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• W2945162776 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. 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. The p53 family of transcription factors regulates highly-diverse cellular functions, including tumor suppression and control of cell specification and identity (1.Levrero M. De Laurenzi V. Costanzo A. Gong J. Wang J.Y. Melino G. The p53/p63/p73 family of transcription factors: overlapping and distinct functions.J. Cell Sci. 2000; 113 (10769197): 1661-1670Crossref PubMed Google Scholar). p53 is a master tumor suppressor that protects organismal fidelity after exposure to cellular stress-like DNA damage. This activity depends on p53's ability to activate transcription of a canonical network of genes involved in DNA damage repair, cell cycle arrest, apoptosis, and senescence (2.Kastenhuber E.R. Lowe S.W. Putting p53 in context.Cell. 2017; 170 (28886379): 1062-107810.1016/j.cell.2017.08.028Abstract Full Text Full Text PDF PubMed Scopus (938) Google Scholar). Many of these canonical transcriptional pathways are individually dispensable for tumor suppression, suggesting p53 regulates a redundant and not yet fully characterized transcriptional network (3.Andrysik Z. Galbraith M.D. Guarnieri A.L. Zaccara S. Sullivan K.D. Pandey A. MacBeth M. Inga A. Espinosa J.M. Identification of a core TP53 transcriptional program with highly distributed tumor suppressive activity.Genome Res. 2017; 27 (28904012): 1645-165710.1101/gr.220533.117Crossref PubMed Scopus (77) Google Scholar, 4.Li T. Kon N. Jiang L. Tan M. Ludwig T. Zhao Y. Baer R. Gu W. Tumor suppression in the absence of p53-mediated cell-cycle arrest, apoptosis, and senescence.Cell. 2012; 149 (22682249): 1269-128310.1016/j.cell.2012.04.026Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar). Genetic inactivation of the p53 tumor suppressor is highly-recurrent across cancer types. p53 mutations, while frequent, vary depending on the tumor type with additional genetic and epigenetic mechanisms proposed to inactivate the p53 pathway in the presence of WT p53 (5.Zhu J. Dou Z. Sammons M.A. Levine A.J. Berger S.L. Lysine methylation represses p53 activity in teratocarcinoma cancer cells.Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (27535933): 9822-982710.1073/pnas.1610387113Crossref PubMed Scopus (29) Google Scholar, 6.Ciriello G. Miller M.L. Aksoy B.A. Senbabaoglu Y. Schultz N. Sander C. Emerging landscape of oncogenic signatures across human cancers.Nat. Genet. 2013; 45 (24071851): 1127-113310.1038/ng.2762Crossref PubMed Scopus (903) Google Scholar7.Kandoth C. McLellan M.D. Vandin F. Ye K. Niu B. Lu C. Xie M. Zhang Q. McMichael J.F. Wyczalkowski M.A. Leiserson M.D.M. Miller C.A. Welch J.S. Walter M.J. Wendl M.C. et al.Mutational landscape and significance across 12 major cancer types.Nature. 2013; 502 (24132290): 333-33910.1038/nature12634Crossref PubMed Scopus (2935) Google Scholar). p63 and p73, p53 family members, function similarly to p53 in stress response, although their precise roles in tumor suppression are unresolved (8.Flores E.R. Tsai K.Y. Crowley D. Sengupta S. Yang A. McKeon F. Jacks T. p63 and p73 are required for p53-dependent apoptosis in response to DNA damage.Nature. 2002; 416 (11932750): 560-56410.1038/416560aCrossref PubMed Scopus (723) Google Scholar, 9.Botchkarev V.A. Flores E.R. p53/p63/p73 in the epidermis in health and disease.Cold Spring Harb. Perspect. Med. 2014; 4 (25085956): a01524810.1101/cshperspect.a015248Crossref PubMed Scopus (79) Google Scholar10.Deyoung M.P. Ellisen L.W. p63 and p73 in human cancer: defining the network.Oncogene. 