Cdc45 is a critical effector of myc-dependent DNA replication stress.

c-Myc oncogenic activity is thought to be mediated in part by its ability to generate DNA replication stress and subsequent genomic instability when deregulated. Previous studies have demonstrated a nontranscriptional role for c-Myc in regulating DNA replication. Here, we analyze the mechanisms by which c-Myc deregulation generates DNA replication stress. We find that overexpression of c-Myc alters the spatiotemporal program of replication initiation by increasing the density of early-replicating origins. We further show that c-Myc deregulation results in elevated replication-fork stalling or collapse and subsequent DNA damage. Notably, these phenotypes are independent of RNA transcription. Finally, we demonstrate that overexpression of Cdc45 recapitulates all c-Myc-induced replication and damage phenotypes and that Cdc45 and GINS function downstream of Myc.

Study of cell cycle checkpoints using Xenopus cell-free extracts.

Cell cycle checkpoints are involved in the coordinated response to DNA damage and thus play a key role in maintaining genome integrity. Several model systems have been developed to study the mechanisms and complexity of checkpoint function. Here we describe the application of cell-free extracts derived from Xenopus eggs as a model system to investigate DNA replication, damage, and checkpoint activation. We outline the preparation of cell-free extracts, DNA substrates and their subsequent use in assays aimed at understanding cell cycle checkpoints, and related processes. Several advances made over the years have enabled the continued use of the Xenopus system to answer a variety of questions in DNA replication, repair and checkpoint signaling. It is anticipated that the versatile Xenopus system is amenable to future modification as well to continue studies attempting to understand these important physiological processes.

RB loss promotes aberrant ploidy by deregulating levels and activity of DNA replication factors.

The retinoblastoma tumor suppressor (RB) is functionally inactivated in many human cancers. Classically, RB functions to repress E2F-mediated transcription and inhibit cell cycle progression. Consequently, RB ablation leads to loss of cell cycle control and aberrant expression of E2F target genes. Emerging evidence indicates a role for RB in maintenance of genomic stability. Here, mouse adult fibroblasts were utilized to demonstrate that aberrant DNA content in RB-deficient cells occurs concomitantly with an increase in levels and chromatin association of DNA replication factors. Furthermore, following exposure to nocodazole, RB-proficient cells arrest with 4 n DNA content, whereas RB-deficient cells bypass the mitotic block, continue DNA synthesis, and accumulate cells with higher ploidy and micronuclei. Under this condition, RB-deficient cells also retain high levels of tethered replication factors, MCM7 and PCNA, indicating that DNA replication occurs in these cells under nonpermissive conditions. Exogenous expression of replication factors Cdc6 or Cdt1 in RB-proficient cells does not recapitulate the RB-deficient cell phenotype. However, ectopic E2F expression in RB-proficient cells elevated ploidy and bypassed the response to nocodazole-induced cessation of DNA replication in a manner analogous to RB loss. Collectively, these results demonstrate that deregulated S phase control is a key mechanism by which RB-deficient cells acquire elevated ploidy.

RB loss abrogates cell cycle control and genome integrity to promote liver tumorigenesis.

