A transcriptionally inactive E2F-1 targets the MDM family of proteins for proteolytic degradation.

E2F-1-activated transcription promotes cell cycle progression and apoptosis. These functions are regulated by several factors including the E2F-1-binding protein MDM2 and the retinoblastoma protein pRb. Using a yeast two-hybrid screen we have identified the MDM2-related protein, MDMX, as an E2F-1-binding protein. In these studies we find that coexpression of MDMX with E2F-1 results in degradation of the MDMX protein. Although this proteolytic degradation can be blocked by the protease inhibitors bafilomycin A(1), N-acetyl-Leu-Leu-Norleu-AL, and N-acetyl-Leu-Leu-Met-AL, MDMX degradation is not inhibited by lactacystin, suggesting that degradation occurs by a proteasome-independent mechanism. Using an E2F-1 deletion mutant (E2F-1(180-437)) we show that E2F-1-targeted degradation of MDMX does not require the E2F-1 DNA binding domain and therefore is independent of E2F-1-driven transcription. We also find that this transcriptionally inactive E2F-1 mutant is capable of degrading the MDMX-related protein MDM2 and the MDMX isoform MDMX-S. Mapping of the E2F-1 C terminus reveals that neither a previously characterized C-terminal MDM2 binding domain nor the pRb binding domain on E2F-1 is required for MDMX and MDM2 degradation.

Transcriptional repression from the c-myc P2 promoter by the zinc finger protein ZF87/MAZ.

ZF87/MAZ is a zinc finger-containing transcription factor that was cloned based on its ability to bind to a site within the c-myc P2 promoter. However, its role in the control of c-myc transcription has not yet been well established. Here we have analyzed the effect of ZF87/MAZ overexpression on transcription from the murine c-myc P2 promoter. It was found that when overexpressed in COS cells, ZF87/MAZ significantly represses transcription from P2. The repression is mediated through the ME1a2 element, located at position -86 relative to the P2 transcriptional start site, and is not mediated through either the E2F or the ME1a1 sites. ZF87/MAZ functions as a true transcriptional repressor since it can repress transcription independently of the c-myc promoter, as part of a fusion with the GAL4 protein. The repressive domain within ZF87/MAZ is located in the amino-terminal half of the protein, a region rich in proline and alanine residues. ZF87/MAZ therefore shares features (i.e. a Pro/Ala-rich region) with those of known transcriptional repressor proteins.

An early S phase checkpoint is regulated by the E2F1 transcription factor.

The E2F1 transcription factor regulates transit of cells through the S phase checkpoint, dependent on its association with cyclin A/cdk2. Expression in cells of a mutant E2F1 lacking the cyclin A/cdk2 binding domain leads to partial arrest of cells at the S phase checkpoint. When subconfluent growing cells expressing this mutant E2F1 are analyzed in detail, it is shown here that they display a significantly reduced incorporation of 3H-thymidine into the DNA of each S phase cell, compared to control cells or to cells overexpressing full-length E2F1. Further, when cells are blocked at the G1/S phase border and released, there is a clear reduction in the amount of 3H-thymidine incorporated into the DNA of S phase cells by 1.5 hours post release. Considering a normal 6 hour S phase duration, the results show that the S phase checkpoint mediated by E2F1 is not a late S phase event but an early one.

Cyclin A levels, the duration of S phase and sensitivity to a chemotherapeutic agent are altered in fibroblasts cultured on a fibronectin matrix.

Culture of murine embryonic fibroblasts, but not vascular smooth muscle cells, on a fibronectin matrix significantly shortens their transit time through the S phase of the cell cycle. This shortening corresponds to an increase in both cyclin A protein levels and active cyclin A/cdk2 complex. The increase in cyclin A protein appears due to a translational/post-translational mechanism since there is no increase in cyclin A mRNA following culture of the cells on fibronectin. Treatment of cells cultured on fibronectin with a short pulse of the S phase chemotherapeutic agent camptothecin, resulted in a relative protection from cell death when compared to cells cultured on tissue culture plastic. Thus, while the cells have increased rate of transit through S phase fibronectin-mediated signaling protects the cells from S phase mediated apoptosis. In addition, fibroblasts constitutively expressing a mutant E2F1 transcription factor (E2F1d87) have a lengthened S phase, due to a truncation of the cyclin A/cdk2 binding domain. Culture of these mutant- expressing cells on fibronectin did not shorten their S phase duration in spite of the fact that cyclin A levels and active cyclin A/cdk2 complex were significantly elevated. Thus, although the fibronectin signaling mechanisms culminating in elevated cyclin A were intact in these mutant E2F1 expressing cells, they were insensitive to the effects of this elevated cyclin A. The effect of the mutant E2F1d87 on slowing transit through S phase appears dominant over the effect of elevated cyclin A.