The results demonstrate a direct role of p53 in the repression of mouse Arf transcription by showing the direct binding of p53 to the mouse Arf locus and that p53 is required for recruiting HDAC and PcG proteins to the Arf locus. Our results are consistent with a model where p53 binds specifically to the Arf locus and then recruits histone deacetylases to deacetylate the Arf locus. Deacetylation of the Arf locus then facilitates the recruitment of PRC to the Arf locus, leading to H3K27 trimethylation and silencing of Arf expression (). This model is supported by three lines of evidence. First, Arf expression is elevated in vivo in multiple p53-deficient tissues or organs, is rapidly elevated upon functional inactivation of p53 and is further repressed upon the activation of endogenous p53 in early passage of WT MEFs. Second, p53 directly binds to Arf locus, and that both transactivation and DNA binding activities of p53 are required for the repression of Arf and, importantly, for the binding to and repression of Arf by both HDAC and PRC. Third, we have also shown that both HDAC and PcG are conversely required for p53 to repress Arf expression.
The findings presented here shed mechanistic insights on the p53-mediated oncogenic checkpoint pathway: one on the mechanism of Arf
activation and the other on the feedback regulation of p53. Although it has been observed for more than a decade that many hyperproliferative oncogenes can activate Arf
), the molecular mechanism underlying oncogenic activation of Arf
is unknown. Our demonstration that Arf
is bound and repressed by p53 during normal cell growth suggests a critical step—dissociating p53 from the Arf
locus—for an oncogene to activate Arf
expression. It will be interesting to determine how an oncogene causes p53 dissociation in the presence of increased level of p53 since Arf
activation would lead to p53 stabilization.
Feedback inhibition is a regulatory strategy commonly used in biochemical reactions such as the inhibition of threonine dehydrase by isoleucine (46
). The accumulation of an end-product inhibits the enzyme involved in its synthesis to avoid excessive accumulation and waste of resources. A similar strategy is also widely employed in cell regulations, especially those involved in cell growth and proliferation, to ensure a balanced homeostasis and cell physiology. Feedback inhibition is particularly needed for the control of the function of a gene such as p53 whose activity, if not feedback inhibited, could lead to an irreversible consequence to the cell such as permanent cell cycle arrest or cell death. At low, non-lethal levels of DNA damage, cell cycle progression is delayed by the activation of p53 and then p21 to give cells time to repair the DNA and then resumed when the repair is completed. The resumption of cell cycle progression is achieved through the p53-MDM2 feedback loop in which p53 activates the transcription of its primary inhibitor (6
gene expression exhibits a strong inverse correlation with the functional status of p53 in both human (19
) and mouse cells (33
), suggesting a possible feedback repression of ARF
expression by p53. Ours results provide a molecular basis supporting this feedback regulation. The significance of evolving this second feedback inhibition loop is that the first p53-MDM2 negative feedback loop would not be effective to inhibit p53 to resume the cell cycle if ARF
expression is not repressed: the continuously synthesized ARF would bind to and prevent MDM2 from degrading p53 ().
Our study also adds to the understanding of p53-mediated transcriptional repression, an area that is much less understood than p53-mediated transcriptional activation although an estimated 15% of genes containing a p53 response element can be repressed by p53 [see recent review by (2
)]. p53 has been reported to directly bind with mSin3A, a transcriptional co-repressor and a member of class I HDAC complexes and recruit HDACs to a specific promoter such as Map4
). We also confirmed the association between p53 and HDAC by detecting p53-HDAC1 binding in WT MEFs (Supplementary Figure D
). Our study provides two separate lines of evidence supporting a role of histone deacetylation in p53-mediated repression of Arf
. First, we showed that treatment of MEFs with a low concentration of TSA drastically increased Arf
mRNA (>15-fold) and this effect is seen as early as 6 hours (). Second, we demonstrate that HDAC1 directly binds to and deacetylates Arf
in a p53 dependent manner. We further identify a new mechanism—recruiting PRC—for p53-mediated transcriptional repression. To the best of our knowledge, this represents the first evidence that p53-mediated repression involves polycomb repressive complex which contains histone modifying activities known to function in silencing gene expression. Conversely, identification of a sequence-specific binding factor—p53—in the recruitment of PRC to a specific locus also helps to better understand how PcG is recrutied to their targets, a puzzling issue associated with the repression of many PRC-regulated genes in mammalian cells.