Control of p53-dependent transcription and enhancer activity by the p53 family member p63

Abstract

Transcriptional activation by p53 provides powerful, organism-wide tumor suppression. We hypothesized that the local chromatin environment, including differential enhancer activities, contributes to various p53-dependent transcriptional activities in different cell types during stress-induced signaling. In this work, using ChIP-sequencing, immunoblotting, quantitative PCR, and computational analyses across various mammalian cell lines, we demonstrate that the p53-induced transcriptome varies by cell type, reflects cell type-specific activities, and is considerably broader than previously anticipated. We found that these molecular events are strongly influenced by p53's engagement with differentially active cell type-specific enhancers and promoters. We also observed that p53 activity depends on the p53 family member tumor protein p63 in epithelial cell types. Notably, we demonstrate that p63 is required for epithelial enhancer identity, including enhancers used by p53 during stress-dependent signaling. Loss of p63, but not p53, caused site-specific depletion of enhancer-associated chromatin modifications, suggesting that p63 functions as an enhancer maintenance factor in epithelial cells. Additionally, a subset of epithelial-specific enhancers depends on the activity of p63 providing a direct link between lineage determination and enhancer structure. These results suggest that a broad, cell-intrinsic mechanism controls p53-dependent cellular stress response through differential regulation of cis-regulatory elements.

Locally acting transcription factors are required for p53-dependent cis-regulatory element activity

“…This observation is consistent with recent reports of basal enhancer RNA transcription and histone modification enrichment at potential CREs in the absence of p53 (2,48,54). We therefore wanted to determine whether these p53-bound regions act as CREs for the endogenous expression of GDF15.…”

“…Despite this activity, the large majority of p53 genomic binding events occur within regions that are accessible before p53 engagement (10,48,54,59), similar to what is observed for glucocorticoid receptor binding (66). These regions also contain chromatin modifications associated with active CRE, including H3K27ac and H3K4me1/2 before p53 binding (1,48,54,59,60,60). Further, p53 depletion does not alter basal CRE-associated chromatin modifications or chromatin structure at the large majority of CRE (48,54).…”

“…p53 is a pioneer transcription factor and can mediate context-dependent chromatin remodeling at CREs (10,48,54). Despite this activity, the large majority of p53 genomic binding events occur within regions that are accessible before p53 engagement (10,48,54,59), similar to what is observed for glucocorticoid receptor binding (66).…”

“…These regions also contain chromatin modifications associated with active CRE, including H3K27ac and H3K4me1/2 before p53 binding (1,48,54,59,60,60). Further, p53 depletion does not alter basal CRE-associated chromatin modifications or chromatin structure at the large majority of CRE (48,54). Enhancer-derived RNA (eRNA) have been identified as a strong predictor of transcription factor binding and CRE activity (67).…”

“…6A.) These data suggest that other factors are likely responsible for establishing and maintaining chromatin structure and basal activity at the majority of p53-bound CREs. Recent reports suggest that p53 binding and activity is strongly influenced by cell type-specific chromatin accessibility (1,2,54), which itself is controlled by differential transcription factor activity (11,12,(68)(69)(70). How p53 functions across various cell and tissue contexts is still a vital and open question, but recent reports suggested that p53 binding and activity can be influenced by cell type (2,54,71).…”