2007; 26 (17334395): 5169-518310.1038/sj.onc.1210337Crossref PubMed Scopus (214) Google Scholar). p63 and p73 are primarily lineage-restricted to epithelial cell types where each serves critical and nonoverlapping roles in cell identity and self-renewal (1.Levrero M. De Laurenzi V. Costanzo A. Gong J. Wang J.Y. Melino G. The p53/p63/p73 family of transcription factors: overlapping and distinct functions.J. Cell Sci. 2000; 113 (10769197): 1661-1670Crossref PubMed Google Scholar, 11.Melino G. Memmi E.M. Pelicci P.G. Bernassola F. Maintaining epithelial stemness with p63.Sci. Signal. 2015; 8 (26221054): re910.1126/scisignal.aaa1033Crossref PubMed Scopus (82) Google Scholar). Mutations in TA- and ΔN-p63 isoforms of p63 are causative for a number of epithelial-associated human developmental disorders independent of p53 activity, and mutations in p63 target genes underlie similar phenotypes (12.Fakhouri W.D. Rahimov F. Attanasio C. Kouwenhoven E.N. Ferreira De Lima R.L. Felix T.M. Nitschke L. Huver D. Barrons J. Kousa Y.A. Leslie E. Pennacchio L.A. Van Bokhoven H. Visel A. Zhou H. et al.An etiologic regulatory mutation in IRF6 with loss- and gain-of-function effects.Hum. Mol. Genet. 2014; 23 (24442519): 2711-272010.1093/hmg/ddt664Crossref PubMed Scopus (39) Google Scholar, 13.Brunner H.G. Hamel B.C. Van Bokhoven H. The p63 gene in EEC and other syndromes.J. Med. Genet. 2002; 39 (12070241): 377-38110.1136/jmg.39.6.377Crossref PubMed Scopus (147) Google Scholar). A significant and still outstanding question involves dissection of specific roles and functional interplay between p53 family members during development, in the regulation of cellular homeostasis, and in the etiology of disease. The similarity between DNA-binding motifs originally suggested that competition for binding sites might play a central role in the regulation of p53 family member activity (14.Moll U.M. Erster S. Zaika A. p53, p63 and p73–solos, alliances and feuds among family members.Biochim. Biophys. Acta. 2001; 1552 (11825686): 47-59PubMed Google Scholar, 15.Billant O. Léon A. Le Guellec S. Friocourt G. Blondel M. Voisset C. The dominant-negative interplay between p53, p63 and p73: a family affair.Oncotarget. 2016; 7 (27589690): 69549-6956410.18632/oncotarget.11774Crossref PubMed Scopus (29) Google Scholar16.McDade S.S. Patel D. Moran M. Campbell J. Fenwick K. Kozarewa I. Orr N.J. Lord C.J. Ashworth A.A. McCance D.J. Genome-wide characterization reveals complex interplay between TP53 and TP63 in response to genotoxic stress.Nucleic Acids Res. 2014; 42 (24823795): 6270-628510.1093/nar/gku299Crossref PubMed Scopus (47) Google Scholar). Indeed, previous studies implicate the ΔN-p63 isoforms, lacking a canonical transactivation domain, as direct repressors of the p53-induced transcriptome through a binding site–competitive mechanism (17.Yang A. Zhu Z. Kapranov P. McKeon F. Church G.M. Gingeras T.R. Struhl K. Relationships between p63 binding, DNA sequence, transcription activity, and biological function in human cells.Mol. Cell. 2006; 24 (17188034): 593-60210.1016/j.molcel.2006.10.018Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 18.Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dötsch V. Andrews N.C. Caput D. McKeon F. p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol. Cell. 1998; 2 (9774969): 305-31610.1016/S1097-2765(00)80275-0Abstract Full Text Full Text PDF PubMed Scopus (1840) Google Scholar19.Mundt H.M. Stremmel W. Melino G. Krammer P.H. Schilling T. Müller M. Dominant negative (ΔN) p63α induces drug resistance in hepatocellular carcinoma by interference with apoptosis signaling pathways.Biochem. Biophys. Res. Commun. 2010; 396 (20403333): 335-34110.1016/j.bbrc.2010.04.093Crossref PubMed Scopus (36) Google Scholar). Multiple p63-dependent mechanisms repressing p53 activity have been identified, including control of H2AZ deposition and HDAC 2The abbreviations used are: HDAChistone deacetylaseDHSDNase-hypersensitive siteANOVAanalysis of varianceqRT-PCRquantitative RT-PCRMEFmouse embryonic fibroblastCEBPBCCAAT enhancer–binding protein βTSStranscription start siteChIP-seqChIP-sequencingSkFibforeskin fibroblastEMTepithelial–to–mesenchymal transitionGAPDHglyceraldehyde-3-phosphate dehydrogenaseQCquality control. recruitment (20.Gallant-Behm C.L. Espinosa J.M. ΔNp63α utilizes multiple mechanisms to repress transcription in squamous cell carcinoma cells.Cell Cycle. 