Discontinuous transcription has been described for different mammalian cell lines and numerous promoters. However, our knowledge of how the activity of individual promoters is adjusted by dynamic signaling inputs from transcription factors is limited. To address this question, we characterized the activity of selected target genes that are regulated by pulsatile accumulation of the tumor suppressor p53 in response to ionizing radiation. We performed time‐resolved measurements of gene expression at the single‐cell level by smFISH and used the resulting data to inform a mathematical model of promoter activity. We found that p53 target promoters are regulated by frequency modulation of stochastic bursting and can be grouped along three archetypes of gene expression. The occurrence of these archetypes cannot solely be explained by nuclear p53 abundance or promoter binding of total p53. Instead, we provide evidence that the time‐varying acetylation state of p53's C‐terminal lysine residues is critical for gene‐specific regulation of stochastic bursting.

KEYWORDScellular heterogeneity/ DNA damage/ p53 signaling/ single-cell analysis/ stochastic transcription/ ABSTRACT Discontinuous transcription has been described for different mammalian cell lines and numerous promoters. However, our knowledge of how the activity of individual promoters is adjusted by dynamic signaling inputs from transcription factor is limited. To address this question, we characterized the activity of selected target genes that are regulated by pulsatile accumulation of the tumor suppressor p53 in response to ionizing radiation. We performed time resolved measurements of gene expression at the single cell level by smFISH and used the resulting data to inform a mathematical model of promoter activity. We found that p53 target promoters are regulated by frequency modulation of stochastic bursting and can be grouped along three archetypes of gene expression. The occurrence of these archetypes cannot solely be explained by nuclear p53 abundance or promoter binding of total p53. Instead, we provide evidence that the time-varying acetylation state of p53's C-terminal lysine residues is critical for gene-specific regulation of stochastic bursting. of 1 37

p53 plays a central role in defending the genomic integrity of our cells. In response to genotoxic stress, this tumour suppressor orchestrates the expression of hundreds of target genes, which induce a variety of cellular outcomes ranging from damage repair to induction of apoptosis. In this review, we examine how the p53 response is regulated on several levels in individual cells to allow precise and context-specific fate decisions. We discuss that the p53 response is not only controlled by its canonical regulators but also by interconnected signalling pathways that influence the dynamics of p53 accumulation upon damage and modulate its transcriptional activity at target gene promoters. Additionally, we consider how the p53 response is diversified through a variety of mechanisms at the promoter level and beyond to induce context-specific outcomes in individual cells. These layers of regulation allow p53 to react in a stimulus-specific manner and fine-tune its signaling according to the individual needs of a given cell, enabling it to take the right decision on survival or death.