Mdm2 and MdmX as Regulators of Gene Expression

Abstract

p53 is an important tumor suppressor, functioning as a transcriptional activator and repressor. Upon receiving signals from multiple stress related pathways, p53 regulates numerous activities such as cell cycle arrest, senescence, and cell death. When p53 activities are not required, the protein is held in check by interacting with 2 key homologous regulators, Mdm2 and MdmX, and a search for inhibitors of these interactions is well underway. However, it is now recognized that Mdm2 and MdmX function beyond simple inhibition of p53, and a complete understanding of Mdm2 and MdmX functions is ever more important. Indeed, increasing evidence suggests that Mdm2 and MdmX affect p53 target gene specificity and influence the activity of other transcription factors, and Mdm2 itself may even function as a transcription co-factor through post-translational modification of chromatin. Additionally, Mdm2 affects post-transcriptional activities such as mRNA stability and translation of a variety of transcripts. Thus, Mdm2 and MdmX influence the expression of many genes through a wide variety of mechanisms, which are discussed in this review.

Introduction

The RING domain proteins Mdm2 and MdmX inhibit the functions of the tumor suppressor p53 under unstressed conditions.¹ In response to cellular stress, p53 functions as a transcription factor to promote multiple cellular outcomes.² The importance of p53 in restraining tumorigenesis is evidenced by its being mutated in more than 50% of human cancers. In the remaining cancers that harbor wild-type p53, overexpression of Mdm2 and MdmX is common in some tumor types such as sarcomas.^3-5

Multiple mechanisms exist by which Mdm2 inhibits p53. First, Mdm2 binds to the p53 transactivation domain and prevents recruitment of transcriptional co-activators to target promoters.^6,7 Second, Mdm2, via its RING domain, polyubiquitinates p53, targeting it for degradation by the ubiquitin-proteasome pathway.^8,9 Third, Mdm2 promotes monoubiquitination of p53, exposing a nuclear export signal, leading to its cytoplasmic translocation.^10,11 Fourth, Mdm2 inhibits p53 interaction with DNA.^12-15 Fifth, Mdm2 inhibits p53 mRNA translation indirectly through promoting the degradation of the ribosomal protein L26,¹⁶ a protein that augments p53 translation.¹⁷

MdmX, the structural homologue of Mdm2, also functions as a negative regulator of p53-mediated transcription.^18,19 The RING domain of MdmX, however, does not possess detectable E3 ligase activity.¹⁹ Although there is generally less mechanistic information as to how MdmX regulates transcription functions of p53, it was shown to be a much less potent inhibitor of p53 interaction with DNA.^14,15

Extensive efforts have focused on developing inhibitors of Mdm2 and MdmX for cancer therapy,²⁰ and it is therefore important that we understand how these 2 proteins function as fully as possible. Furthermore, accumulating evidence suggests the existence of p53-independent functions of Mdm2 and MdmX.^1,3 In this review we focus on the more enigmatic aspects of Mdm2 and MdmX, that is, how the 2 proteins regulate gene expression. We discuss how they function both via affecting p53 target gene selectivity and through a wide array of interactions with proteins and mRNA. Figure 1 summarizes the different modes by which Mdm2 and MdmX regulate gene expression.

Figure 1.

Mdm2 and MdmX regulate gene expression by multiple modes. Mdm2 and MdmX affect p53-mediated target gene selectivity both directly and indirectly. First, they regulate enzymes that catalyze p53 post-translational modifications as well as p53-mediated chromatin remodeling and p53 interacting co-factors that influence target gene selectivity. Through direct binding to p53, they also regulate its activities. Second, Mdm2 and MdmX influence the activity of transcription factors other than p53. Third, Mdm2 also interacts with chromatin. where it influences chromatin modifications. Fourth, Mdm2 regulates the stability and translation of a number of mRNA transcripts.

Mdm2 and MdmX Regulate p53 Target Gene Selectivity

Insights derived from mouse models

It was initially assumed that Mdm2 and MdmX are nondiscriminatory inhibitors of p53 transactivation functions. Multiple lines of evidence now suggest that Mdm2 and MdmX affect target gene selectivity by p53 in a cell and tissue specific manner. It has only recently become possible to study these functions of Mdm2 and MdmX in mice, thanks to conditional mouse models in which p53 is reactivated in adult tissues of mice lacking Mdm2 and/or MdmX as well as p53 expression.