2013; 12 (23324337): 409-41610.4161/cc.23593Crossref PubMed Scopus (12) Google Scholar21.Gallant-Behm C.L. Ramsey M.R. Bensard C.L. Nojek I. Tran J. Liu M. Ellisen L.W. Espinosa J.M. ΔNp63α represses anti-proliferative genes via H2A.Z deposition.Genes Dev. 2012; 26 (23019126): 2325-233610.1101/gad.198069.112Crossref PubMed Scopus (46) Google Scholar, 22.Ramsey M.R. He L. Forster N. Ory B. Ellisen L.W. Physical association of HDAC1 and HDAC 2 with p63 mediates transcriptional repression and tumor maintenance in squamous cell carcinoma.Cancer Res. 2011; 71 (21527555): 4373-437910.1158/0008-5472.CAN-11-0046Crossref PubMed Scopus (83) Google Scholar23.LeBoeuf M. Terrell A. Trivedi S. Sinha S. Epstein J.A. Olson E.N. Morrisey E.E. Millar S.E. Hdac1 and Hdac2 act redundantly to control p63 and p53 functions in epidermal progenitor cells.Dev. Cell. 2010; 19 (21093383): 807-81810.1016/j.devcel.2010.10.015Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Increased p63 activity is thought to drive certain epithelial-derived cancers, particularly squamous cell carcinomas, with both p53-dependent and -independent mechanisms (24.Saladi S.V. Ross K. Karaayvaz M. Tata P.R. Mou H. Rajagopal J. Ramaswamy S. Ellisen L.W. ACTL6A is co-amplified with p63 in squamous cell carcinoma to drive YAP activation, regenerative proliferation, and poor prognosis.Cancer Cell. 2017; 31 (28041841): 35-4910.1016/j.ccell.2016.12.001Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). As the majority of cancers are derived from epithelial tissues, the mechanisms of p53 family–dependent tumor suppression in those tissues are of special interest (7.Kandoth C. McLellan M.D. Vandin F. Ye K. Niu B. Lu C. Xie M. Zhang Q. McMichael J.F. Wyczalkowski M.A. Leiserson M.D.M. Miller C.A. Welch J.S. Walter M.J. Wendl M.C. et al.Mutational landscape and significance across 12 major cancer types.Nature. 2013; 502 (24132290): 333-33910.1038/nature12634Crossref PubMed Scopus (2935) Google Scholar). histone deacetylase DNase-hypersensitive site analysis of variance quantitative RT-PCR mouse embryonic fibroblast CCAAT enhancer–binding protein β transcription start site ChIP-sequencing foreskin fibroblast epithelial–to–mesenchymal transition glyceraldehyde-3-phosphate dehydrogenase quality control. Fate choice after p53 activation, be it apoptosis, temporary/permanent cell cycle arrest, or continued proliferation, is variable across cell types suggesting that p53-dependent transcription is also cell type–dependent (2.Kastenhuber E.R. Lowe S.W. Putting p53 in context.Cell. 2017; 170 (28886379): 1062-107810.1016/j.cell.2017.08.028Abstract Full Text Full Text PDF PubMed Scopus (938) Google Scholar). A bevy of sensitive methodological approaches has been used to identify p53-binding sites and gene targets across transformed and primary cell lines in an effort to explain these terminal cell fate choices and p53-dependent tumor suppression. A recent set of meta-analyses has suggested that p53 binding to the genome is invariant (25.Verfaillie A. Svetlichnyy D. Imrichova H. Davie K. Fiers M. Kalender Atak Z. Hulselmans G. Christiaens V. Aerts S. Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic.Genome Res. 2016; 26 (27197205): 882-89510.1101/gr.204149.116Crossref PubMed Scopus (43) Google Scholar), proposing that p53 acts independently to drive gene expression of a core tumor suppressor network across all cell types due to the low enrichment of other transcription factor motifs at p53-bound enhancers and the reported pioneer factor activity of p53 (3.Andrysik Z. Galbraith M.D. Guarnieri A.L. Zaccara S. Sullivan K.D. Pandey A. MacBeth M. Inga A. Espinosa J.M. Identification of a core TP53 transcriptional program with highly distributed tumor suppressive activity.Genome Res. 2017; 27 (28904012): 1645-165710.1101/gr.220533.117Crossref PubMed Scopus (77) Google Scholar, 25.Verfaillie A. Svetlichnyy D. Imrichova H. Davie K. Fiers M. Kalender Atak Z. Hulselmans G. Christiaens V. Aerts S. Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic.Genome Res. 2016; 26 (27197205): 882-89510.1101/gr.204149.116Crossref PubMed Scopus (43) Google Scholar, 26.Younger S.T. Rinn J.L. p53 regulates enhancer accessibility and activity in response to DNA damage.Nucleic Acids Res. 2017; 45 (28973438): 9889-990010.1093/nar/gkx577Crossref PubMed Scopus (32) Google Scholar). Conversely, a series of recent p53 ChIP-seq experiments observed cell type–specific p53 binding and gene targets (27.Nguyen T.T. Grimm S.A. Bushel P.R. Li J. Li Y. Bennett B.D. Lavender C.A. Ward J.M. Fargo D.C. Anderson C.W. Li L. Resnick M.A. Menendez D. Revealing a human p53 universe.Nucleic Acids Res. 2018; 46 (30107566): 8153-816710.1093/nar/gky720Crossref PubMed Scopus (49) Google Scholar, 28.Hafner A. Lahav G. Stewart-Ornstein J. Stereotyped p53 binding tuned by chromatin accessibility.bioRxiv. 2017; (10.1101/177667)Google Scholar). Because of the high importance of these conflicting observations and the mutual exclusivity of the models, additional experimental evidence and models are required to unravel these disparate p53 regulatory mechanisms. We previously proposed that the local chromatin environment, including variable chromatin accessibility and enhancer activity, contributes to novel p53 activities across cell types (29.Sammons M.A. Zhu J. Drake A.M. Berger S.L. TP53 engagement with the genome occurs in distinct local chromatin environments via pioneer factor activity.Genome Res. 2015; 25 (25391375): 179-18810.1101/gr.181883.114Crossref PubMed Scopus (71) Google Scholar). To address this question, we performed genomewide transcriptome, epigenome, and p53 cistrome profiling in primary foreskin fibroblasts (SkFib) and mammary basal epithelial (MCF10A) cell lines, two cell types with varying enhancer activity at p53-binding sites (29.Sammons M.A. Zhu J. Drake A.M. Berger S.L. TP53 engagement with the genome occurs in distinct local chromatin environments via pioneer factor activity.Genome Res. 2015; 25 (25391375): 179-18810.1101/gr.181883.114Crossref PubMed Scopus (71) Google Scholar). Our results directly implicate differential cis-regulatory element activity as a mediator of the p53 network, with both differential promoter and enhancer activity contributing to p53-dependent gene expression variability. Furthermore, we have identified the p53 family member p63 as one factor that drives the epithelial-specific p53 transcriptome through an enhancer maintenance activity. We further propose that p63 serves as a pioneer factor for a set of epithelial-specific enhancers. Thus, these data support a mechanism whereby cooperating transcription factors control cell type–dependent cis-regulatory networks that regulate p53 activity. Gene activation downstream of p53 stabilization has been extensively studied, but whether p53 transcriptionally activates the same genes across all cell types is still unresolved. Previous work suggested that p53-dependent transcriptional activity might vary between epithelial cells and fibroblasts due to differences in predicted enhancer activity between the two cell types (29.Sammons M.A. Zhu J. Drake A.M. Berger S.L. TP53 engagement with the genome occurs in distinct local chromatin environments via pioneer factor activity.Genome Res. 2015; 25 (25391375): 179-18810.1101/gr.181883.114Crossref PubMed Scopus (71) Google Scholar). We chose two widely used model cell lines (MCF10A mammary epithelial cells and dermal foreskin fibroblasts) to address this unresolved question. The two cell types differ in developmental origin, with dermal fibroblasts and