The p53 tumor suppressor is a homotetrameric, sequence-specific transcription factor that has crucial roles in apoptosis, cell cycle arrest, cellular senescence, and DNA repair. It is maintained at low levels in unstressed cells, but stabilized and activated upon DNA damage by means of extensive post-translational modification (PTM). Our research has focused on identifying and exploring the biological roles of p53 PTMs to better understand how they modulate p53 function. Reciprocal negative regulation of p53 and Nanog maintains differentiation p53 supports the differentiation of embryonic stem cells (ESC) into differentiated states through suppression of NANOG, a gene required for ESC self-renewal. Previously, we showed that p53 Ser 315 phosphorylation was important for the suppression of Nanog expression during mouse ESC differentiation in a model containing a chimeric humanized p53 gene. p53 also suppresses dedifferentiation by maintaining suppression of NANOG in differentiated cells, a mechanism of tumor suppression demonstrated to be important in several human cancers, including gliomas and breast cancer. We investigated the roles of induced expression of Nanog in tumorigenesis and metastasis using an engineered mouse model. In a recent article published in Oncogene, we demonstrated that co-expression of Nanog and the oncogene Wnt in the mammary tissues of mice promoted tumorigenesis and metastasis. In this context, overexpression of Nanog activated the focal adhesion and calcium signaling pathways and suppressed the p53 signaling pathway, leading to increased tumor cell mobility, invasiveness and metastasis. Analysis of changes in gene expression between control tumors and tumors expressing high levels of Nanog revealed that the promoters of the most highly up-regulated genes exhibited the presence of Nanog transcription factor binding sites as well as the presence of both activating (H3K4me3) and repressive (H3K27me3) histone modifications. These results suggest that expression of Nanog in differentiated cells leads to inappropriate expression of genes with "poised" promoters that contribute to the metastasis of tumor cells. Global effects of p53 PTM Mouse models containing missense mutations at p53 PTM sites have been used to investigate the complex effects of p53 PTMs in a physiological setting. In these studies, we have primarily focused on knock-in mice containing mutation of Ser18 (human Ser15) to alanine in both alleles of endogenous p53, thereby preventing phosphorylation of this site. Our previous quantitative mass spectrometry studies of these mice demonstrated that the knock-in mutation affected proteins with roles in energy and metabolism pathways following ionizing radiation. As p53 has been shown to have important roles in regulation of metabolism and energy pathways, further investigation into modulation of these effects by phosphorylation is warranted. Thus, we are currently initiating studies to further understand the modulation of p53-dependent effects on metabolism in these knock-in mice. As a complement to mouse models, we are developing techniques to investigate global effects of p53 PTM in human cells. Recent advances in genomic editing provide practical methods for introducing specific modifications into genes in human cells. As protein translation accounts for a significant expenditure of energy by the cell, we are developing a sensitive method for analyzing protein translation in limited-availability samples. These methods will allow a more comprehensive investigation of the interrelationships between p53 PTMs and metabolism. Effects of p53 N-terminal phosphorylation on its protein-protein interactions One of the naturally expressed isoforms of p53, deltaNp53, lacks the first transactivation domain (TAD1) of p53 but does contain the second transactivation domain (TAD2). The expression and stability of the two proteins are affected differently by cell type, cell cycle phase and exposure to various stresses. p53 and deltaNp53 form heterotetramers and the relative abundance of deltaNp53 influences the transactivation activity and target gene specificity of p53. Our characterization of the binding of TAD1 and TAD2 of p53 to the Taz2 domain of the transcriptional coactivator p300 demonstrated that although the two domains bound to Taz2 with equal affinity, the binding of TAD1 was affected by p53 phosphorylations, whereas the binding of TAD2 was unaffected. To better understand the differences between the complexes of Taz2 with TAD1 and TAD2, we have determined the solution structure of a p53 TAD2 peptide in complex with Taz2. Upon binding to Taz2, p53 TAD2 forms a short alpha-helix, similar to the complex-dependent formation of a TAD1 alpha helix in the TAD1-Taz2 complex. Concomitant mutagenesis and binding studies have helped further characterize the complex. Comparison of the structures of the two complexes sheds light on how these two similar domains within p53 may function differently in co-activator recruitment after stress and suggests reasons for differences in transactivation between p53 and deltaNp53. In addition, as several new structures of p53 TAD2 complexes have been published, comparison of these structures with the TAD2-Taz2 complex will provide new understanding of the importance of flexibility in this domain for the formation of critical protein-protein interactions. Modulation of DNA binding by post-translational modification The frequency of DNA strand breaks produced by the decay of Auger electron-emitting radionuclides is inversely proportional to the distance of DNA nucleotides from the decay site. Thus, it provides a very sensitive measure of changes in the local conformation of the DNA. In a collaborative project with Victor Zhurkin (LCB) and Igor Panyutin (CC), we used radioprobing to study the conformation of DNA in complex with p53. This work, published in the International Journal of Radiation Biology, demonstrated that the most significant changes in the break frequency distributions were detected close to the center of the binding site, consistent with increased DNA twisting in this region as well as local DNA bending and sliding. Future studies will examine the effects of post-translational modification of the p53 DNA binding domain, including acetylation, on p53-induced DNA bending. Functional effects and interplay of p53 C-terminal modifications The C-terminus of p53 exhibits a diverse array of post-translational modifications, including phosphorylation, methylation, acetylation, ubiquitinylation, sumoylation, and neddylation, that are primarily localized to the terminal thirty residues of the protein. We are interested in understanding the specific effects of individual site-specific modifications and the interplay between them. We have investigated the effects of mono- and dimethylation of p53 Lys382, a site that alternatively can be methylated, acetylated, or ubiquitinylated. Mono-methylation of p53 Lys382 results in repression of the activity of p53 as a transcription factor and we have continued to investigate the mechanism of repression. Dimethylation of p53 Lys382 is critical for the interaction of p53 with the tandem Tudor domain (TD) of the DNA damage response mediator 53BP1. We are currently exploring the role of additional modifications within the C-terminal regulatory domain that may combine with Lys382 dimethylation to further modulate the binding of p53 to the TD domain. Further experiments will provide insight into the interactions of 53BP1 with p53 that facilitate repair of DNA damage.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005599-23
Application #
8762998
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
23
Fiscal Year
2013
Total Cost
$394,528
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Lu, X; Mazur, S J; Lin, T et al. (2014) The pluripotency factor nanog promotes breast cancer tumorigenesis and metastasis. Oncogene 33:2655-64
Feng, Hanqiao; Jenkins, Lisa M Miller; Durell, Stewart R et al. (2009) Structural basis for p300 Taz2-p53 TAD1 binding and modulation by phosphorylation. Structure 17:202-10
Jenkins, Lisa M Miller; Yamaguchi, Hiroshi; Hayashi, Ryo et al. (2009) Two distinct motifs within the p53 transactivation domain bind to the Taz2 domain of p300 and are differentially affected by phosphorylation. Biochemistry 48:1244-55
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