Interferon regulatory factor 1 (IRF-1) and p53 control distinct sets of downstream genes; however, these two antioncogenic transcription factors converge to regulate p21 gene expression and to inhibit tumor formation. Here we investigate the mechanism by which IRF-1 and p53 synergize at the p21 promoter and show that stimulation of p21 transcription by IRF-1 does not require its DNA-binding activity but relies on the ability of IRF-1 to bind the coactivator p300 and to stimulate p53-dependent transcription by an allosteric mechanism. Deletion of the p300-binding sites in IRF-1 eliminates the ability of IRF-1 to stimulate p53 acetylation and associated p53 activity. Complementing this, small peptides derived from the IRF-1-p300 interface can bind to p300, stabilize the binding of p300 to DNA-bound p53, stimulate p53 acetylation in trans, and up-regulate p53-dependent activity from the p21 promoter. The nonacetylatable p53 mutant (p53-6KR) cannot be stimulated by IRF-1, further suggesting that p53 acetylation is the mechanism whereby IRF-1 modifies p53 activity. These data expand the core p300-p53 protein LXXLL and PXXP interface by including an IRF-1-p300 interface as an allosteric modifier of DNA-dependent acetylation of p53 at the p21 promoter.

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PIDD has been implicated in survival and apoptotic pathways in response to DNA damage, and a role for PIDD was recently identified in non-homologous end-joining (NHEJ) repair induced by c-irradiation. Here, we present an interaction of PIDD with PCNA, first identified in a proteomics screen. PCNA has essential functions in DNA replication and repair following UV irradiation. Translesion synthesis (TLS) is a process that prevents UV irradiation-induced replication blockage and is characterized by PCNA monoubiquitination and interaction with the TLS polymerase eta (polg). Both of these processes are inhibited by p21. We report that PIDD modulates p21-PCNA dissociation, and promotes PCNA monoubiquitination and interaction with polg in response to UV irradiation. Furthermore, PIDD deficiency leads to a defect in TLS that is associated, both in vitro and in vivo, with cellular sensitization to UV-induced apoptosis. Thus, PIDD performs key functions upon UV irradiation, including TLS, NHEJ, NF-jB activation and cell death. Cell Death and Differentiation (2011) 18, 1036-1045 doi:10.1038/cdd.2011; published online 18 March 2011 DNA lesions can result from endogenous metabolic processes as well as from exogenous DNA damaging agents. In both cases, different cellular responses are engaged (cell cycle arrest, repair pathways or apoptosis) to maintain genetic integrity. PCNA (proliferating cell nuclear antigen) acts as a DNA sliding clamp, which helps loading of replicative DNA polymerases. PCNA is involved in several forms of DNA repair, such as NER (nucleotide excision repair), BER (base excision repair) and MMR (mismatch repair), and also in other aspects of DNA metabolism, such as replication, chromatin assembly and cohesion. 1 PCNA mediates these different functions through interactions with proteins specific to each process. 2 Many of these PCNA interacting proteins (PIPs) contain a so-called PIP-box. 1,3 Competition between different partners for the same binding site on PCNA is one of the mechanisms that coordinate the functions of PCNA in DNA replication and repair. PCNA is loaded around the DNA by the conserved chaperone-like clamp loader complex RFC (replication factor C), consisting of five subunits (RFC1 to RFC5). 4,5 The RFC subunits all contain a PIP-box and thereby physically interact with PCNA. 6 Once PCNA is linked to the DNA, the RFC complex is ejected from the clamp to allow DNA polymerase access to the clamp. 7 The protein with the strongest affinity for PCNA is the PIPbox containing p21 (Cip1/Waf1), 8 a potent cyclin-dependent kinase (CDK) inhibitor involved in cell cycle regulation, except in response to UV, where cell cycle arrest is independent of p21. 9 P21 binds to PCNA and inhibits its activity, 10,11 mainly because p21 obstructs the interaction of PCNA with DNA replication and repair factors. [12][13][14][15] In vitro studies have shown that p21 also inhibits the loading of PCNA onto DNA, 7 thereby compromising DNA repair. 9 UV is one of the major exogenous sources of DNA damage. The most common...

Our understanding of the post-translational processes involved in regulating the interferon regulatory factor-1 (IRF-1) tumor suppressor protein is limited. The introduction of mutations within the C-terminal Mf1 domain (amino acids 301-325) impacts on IRF-1-mediated gene repression and growth suppression as well as the rate of IRF-1 degradation. However, nothing is known about the proteins that interact with this region to modulate IRF-1 function. A biochemical screen for Mf1-interacting proteins has identified an LXXLL motif that is required for binding of Hsp70 family members and cooperation with Hsp90 to regulate IRF-1 turnover and activity. These conclusions are supported by the finding that Hsp90 inhibitors suppress IRF-1-dependent transcription shortly after treatment, although at later time points inhibition of Hsp90 leads to an Hsp70-dependent depletion of nuclear IRF-1. Conversely, the half-life of IRF-1 is increased by Hsp90 in an ATPase-dependent manner leading to the accumulation of nuclear but not cytoplasmic IRF-1. This study begins to elucidate the role of the Mf1 domain of IRF-1 in orchestrating the recruitment of regulatory factors that can impact on both its turnover and transcriptional activity.Interferon regulatory factor-1 (IRF-1), 3 the founding member of the interferon regulatory factor family, is a transcription factor that regulates a diverse range of target genes during the response to stimuli such as pathogen infection (1), DNA damage (2, 3), and hypoxia (4). In addition, the loss of IRF-1 can cooperate with c-Ha-ras (5) in cellular transformation; it becomes up-regulated in cells that bear oncogenic lesions (6), and deletions of IRF-1 are associated with the development of gastric and esophageal tumors, as well as some leukemias (7-9). On the basis of these observations IRF-1 has been characterized as a tumor suppressor protein. Although initially identified as a component of the IFN␤-enhanceosome complex, IRF-1 has since been demonstrated to regulate the expression of a large cohort of interferon-responsive genes involved in negative growth control (10 -12).Structurally, IRF-1 includes several domains; prominent among these is a highly conserved N-terminal sequence-specific DNA-binding domain, a transactivation domain, and a C-terminal regulatory domain known as the enhancer (13). The enhancer was originally identified as a region required for maximal IRF-1-mediated transactivation, although it does not have intrinsic transactivation potential (13). More recent structurefunction analysis has shown that the enhancer is involved in the recruitment of coactivators to IRF-1 target promoters (14) and that it can facilitate IRF-1-mediated growth suppression (15), as well as being an important determinant of the rate at which IRF-1 is degraded (15-17). Housed within the enhancer is a multifunctional subdomain that we have named Mf1 (Multifunctional 1; amino acids 301-325). This domain impacts on IRF-1-mediated transrepression of the CDK2 gene (14) and is required for maximal IRF-1-mediated...