The aging population is rapidly expanding, and with it, the prevalence of chronic diseases such as diabetes, cancer, and Alzheimer's disease. This project focuses on elucidating the basic mechanisms whereby a naturally occurring p53 isoform, ?Np53, alters wild-type p53 (WTp53) function. In mice, co-expression of WTp53 and ?Np53 results in an accelerated aging phenotype with premature development of aging pathologies such as osteoporosis and Alzheimer's disease. The basic mechanisms driving these ?Np53- dependent physiological changes remain poorly understood. Human ?Np53 lacks the first N-terminal transactivation domain of WTp53 (residues 1-39), and is preferentially translated during cell stress. Modulation of different p53-dependent stress responses is likely achieved through expression of alternative p53 isoforms that can change p53's DNA binding and/or gene expression patterns. ?Np53 oligomerizes with WTp53 to form hetero-tetramers with altered function compared to WTp53 tetramers. Co-expression of ?Np53 and WTp53 results in the formation of a mixed population ?Np53:WTp53 tetramers, including ?contaminating? WTp53 tetramers. This precludes a reliable functional comparison of ?Np53:WTp53 tetramers vs. WTp53. To circumvent this issue, we developed a strategy?based upon the native p53 tetramer structure?in which ?Np53 is tethered to WTp53 (?Np53:WTp53), resulting in a pure population of tetramers with a 2:2 ratio of ?Np53 to WTp53. This innovative strategy will allow us to link transcriptional and/or phenotypic changes directly to ?Np53:WTp53 for the first time.
In Aim 1 of this project, I will use ChIP-seq to determine how genomic occupancy of ?Np53:WTp53 changes relative to WTp53. These data will be matched with GRO-seq data to correlate occupancy changes with global transcriptional changes in genome-edited iPS cells that express ?Np53:WTp53 or WTp53 from the endogenous TP53 locus.
In Aim 2, ?Np53:WTp53 or WTp53 iPS cells will be differentiated into fibroblasts to assess whether ?Np53:WTp53-specific changes affect differentiation in human cells; furthermore, differentiated fibroblasts will be propagated and tracked to determine whether chronic ?Np53:WTp53 expression (i.e. across many population doublings) results in more rapid induction of senescence (or other phenotypic changes) relative to cells expressing WTp53. A key aspect of this work will be the use of stable CRISPR-Cas9 ?knock-in? iPS cell lines expressing ?Np53:WTp53 (or controls) under the control of its native genomic locus. This will allow us to accurately determine how ?Np53 alters WTp53 function in a physiologically relevant context. Overall, this study should provide insight into the basic mechanisms by which ?Np53 alters WTp53 function as a transcription factor, and how ?Np53 may impact the well-established roles for p53 in cancer, aging, or stem cell biology.
The p53 protein is broadly implicated in human development and disease; its naturally occurring isoform, called ?Np53, is linked to age-related pathologies such as diabetes and Alzheimer's disease through mechanisms that are poorly understood. Using a combination of cutting-edge technologies and innovative cellular tools, we seek to define the molecular mechanisms through which ?Np53 controls p53 function in human cells. This basic insight could ultimately yield new therapeutic strategies relevant to cancer, developmental disorders, and age-related pathologies.