BACKGROUND & AIMS: The retinoblastoma (RB) tumor suppressor is functionally inactivated in most hepatocellular carcinomas (HCC), although the mechanisms by which RB suppresses liver tumorigenesis are poorly defined. We investigated the impact of RB loss on carcinogen-induced liver tumorigenesis. METHODS: Mice harboring liver-specific RB ablation and normal littermates were exposed to the hepatocarcinogen diethylnitrosamine (DEN). The influence of RB loss on liver tumorigenesis was assessed by evaluating tumor multiplicity, proliferation, and genome integrity within tumors arising in RB-deficient and wild-type livers. In silico analyses were used to probe the association between gene expression signatures for RB loss and chromosomal instability and the ability of genes up-regulated by RB loss to predict the survival of human HCC patients. RESULTS: RB deficiency significantly increased tumor multiplicity in livers exposed to DEN. Although hepatocytes in nontumor regions of DEN-exposed livers were quiescent regardless of RB status, tumors arising in RB-deficient livers were significantly more proliferative than those in normal livers and expressed high levels of RB/E2F target genes. Analysis of genes up-regulated by RB loss demonstrated significant overlap with a gene expression signature associated with chromosomal instability. Correspondingly, tumors arising in RB-deficient livers were significantly more likely to harbor hepatocytes exhibiting altered ploidy. Finally, gene expression analysis of human HCCs demonstrated that elevated expression of RB-regulated genes independently predicts poor survival. CONCLUSIONS: RB deletion in the mouse liver enhances DEN-induced tumorigenesis, associated with increased hepatocyte proliferation and compromised genome integrity. Evaluation of RB status may be a useful prognostic factor in human HCC.

The KLF2 transcription factor does not affect the formation of preadipocytes but inhibits their differentiation into adipocytes.

Kruppel-like transcription factor 2 (KLF2), a critical gene for mouse embryogenesis, was recently identified as an inhibitor of adipogenesis. However, it is still unknown whether KLF2 is a natural repressor of adipocyte differentiation and if KLF2 affects the formation of preadipocytes. It may also be important for preadipocyte formation, as KLF2 is crucial for lung development and blood vessel formation. In this study, we show that differentiation of preadipocytes not only results in a concomitant decrease in the levels of KLF2 protein but also significantly reduces KLF2 promoter activity. We have generated tet-responsive lines of 3T3L1 that express physiological levels of KLF2 and show that reexpression of KLF2 prevents preadipocyte differentiation, thereby confirming the inhibition of adipogenesis by KLF2, partially via the restoration of Pref-1. In addition, we studied the contribution of KLF2-negative cells to the formation and subsequent differentiation of preadipocytes. We demonstrate that embryoid bodies derived from KLF2(-)(/)(-) ES cells can differentiate into adipocytes as evidenced by the accumulation of lipids and expression of several biochemical markers. Moreover, mouse embryonic fibroblasts (MEFs) derived from KLF2(-)(/)(-) mouse embryos differentiate efficiently into adipocytes. Interestingly, quantification of lipid accumulation in MEFs indicated that KLF2(-)(/)(-) cells are more prone to differentiate at the early stage of the process, suggesting that KLF2 is a natural repressor of differentiation in vivo. Taken together, these studies demonstrate that KLF2 does not affect the commitment of multipotent stem cells into the preadipocytic lineage but rather maintains their preadipocyte state and thereby negatively regulates their transition into adipocytes.

WWP1-dependent ubiquitination and degradation of the lung Krüppel-like factor, KLF2.

The zinc-finger transcription factor Krüppel-like factor-2 plays an important role in pulmonary development, inhibition of adipocyte differentiation, and maintaining quiescence in single-positive T cells. KLF2 levels rapidly decrease during adipogenesis and activation of T cells, but the pathways involved remain unclear. Previously, we identified WWP1, a HECT-domain E3-ubiquitin ligase, as an interacting partner of KLF2. This led us to speculate that KLF2 may be targeted for ubiquitination. Here, we demonstrate that WWP1 interacts with KLF2 in vivo and mediates both poly-ubiquitination and proteasomal degradation of KLF2. Deleting the inhibitory domain of KLF2 abrogated KLF2-WWP1 interactions and abolished WWP1-mediated poly-ubiquitination and down-regulation of KLF2. Furthermore, lysine-121 in the inhibitory domain of KLF2 is critical for ubiquitin-conjugation. Finally, the catalytic cysteine of WWP1 is not required for KLF2-ubiquitination. Our experiments demonstrate for the first time that WWP1 promotes ubiquitination and degradation of KLF2 and is not involved in the ubiquitin-transfer reaction.