Wilms tumor is a pediatric kidney cancer associated with inactivation of the WT1 tumor-suppressor gene in 5 to 10% of cases. Using a high-resolution screen for DNA copy-number alterations in Wilms tumor, we identified somatic deletions targeting a previously uncharacterized gene on the X chromosome. This gene, which we call WTX, is inactivated in approximately one-third of Wilms tumors (15 of 51 tumors). Tumors with mutations in WTX lack WT1 mutations, and both genes share a restricted temporal and spatial expression pattern in normal renal precursors. In contrast to biallelic inactivation of autosomal tumor-suppressor genes, WTX is inactivated by a monoallelic "single-hit" event targeting the single X chromosome in tumors from males and the active X chromosome in tumors from females.

E2F-mediated transcription is thought to involve binding of an E2F-pocket protein complex to promoters in the G 0 phase of the cell cycle and release of the pocket protein in late G 1 , followed by release of E2F in S phase. We have tested this model by monitoring protein-DNA interactions in living cells using a formaldehyde cross-linking and immunoprecipitation assay. We find that E2F target genes are bound by distinct E2F-pocket protein complexes which change as cells progress through the cell cycle. We also find that certain E2F target gene promoters are bound by pocket proteins when such promoters are transcriptionally active. Our data indicate that the current model applies only to certain E2F target genes and suggest that Rb family members may regulate transcription in both G 0 and S phases. Finally, we find that a given promoter can be bound by one of several different E2F-pocket protein complexes at a given time in the cell cycle, suggesting that cell cycle-regulated transcription is a stochastic, not a predetermined, process.

Uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells is a problem common to many investigators. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcription factor binding. By using a formaldehyde crosslinking procedure and Gal4 fusion proteins, we have analyzed the timing and functional consequences of binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes. We demonstrate that the endogenous cad promoter can be immunoprecipitated with antibodies against Myc and USF1. We further demonstrate that although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain. We also show that the amount of Myc bound to the cad promoter f luctuates in a growth-dependent manner. Thus, our data analyzing both DNA binding and promoter activity in intact cells suggest that cad is a Myc target gene. In addition, we show that Myc binding can occur at many sites in vivo but that the position of the binding site determines the functional consequences of this binding. Our data indicate that a post-DNA-binding mechanism determines Myc target gene specificity. Importantly, we have demonstrated the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian genes.

We show that the Mre11 complex associates with E2F family members via the Nbs1 N terminus. This association and Nbs1 phosphorylation are correlated with S-phase checkpoint proficiency, whereas neither is sufficient individually for checkpoint activation. The Nbs1 E2F interaction occurred near the Epstein-Barr virus origin of replication as well as near a chromosomal replication origin in the c-myc promoter region and was restricted to S-phase cells. The Mre11 complex colocalized with PCNA at replication forks throughout S phase, both prior to and coincident with the appearance of nascent DNA. These data suggest that the Mre11 complex suppresses genomic instability through its influence on both the regulation and progression of DNA replication.The Mre11 complex (composed of Mre11, Rad50, and Nbs1) and the ataxia-telangiectasia mutated (ATM) protein kinase are required to activate a DNA damage-induced Sphase checkpoint in mammalian cells (46 (12,52,58,66). Mutant cells fail to repress the firing of DNA replication origins in the presence of ionizing radiation (IR)-induced DNA damage, a phenomenon termed radioresistant DNA synthesis (RDS) (28, 42). Hence, the Mre11 complex can act as a negative regulator of DNA replication origins in response to DNA damage.The Mre11 complex is also important for recombinational DNA repair, as established by genetic analyses with Saccharomyces cerevisiae (21). Both the conservation of Mre11 and Rad50 and in vitro studies of the human Mre11 complex strongly suggest that the human Mre11 complex also functions in DNA recombination (43,44,63). DNA recombination and DNA replication functions are intrinsically linked; thus, Mre11 complex recombination functions are implicated in S-phase progression in addition to its role in S-phase regulation. In vertebrates, null mutants of the Mre11 complex are inviable (33,68,73), and DT40 cells depleted of Mre11 die with chromosome damage indicative of failure to resolve double-strand breaks arising during DNA replication (69). This suggests that the complex's recombination functions are required for DNA replication in a manner analogous to that of Rad51 (45,69). In Rad51-deficient cells, spontaneous chromosomal breakage during DNA replication leads to cell death (32,54,56,64). It is not clear whether the Mre11 complex's influence on the S-phase checkpoint is related to its DNA recombination functions.The Nbs1 protein is an important link between the Mre11 complex and the ATM-controlled S-phase checkpoint. ATM phosphorylates Nbs1 (20,31,67,72), and this event is required for checkpoint activation (31, 72). Its role in cell cycle regulation is consistent with the fact that Nbs1 contains a forkheadassociated (FHA) domain and a BRCA1 C-terminal (BRCT) domain (66), each of which is found in a number of proteins that effect DNA damage-dependent checkpoint functions (4,10,22,57,59).We identified the E2F1 transcription factor in a screen for proteins that interacted with the Nbs1 N-terminal region and established evidence that this interaction occurs on chr...

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