A hint about the differential effects of Mdm2 and MdmX on p53-mediated transcription is given by the phenotypes of the null mice. Mdm2 null mice die at 5.5 days post coitum (dpc).^21,22 Bax deletion, although not suppressing the lethality of Mdm2 null mice, delays the death of the embryo, and the death is due to cell cycle arrest rather than apoptosis.²³ MdmX null mice die later in development, at 7.5 to 12 dpc, due to proliferative arrest.^24,25 Deletion of the cell cycle arrest gene p21 partially rescues the phenotype.^23,26 These results suggest that p53 activity in the absence of Mdm2 or MdmX is qualitatively different, activating specific outcomes. However, these experiments do not answer whether the activation of cell cycle arrest leading to death in mouse embryos lacking both Mdm2 and Bax results from a timing delay in activation of p53 or is due to another reason. To be more specific, it may be that activation of p53 at 7.5 dpc preferentially promotes cell cycle arrest, whereas activation of p53 earlier in development will result in death by apoptosis.

A more careful examination of the effects of Mdm2 and MdmX loss on p53 activity was carried out in mouse embryo fibroblasts (MEFs) into which a temperature-sensitive (TS) p53 (p53^A135V) was engineered.²⁷ This analysis showed that the phenotypes discussed above are not due to the timing of p53 activation but rather are due to specific effects of Mdm2 and MdmX loss. p53^A135V was introduced into MEFs that were isolated from mice lacking p53 and Mdm2 (TSΔ2), p53 and MdmX (TSΔX), or p53 alone (TS). In such mice, at the permissive temperature (32°C), p53 levels differ between the cell lines, with levels highest in TSΔ2, medium in TS, and lowest in TSΔX. The low p53 levels in TSΔX are due to Mdm2-mediated degradation, as cells engineered from the triple knock-outs (TSΔ2ΔX) do not exhibit low p53 levels. p53 reactivation in Mdm2 null MEFs results in apoptotic cell death, but not in MdmX nulls. TSΔ2ΔX behave likeTSΔ2, whereas TSΔX cells exhibit primarily G1 arrest following p53 reactivation despite the low p53 levels. An examination of p53 target gene mRNAs revealed that in the TSΔX cells, p53 activates Mdm2 and the cell cycle arrest gene p21, but not cyclin G1, Bax, Perp, and Noxa. TSΔ2, in contrast, activates the proapoptotic genes Perp and Noxa but not cyclin G1 and Bax, whereas p21 is only slightly elevated. Thus, Mdm2 and MdmX determine the outcome of p53 activation through affecting target gene selectivity by p53. A possible mechanistic explanation is that rather than directly influencing target gene activation, Mdm2 and MdmX simply affect p53 levels, and these, in turn, affect the outcome—cell cycle arrest when p53 levels are low versus apoptosis when p53 levels are high.

A recently published mouse model of inducible p53 in the background of either MdmX or Mdm2 knockout has enabled analysis of transcriptional responses to p53 activation in the absence of Mdm2 or MdmX in vivo. Knockin mice that express 4-hydroxy-tamoxifen (4-OHT)-dependent p53 (p53ER^TAM) from its endogenous locus in response to 4-OHT were generated in wild-type (WT), MdmX null, or Mdm2 null backgrounds.²⁸ Systemic reactivation of p53 in the adult MdmX null mice leads to activation of p53 target genes in a tissue specific manner. p21 is induced in all tested tissues except the small intestine, whereas the proapoptotic target Puma induction is restricted to the bone marrow, spleen, thymus and intestine—the classic radiosensitive tissues. Accordingly, tissues expressing Puma exhibit increased apoptosis. However, in spite of these responses to p53 activation, the mice are viable and recover well from 7-day sustained p53 activation. The intestines maintain function despite ongoing apoptosis, probably due to the continued proliferation resulting from lack of p21 activation in this tissue. This response is surprising in light of the previously published results of p53 activation in Mdm2 null background, where the mice cannot sustain a transient p53 restoration by 4-OHT administration and suffer severe loss of intestinal barrier function leading to death within 6 days.²⁹ In this mouse model of p53 reactivation, restored p53 levels are higher in the absence of MdmX than in WT cells,²⁸ in contrast to the model discussed above from Barboza et al.,²⁷ where levels of TS p53 are lower in the absence of MdmX than in its presence. Thus, inherent differences between the experimental systems may influence the outcome of p53 activation, from which we can learn about the factors influencing the response to p53 activation in different contexts.

Studies with human cancer cells

Global alterations in gene expression following MdmX and Mdm2 knockdown in MCF7 breast cancer cells containing WT p53 have been analyzed.^30,31 In this system, knockdown of either Mdm2 or MdmX leads to p21 induction and cell cycle arrest at G1 phase but does not increase cell death. Apoptosis is increased in cells harboring siRNAs directed against Mdm2 or MdmX in response to DNA damage with doxorubicin or cisplatin. The authors focused on genes that are commonly affected by the down-regulation of Mdm2 and MdmX, identifying 394 genes, of which 222 are up-regulated in response to the knockdown. The list of up-regulated genes is enriched for cell cycle arrest-promoting p53 target genes, including p21, BTG2, and ACTA2. The remaining 172 genes are down-regulated in the knockdown cells and are enriched for E2F1 target genes, consistent with repression of E2F1 activity by elevated p21. It would be interesting to analyze the microarray data for genes differentially affected in cells harboring siRNAs directed against Mdm2 versus MdmX.

