The tumor suppressor p53 is implicated as playing a key role in human cancer. p53 exerts much of its activity as a transcription factor that regulates gene expression. Its C-terminus domain (CTD) is highly enriched in basic amino acids and is extensively post-translationally modified. The C-terminus has been studied extensively as playing a role in interactions with both DNA and protein, thereby regulating the activity of p53 in gene expression. Nevertheless, a full understanding of how the CTD contributes to p53 function has remained elusive. The proposed research will build on preliminary and published work from the Manfredi and Prives laboratories to clarify the roles of the CTD in organismal homeostasis, tumor suppression, and oncogenesis. The three Specific Aims will provide unprecedented illumination of the roles of the CTD in vitro, in cells and in mice.
Aim 1 builds on data from a knock-in mouse generated by Manfredi where deletion of the CTD results in postnatal lethality (Hamard et al. 2013. Genes Dev 27, 1868). The Manfredi lab will analyze how loss of the p53 CTD affects gene expression in different tissues and will identify cellular proteins that bind and regulate the CTD in a tissue specific manner.
Aim 2 is based on recent findings from the Prives lab (Laptenko et al. 2015. Mol Cell 57, 1034) showing that the CTD is required for p53 to bind to sites within select target genes and that the CTD causes structural changes in the central core domain that stabilize p53-DNA complex formation. The Prives lab will obtain data about the repertoire of DNA sequences that require the CTD for optimal binding, and will determine the core-domain base contacts within DNA that require the CTD. A core domain mutant p53 (termed RE) can rescue the defects of deletion of CTD in vitro and Prives will use gene editing to alter human cell lines to express either p53 ?CTD or RE/?CTD from the endogenous p53 locus. The Manfredi group will generate a RE/?CTD mouse and determine whether this affects the pathology and gene expression that they previously reported.
Aim 3 will provide insight into how the CTD impacts tumor-derived mutant p53 to promote oncogenesis (gain-of-function activity), building on previous studies of the Prives group (Freed-Pastor et al. 2012. Cell 148, 244). Recent unpublished work from the Prives group showed that mutant p53 can induce expression of VEGFR2, the VEGF receptor that is needed for tumor angiogenesis. They found that mutant p53 binds to and is needed for loss of nucleosomes at the VEGFR2 promoter that requires the SWI/SNF chromatin remodeling complex. To ascertain whether and how the CTD plays a role in mutant p53 gain of oncogenic function, Prives will determine whether mutant p53 requires the CTD for VEGFR2 expression, whether the CTD is needed for remodeling the VEGR2 promoter, and whether it is needed to cooperate with SWI/SNF. Prives will also identify genes globally regulated by mutant p53 that require the CTD and which pathways are CTD-dependent. Manfredi will generate knock-in mutant p53 mice with or without the CTD and explore its role in tumorigenesis in vivo. A long term goal would be to link such discoveries to cancer.
The proposed research is focused on understanding the molecular basis of the tumor suppressor activity of wild-type p53 and the oncogenic functions of tumor-derived mutant p53. The long-term goal is to utilize this knowledge to improve diagnosis and design novel targeted therapies for a wide variety of human cancers.
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|Manfredi, James J (2017) Tumor suppression by p53 involves inhibiting an enabler, FGF13. Proc Natl Acad Sci U S A 114:632-633|
|Kribelbauer, Judith F; Laptenko, Oleg; Chen, Siying et al. (2017) Quantitative Analysis of the DNA Methylation Sensitivity of Transcription Factor Complexes. Cell Rep 19:2383-2395|
|Laptenko, Oleg; Tong, David R; Manfredi, James et al. (2016) The Tail That Wags the Dog: How the Disordered C-Terminal Domain Controls the Transcriptional Activities of the p53 Tumor-Suppressor Protein. Trends Biochem Sci 41:1022-1034|
|Laptenko, Oleg; Shiff, Idit; Freed-Pastor, Will et al. (2015) The p53 C terminus controls site-specific DNA binding and promotes structural changes within the central DNA binding domain. Mol Cell 57:1034-1046|