Degradation of survivin by the X-linked inhibitor of apoptosis (XIAP)-XAF1 complex.

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a putative tumor suppressor in which expression is significantly reduced in human cancer cell lines and primary tumors. The proapoptotic effects of XAF1 have been attributed to both caspase-dependent and -independent means. In particular, XAF1 reverses the anti-caspase activity of XIAP, a physiological inhibitor of apoptosis. We further investigated the function of XAF1 by examining its relationship with other IAPs. Immunoprecipitation studies indicate that XAF1 binds to XIAP, cIAP1, cIAP2, Livin, TsIAP, and NAIP but not Survivin, an IAP that prevents mitotic catastrophe and in which antiapoptotic activity is exerted through direct XIAP interaction and stabilization. We found that overexpressed XAF1 down-regulates the protein expression of Survivin. Under these conditions, Survivin expression was restored in the presence of the proteasome inhibitor MG132 or a XIAP RING mutant that is defective in ubiquitin-protein isopeptide ligase (E3) activity, suggesting that XAF1 interaction activates E3 activity of XIAP and targets Survivin by direct ubiquitination. In addition, RNA interference targeting endogenous XIAP protected Survivin degradation by XAF1. Furthermore, interferon-beta-mediated XAF1 induction promoted formation of an endogenous XIAP-XAF1-Survivin complex. This complex facilitated Survivin degradation, which was prevented in XAF1(-/-) stable clones. Altogether, our study demonstrates that XAF1 mediates Survivin down-regulation through a complex containing XIAP, supporting dual roles for XAF1 in apoptosis and mitotic catastrophe.

Silencing of the XAF1 gene by promoter hypermethylation in cancer cells and reactivation to TRAIL-sensitization by IFN-beta.

BACKGROUND: XIAP-associated factor 1 (XAF1) is a putative tumor suppressor that exerts its proapoptotic effects through both caspase-dependent and -independent means. Loss of XAF1 expression through promoter methylation has been implicated in the process of tumorigenesis in a variety of cancers. In this report, we investigated the role of basal xaf1 promoter methylation in xaf1 expression and assessed the responsiveness of cancer cell lines to XAF1 induction by IFN-beta. METHODS: We used the conventional bisulfite DNA modification and sequencing method to determine the methylation status in the CpG sites of xaf1 promoter in glioblastoma (SF539, SF295), neuroblastoma (SK-N-AS) and cervical carcinoma (HeLa) cells. We analysed the status and incidence of basal xaf1 promoter methylation in xaf1 expression in non-treated cells as well as under a short or long exposure to IFN-beta. Stable XAF1 glioblastoma knock-down cell lines were established to characterize the direct implication of XAF1 in IFN-beta-mediated sensitization to TRAIL-induced cell death. RESULTS: We found a strong variability in xaf1 promoter methylation profile and responsiveness to IFN-beta across the four cancer cell lines studied. At the basal level, aberrant promoter methylation was linked to xaf1 gene silencing. After a short exposure, the IFN-beta-mediated reactivation of xaf1 gene expression was related to the degree of basal promoter methylation. However, in spite of continued promoter hypermethylation, we find that IFN-beta induced a transient xaf1 expression, that in turn, was followed by promoter demethylation upon a prolonged exposure. Importantly, we demonstrated for the first time that IFN-beta-mediated reactivation of endogenous XAF1 plays a critical role in TRAIL-induced cell death since XAF1 knock-down cell lines completely lost their IFN-beta-mediated TRAIL sensitivity. CONCLUSION: Together, these results suggest that promoter demethylation is not the sole factor determining xaf1 gene induction under IFN-beta treatment. Furthermore, our study provides evidence that XAF1 is a crucial interferon-stimulated gene (ISG) mediator of IFN-induced sensitization to TRAIL in cancer.

Molecular and functional analyses of incompatibility genes at het-6 in a population of Neurospora crassa.

Two closely linked genes, un-24 and het-6, associated with the het-6 heterokaryon incompatibility functional haplotype were examined in 40 Neurospora crassa strains from a Louisiana sugarcane field. Partial diploid analyses were used to determine that half of the strains were functionally Oak Ridge (OR) and half were non-OR and indistinguishable from the standard Panama (PA) form. PCR-based markers were developed to identify polymorphisms within both un-24 and het-6. Two common forms of each gene occur based on these molecular markers. Rare forms of both un-24 and het-6 were identified as variants of the non-OR form by a DNA transformation assay. The heterokaryon incompatibility function of haplotypes, based on partial diploid analyses, was perfectly correlated with the PCR-based markers at both loci. This correlation indicates that the two loci are in severe linkage disequilibrium in this population sample and may act as an incompatibility gene complex. Southern hybridizations using OR- and PA-derived cloned probes from the region that spans un-24 and het-6 showed that the apparent absence of recombination in this approximately 25-kbp region is associated with low levels of overall sequence identity between the PA and OR forms.

Vegetative incompatibility in the het-6 region of Neurospora crassa is mediated by two linked genes.

Non-self-recognition during asexual growth of Neurospora crassa involves restriction of heterokaryon formation via genetic differences at 11 het loci, including mating type. The het-6 locus maps to a 250-kbp region of LGIIL. We used restriction fragment length polymorphisms in progeny with crossovers in the het-6 region and a DNA transformation assay to identify two genes in a 25-kbp region that have vegetative incompatibility activity. The predicted product of one of these genes, which we designate het-6(OR), has three regions of amino acid sequence similarity to the predicted product of the het-e vegetative incompatibility gene in Podospora anserina and to the predicted product of tol, which mediates mating-type vegetative incompatibility in N. crassa. The predicted product of the alternative het-6 allele, HET-6(PA), shares only 68% amino acid identity with HET-6(OR). The second incompatibility gene, un-24(OR), encodes the large subunit of ribonucleotide reductase, which is essential for de novo synthesis of DNA. A region in the carboxyl-terminal portion of UN-24 is associated with incompatibility and is variable between un-24(OR) and the alternative allele un-24(PA). Linkage analysis indicates that the 25-kbp un-24-het-6 region is inherited as a block, suggesting that a nonallelic interaction may occur between un-24 and het-6 and possibly other loci within this region to mediate vegetative incompatibility in the het-6 region of N. crassa.

An osmotic-remedial, temperature-sensitive mutation in the allosteric activity site of ribonucleotide reductase in Neurospora crassa.

An osmotic-remedial, temperature-sensitive conditional mutant (un-24) was generated by Repeat Induced Point mutation (RIP) from a cross between a wild-type N. crassa strain and a strain carrying a approximately 250-kb duplication of the left arm of linkage group II (LGII). The mutation was mapped to the duplicated segment, within 2.6 map units of the heterokaryon incompatibility locus het-6. DNA transformation identified a 3.75-kb fragment that complemented the temperature-sensitive phenotype. A large ORF within this fragment was found to have a high degree of sequence identity to the large subunit of ribonucleotide reductase (RNR) from diverse organisms. Conserved amino acids at the active site and the allosteric activity sites are also evident. An unusual feature of the Neurospora sequence is a large insertion near the C-terminus relative to otherwise homologous sequences from other organisms. Three transition mutations, indicative of RIP, were identified in the N-terminal region of the temperature-sensitive mutant allele. One of these mutations results in a non-conservative amino acid substitution within the four-helix bundle that is important in the allosteric control of ribonucleotide reductase activity. This substitution appears to disrupt proper folding of the allosteric activity site during synthesis of the protein.