Work in our laboratory has shown that levels of Mdm2 or MdmX can have profound impact on the p53 response. We examined the effect of excess Mdm2 on p53-mediated target gene activation in H1299 lung cancer cells with tetracycline-regulatable p53 that were engineered to constitutively express excess Mdm2.³² The excess Mdm2 does not affect p53 levels or localization but does affect target gene activation. Specifically, upon p53 induction in cells without excess Mdm2, expression of p21, Bax, 14-3-3σ, and PIG-3 genes is increased. In cells expressing extra Mdm2, however, 14-3-3σ and PIG-3 mRNA levels are markedly reduced, whereas p21 and Bax expression is unaffected. The outcome of p53 activation is thus altered by the excess Mdm2, from primarily G1 arrest with little G2 arrest in control cells to significantly increased G2 arrest in cells expressing excess Mdm2. Conversely, our recent study demonstrated that down-regulation of MdmX impairs stress-induced transcriptional activation of the p53 target genes Mdm2 and Wip1 but not several other p53 targets such as p21, 14-3-3σ, cyclin G1, Bax, PIG-3, and Noxa. ¹⁵ Mechanistically, down-regulation of MdmX in MCF7 cells results in impaired recruitment of p53 to the Mdm2 promoter.

Mdm2 regulates p53 post-translational modifications

Another way by which Mdm2 affects p53-regulated gene expression is through regulating the proteins that post-translationally modify p53. In fact, post-translational modifications of p53 have been well documented to influence its target gene activation.^33-35 Complicating the issues here is the fact that in some cases modifiers of p53 are themselves targets of ubiquitination and degradation by Mdm2, such as the histone acetyl transferases PCAF³⁶ and Tip60.³⁷

Perhaps the best-studied interplay between Mdm2 and p53 modifiers involves histone acetyl transferases. Mdm2 inhibits p300^38,39 and PCAF⁴⁰-mediated acetylation of p53. Although Kobet et al. ³⁸ concluded that Mdm2 binds to p300 and to p53 via separate domains, forming a ternary complex, Matt et al. ⁴¹ reported that there is no specific interaction between Mdm2 and p300. Teufel et al. ⁷ reconciled this dispute by describing a multidomain interaction between p300 and p53, mapping 4 regions on p300 that interact with TAD1 (residues 20-40) and TAD2 (residues 41-60), that comprise the 2 transcriptional activation domains of p53. Structural analysis supports a model of ternary complex formation with Mdm2 interacting with TAD1 and p300 interacting with TAD2.⁴² Further support for the multidomain interaction of p300 with p53 comes from the observation that whereas the interaction of Mdm2 with p53 is inhibited dramatically by phosphorylation of p53(T18), the interaction of p53 with p300 or CBP is inhibited in an additive fashion by phosphorylation of p53 on multiple residues.^43,44

The inhibition of p53 acetylation by Mdm2 is affected by the protein SOX4, which is often up-regulated in cancers. SOX4 shifts the balance between ubiquitination and acetylation of p53. By binding to p53, SOX4 inhibits ubiquitination of p53 by Mdm2, whereas by interacting with p300/CBP it promotes p53 acetylation.⁴⁵

Not only does Mdm2 inhibit p53 acetylation, it also promotes HDAC1-mediated p53 deacetylation via multiple mechanisms. Initially, Mdm2 was shown to associate with HDAC1 in cells and to promote p53 deacetylation and ubiquitination.⁴⁶ It was later shown that other factors affect the Mdm2 and HDAC1-mediated p53 deacetylation. The matrix attachment region (MAR) binding protein SMAR1 promotes p53 deacetylation by forming a ternary complex with Mdm2 and S15-phosphorylated p53, leading to HDAC1 recruitment and deacetylation of p53.⁴⁷ Kap1 is an adaptor protein that interacts with p53 in an Mdm2-dependent manner. Kap1 cooperates with Mdm2 both to promote HDAC1 interaction with p53, leading to p53 deacetylation, and to enhance Mdm2-mediated ubiquitination of p53.⁴⁸ A subsequent study reported that Kap1 forms a ternary complex with p53 and Mdm2 following some forms of stress and thus influences p53 target gene selectivity.⁴⁹

Acetylation is not the only p53 post-translational modification regulated by Mdm2. The protein kinase HIPK2, which phosphorylates p53 on S46, thus promoting apoptosis, is also a target of Mdm2. Overexpression of mutant Mdm2 that cannot bind to and degrade p53 but can still degrade HIPK2 results in lower p53 S46 phosphorylation and compromised cell death in response to lethal dose of doxorubicin.⁵⁰ HIPK2 promotes apoptosis also by a p53-independent mechanism, through the inhibition of the transcriptional co-repressor CtBP.⁵¹ Therefore, by down-regulating HIPK2, Mdm2 may affect transcription by p53 as well as by CtBP.

