Cell type-dependent control of p53 transcription and enhancer activity by p63

Transcriptional activation by p53 provides powerful, organism-wide tumor suppression. In this work, we demonstrate that the p53-induced transcriptome varies based on cell type, reflects cell type-specific activities, and is considerably more broad than previously anticipated. This behavior is strongly influenced by p53 engagement with differentially active cell type-specific enhancers and promoters. In epithelial cell types, p53 activity is dependent on the p53 family member p63, which displays widespread enhancer binding. 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, leads to 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 is dependent on the activity of p63 providing a direct link between lineage determination and enhancer structure. These data suggest a broad, cell-intrinsic mechanism for regulating the p53-dependent cellular response to stress through differential regulation of cis-regulatory elements.


INTRODUCTION
The p53 family of transcription factors regulate highly diverse cellular functions, including tumor suppression and control of cell specification and identity (1). p53 is a master tumor suppressor that protects organismal fidelity after exposure to cellular stress like DNA damage. This activity is dependent 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). Surprisingly, many of these transcriptional pathways are individually dispensable for tumor suppression, suggesting p53 regulates a redundant and not-yet fully characterized transcriptional network (3,4).
Genetic inactivation of the p53 tumor suppressor pathway 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 wild-type p53 (5)(6)(7). p63 and p73, p53 family members, function similarly to p53 in stress response, although their precise roles in tumor suppression are unresolved (8)(9)(10). p63 and p73 are primarily lineage restricted to epithelial cell types where each serves critical and non-overlapping roles in cell identity and self-renewal (1,11). Mutations in TA and ∆Np63 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 (12,13). 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 suggested that competition for binding sites serves a central role in the regulation of p53 family member activity (14)(15)(16).
Indeed, previous studies implicate the ∆Np63 isoforms, lacking transactivation domains, as direct repressors of the p53-induced transcriptome through a binding site competitive mechanism (17)(18)(19). Amplification of p63 is thought to drive a number of epithelial cancers, particularly squamous cell carcinomas, although both p53-dependent and independent mechanisms have been proposed for this driver activity (20). As the majority of cancers are derived from epithelial tissues, the mechanisms of p53 family dependent tumor suppression in those tissues is of special interest (7).
Fate choice after p53 activation, be it apoptosis, temporary/permanent cell cycle arrest, or continued proliferation, is variable across cell types suggesting that p53dependent transcription is also cell type-dependent (2). A bevy of sensitive methodological approaches have 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 have suggested that p53 binding to the genome is invariant (21), 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 (3,21,22). Conversely, similar experimental approaches demonstrate cell type-specific p53 binding and gene targets although the mechanisms driving these differential activities are unknown (BioRxiv: https://doi.org/10.1101/177667). Additional experimental evidence and models are required to unravel these mutually exclusive 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 (23). To address this question, we performed genomewide transcriptome, epigenome, and p53 cistrome profiling in primary foreskin fibroblasts (SkFib) and breast epithelial (MCF10A) cell lines, two cell types with varying enhancer activity at p53 binding sites (23). Our results directly implicate differential cis-regulatory element activity as a mediator of the p53 network, with both promoter and enhancer activity contributing to p53-dependent gene expression differences. Further, 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 licensing factor for a set of epithelial-specific enhancers. Thus, these data support a mechanism whereby co-operating transcription factors control cell typedependent cis-regulatory networks that regulate p53 activity.

RNA-sequencing sample and library preparation
Total mRNA was extracted with E.Z.N.A. Total RNA kit (Omega; R6834-02) and Poly(A)+ RNA was isolated by double selection with poly-dT beads, using 2.5 µg total RNA, which is then followed by first-and second-strand synthesis. Sequencing libraries were prepared using NEXTflex Rapid Illumina DNA-Seq Library Prep Kit (Bioo Scientific). Samples were single-end sequenced on an NextSeq 500. RNA-seq reads were aligned via STAR (24) to Ensembl v.75/hg19. Count tables were generated using STAR-aligned BAM files and HTseq (25). Count tables were then used to call differentially expressed genes using DEseq2 (26).

