The MDMX gene product is related to the MDM2 oncoprotein, both of which interact with the p53 tumor suppressor. We have identified a novel transcript of the MDMX gene that is expressed in a variety of cell lines, and in particular, in growing and transformed cells. This transcript is identical to the published sequence yet it has a short internal deletion of 68 base pairs. This deletion produces a shift in the reading frame after codon 114, resulting in the inclusion of a stop codon at amino acid residue 127 (full-length MDMX is 489 residues). This truncated MDMX protein is termed MDMX-S ("short form"), represents only the p53-binding domain, and appears to bind p53 better than full-length MDMX. The MDMX-S protein can be detected in cell extracts and when overexpressed is much more effective than MDMX at inhibiting p53-mediated transcriptional activation and induction of apoptosis. Since MDMX-S lacks the central and carboxyl-terminal regions contained within full-length MDMX, it is likely to play a key role in the regulation of cell proliferation and apoptosis in a way distinct from MDMX.The p53 tumor suppressor protein plays an important role in regulating movement through a number of cell cycle checkpoints (1-3, 38). The activity of p53 is in turn modulated through the action of the MDM2 protein which is amplified in a variety of tumors (4 -6). MDM2 can associate with p53 (7) and directly inhibit p53's ability to activate transcription of target genes, such as p21. MDM2 performs this function in a number of ways. First, the amino-terminal domain MDM2 binds tightly to the transcriptional transactivation domain of p53 (4 -6, 8, 9). Through this interaction, MDM2 blocks the ability of p53 to activate transcription of specific target genes by repressing the formation of the preinitiation complex mediated through the TFIIE and TATA-binding protein subunits of RNA polymerase II (10, 11). The second blocking activity of MDM2 is that it targets p53 for proteolytic degradation by the ubiquitin-proteasome pathway (12)(13)(14)40). Third, MDM2 also functions to shuttle p53 out of the nucleus and into the cytoplasm, via a nuclear export signal located within the MDM2 protein (33).While MDM2 was first identified as a protein that inhibits the action of p53 it has recently been shown to affect other components of the G 1 /S transition. MDM2 was found to associate with both the E2F1 transcription factor (15) and the retinoblastoma tumor suppressor (pRb) 1 (16). The interaction with pRb was found to be repressive, in that the association of MDM2 with the carboxyl-terminal domain of pRb inhibited the growth regulatory function of pRb (16). The interaction with E2F1, over the amino terminus of MDM2 and the carboxyl terminus of E2F1, was stimulatory in the sense that MDM2 enhanced the ability of E2F1 to activate target gene expression (15). Thus MDM2 appears to regulate at least three of the important players in the G 1 /S transition: p53, pRb, and E2F1. It has therefore been concluded that MDM2 functions in the fashion of a domi...

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.The c-myc proto-oncogene is known as a critical factor in the control of both cell proliferation (1-5) and apoptosis (6). In particular, c-myc is known as a cooperating oncogene and is thought to be an important contributing factor to neoplastic transformation and the generation of many cancers (1-5). That c-myc plays such a key role in regulating cell proliferation and cell death is consistent with the observations that its expression is tightly controlled in normal cells. Expression of both c-myc mRNA and protein is extremely low in quiescent cells, and it increases in proliferating cells (1-5). Since c-myc plays an important role in regulating cell proliferation, much effort has gone into understanding how its expression is modulated.c-myc gene expression is controlled at least in part at the level of transcription initiation, and extensive research has gone into identifying the transcriptional control regions within the c-myc promoter. This has been complicated by the fact that c-myc has multiple promoters, which are termed P0, P1, P2, and P3 (4, 5). Yet in many normal and transformed cells, a majority of transcripts initiate at the P2 promoter (4, 5). A large number of cis-acting sequences exist both 5Ј and 3Ј to the P2 transcription start site (5, 7). These sequences, although some quite distant from P2, regulate transcription initiation from P2 and include binding sites for the FUSE factor (8, 9), Fos/Jun (10, 11), Cut (12), YY1 (13), v-Abl (14, 15), Blimp-1 (16), NF-B/Rel (17), CTCF (18), and CF1 (19). The expression of some of these factors are cell-and tissue-type specific. Additionally, some of these factors, such as Fos/Jun, Blimp-1, and CTCF, function to repress the transcription of c-myc.cis-Acting sequences more proximal to the P2 promoter are comprised of an initiator sequence at the start site, a well defined TATA sequence at position Ϫ29, and three elements termed ME1a1, E2F, and ME1a2 at positions Ϫ46, Ϫ64, and Ϫ85 relative to P2 (4, 20 -23). ...