Mdm2 inhibits p53-promoted chromatin remodeling

Chromatin remodeling is an essential step in transcriptional regulation and plays a key role in development and cancer.⁵² Human SWI/SNF chromatin remodeling complexes contain 1 of 2 ATPase subunits: Brahma (Brm) or Brahma-Related Gene 1 (Brg1).⁵³ Both Brm- and Brg1-containing complexes differentially regulate p53 activity. Brg1-containing complexes are recruited by p53 to a subset of p53 target gene promoters, including the p21 promoter, but not to the Mdm2 promoter.⁵⁴ Mdm2 overexpression in normal cells was shown to arrest cells in G1.⁵⁵ This growth inhibitory function of Mdm2 requires both p53 and Brg1.⁵⁶ Mdm2 protein can compete Brg1-containing complexes off p53, allowing the released Brg1-containing complexes to inhibit the expression of cyclin A and arrest the cell cycle. Thus, 2 epigenetic processes that are involved in p53-mediated gene regulation (histone post-translational modifications and chromatin remodeling) are influenced by Mdm2.

Binding partners that regulate p53 activity can themselves be Mdm2 targets

The E3 ligase activity of Mdm2 can also influence p53 transcriptional activity through targeting its cofactors. The first case involves Junction-Mediating and Regulatory protein (JMY), a cofactor of p300 that augments p53 mediated transcriptional activation and apoptosis.⁵⁷ More recently, cytoplasmic JMY has been implicated in the nucleation of actin filaments promoting cell motility.⁵⁸ Mdm2 interacts with JMY and targets it for proteasomal degradation, reducing the level of nuclear JMY. Following DNA damage, the Mdm2:JMY interaction is reduced and JMY accumulates in cells. This accumulation is enhanced in cells treated with the Mdm2:p53 inhibitor Nutlin-3. Accumulated JMY then functions in augmenting the apoptotic response, as demonstrated by down-regulation of JMY leading to decrease in apoptosis and increased G1 arrest in UV-treated cells.⁵⁹

The second instance involves heterogeneous nuclear ribonucleoprotein K (hnRNP K), a poly-C binding protein that can interact with both DNA and RNA via its hnRNP K homology (KH) domain. hnRNP K is involved in transcription control in multiple ways, regulating the expression of c-Myc, SRC, and BRCA1, among others.⁶⁰ hnRNP K accumulates in cells following DNA damage and is recruited to p53 target promoters in a p53-dependent manner. It is required for efficient recruitment of p53 to its target promoters and for induction of p53 target genes, and siRNA to hnRNP K severely impairs cell cycle arrest following DNA damage. hnRNP K is a target of Mdm2-mediated ubiquitination, and its stabilization following DNA damage requires inhibition of its ubiquitination.⁶¹ How hnRNP K functions in p53 recruitment and target gene activation is still unclear. It also remains to be determined whether hnRNP K is required for induction of all p53 targets or a specific subset.

Beyond p53: Mdm2 and MdmX Regulate Multiple Transcription Factors

Mice in which p53 is knocked out alone⁶² or together with Mdm2^21,22 or with MdmX^24,25 have similarly increased incidence of cancers and early death. These elegant models indicate that if there are p53-independent functions of Mdm2 or MdmX, they are not significant to the life of the mouse. Nevertheless, other models suggest that the story may be not so simple. For example, a study of tumor onset and spectrum comparing p53 null mice with p53;Mdm2 double null or p53 null Mdm2 heterozygous mice showed that deletion of 1 (but not 2) Mdm2 allele alters the tumor onset and spectrum, increasing sarcoma incidence.⁶³ An additional example is provided by a careful examination of the timing of tumor formation in mice lacking p53 and MdmX, or p53 alone, revealing an earlier onset of tumors in the p53;MdmX double knock-out mice. Analysis in MEFs showed that MdmX promotes bipolar mitoses and genetic stability in a p53-independent manner.^64,65 This function is specific to MdmX, as Mdm2 co-deletion does not affect the growth of p53 null MEFs.⁶⁶

