Cell cycle regulatory genes are principal targets of p53-dependent, RB-mediated transcriptional repression following DNA damage
Authors: Kevin B. Spurgers, Georgette Rolle, David L. Gold, Nicole Bohnenstiehl, Kevin R. Coombes, Ray E. Meyn, Christopher J. Logothetis, Timothy J. McDonnell
Journal: Cancer Research (2006)

2498   Historically, most studies have sought to explain p53 function through the transactivation of downstream target genes. It has been underappreciated that p53 can also repress specific genes to affect a particular cellular response. Although little is known about the menu of genes repressed by p53, evidence suggests that repression is important for p53-induced cell death and cell cycle arrest. Beyond these functions, p53 can affect other cellular processes including DNA repair, senescence and differentiation. We hypothesized that, in addition to the transactivation of target genes, p53 represses diverse sets of genes to affect various cellular processes. Affymetrix U133A microarrays were used to identify p53-repressed genes in PC3 prostate cancer cells following adenoviral p53 gene transfer (Ad-p53). Using functional over-representation analysis, we further sought to objectively identify cellular processes influenced by this program of transcriptional repression. After objective validation of the array data, using RT-PCR of 20 randomly selected genes, we estimated a confirmation rate of >95.5% for the complete data set. Notably, 40% of the 111 identified p53-repressed genes regulate cell cycle progression. This is a statistically significant enrichment of genes with this function in our menu of p53-repressed targets (P = 5E-28). These genes exhibit p53-dependent repression in etoposide treated LNCaP cells before cell cycle arrest occurs. Although the repression is p53-dependent, experiments with cycloheximide indicate this process requires new protein synthesis. In some cases, the repression of p53 target genes is known to require transactivation of p21, the expression of which can activate the RB protein family and promote the formation of RB/E2F repressor complexes. Interestingly, 70% of the repressed cell cycle genes identified here are known E2F targets. We demonstrate that inhibition of RB proteins with E1a prevents the repression of several cell cycle genes after DNA damage. RB family proteins are known to recruit histone deacetylase activity (HDAC) to repressed promoters. Consistent with this scenario, inhibition of HDAC also prevents the repression of cell cycle p53 targets. Through the corepressor sin3a, p53 can recruit HDAC to the promoters of repressed genes. We demonstrate that engineered p53 constructs that do not bind sin3a, fail to repress cell cycle targets but still transactivate p21. We conclude that p53-induced cell cycle arrest is a function of not only the transactivation of cell cycle inhibitors (e.g., p21), but also the repression of targets that promote cell proliferation at several distinct phases of the cell cycle. Our findings suggest that the mechanism of repression involves RB/E2F repressor complexes and recruitment of HDAC. It is possible that p53 is directly involved in the recruitment of HDAC to these repressed promoters.