Quantitative Real-Time PCR
Total RNA was extracted as per RNA-sequencing protocol and cDNA was synthesized using 1ug of total RNA as a template and the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems; 413760). Relative standard qPCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad; 172-5124) with primers shown in Supplementary Table S8 on an ABI 7900HT instrument.
Lentivirus containing supernatants were collected at 24 hours and 48 hours posttransfection, and filtered through a 0.45 µm membrane and stored in aliquots at -80°C.
MCF10A or SkFib cells were transfected with lentivirus supplemented with 8 µg/ml polybrene. At 24 hour post-infection with lentivirus, media was replaced with the proper puromycin selection (0.5 µg/ml for MCF10A and 2 µg/ml for SkFib).

ChIP-sequencing sample and library preparation
Cells were crosslinked at 80% confluency with 1% formaldehyde for 10 min at room temperature. Crosslinking was quenched with 125mM glycine and the resulting pellet was washed twice with cold PBS and lysed as previously described (27). Samples were subjected to sonication with Diagenode Bioruptor Plus for 40 cycles (30 sec on/off at high setting) for shearing chromatin to 150-500 bp average size. Immunoprecipitation reactions for SkFib, MCF10A, and HCT116 cell lines were performed with Diagenode IP-Star Compact Automated System, with the exception of p53 ChIP experiments.
Mouse embryonic fibroblast and p53 ChIP-seq were performed as described (23).
Sequencing libraries were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs). Prior to sequencing, library quality control was performed with Qubit (Thermo Fisher Scientific), Bioanalyzer (Agilent) and qPCR quantification. All ChIP samples, including input, were single-end sequenced on an NextSeq 500 at the University at Albany Center for Functional Genomics. Uniquely aligned reads (up to one mismatch) were aligned to NCBI37/hg19 using Bowtie2 (28).

ChIP-seq Peak Calling and Differential Binding Analysis
Significant regions of transcription factor (p53, p63) and histone modification (H3K4me2, H3K4me3, H3K27ac) enrichment were called using macs v.2.1.0 (29) preserving only peaks with an adjusted P-value < 0.01. All peak calling was performed with sheared chromatin input. p53 and p63 motif analysis was performed using p53scan (30) and all macs-derived peaks lacking a p53/p63 consensus motif were removed from further analysis. A full list of p53/p63 peaks can be found under GEO Accession Number GSE111009. Global transcription factor motif enrichment experiments were performed using the findMotifsGenome.pl script from HOMER (31). Differential enrichment of p53 ChIP-seq datasets across cell types was performed using DiffBind (32).

Analysis of histone modification enrichment
Chromatin enrichment analyses were performed using the annotatePeaks.pl script of HOMER. Read-depth-scaled chromatin tag enrichment within a specified window was input-normalized (target -input) and then further scaled to total genomewide enrichment of peaks for that histone modification. H3K27ac ChIP-seq data from MCF7 cells was produced by the ENCODE Project (ENCFF855RCK and ENCFF761EUR).

Computational Analysis and Plotting
Peak file intersections were performed using the intersectBed package of BedTools (33), scoring positive intersections as those with at least 1bp of overlap (-f 1E-9). The original peak file (-a) was reported only once (-u) if any intersection exists with the query file (-b). The closestBed package of BedTools was used to measure distances between features with the distance reported using the -d option. Figures were generated using Graphpad Prism or Datagraph. Bigwig (bw) files were generated with the bamCoverage package of deepTools v.2.5.4 (34) with a bin size of 1 (--binSize 1) and read extension (--extendReads 300). Heatmaps were then generated using bigwig files for a -/+ 1000bp region from the p53/p63 peak center using the computeMatrix (reference-point, -a 1000, -b 1000, --binSize 10) and plotHeatmap functions. Genome browser tracks (bedGraph) were generated using the makeUCSCfile.pl package of HOMER (31). K-means clustering was performed using cluster 3.0 for Mac.

