Specific Aim 1: Investigate p53 and miRNAs as molecular nodes in replicative stress and stem cell biology Hypothesis: Differential mechanisms regulate the expression levels of p53 isoforms to coordinate replicative senescence The expression level of delta133p53, an inhibitory p53 isoform that is produced via transcription from an alternative promoter in intron 4, is downregulated during replicative senescence in human fibroblasts at the protein level rather than at the transcriptional level. In contrast to full-length p53 and p53beta that are subject to proteasomal degradation, delta133p53 was revealed to be degraded through selective autophagy, which involves the ubiquitination of substrate proteins and p62/SQSTM1 (an adaptor for ubiquitinated substrates). We showed that delta133p53 was ubiquitinated at the C-terminal lysine residues and that this ubiquitination was essential for autophagic degradation of delta133p53. A chaperone-associated E3 ubiquitin ligase STUB1 was downregulated during replicative senescence and interacted with delta133p53. Consistently, siRNA knockdown of STUB1 induced recruitment of delta133p53 to autophagosomes, autophagic degradation of delta133p53 and thereby senescence, suggesting that STUB1 is an endogenous regulator of delta133p53 degradation and cellular senescence. During replicative senescence, while full-length p53 levels and activity are maintained, the isoform-specific autophagic degradation of delta133p53 leads to relief from its inhibitory effect on full-length p53 and the full activation of full-length p53-mediated senescence programs. Together with our previous report that the downregulation of the splicing factor SRSF3 induces the alternative RNA splicing generating p53beta (a p53 isoform cooperating with full-length p53) during replicative senescence, our findings support the hypothesis that multiple p53 isoforms under different regulatory controls constitute a regulatory network to modulate the functions of full-length p53 in p53-mediated biological processes. Hypothesis: p53 isoform switching is a physiological mechanisms of replicative senescence Circulating CD8+ T lymphocytes undergo a series of differentiation stages toward replicative senescence in vivo with specific changes in cell surface antigens (e.g., loss of CD28 and gain of CD57). We previously found that: i) CD8+ T lymphocytes with CD28-CD57+ and senescent phenotypes accumulated in a donor age-dependent manner;ii) these in vivo-accumulated senescent CD8+ T lymphocytes expressed diminished levels of delta133p53 and increased levels of p53beta;and iii) reconstituted expression of delta133p53, as well as that of CD28, restored cell proliferation and extended replicative lifespan. We have recently revealed that delta133p53 and CD28 upregulates each other: i) full-length p53 transcriptionally represses CD28 expression and the inhibition of full-length p53 by delta133p53 leads to transcriptional upregulation of CD28;and ii) CD28-mediated upregulation of delta133p53 at the protein level may involve autophagic degradation of delta133p53 as described above. Our new data showing delta133p53-induced restoration of the central memory markers (i.e., CD27 and CD62L) and attenuation of the terminal differentiation markers (i.e., PD-1 and LAG-3) suggest that enhanced expression of delta133p53 may be a strategy for """"""""functionally rejuvenating"""""""" CD8+ T lymphocytes toward the restoration of immune functions in the elderly or patients with HIV infection. We have also found that CD8+ T lymphocytes isolated from human lung cancer tissues, like circulating CD8+ T lymphocytes, contain senescent CD28-CD57+ populations with diminished delta133p53 and increased p53beta expression, suggesting their role in the tumor microenvironment. Hypothesis: p53 isoforms are physiological regulators of human pluripotent stem cells We found that: i) overexpression of delta133p53 in human fibroblasts increased the efficiency of reprogramming to iPS cells;and ii) undifferentiated human pluripotent stem cells (i.e., ES and iPS cells) are the cell type that expresses the most abundant levels of endogenous delta133p53 protein. To elucidate the roles of upregulated delta133p53 in self-renewal and pluripotency of undifferentiated ES and iPS cells, we are currently establishing methods to knock down endogenous expression of delta133p53, including lentiviral shRNA vectors, TALEN and CRISPR/Cas9 systems. To investigate the roles of p53 isoforms in ES/iPS differentiation, we are examining the expression levels of endogenous delta133p53 and p53beta during differentiation from ES/iPS to neural stem cells to neurons and glial cells. We will also examine whether and how delta133p53 and p53beta expression modulates the differentiation processes.
Specific Aim 2 : Define the Role of p53 Isoforms and Mutant Variants in Control of Cellular Division of Normal and Cancer Cells Hypothesis: p53 isoforms and mutants have gain-of-function activities Cancer-associated mutants of full-length p53 can promote tumorigenesis in the absence of wild-type p53 (so-called """"""""gain-of-function"""""""" mutants). We hypothesize that wild-type and mutant p53 isoforms also have gain-of-function activities. We have constructed the inducible expression vectors driving wild-type delta133p53 and mutants (V157F, R175H, R249S and R275H) and transduced them to p53-null cell lines (fibroblasts and lung cancer cell lines). We are examining the expression profiles of mRNA, microRNA and non-coding RNA upon induction of these wild-type and mutant delta133p53 to identify signaling pathways regulated by their gain-of-function activities. Asymmetric cell division is a stem cell-associated phenotype possibly regulated by wild-type and/or mutant delta133p53 and under investigation in these cells (in collaboration with Brid Ryan).
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