# How does the naturally-occurring delta-Np53 isoform influence aging?

> **NIH NIH R03** · UNIVERSITY OF COLORADO · 2020 · $77,000

## Abstract

SUMMARY
The R03 project described here focuses on a naturally occurring p53 isoform, called ΔNp53, with well-
established links to mammalian aging. The ΔNp53 isoform was first characterized with mouse knock-in studies.
Expression of ΔNp53 together with WTp53 caused accelerated aging, accompanied by age-related pathologies
such as osteoporosis and memory loss. Importantly, expression with WTp53 is required for the accelerated
aging phenotype, indicating that mixed ΔNp53:WTp53 tetramers are essential. This presents a significant barrier
to understanding how ΔNp53 specifically affects cellular function, because co-expression of ΔNp53 and WTp53
will result in tetramer heterogeneity, including formation of native, WTp53 tetramers. To circumvent this problem,
we expressed ΔNp53:WTp53 as a single transcript that ensured a 2:2 stoichiometry (ΔNp53:WTp53) within the
tetramer. This circumvented confounding issues associated with ΔNp53 and WTp53 co-expression and allowed
clear delineation between cellular effects triggered by WTp53 vs. ΔNp53:WTp53.
 In a "proof of concept" study (published 2013), we confirmed that this tethering strategy would yield
reliable, biologically relevant data. The experiments were done in p53-null H1299 cells and p53 expression
(WTp53, ΔNp53:WTp53, or WTp53:WTp53) was induced by transient transfection. After normalization for total
p53 protein levels, gene expression changes were assessed using microarrays, which measure steady-state
mRNA levels, and comparative metabolomics experiments were completed. We now seek to employ more
rigorous and informative "next generation" methods to address how the naturally occurring ΔNp53
isoform alters WTp53 function. Instead of transfection into p53-null cells, we will use genome-edited CRISPR-
Cas9 knock-in cell lines (MCF10a, which normally express WTp53) in which ΔNp53:WTp53, WTp53:WTp53, or
WTp53 (control) have been inserted into the native TP53 locus. Instead of microarrays, steady-state mRNA
levels will be assessed with RNA-Seq (Aim 2), and we will assess global transcriptional changes using PRO-
Seq (Aim 1)—which directly and precisely measures nascent transcription—and assess potential changes in
p53 genomic occupancy using ChIP-Seq (Aim 1). One shared method (i.e. used in the 2013 study and also
proposed here) will be the metabolomics analyses (Aim 2), which will be completed by Metabolon Inc., as before.
 In addition to application of cutting-edge methods and our innovative strategy to define the biological
effects of ΔNp53 specifically, a key aspect of this study is chronic, yet transient induction of p53 over time (Aim
2). Brief periods of cell stress (i.e. p53 induction) have been shown to elevate human ΔNp53 protein levels, and
such transient increases are a basic feature in our experimental design. If successful, these studies will identify
key transcriptional and/or metabolic pathways that are altered by ΔNp53:WTp53 expression. Given its
established links to mammalian aging, thi...

## Key facts

- **NIH application ID:** 9851777
- **Project number:** 5R03AG061466-02
- **Recipient organization:** UNIVERSITY OF COLORADO
- **Principal Investigator:** Robin DeAnne Dowell-Deen
- **Activity code:** R03 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $77,000
- **Award type:** 5
- **Project period:** 2019-02-01 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9851777

## Citation

> US National Institutes of Health, RePORTER application 9851777, How does the naturally-occurring delta-Np53 isoform influence aging? (5R03AG061466-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9851777. Licensed CC0.

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