mammary epithelial cells arising from mesoderm and ectoderm, respectively. Importantly, both cell types are nontumorigenic in mouse models, suggesting they represent a nontransformed state. We performed three biological replicates of poly(A)-enriched, strand-specific RNA-seq in both cell lines after p53 activation in response to a 6-h treatment with 5 μm Nutlin-3A. Nutlin-3A is a highly-specific MDM2 antagonist that leads to p53 stabilization without activation of p53-independent DNA damage pathways that can confound analysis (30.Vassilev L.T. Vu B.T. Graves B. Carvajal D. Podlaski F. Filipovic Z. Kong N. Kammlott U. Lukacs C. Klein C. Fotouhi N. Liu E.A. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2.Science. 2004; 303 (14704432): 844-84810.1126/science.1092472Crossref PubMed Scopus (3784) Google Scholar). Raw data were aligned using STAR, and differentially expressed genes were called using DESeq2. Use of a stringent cutoff (2-fold increase in expression after Nutlin-3A treatment, q value <0.05) revealed different patterns of gene expression for MCF10A versus SkFib (Fig. 1A). Genes up-regulated in both cell lines are significantly associated with the core p53 response and programmed cell death (Fig. 1B and Table S1), as expected. MCF10A displayed a larger group of Nutlin-3A–induced targets enriched in gene ontology groups related to establishment of the epithelial barrier and p53-dependent processes like programmed cell death and homeostasis (Fig. 1, A and B). SkFib showed a markedly smaller, albeit specific, p53-activated transcriptome (Fig. 1A). These genes are associated with other cell stress-related pathways, including those associated with the hypoxia response and catabolic processes. Allowing for any change in expression while maintaining a corrected p value of less than 0.05 yielded an increased number of commonly and differentially regulated genes, but the trend that p53-dependent gene targets are more abundant in MCF10A cells remained consistent across analytic methods (Fig. S1A). Orthogonal validation by qRT-PCR confirmed a stringent cell-type specificity for the tested set of genes identified by RNA-seq (Fig. 1, C–E). Up-regulation of canonical p53 targets like BTG2 and CDKN1A are indeed similar across cell types (Fig. 1C). Many of the cell type–specific targets were undetected or detected at extremely low levels in the opposing cell type (Fig. 1, D and E). The majority of the MCF10A-specific targets represent novel p53-dependent genes not previously identified in a large-scale meta-analysis of human p53 gene targets (32.Fischer M. Census and evaluation of p53 target genes.Oncogene. 2017; 36 (28288132): 3943-395610.1038/onc.2016.502Crossref PubMed Scopus (466) Google Scholar). Over 20% of newly identified SkFib-specific targets have not been observed previously (Fig. 1F and Tables S2 and S3). These initial analyses reveal that the p53-activated transcriptome varies between nontransformed cell types and may reflect tailored, cell type–dependent responses after cellular stress. To confirm the p53-dependent nature of these Nutlin-3A–induced genes, p53 mRNA and protein expression were depleted using shRNA in both MCF10A and SkFib (Fig. 1, G and H, and J and K). Knockdown of p53 in SkFib (Fig. 1, G and H) led to sharp reduction in both basal and Nutlin-3A–induced expression of the SkFib-specific genes, GDNF and TRIM55 (Fig. 1I). Depletion of p53 in MCF10A (Fig. 1, J and K) caused a loss of Nutlin-3A–induced RIC3 and IL1B expression relative to a nontargeting control shRNA (Fig. 1L). Taken together, our RNA-seq analysis and p53-depletion experiments indicate differential activation of p53-dependent targets across nontransformed cell lines. The most straightforward potential mechanism driving our observation of cell type–dependent transcriptional activation by p53 involves differential binding of p53 to regulatory regions controlling those genes. Recent analyses reached opposing conclusions with regard to the cell type–dependence of p53 engagement with the genome (3.Andrysik Z. Galbraith M.D. Guarnieri A.L. Zaccara S. Sullivan K.D. Pandey A. MacBeth M. Inga A. Espinosa J.M. Identification of a core TP53 transcriptional program with highly distributed tumor suppressive activity.Genome Res. 2017; 27 (28904012): 1645-165710.1101/gr.220533.117Crossref PubMed Scopus (77) Google Scholar, 25.Verfaillie A. Svetlichnyy D. Imrichova H. Davie K. Fiers M. Kalender Atak Z. Hulselmans G. Christiaens V. Aerts S. Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic.Genome Res. 2016; 26 (27197205): 882-89510.1101/gr.204149.116Crossref PubMed Scopus (43) Google Scholar, 27.Nguyen T.T. Grimm S.A. Bushel P.R. Li J. Li Y. Bennett B.D. Lavender C.A. Ward J.M. Fargo D.C. Anderson C.W. Li L. Resnick M.A. Menendez D. Revealing a human p53 universe.Nucleic Acids Res. 2018; 46 (30107566): 8153-816710.1093/nar/gky720Crossref PubMed Scopus (49) Google Scholar, 28.Hafner A. Lahav G. Stewart-Ornstein J. Stereotyped p53 binding tuned by chromatin accessibility.bioRxiv. 2017; (10.1101/177667)Google Scholar). We therefore assessed whether differences in p53 engagement with the genome in our model cell lines might explain our observations of cell type–dependent transcriptomes. Two biological replicates of p53 ChIP-seq were performed in either DMSO or Nutlin-3A–treated MCF10A and SkFib. Regions of significant p53 enrichment relative to input (peaks) were called using MACS2 (33.Zhang Y. Liu T. Meyer C.A. Eeckhoute J. Johnson D.S. Bernstein B.E. Nusbaum C. Myers R.M. Brown M. Li W. Liu X.S. Model-based analysis of ChIP-Seq (MACS).Genome Biol. 2008; 9 (18798982): R13710.1186/gb-2008-9-9-r137Crossref PubMed Scopus (8536) Google Scholar). Only peaks identified in both replicates were considered for further analysis (see under “Experimental procedures”). Consistent with the cell type-dependence of our gene expression observations, our ChIP-seq approach revealed a highly-enriched set of MCF10A-specific p53-binding sites and a smaller number of SkFib-specific sites (Fig. 2A). A highly-similar set of differential p53-binding events was also observed using the DiffBind statistical framework (Fig. S1B), suggesting the differential binding is unlikely to be an artifact of any one peak calling or replication strategy. Taken together, our peak calling approaches indicate differential genome engagement of p53 in MCF10A versus SkFib. At commonly bound sites, basal and Nutlin-3A–induced p53 enrichment is higher in MCF10A relative to SkFib (Fig. 2B). Despite higher overall occupancy in MCF10A, SkFib show higher relative fold-change in p53 enrichment between DMSO and Nutlin-3A treatment (Fig. 2B). Relatedly, absolute p53 occupancy differences do not correlate with the ability to activate common gene targets as Nutlin-3A induced gene fold-changes are higher in SkFib compared with MCF10A (Fig. 2C). Cell type–specific binding events displayed lower absolute p53 occupancy than at common sites, suggesting that common sites might represent more stable or higher affinity p53-binding sites (Fig. 2, B, D and E). This observation is in line with previous observations in transformed cancer cell lines and suggests a core set of p53-binding events is well-conserved across cell types (3.Andrysik Z. Galbraith M.D. Guarnieri A.L. Zaccara S. Sullivan K.D. Pandey A. MacBeth M. Inga A. Espinosa J.M. Identification of a core TP53 transcriptional program with highly distributed tumor suppressive activity.Genome Res. 2017; 27 (28904012): 1645-165710.1101/gr.220533.117Crossref PubMed Scopus (77) Google Scholar, 25.Verfaillie A. Svetlichnyy D. Imrichova H. Davie K. Fiers M. Kalender Atak Z. Hulselmans G. Christiaens V. Aerts S. Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic.Genome Res. 2016; 26 (27197205): 882-89510.1101/gr.204149.116Crossref PubMed Scopus (43) Google Scholar, 27.Nguyen T.T. Grimm S.A. Bushel P.R. Li J. Li Y. Bennett B.D. Lavender C.A. Ward J.M. Fargo D.C. Anderson C.W. Li L. Resnick M.A. Menendez D. Revealing a human p53 universe.Nucleic Acids Res. 2018; 46 (30107566): 8153-816710.1093/nar/gky720Crossref PubMed Scopus (49) Google Scholar). We then wanted to validate our observations of differential p53 binding using standard ChIP-qPCR methods. p53 binding at the CDKN1A promoter in both MCF10A and SkFib increased in response to Nutlin-3A treatment and was sensitive to depletion of p53 by shRNA (Fig. 2F). ChIP signal at the CDKN1A promoter was more highly enriched relative to a region ≈4 kb upstream of the binding event, confirming the specificity of p53 binding. Examination of p53 binding at two genomic locations in SkFib and MCF10A confirmed our ChIP-seq studies. p53 binding was enriched at the BDNF locus in SkFib and was sensitive to p53 depletion, whereas the signal observed in MCF10A was at a background level and not affected by either Nutlin-3A treatment or p53 knockdown (Fig. 2G). Similar cell-type specificity was observed for an MCF10A-specific p53-binding event (Fig. 2H). Taken together, these data provide evidence that the p53-dependent transcriptome is cell type–specific and that p53 engagement with the genome is variable across cell types. We next wanted to identify potential mechanisms driving cell-type specificity within the p53-dependent transcriptome and cistrome. Previous analyses of p53 genomic occupancy suggested that gene proximal binding of p53 correlates with changes in gene activation (3.Andrysik Z. Galbraith M.D. Guarnieri A.L. Zaccara S. Sullivan K.D. Pandey A. MacBeth M. Inga A. Espinosa J.M. Identification of a core TP53 transcriptional program with highly distributed tumor suppressive activity.Genome Res. 2017; 27 (28904012): 1645-165710.1101/gr.220533.117Crossref PubMed Scopus (77) Google Scholar, 32.Fischer M. Census and evaluation of p53 target genes.Oncogene. 2017; 36 (28288132): 3943-395610.1038/onc.2016.502Crossref PubMed Scopus (466) Google Scholar) but that many p53-dependent gene targets are likely regulated by more distal p53-binding events. MCF10A-specific genes are more likely to have a p53-binding event near their TSS compared with p53 peaks in SkFib (Fig. S1C). Conversely, SkFib-specific genes are not associated with either a higher number of SkFib-specific p53-binding events or more proximal p53-binding events than in MCF10A (Fig. S1C). These observations suggest that although cell type–specific p53 binding correlates with an increase in p53 gene targets in MCF10A, differential p53 binding alone is insufficient to explain differential gene expression. We and others previously proposed that differential regulatory region activity may control p53-dependent gene expression (29.Sammons M.A. Zhu J. Drake A.M. Berger S.L. TP53 engagement with the genome occurs in distinct local chromatin environments via pioneer factor activity.Genome Res. 2015; 25 (25391375): 179-18810.1101/gr.181883.114Crossref PubMed Scopus (71) Google Scholar, 34.Sullivan K.D. Galbraith M.D. Andrysik Z. Espinosa J.M. Mechanisms of transcriptional regulation by p53.Cell Death Differ. 2018; 25 (29125602): 133-14310.1038/cdd.2017.174Crossref PubMed Scopus (244) Google" @default.
• W2945162776 created "2019-05-29" @default.
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• W2945162776 creator A5086901195 @default.
• W2945162776 date "2019-07-01" @default.
• W2945162776 modified "2023-10-14" @default.
• W2945162776 title "Control of p53-dependent transcription and enhancer activity by the p53 family member p63" @default.
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• W2945162776 doi "https://doi.org/10.1074/jbc.ra119.007965" @default.
• W2945162776 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6615668" @default.
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