The naturally occurring ?40p53 isoform inhibits eRNA transcription and enables context-specific regulatory inputs during p53 activation [5FU_RNA]

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 ?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, 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. Overall design: CRISPR/Cas9 edited MCF10A cells were treated with 375uM 5-Fluorouracil (5FU) and 0.375% DMSO (control) for 20 hours with a total of 2 biological replicates. Total RNA was isolated and enriched for mRNA and directional RNA-seq libraries were constructed and sequenced.