An Investigation of How the Naturally Occurring Isoform ∆40p53 Alters WTp53 Activity

The aging population is rapidly expanding, and with it the prevalence of chronic diseases such as diabetes, cancer, and Alzheimer&rsquo;s disease. As our understanding of the biology of aging advances, the complexity of the aging process becomes more apparent. The naturally occurring p53 isoform ∆40p53 lacks the first N-terminal transactivation domain of WTp53 and 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 &ldquo;contaminating&rdquo; WTp53 tetramers. To circumvent this issue, I implemented a strategy&mdash;based upon the native p53 tetramer structure&mdash;in which ∆40p53 was tethered to WTp53 (∆40p53:WTp53) as a single transcript, resulting in tetramers with a defined 2:2 ratio of &Delta;40p53 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, I 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.</p

The Δ40p53 isoform inhibits p53-dependent eRNA transcription and enables regulation by signal-specific transcription factors during p53 activation

The naturally occurring &Delta;40p53 isoform heterotetramerizes with wild-type p53 (WTp53) to regulate development, aging, and stress responses. How &Delta;40p53 alters WTp53 function remains enigmatic because their co-expression causes tetramer heterogeneity. We circumvented this issue with a well-tested strategy that expressed &Delta;40p53:WTp53 as a single transcript, ensuring a 2:2 tetramer stoichiometry. Human MCF10A cell lines expressing &Delta;40p53:WTp53, WTp53, or WTp53:WTp53 (as controls) from the native TP53 locus were examined with transcriptomics (precision nuclear run-on sequencing [PRO-seq] and RNA sequencing [RNA-seq]), metabolomics, and other methods. &Delta;40p53:WTp53 was transcriptionally active, and, although phenotypically similar to WTp53 under normal conditions, it failed to induce growth arrest upon Nutlin-induced p53 activation. This occurred via &Delta;40p53:WTp53-dependent inhibition of enhancer RNA (eRNA) transcription and subsequent failure to induce mRNA biogenesis, despite similar genomic occupancy to WTp53. A different stimulus (5-fluorouracil [5FU]) also showed &Delta;40p53:WTp53-specific changes in mRNA induction; however, other transcription factors (TFs; e.g., E2F2) could then drive the response, yielding similar outcomes vs. WTp53. Our results establish that &Delta;40p53 tempers WTp53 function to enable compensatory responses by other stimulus-specific TFs. Such modulation of WTp53 activity may be an essential physiological function for &Delta;40p53. Moreover, &Delta;40p53:WTp53 functional distinctions uncovered herein suggest an eRNA requirement for mRNA biogenesis and that human p53 evolved as a tetramer to support eRNA transcription. &nbsp;</p

Suppression of p53 response by targeting p53-Mediator binding with a stapled peptide

DNA-binding transcription factors (TFs) remain challenging to target with molecular probes. Many TFs function in part through interaction with Mediator, a 26-subunit complex that controls RNA polymerase II activity genome-wide. We sought to block p53 function by disrupting the p53-Mediator interaction. Through rational design and activity-based screening, we characterize a stapled peptide, with functional mimics of both p53 activation domains, that blocks p53-Mediator binding and selectively inhibits p53-dependent transcription in human cells; importantly, this &ldquo;bivalent&rdquo; peptide has negligible impact, genome-wide, on non-p53 target genes. Our proof-of-concept strategy circumvents the TF entirely and targets the TF-Mediator interface instead, with desired functional outcomes (i.e., selective inhibition of p53 activation). Furthermore, these results demonstrate that TF activation domains represent viable starting points for Mediator-targeting molecular probes, as an alternative to large compound libraries. Different TFs bind Mediator through different subunits, suggesting this strategy could be broadly applied to selectively alter gene expression programs. &nbsp;</p

A novel comparative design procedure for reconfigurable assembly fixtures

While market requirements demand that manufacturing systems increase their responsiveness, assembly fixtures remain limited in corresponding to the same demand. Fixture designers as practitioners are left without guidance to design reconfigurable fixtures. This study proposes a comparative design procedure for reconfigurable assembly fixtures that can adapt to manufacturing system characteristics by using efficiency metrics. In this study, a theoretical analysis based on manufacturing systems is presented to establish efficiency metrics. Later, these metrics are utilized in a design procedure that offers guidance and determines the efficiency of fixtures in conceptual and detailed design stages. Finally, an experimental verification is presented

Wilderness dermatology: mountain exposures

Exploring the mountains is a highly rewarding past time; however, certain high-altitude exposures can lead to dermatologic manifestations. In this review article, the authors will describe cold, solar, and severe weather that one may experience when spending time outdoors. Factors such as increased ultraviolet radiation, temperature extremes, and low partial pressure of oxygen, along with human physiologic parameters also contribute to disease severity and presentation. This review article will address the diagnosis, treatment, and prevention of high-altitude dermatology exposures

Wilderness dermatology: mountain exposures

Exploring the mountains is a highly rewarding past time; however, certain high-altitude exposures can lead to dermatologic manifestations. In this review article, the authors will describe cold, solar, and severe weather that one may experience when spending time outdoors. Factors such as increased ultraviolet radiation, temperature extremes, and low partial pressure of oxygen, along with human physiologic parameters also contribute to disease severity and presentation. This review article will address the diagnosis, treatment, and prevention of high-altitude dermatology exposures

Suppression of p53 response by targeting p53-Mediator binding with a stapled peptide

DNA-binding transcription factors (TFs) remain challenging to target with molecular probes. Many TFs function in part through interaction with Mediator, a 26-subunit complex that controls RNA polymerase II activity genome-wide. We sought to block p53 function by disrupting the p53-Mediator interaction. Through rational design and activity-based screening, we characterize a stapled peptide, with functional mimics of both p53 activation domains, that blocks p53-Mediator binding and selectively inhibits p53-dependent transcription in human cells; importantly, this "bivalent" peptide has negligible impact, genome-wide, on non-p53 target genes. Our proof-of-concept strategy circumvents the TF entirely and targets the TF-Mediator interface instead, with desired functional outcomes (i.e., selective inhibition of p53 activation). Furthermore, these results demonstrate that TF activation domains represent viable starting points for Mediator-targeting molecular probes, as an alternative to large compound libraries. Different TFs bind Mediator through different subunits, suggesting this strategy could be broadly applied to selectively alter gene expression programs