One of the main engines that drives cellular transformation is the loss of proper control of the mammalian cell cycle. The cyclin-dependent kinase inhibitor p21 (also known as p21 WAF1/Cip1 ) promotes cell cycle arrest in response to many stimuli. It is well positioned to function as both a sensor and an effector of multiple anti-proliferative signals. This Review focuses on recent advances in our understanding of the regulation of p21 and its biological functions with emphasis on its p53-independent tumour suppressor activities and paradoxical tumour-promoting activities, and their implications in cancer.Higher eukaryotes have evolved multiple checkpoint mechanisms to monitor and respond to cellular perturbations, halting cellular progression until errors are fixed or the environment becomes permissible to the faithful transmission of genetic material 1 . Perturbations in checkpoint mechanisms are detrimental to the integrity of the genome, promote cancer development 2 and significantly affect the efficacy of anticancer treatment 3 . The tumour suppressor protein p53 mediates the DNA damage-induced checkpoint through the transactivation of various growth inhibitory or apoptotic genes. Among these, the small 165 amino acid protein p21 (also known as p21 WAF1/Cip1 ) mediates p53-dependent G1 growth arrest 4,5 . Earlier studies supported the view that p21 suppresses tumours by promoting cell cycle arrest in response to various stimuli. Additionally, substantial evidence from biochemical and genetic studies indicates that p21 acts as a master effector of multiple tumour suppressor pathways for promoting anti-proliferative activities that are independent of the classical p53 tumour suppressor pathway (FIG. 1). Despite its profound role in halting cellular proliferation and its ability to promote differentiation and cellular senescence, recent studies suggest that, under certain conditions, p21 can promote cellular proliferation and oncogenicity 6 . Consequently, p21 is often misregulated in human cancers, but its expression, depending on the cellular context and circumstances, suggests that it can act as a tumour suppressor or as an oncogene (TABLE 1). p21 mediates its various biological activities primarily by binding to and inhibiting the kinase activity of the cyclin-dependent kinases (CDKs) CDK2 and CDK1 (also known as CDC2) leading to growth arrest at specific stages in the cell cycle (FIG. 2). In addition, by binding to proliferating cell nuclear antigen (PCNA), p21 interferes with PCNA-dependent DNA NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript polymerase activity, thereby inhibiting DNA replication and modulating various PCNAdependent DNA repair processes. In this Review we discuss recent advances concerning the complex role of p21 in the development of cancer. We describe the various effector functions of p21 that allow it to exert its biological activities. We further describe our current understanding of the various mechanisms that control p21 expression, both transcri...

[Keywords: CRL4; DDB1; PCNA; p21 ubiquitylation; Cdt2; DCAFs] Supplemental material is available at http://www.genesdev.org.

SUMMARY PR-Set7/Set8 is a cell cycle-regulated enzyme that monomethylates lysine 20 of histone H4 (H4K20). Set8 and monomethylated H4K20 are virtually undetectable during G1 and S phases of the cell cycle but increase in late S and in G2. We identify CRL4Cdt2 as the principal E3 ubiquitin ligase responsible for Set8 proteolytic degradation in the S-phase of the cell cycle, which requires Set8-PCNA interaction. Inactivation of the CRL4-Cdt2-PCNA-Set8 degradation axis results in (a) DNA damage and the induction of tumor suppressor p53 and p53-transactivated pro-apoptotic genes, (b) delayed progression through G2 phase of the cell cycle due to activation of the G2/M check-point, (c) specific repression of histone gene transcription and depletion of the histone proteins, and (d) repression of E2F1-dependent gene transcription. These results demonstrate a central role of CRL4Cdt2-dependent cell cycle regulation of Set8 for the maintenance of a stable epigenetic state essential for cell viability.

SUMMARY ADP-ribosylation of proteins is emerging as an important regulatory mechanism. Depending on the family member, ADP-ribosyltransferases conjugate either a single ADP-ribose to a target, or generate ADP-ribose chains. Here we characterize Parp9, a mono-ADP-ribosyltransferase reported to be enzymatically inactive. Parp9 undergoes heterodimerization with Dtx3L, a histone E3 ligase involved in DNA damage repair. We show that the Dtx3L/Parp9 heterodimer mediates NAD+- dependent mono-ADP-ribosylation of ubiquitin, exclusively in the context of ubiquitin processing by E1 and E2 enzymes. Dtx3L/Parp9 ADP-ribosylates the carboxyl group of Ub Gly76. Because Gly76 is normally used for Ub conjugation to substrates, ADP-ribosylation of the Ub carboxyl terminus precludes ubiquitylation. Parp9 ADP-ribosylation activity therefore restrains the E3 function of Dtx3L. Mutation of the NAD+ binding site in Parp9 increases the DNA repair activity of the heterodimer. Moreover, poly-ADP-ribose binding to the Parp9 macrodomains increases E3 activity. Dtx3L heterodimerization with Parp9 enables NAD+ and poly- ADP-ribose regulation of E3 activity.

One of the fundamental challenges facing the cell is to accurately copy its genetic material to daughter cells. When this process goes awry, genomic instability ensues in which genetic alterations ranging from nucleotide changes to chromosomal translocations and aneuploidy occur. Organisms have developed multiple mechanisms that can be classified into two major classes to ensure the fidelity of DNA replication. The first class includes mechanisms that prevent premature initiation of DNA replication and ensure that the genome is fully replicated once and only once during each division cycle. These include cyclin-dependent kinase (CDK)-dependent mechanisms and CDK-independent mechanisms. Although CDK-dependent mechanisms are largely conserved in eukaryotes, higher eukaryotes have evolved additional mechanisms that seem to play a larger role in preventing aberrant DNA replication and genome instability. The second class ensures that cells are able to respond to various cues that continuously threaten the integrity of the genome by initiating DNA-damage-dependent "checkpoints" and coordinating DNA damage repair mechanisms. Defects in the ability to safeguard against aberrant DNA replication and to respond to DNA damage contribute to genomic instability and the development of human malignancy. In this article, we summarize our current knowledge of how genomic instability arises, with a particular emphasis on how the DNA replication process can give rise to such instability.