Furthermore, in A549 lung cancer cells as well as HeLa cells, the mRNA and protein levels of p53 and p21Waf1/Cip1 were stabilized specifically upon DP1 depletion, whereas p53-regulated apoptotic factor BAX mRNA was unchanged. Finally, the impairment of DP1, but not DP2, increased senescence in HeLa, A549 and WI-38 diploid fibroblasts but not in p53 null Saos-2 osteosarcoma cells. Taken together, these results suggest that DP1, but not DP2, is uniquely involved in the regulation of the p53 and p21Waf1/Cip1 pathway, thereby augmenting senescence in human somatic cells. Copyright © 2011 John Wiley & Sons, Ltd.
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ABSTRACT: E2F transcription factors play pivotal roles in controlling the expression of genes involved in cell-cycle progression. Different viruses affect E2F1/retinoblastoma (Rb) interactions by diverse mechanisms releasing E2F1 from its suppressor Rb, enabling viral replication. We show that in T cells infected with human herpesvirus 6A (HHV-6A), the E2F1 protein and its cofactor DP1 increased, whereas the Rb protein underwent massive degradation without hyperphosphorylation at three sites known to control E2F/Rb association. Although E2F1 and DP1 increased without Rb suppression, the E2F1 target genes-including cyclin A, cyclin E, and dihydrofolate reductase-were not up-regulated. To test whether the E2F1/DP1 complexes were used for viral transcription, we scanned the viral genome for genes containing the E2F binding site in their promoters. In the present work, we concentrated on the U27 and U79 genes known to act in viral DNA synthesis. We constructed amplicon-6 vectors containing a GFP reporter gene driven by WT viral promoter or by promoter mutated in the E2F binding site. We found that the expression of the fusion U27 promoter was dependent on the presence of the E2F binding site. Test of the WT U79 promoter yielded >10-fold higher expression of the GFP reporter gene than the mutant U79 promoter with abrogated E2F binding site. Moreover, by using siRNA to E2F1, we found that E2F1 was essential for the activity of the U79 promoter. These findings revealed a unique pathway in HHV-6 replication: The virus causes Rb degradation and uses the increased E2F1 and DP1 factors to transcribe viral genes.Proceedings of the National Academy of Sciences 12/2013; 111(1). DOI:10.1073/pnas.1308854110 · 9.67 Impact Factor
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