The MDMX gene product is related to the MDM2 oncoprotein, both of which interact with the p53 tumor suppressor. A novel transcript of the MDMX gene has been previously identified that has a short internal deletion of 68 base pairs, producing a shift in the reading frame after codon 114, resulting in the inclusion of 13 novel amino acids (after residue 114) followed by a stop codon at amino acid residue 127. This truncated MDMX protein, termed MDMX-S, represents only the p53 binding domain and binds and inactivates p53 better than full-length MDMX or MDM2. Here we show that when expressed in cells, MDMX-S is targeted more efficiently to the nucleus than MDMX. MDMX-S suppresses p53-mediated transcription from a p53 target promoter better than full-length MDMX. The DNA damage inducibility of these p53 responsive promoters was suppressed better by MDMX-S than by MDMX. Analysis of the MDMX-S protein indicated that the 13 novel amino acids at its carboxy terminus was responsible for high affinity binding to p53 in vitro and for high level expression of the protein in cells. Deletion of this 13 amino acid sequence resulted in a protein that was not able to bind p53 and was not able to be expressed well in cells. Taken together, these data point to an important domain within MDMX-S that enables it to function well in vivo to block p53 activity. Published 2003 Wiley-Liss, Inc.

The E2F1 transcription factor can initiate proliferation or apoptosis, the latter by both transcription-dependent and -independent mechanisms. Recently, an E2F1 mutant lacking the DNA binding domain, E2F1(180-437), has been implicated in degradation of MDMX and MDM2 proteins via lysosomal proteases. MDM proteins block p53 dependent apoptosis by directly inhibiting p53 stability and function. Here we demonstrate E2F1(180-437) induces death in HEK293 cells independent of E2F1 transcriptional activation and p53 stabilization. E2F1(180-437) elevates the activity of the calcium-activated protease, calpain, which is required for E2F1 induced proteolysis of MDMX and E2F1 induced cell loss. To determine if E2F1 could be activating proteolysis via calpains in neurodegeneration, we examined MDMX immunofluorescence in simian immunodeficiency virus encephalitis (SIVE). We found a reciprocal relationship between E2F1 and MDMX staining: in SIVE where E2F1 immunostaining is increased, MDMX is decreased, while in controls where E2F1 immunostaining is low, MDMX is high. Together these experiments support a new function for E2F1 in the activation of calpain proteases and suggest a role for this pathway in SIVE.

Several proteins with important roles in oncogenesis have been shown to regulate the function of the E2F-1 transcription factor, which is known to activate the expression of genes required for proliferation and apoptosis. Here we identify the MDMX oncoprotein as an E2F-1-binding factor, from a yeast-two hybrid screen using a portion of the E2F-1 protein as "bait." We demonstrate that the region within MDMX needed for the E2F-1:MDMX interaction is located in the central part of the protein, C-terminal of the p53-binding domain. The region within E2F-1 needed for this association is adjacent to the DNA binding domain. Further, when expressed in vivo or in vitro the MDMX protein migrates as two isoforms on SDS-PAGE, the faster migrating isoform having the stronger affinity for the E2F-1 proteins. It appears that this interaction reduces the ability of E2F-1 to bind DNA. Expression of MDMX along with E2F-1 and Dp-1 in Saos2 cells reduces the ability of E2F-1 to bind to its consensus DNA sequence, without altering E2F-1 protein levels. These data indicate that the MDMX protein is capable of associating with E2F-1 and negatively regulating its DNA binding ability.