Perhaps the most obvious candidate for Mdm2-mediated regulation is p73, the p53 homologue^67,68 that, like p53, can promote cell cycle arrest and apoptosis.^67,69 There are multiple isoforms of p73. Those expressed from its P1 promoter contain a transactivation domain (TAp73) and display functional similarities to p53.⁷⁰ Mdm2 was shown to bind to p73 and inhibit its transcriptional activity without promoting its degradation.^71-74 In another study, however, overexpressed Mdm2 was shown to stabilize p73 and enhance its transcriptional activity.⁷⁵ A later report demonstrated that Mdm2 mediates the NEDD8 modification of TAp73, promoting its nuclear export and inhibiting its activity.⁷⁶ Nutlin-3 disrupts the interaction between Mdm2 and TAp73, leading to stabilization and activation of p73.⁷⁷ Mdm2 protects activated T cells from p73-mediated cell death.⁷⁸ Upon T-cell activation, NF-κB signaling activates the Mdm2 P1 promoter. The induced Mdm2 interacts with p73 and inhibits the activation of its proapoptotic target gene Bim, thus preventing cell death.

But the story does not end with p73, as there are several examples of other p53-independent factors that are regulated by Mdm2. A study into the links between Mdm2 and NF-κB found that Mdm2 can both inhibit p53-mediated repression of p65 and directly activate p65 transcription via interaction with SP1 sites in cells lacking p53.⁷⁹ An examination of Mdm2 overexpression in rhabdomyosarcoma cell lines revealed differential responses depending on the state of NF-κB.⁸⁰ In Rh30 cells, Mdm2 promotes the expression of p65 as described above, enhancing cell growth. In RD cells, which have constitutive expression of p65, Mdm2 represses p65, inhibiting cell growth and promoting apoptosis.

Unlike p73 and NF-κB signaling that were shown to be affected by Mdm2, transforming growth factor β (TGFβ) signaling is regulated by both Mdm2 and MdmX. TGFβ is a family of 5 cytokines that play roles in cell proliferation, migration, differentiation, and apoptosis. All TGFβs activate the TGFβ Ser/Thr kinase receptors, which signal through phosphorylation of Receptor Smad proteins. Phosphorylated Receptor Smads translocate to the nucleus, bind Smad4 and other cofactors, and function as transcription factors, leading to both tumor promoting as well as suppressing outcomes, depending on the cellular context. Among the genes regulated by Smad proteins are the CDK inhibitors p21 and p27. ⁸¹

Mdm2 was initially shown to inhibit TGFβ signaling independent of p53 in a screen for TGFβ suppressor cDNAs.⁸² Further investigation revealed that in an overexpression system, Mdm2 suppresses the activity of Smads1, 2, 3, and 4. Moreover, both Mdm2 and MdmX exhibit inhibitory functions toward Smad4 through affecting its subcellular localization, leading to its exclusion from the nucleus. Curiously, neither Mdm2 nor MdmX forms a complex with Smad4, leaving the mechanism for cytoplasmic sequestration upon co-expression unsolved.⁸³ A second study showed that MdmX but not Mdm2 inhibits co-expressed Smad3 and Smad4 transcriptional activity, although it should be noted that Mdm2 still exhibits some inhibitory effect on endogenous Smads.⁸⁴ The authors did not see any effect of Mdm2 or MdmX on Smad localization. A possible explanation for the discrepancy is that Yam et al. ⁸³ performed their analyses in HeLa cells, whereas Kadakia et al. ⁸⁴ used H1299 cells. Overexpression of p300, a cofactor for Smad transcriptional activation, reverses the Smad inhibition exerted by overexpressed MdmX.⁸⁴

Considering the tissue-specific effects of Mdm2 and MdmX knockout mice discussed above, it comes as no surprise that studies on specific cancers or cell types reveal additional unique roles for Mdm2 in the regulation of cell physiology.

Overexpression of Mdm2 in the myoblast cell line C2C12 induces adipocyte differentiation through inhibition of MyoD-dependent transcriptional activation,⁸⁵ and overexpression of SP1 or pRb can rescue MyoD activity.⁸⁶ Further analysis of the role of Mdm2 in adipogenesis revealed that Mdm2 is required for cAMP-regulatory element binding protein (CREB)-dependent transactivation during adipocyte differentiation, in a p53-independent way.⁸⁷

In pancreatic cancer cells with activated K-Ras and mutant p53, Mdm2 is overexpressed due to the hyperactive Ras signaling and is essential for cell viability.⁸⁸ Analysis of cell cycle regulating proteins revealed that Mdm2 is required for the expression of cyclin D1, c-Jun, and c-Myc in these cells, suggesting a potential mechanism for the p53-independent growth promoting activity of Mdm2.

