The p53 tumor suppressor is an important transcription factor that has been highly conserved throughout vertebrate evolution. The wild-type p53 protein acts as a controller of cell growth and cell death. The negative feedback regulator of p53, Mdm2, is critically important to sustain development. p53 activates expression of mdm2 and the Mdm2 protein in turn inactivates p53 by either repressing its transcriptional activity or facilitating its degradation by the proteasome. The Bargonetti laboratory and colleagues are among only a few groups that have provided evidence that Mdm2 forms both soluble as well as chromatin-bound complexes with p53 in human cells. Several lines of evidence suggest that this process exemplifies an important basic transcription regulation program where Mdm2 influences the recruitment of factors to chromatin to prevent functional transcription, but this has yet to be fully elucidated. Human cell lines are used to investigate the chemical nature and function of p53-Mdm2 chromatin-associated complexes and their impact on transcription. The Bargonetti group is carrying out basic research in combination with a strong broader impact objective to produce the next generation of multicultural and multiethnic scientists. The core objectives of the project are the following: 1) Determine if Mdm2 blocks transcription initiation or elongation at p53 target genes. 2) Characterize the role of Mdm2 on histone modifications at p53 target genes. 3) Determine if Mdm2 and p300 act as competitors for the p53 transcription factor in Mdm2 over-expression cell culture models. 4) Examine the regulation of chromatin-associated p53 by Mdm2 proteins produced from alternatively spliced mdm2. Based on the results obtained from this project the ability of the alternate forms of Mdm2 to influence p53 transcriptional activity, recruitment of p300 and PCAF, and regulation of local and distal histone modification will be determined. 5) Integrate research and teaching as a method to excite the next generation, multiethnic, science work force.
The project being carried out by the Bargonetti laboratory group integrates research and teaching as a method to excite the next generation, multiethnic, science work force. Undergraduate students as well as graduate students are part of the research team. Under-represented minority students, as well as non-minority students, carry out the research and are mentored by Dr. Bargonetti in a multicultural environment where high standards are set for all and mutual respect for all cultures is the expectation. All students participating in this project are encouraged to move on to notable Ph.D. programs, post-doctoral positions and tenure track faculty jobs.
The NSF-funded project "Growth Control Regulated by p53 and Mdm2" has achieved its scientific and broader impact goals. Scientifically, the high impact has been to define two new Mdm2 growth-activating pathways that do not directly target the tumor suppressor p53 for degradation. The Bargonetti laboratory has discovered that one p53-independent activity of Mdm2, which is referred to as the non-canonical Mdm2 function, is contributed by Mdm2 isoforms that are not full length Mdm2 (Mdm2-FL). The alternative Mdm2 isoforms are encoded by alternative splice variants of the mdm2 RNA message. The Bargonetti laboratory discovered high expression of the mdm2-C transcript encoded from the gene’s second promoter region, when this promoter contains a G single nucleotide polymorphism. They were the first group to ever detect an endogenously produced Mdm2 alternative isoform protein product. They detected the endogenous production of the Mdm2-C isoform by creating a polyclonal antibody directed at the isoform specific peptide region. This region was highly immunogenic, and this peptide has the potential to be useful for the generation of highly specific mouse monoclonal antibodies. Antibodies to Mdm2-C will be important for future research on alternative mechanisms of growth control. The Bargonetti laboratory discovered that Mdm2-C promotes colony formation of human cells in the absence of p53. They also found that irrespective of the expression of p53, Mdm2-C activated human cell proliferation and promoted human cell survival. The other new Mdm2 pathway that the Bargonetti laboratory identified inhibits transcriptional elongation at the chromatin level. Chromatin mediated inhibition of transcription elongation by Mdm2 has never been previously described. Their work on this NSF-funded project has been instrumental in describing that Mdm2 isoforms increase the proteomic diversity of actively proliferating human cells that have aberrant checkpoints. The Mdm2 proteomic diversity activates multiple growth activating pathways that remain to be understood. The work from the Bargonetti laboratory has set the stage for discovering the multiple Mdm2 isoform growth activating pathways. The achieved broader impact goals mentioned above came from training the next generation of multi-ethnic scientists. A large group of multi-ethnic scientists were trained while they successfully made discoveries concerning the Mdm2 pathway. Dr. Bargonetti directly trained nine women who carried out the experiments that achieved the high impact scientific goals described above. Four women were undergraduates and five were PhD students. This scientific program vastly increased the number of American under-represented minority women scientists. According to NSF statistics, in 2006, Blacks and Hispanics made up just 7% of working scientists and engineers in America. All under-represented minority groups together make up just 6% of graduate enrollment in all science and engineering fields. The mentor Dr. Bargonetti has used this project to train six under-represented minority women. Three of the six minority women were undergraduate students. The three minority undergraduate students are Hispanic and have gone on to highly competitive PhD programs. These programs include Princeton University, Albert Einstein College of Medicine, and Memorial Sloan Kettering Cancer Center. The other three minority women scientists (two Hispanic and one Black) are PhD students attending the City University of New York (CUNY) Biology PhD program. Two additional female PhD students trained on this project; one Israeli woman who obtained her PhD in 2011 and is currently a Post-doctoral fellow at Weill Cornell Medical College, and one Indian woman who is a current student. The CUNY PhD students in the Bargonetti laboratory are in varied stages of their career, with the current most senior minority student scheduled to defend her thesis at the end of November 2012. The students trained through this project will be the next generation of multi-ethnic scientists and they are outstanding. They understand how to ask important scientific questions and how to design experiments to tackle tuff scientific problems. The students have been trained in how to communicate their science and will contribute to the broader impact National Science Foundation initiatives by teaching future generations and communicating scientific knowledge to the scientific community and to the public.
