Project description:This study is to find the cellular and molecular mechanisms by which a naturally-occurring deltaNp53 isoform causes accelerated aging in humans. The biological function of deltaNp53, which lacks only 40 N-terminal amino acids, represents an example of p53 as a regulator of mammalian aging. When expressed together with WTp53 in mice, ÎNp53 causes an aging phenotype such as shorter life span, reduced body mass, organ atrophy and osteoporosis. Because p53 must form a tetramer to regulate transcription, we generated p53 clones (based upon the structure of the native p53 tetramer) containing one ÎNp53 linked with one WTp53 to form a functional ÎNp53:WTp53 tetramer with 1:1 stoichiometry. Thus, our strategy ensured each p53 tetramer contained 2 ÎNp53 and 2 WTp53 proteins. Importantly, ÎNp53:WTp53 form stable tetramers, based upon gel filtration chromatography and structural analysis using electron microscopy. Furthermore, the ÎNp53:WTp53 tetramer activates transcription equally well compared with WTp53 tetramers in an in vitro reconstituted transcription system. Having verified the stoichiometry, stability, structure, and activity of these ÎNp53:WTp53 tetramers, here we used microarray analysis to compare global gene expression patterns in p53-null H1299 cells expressing either WTp53 or ÎNp53:WTp53. As expected, global gene expression was largely similar, since the differences between ÎNp53:WTp53 tetramers and WTp53 tetramers are slight: only 2 of 4 p53 proteins will be different in the ÎNp53:WTp53 tetramer. Among only several dozen genes that were selectively up- or down-regulated 2-fold or greater, many genes known to regulate mammalian aging were altered in cells expressing ÎNp53:WTp53, including insulin signaling pathway members (IRS1, INPP5D, PLK3, MAP3K1, FGF5) and regulators of glucose metabolism (SLC2A2, CRYAB, LRCH1). Expression of other key metabolic genes were also altered in cells expressing ÎNp53:WTp53 tetramers, suggesting that global me tabolic changes might contribute to ÎNp53:WTp53 pathology. In collaboration with Metabolon (Durham, NC), we identified approximately one hundred metabolites that were significantly up- or down-regulated in H1299 cells expressing ÎNp53:WTp53. The metabolome analysis was a powerful complement to the gene expression data, and further suggested that the mTOR pathway (e.g. across-the-board up-regulation of amino acid levels) and mitochondrial function (e.g. up-regulation of carnitine, important for a-oxidation of fatty acids) was altered in cells expressing ÎNp53:WTp53. These findings were subsequently validated using biochemical and cell-based approaches. Furthermore, whereas equal expression of ÎNp53 and WTp53 cause accelerated aging in mammals, due to alternative splicing and translation initiation ÎNp53 is a naturally-occurring isoform whose expression levels can change throughout the lifetime. Thus, the cellular and molecular mechanisms identified from this work will likely reflect changes common to normal, physiological aging. Comparison of global gene expression profiles in WTp53 or ÎNp53:WTp53 expressed H1299 RNA from FACS sorted GFP positive hybrid to Affymatrix Gene The exp 0, exp 4 and exp 5 are independent replicate/transfections/biological experiments. The exp d, exp e and exp f are control experiments.

Project description:The naturally occurring p53 isoform ∆40p53 lacks the first N-terminal transactivation domain of WTp53 and is implicated in mammalian aging. ∆40p53 is preferentially translated during cell stress. The ∆40p53 isoform oligomerizes with WTp53 to form hetero-tetramers with altered function compared to WTp53 tetramers. Co-expression of ∆40p53 and WTp53 results in the formation of a mixed population ∆40p53:WTp53 tetramers, including “contaminating” WTp53 tetramers. Here we implemented a strategy—based upon the native p53 tetramer structure—in which ∆40p53 was tethered to WTp53 (∆40p53:WTp53) as a single transcript, resulting in tetramers with a defined 2:2 ratio of Δ40p53 to WTp53. Using CRISPR/Cas9, human cell lines were generated in which ∆40p53:WTp53 (and controls) was inserted at the native TP53 locus. This enabled ∆40p53:WTp53 expression and induction in a physiologically relevant manner. As with WTp53, activation of ∆40p53:WTp53 with Nutlin-3a causes increased genome occupancy. However, unlike WTp53, precision run-on nuclear sequencing (PRO-seq) showed that increased ∆40p53:WTp53 occupancy does not correlate with robust transcriptional activation. Activated ∆40p53:WTp53 only induces transcription of a small subset of WTp53 enhancer RNAs (eRNAs) and other annotated regions. Defective eRNA production correlates with loss of downstream mature mRNA and p53-dependent cell-cycle arrest. Pathway analysis of ∆40p53:WTp53 induced transcriptional changes identified increased activation of stress and aging related pathways in ∆40p53:WTp53 compared to WTp53. However, these pathways never mature to full activation under Nutlin-3a treatment. Therefore, we propose a model where activation of ∆40p53 results in the transcription of a subset of WTp53 targets, setting the stage for synergistic pathway activation with other stress specific transcription factors driving cellular response in ways that differ from WTp53. Implications of these results provide an additional mechanistic layer of p53 regulation, in which expression of ∆40p53 isoform allows for modulation of WTp53 activities in a context dependent manner, tipping the scales towards activation of aging-associated pathways.

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Metabolic and gene expression changes induced by the naturally occurring ΔNp53 isoform link mTOR pathway and mitochondrial alterations to the progeroid phenotype.

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