One of the most studied transcription factors regulated by Mdm2 is E2F1. Unfortunately, as often the case in science (indeed epitomized by p53 itself), the more you learn about a topic, the more complex it becomes. E2F family transcription factors are regulators of cell cycle genes. E2Fs are primarily regulated by their inhibitory binding partner pRb (although some family members are pRb independent), which becomes phosphorylated by cyclin-dependent kinases (CDKs) leading to its dissociation from E2Fs, allowing them to transactivate their targets (reviewed in Chen et al. ⁸⁹). The p53 target gene p21 encodes a CDK inhibitor and thus links p53 to the pRb pathway. However, the links between the 2 pathways are much more complex, with pRb, p53, Mdm2, and ARF forming a network of binding interactions affecting each other’s activities.⁹⁰ Mdm2 interacts with E2F1 and stimulates the transcriptional activity of E2F1 and its binding partner DP1.⁹¹ Moreover, Mdm2 mediates E2F1 activation through interacting with the E2F1 inhibitor pRb, in a p53-independent manner,^92,93 and targeting pRb for degradation.⁹⁴ ARF overexpression leads to higher levels of pRb, suggesting that ARF inhibits Mdm2-mediated pRb degradation.⁹⁴ Interestingly, Mdm2 was also shown to promote E2F1 degradation,⁹⁵ under conditions in which E2F1 normally induces apoptosis. Casein kinase 1 (CK1) complexes with Mdm2 to promote the degradation of p53 and the stabilization of E2F1 in unstressed cells.⁹⁶ Therefore, Mdm2 regulates E2F1 activity under different conditions both positively and negatively, directly and indirectly, by protein-protein interaction and E3 ligase activity.

Recently Mdm2 was reported to influence transcription by affecting protein folding. Mdm2 possesses an ATP binding activity through the P-loop motif within its RING domain.^97,98 Mdm2 functions as a chaperone toward p53 and E2F1, and this activity is influenced by ATP binding, in which binding of the nucleotide to Mdm2 stimulates p53 folding into a DNA binding-competent form.⁹⁹ E2F1, in contrast, is misfolded in response to Mdm2 ATP binding, and thus its DNA binding activity is reduced. Although E2F1 is an in vitro ubiquitination target of Mdm2, this activity of Mdm2 is not involved in the chaperone function.¹⁰⁰

A surprising role for Mdm2 in the expression of human papillomavirus (HPV) genes has been described.¹⁰¹ Mdm2 with intact E3 ligase activity is required for the gene expression mediated by the HPV-16 protein E2. Mdm2 directly interacts with E2 and is recruited to HPV-16 type promoter DNA, where it promotes E2-mediated transcriptional activation.

In summary, Mdm2 and MdmX influence target gene activation both by p53 and by a variety of transcription factors and signaling cascades. With the growing number of Mdm2 targets being discovered, we are likely to discover additional links with pathways important for life and death of a cell.

Linking Mdm2 to Chromatin

Evidence collected over the past years suggests that Mdm2 itself can affect transcription by localizing to promoter DNA, in both p53-dependent and -independent manners. Early on it was shown in an in vitro transcription system that Mdm2 fused to a heterologous DNA binding domain represses transcription.¹⁰² A region between aa 50-222 is required for this inhibition and directly interacts with basal transcription machinery components (TFIIE and TBP) in a p53-independent manner. That study examined Mdm2 (aa 1-324) function on naked DNA. In subsequent study, a heterologous DNA binding domain (Gal4) fused to full-length Mdm2 confirmed repression of p53-independent transcription of a luciferase reporter in cells. However, in this case, Mdm2 RING domain deletion abolishes the repressive ability of Mdm2.¹⁰³ These data suggest that Mdm2 can function as a co-repressor if recruited to a promoter via its interaction with p53 as well as with other transcription factors that may possess DNA binding activity and can interact with Mdm2. Taken together, these studies suggest that there may be 2 independent domains in Mdm2 that are involved in transcriptional repression: one that resides within amino acids 50-222 and another within the RING domain.

In more physiological settings, Mdm2 was indeed shown to interact with endogenous promoters. Chromatin immunoprecipitation (ChIP) in cells overexpressing Mdm2^32,104 as well as in cells with high^103,105 or normal¹⁰⁶ levels of endogenous Mdm2 demonstrated that Mdm2 can be detected on p53 target gene promoters. The association of endogenous Mdm2 with p53 response elements is p53 dependent, as Mdm2 was not detected on these gene promoters in cells lacking p53¹⁰⁶. Further supporting this observation, Mdm2 was shown to co-localize with p53 on the p21 gene promoter in a sequential ChIP experiment.¹⁰³ Following DNA damage, Mdm2 dissociates from p53 target gene promoters.^104,106 A global Mdm2 ChIP, however, has not been reported. Hence, we cannot conclude whether Mdm2 localization to chromatin is solely a p53-dependent event.

