E2F regulates DDB2: consequences for DNA repair in Rb-deficient cells

Abstract

DDB2, a gene mutated in XPE patients, is involved in global genomic repair especially the repair of cyclobutane pyrimidine dimers (CPDs), and is regulated by p53 in human cells. We show that DDB2 is expressed in mouse tissues and demonstrate, using primary mouse epithelial cells, that mouse DDB2 is regulated by E2F transcription factors. Retinoblastoma (Rb), a tumor suppressor critical for the control of cell cycle progression, regulates E2F activity. Using Cre-Lox technology to delete Rb in primary mouse hepatocytes, we show that DDB2 gene expression increases, leading to elevated DDB2 protein levels. Furthermore, we show that endogenous E2F1 and E2F3 bind to DDB2 promoter and that treatment with E2F1-antisense or E2F1-small interfering RNA (siRNA) decreases DDB2 transcription, demonstrating that E2F1 is a transcriptional regulator for DDB2. This has consequences for global genomic repair: in Rb-null cells, where E2F activity is elevated, global DNA repair is increased and removal of CPDs is more efficient than in wild-type cells. Treatment with DDB2-siRNA decreases DDB2 expression and abolishes the repair phenotype of Rb-null cells. In summary, these results identify a new regulatory pathway for DDB2 by E2F, which does not require but is potentiated by p53, and demonstrate that DDB2 is involved in global repair in mouse epithelial cells.

E2F4 Function in G2 Maintaining G2-arrest to Prevent Mitotic Entry with Damaged DNA

“…Endogenous E2F1 and E2F3 bind to the DDB2 promoter and treatment with E2F1-antisense or E2F1-siRNA decreases DDB2 transcription, demonstrating that E2F1 is a transcriptional regulator of DDB2. 51 Several expression profiling experiments have documented that similar to p130, E2F4 regulates many genes that are critical for mitotic entry. Our results are consistent with previous studies based on combined cross-linking DNA immunoprecipitation and microarray experiments, 52 which have identified some of the same targets we have uncovered as E2F4 target genes with a putative role in various phases of the cell cycle including G 2 and M. 53 Similar targets were independently identified in studies examining specific p130 or Rb-regulated gene expression.…”

Post-Transcriptional Regulation of E2F Transcription Factors: Fine-Tuning DNA Repair, Cell Cycle Progression and Survival in Development & Disease

“…Further, deletion of pRb increases DDB2 mRNA and protein levels, together with ability of these cells to repair DNA damage. The latter is associated with more efficient CPD removal relative to that in pRb-expressing cells (Prost et al, 2007). Solid tumours frequently exhibit hypoxic cores, which contribute to genetic instability within the tumour microenvironment (Bindra et al, 2005).…”

“…During the latter, E2F-1 can play roles to induce cell cycle arrest and upregulate DNA repair, by directing expression of multiple genes. These genes are involved in mismatch repair (MSH2, MLH1), nucleotide excision repair (DDB2, RPA), homologous recombination repair (RAD51, RAD54, RECQL), base excision repair (UNG, APE) & non-homologous end joining (Chang et al, 2006;Ishida et al, 2001;Polager and Ginsberg, 2008;Prost et al, 2007). In humans, E2F-1 is a 437 amino acid protein, which shows constitutive and rapid nucleocytoplasmic shuttling in a variety of cells .…”

Independent regulation of P53 stabilisation and activation after Rb deletion in primary epithelial cells

“…One of the critical effects of Rb deletion is to increase E2F activity (2,16). It has been reported that overexpression of E2F activates p19ARF (17).…”