MDM2-Driven Ubiquitination Rapidly Removes p53 from Its Cognate Promoters

Abstract

MDM2 is the principal antagonist of the tumor suppressor p53. p53 binds to its cognate DNA element within promoters and activates the transcription of adjacent genes. These target genes include MDM2. Upon induction by p53, the MDM2 protein binds and ubiquitinates p53, triggering its proteasomal degradation and providing negative feedback. This raises the question whether MDM2 can also remove p53 from its target promoters, and whether this also involves ubiquitination. In the present paper, we employ the MDM2-targeted small molecule Nutlin-3a (Nutlin) to disrupt the interaction of MDM2 and p53 in three different cancer cell lines: SJSA-1 (osteosarcoma), 93T449 (liposarcoma; both carrying amplified MDM2), and MCF7 (breast adenocarcinoma). Remarkably, removing Nutlin from the culture medium for less than five minutes not only triggered p53 ubiquitination, but also dissociated most p53 from its chromatin binding sites, as revealed by chromatin immunoprecipitation. This also resulted in reduced p53-responsive transcription, and it occurred much earlier than the degradation of p53 by the proteasome, arguing that MDM2 removes p53 from promoters prior to and thus independent of degradation. Accordingly, the short-term pharmacological inhibition of the proteasome did not alter the removal of p53 from promoters by Nutlin washout. However, when the proteasome inhibitor was applied for several hours, depleting non-conjugated ubiquitin prior to eliminating Nutlin, this compromised the removal of DNA-bound p53, as did an E1 ubiquitin ligase inhibitor. This suggests that the ubiquitination of p53 by MDM2 is necessary for its clearance from promoters. Depleting the MDM2 cofactor MDM4 in SJSA cells did not alter the velocity by that p53 was removed from promoters upon Nutlin washout. We conclude that MDM2 antagonizes p53 not only by covering its transactivation domain and by destabilization, but also by the rapid, ubiquitin-dependent termination of p53–chromatin interactions.

Progress in Anticancer Drug Development Targeting Ubiquitination-Related Factors

“…This strategy appears effective to treat B-cell lymphoma and malignant hematological diseases ( Table 1 ) [ 39 , 40 ]. Furthermore, UAE inhibition can stabilize some tumor suppressors, such as p53, thus delaying the progress of cancer development [ 41 , 42 ]. There is also evidence showing that this inhibition activates the NF-κB signaling pathway so as to suppress malignant tumor growth [ 43 ].…”

Cytoplasmic localization of Mdm2 in cells expressing mutated NPM is mediated by p53

“…This interaction mediates stepwise p53 ubiquitinylation. Subsequently, Mdm2 with Mdm4 facilitates degradation of polyubiquitinylated p53 by proteasome [12][13][14]. The interaction between N termini of Mdm2 and p53 is accompanied by inhibition of p53 transcriptional activity [15].…”

p53 Function and Dysfunction in Human Health and Diseases

“…The study employed the MDM2-targeted small molecule Nutlin-3a to disrupt the interaction of MDM2 and p53 in three different cancer cell lines. The results strongly suggest that MDM2-mediated ubiquitination not only triggers p53 degradation but also the removal of p53 from its bond with promoter DNA within minutes [ 16 ]. The question remains whether this activity of MDM2, i.e., its ability to dissociate p53 from DNA, can be regulated under physiological circumstances.…”