Chromatin bound Mdm2 may suppress p53 through multiple mechanisms. As described above, the classic model for Mdm2 inhibition of p53 transcription activity is by inhibiting its transactivation domain from recruiting transcriptional co-activators such as p300/CBP. Additionally, Mdm2 can recruit transcriptional co-repressors as well as attach ubiquitin moieties to histones, modifying the chromatin state at its vicinity.

Mdm2 forms a complex with SUV39H1 and EHMT1 histone methyl transferase proteins that catalyze the repressive histone H3 Lys9 (H3K9) methylation.¹⁰⁷ Mdm2 recruits SUV39H1 and EHMT1 to p53, forming a complex that possesses H3K9 methylation ability in vitro. Interestingly, H3K9 methylation of the p53 target promoters p21 and Puma is increased following activation of p53, and Mdm2 expression promotes this modification. This suggests that SUV39H1 and EHMT1-mediated H3K9 methylation may play a role in the Mdm2:p53 negative feedback loop. ARF interaction with Mdm2 inhibits interaction with SUV39H1 and EHMT1, suggesting that oncogenic stress regulates Mdm2-interaction with histone methyl transferases. It is interesting to note that MdmX does not interact with SUV39H1 and EHMT1.¹⁰⁷ Whether Mdm2 associates with chromatin while performing this function needs to be directly tested.

A more direct function of Mdm2 in influencing gene expression is through monoubiquitination of histone H2B. Mdm2 localizes with p53 to its response element in the p21 gene, which promotes monoubiquitination of histone H2B.¹⁰³ The function of this modification is not well understood. On one hand, Mdm2 tethered to DNA represses basal transcription from a luciferase reporter in a RING domain-dependent manner,¹⁰³ suggesting a repressive role for this modification as has been reported in yeast.¹⁰⁸ On the other hand, analysis of the genome-wide occurrence of monoubiquitinated histone H2B (ubH2B) showed correlation with actively transcribed genes. Analysis of ubH2B dynamics along the p21 gene revealed association throughout the whole coding region that is stimulated by p53 induction and quickly removed when p53 levels are reduced.¹⁰⁹ Thus, Mdm2 may exert positive as well as negative effects on transcription, both mediated by the Mdm2 RING domain. It is important to note, however, that Mdm2 is not the primary E3 ligase for H2B. The RNF20/40 complex is thought to be the main modifier, although BRCA1 can also promote this modification.¹¹⁰

Mdm2 Regulates mRNA Stability and Translation

As if there were not enough complexity in the multiple roles Mdm2 plays in regulating both p53-dependent and -independent transcription, we now know there are additional aspects of gene expression that are affected by Mdm2. Notably, a growing number of mRNAs have been reported to be regulated by Mdm2 post-transcriptionally (Table 1). The Mdm2 RING domain was shown to specifically interact with RNA in vitro in 1996.¹¹¹ However, we are just beginning to discover which mRNA transcripts Mdm2 interacts with in the cell and the biological consequences of such interactions.

Table 1.

Mdm2-Mediated Regulation of mRNA Translation and Stability

mRNA	Mechanism	References
p53	Mdm2 RING domain interacts with p53 mRNA and enhances its translation	Candeias et al. 112
		Gajjar et al.113
p53	Mdm2 polyubiquitinates the p53 translation enhancer, L26	Ofir-Rosenfeld et al. 16
XIAP	Mdm2 RING domain interacts with the 5′-UTR IRES sequence, enhancing XIAP translation	Gu et al. 123
MYCN	Mdm2 RING domain interacts with AU-rich elements within the 3′-UTR of MYCN mRNA, enhancing its stability	Gu et al. 128
VEGF	Mdm2 RING domain interacts with VEGF 3′-UTR and increases its stability	Zhou et al. 130

p53 mRNA was the first transcript discovered to be regulated by Mdm2. The Mdm2 binding domain-encoding sequence in the p53 mRNA interacts with Mdm2, leading to enhanced translation of p53 as well as inhibition of Mdm2 E3 ligase activity.¹¹² The stimulation of p53 translation does not depend on Mdm2 E3 ligase activity since an inactive mutant, Mdm2^C464A, enhances p53 translation almost as well as WT Mdm2. In a recent follow-up study, the p53 mRNA: Mdm2 interaction was shown to depend on ATM-mediated phosphorylation of Mdm2 at S395, which contributes to p53 induction following DNA damage.¹¹³ Its importance was exemplified by the discovery that whereas Mdm2 siRNA increases apoptosis in unstressed conditions, it actually protects cells from undergoing apoptosis following DNA damage in an ATM-dependent manner. Interestingly, the p53 mRNA sequence encoding its Mdm2 binding domain is the same sequence that interacts with Mdm2, whereas the Mdm2 domain that binds the p53 mRNA is the RING finger domain, which is responsible for ubiquitinating p53. This suggests that the 2 functions of Mdm2 in the regulation of p53 may have co-evolved in p53 and Mdm2.¹¹⁴

Mdm2 regulates p53 mRNA through an additional, indirect mechanism, mediated by nucleolin and the ribosomal protein L26. In unstressed cells, nucleolin interacts with a double-stranded RNA structure formed by the 5′- and 3′-UTRs of the p53 mRNA, thereby inhibiting translation, possibly through stabilizing the repressive RNA structure.^17,115 Following DNA damage, L26 interacts with the same double-stranded p53 mRNA structure and augments its translation,^17,116 at least in part by interacting with nucleolin and preventing its homodimerization.¹¹⁵ Under unstressed conditions, Mdm2 ubiquitinates L26 and targets it for degradation, allowing nucleolin to repress p53 translation. Furthermore, the Mdm2 acidic domain interacts with L26 and prevents it from binding and augmenting translation of p53 mRNA. Following DNA damage, this inhibition is relieved, and L26 is free to augment p53 translation.¹⁶

Another mRNA regulated by Mdm2 is the X-linked inhibitor-of-apoptosis protein, XIAP. XIAP inhibits caspases 3, 7, and 9,^117-119 the caspases responsible for initiating apoptosis in response to cellular stress via the intrinsic pathway.^120,121 Transfected Mdm2 leads to an increase in XIAP protein levels.⁷⁹ The XIAP mRNA contains an internal ribosome entry site (IRES) sequence in its 5′-UTR.¹²² Mdm2 interacts with the XIAP IRES through its RING domain, thereby enhancing its translation. Importantly, cancer cells overexpressing Mdm2 are resistant to IR-induced apoptosis, which is dependent on the Mdm2:XIAP IRES interaction but independent of p53, as it was observed in p53 null cells.¹²³

Another gene whose mRNA is regulated by Mdm2 is MYCN, a gene often amplified in neuroblastoma that is associated with poor prognosis.^124,125 Mdm2 is also often amplified in neuroblastomas¹²⁶ and was shown to be a direct transcriptional target of MYCN in this tumor type.¹²⁷ Forming a positive feedback loop, Mdm2 interacts with the MYCN mRNA and leads to its stabilization.¹²⁸ Mechanistically, the stabilization of MYCN mRNA is mediated by the Mdm2 RING domain interaction with AU-rich elements within the 3′-UTR of MYCN mRNA and correlates with increased translation of MYCN. In contrast to Mdm2 regulation of XIAP, however, Mdm2 does not regulate IRES-dependent translation of MYCN; rather the enhanced translation seems to be a result of increased MYCN mRNA stability. Knockdown of Mdm2 in neuroblastoma cells down-regulates MYCN and inhibits colony formation (while not activating p53) and also protects p53 null neuroblastoma cells from resistance to IR.¹²⁸

Vascular endothelial growth factor (VEGF) is an important inducer of angiogenesis and is essential for solid tumor development.¹²⁹ The Mdm2 RING domain interacts with the VEGF mRNA 3′-UTR and increases its stability, leading to increased levels of VEGF protein, similar to its regulation of MYCN. In a xenograft mouse model, knockdown of Mdm2 in p53-null neuroblastoma cells reduces tumor size and microvessel density.¹³⁰ The possible involvement of Mdm2-mediated regulation of MYCN in that xenograft experiment was not addressed.

There are still many unanswered questions regarding regulation of post-transcriptional gene expression by Mdm2. The precise mechanisms of Mdm2-mediated mRNA stability and translation enhancement are not yet fully delineated. For example, it would be interesting determine the structure of the p53 double-stranded UTR complex and how nucleolin and L26 affect it. More globally, an anti-Mdm2 RNA immunoprecipitation followed by a microarray or RNA-seq analysis may give us a more extensive picture of the transcripts bound and potentially regulated by Mdm2.

MdmX has not been shown as of yet to regulate mRNA stability or translation, but a thorough examination has not been reported. The RING domain is the primary region by which Mdm2 regulates mRNAs. Considering the high degree of similarity between the Mdm2 and MdmX RING domains, it is possible that MdmX carries out similar functions. The fact that both RING domains bind nucleotides with similar affinity and specificity might suggest that they have similar or related interactions with RNA.^97,98

In conclusion, Mdm2 and MdmX modulate gene expression by multiple pathways, with outcomes and mechanisms not fully elucidated. Both global approaches for studying the effects of these proteins on gene expression as well as biochemical mechanistic studies will help us answer the “what” and “how” questions. The answers are likely to be quite complex, as the Mdm proteins appear to have tissue-specific effects that may also depend on the developmental stage. Future research will very likely reveal new and interesting modes by which the 2 proteins regulate gene expression separately and together.