Statistical Testing
Statistical testing was performed using Graphpad Prism (v. 7) or using built-in statistical parameters in R (v.3.4.0).

p53-dependent transcription and DNA binding are cell type-dependent
We used polyA-enriched RNA-seq to assess gene expression dynamics in model cell lines (MCF10A and SkFib) after p53 activation in response to Nutlin-3A treatment ( Figure 1A, Supplementary Figure S1A). We reasoned Nutlin-3A, a non-genotoxic activator of p53, would better reflect p53-dependent transcriptional activity relative to genotoxic activation methods as DNA damage-dependent, but p53-independent, signaling would not confound our analysis (36, 37). Basal and nutlin-3A-induced p53 protein levels are substantially elevated in MCF10A versus SkFib. Consistent with a cell type-dependent activity of p53, MCF10A cells displayed a broad and specific set of Nutlin-3A-induced genes, whereas SkFib showed a markedly smaller, albeit specific, p53-activated transcriptome ( Figure 1B Recent analyses reached opposing conclusions with regard to the cell typedependence of p53 engagement with the genome (21,39). We thus assessed whether differences in p53 engagement with the genome in our model cell lines might explain our observations of cell type-dependent transcriptomes. Consistent with cell typedependence, genomewide ChIP-seq revealed a highly-enriched set of MCF10A-specific p53 binding sites and a smaller number of SkFib-specific sites, mirroring our gene expression observations ( Figure 1G). We only considered p53 binding sites that replicated across two independent biological replicates. Cell type-dependent differential p53 binding was further validated using the DiffBind statistical framework MCF10A-specific genes have a higher percentage of proximal p53 binding relative to SkFib. Notably, we observe both MCF10A and SkFib p53 binding proximal to SkFibspecific genes (Supplementary Figure S1M). Cell type-specific binding events displayed lower p53 occupancy than at common sites, suggesting that common sites might represent more stable or higher affinity p53 binding sites (Supplementary Figure S1N). This observation is in line with previous results suggesting a core p53 program is well conserved across cell types (3,21,40). Taken together, these data provide evidence that the p53-dependent transcriptome is indeed cell type-specific and that p53 engagement with the genome is variable across cell types.
Promoter and enhancer chromatin states define differential p53 binding and transcriptional activity.
We next wanted to identify potential mechanisms driving the strong cell type-specificity within the p53 transcriptome and cistrome. The most straightforward hypothesis is that differential p53 binding drives cell type-specific transcriptome differences, but our data suggest that this might only explain MCF10A behavior ( Figure 1G, Supplementary Figure S1M). Thus, we examined GDNF, a SkFib-specific gene, and observed proximal p53 binding 8.5kb upstream of the GDNF transcriptional start site (Figure 2A). We also noted p53 binding to this region in MCF10A, even though p53-dependent GDNF expression is SkFib-specific ( Figure 2A). Similar p53 proximal binding was observed at SkFib-specific genes TRIM55 and MAMDC2 (Supplementary Figure S2A), suggesting that differential proximal p53 binding per se does not explain the transcriptional specificity observed in SkFib relative to MCF10A.
We and others previously proposed that differential enhancer activity may regulate p53-dependent gene expression (23,41). As enhancers also have a strong cell type-dependence (42, 43), we performed ChIP-seq experiments for canonical promoter (H3K4me3) and enhancer-associated (H3K4me2, H3K27ac) histone modifications to determine whether differentially active promoters or enhancers might partially explain the observed cell type specificity of the p53 transcriptome. The local chromatin environment at the GDNF proximal p53 binding site is enriched for H3K27ac and H3K4me2, but lacks H3K4me3, suggestive of an active enhancer in both cell types (dashed box, Figure 2A). Consistent with high basal and p53-inducible expression of GDNF in SkFib, the GDNF promoter is strongly enriched for the canonical promoterassociated histone modification H3K4me3 (solid box, Figure 2A) which is completely absent from MCF10A. We observed differential promoter/enhancer activity at other SkFib-specific p53 target promoters/enhancers (Supplementary Figure S2A) without differences in p53 binding. Differential promoter/enhancer activity may therefore be a general mechanism by which cell types permit activation of particular p53 target genes.
We next investigated putative mechanisms driving our observation of epithelialspecific p53 target gene activation. p53 binding is pervasive in MCF10A relative to SkFib ( Figure 1G Figure  S4E). Globally, p63 binds to significantly more genomic loci than does p53 ( Figure 3B), and over 75% of epithelial-specific p53 binding events overlap p63 binding. Shared binding sites display higher occupancy and are more likely to have enhancer-associated chromatin modifications than do sites unique to either p53 or p63 ( Figure 3C, Supplementary Figure S4F), suggesting that both p53 and p63 might be necessary for full enhancer activity.
Overall, p63 binding correlates with epithelial-specific enhancer modifications and co-occupies the majority of p53 sites ( Figure 3B-E, Supplementary Figure S4G).
The modal binding of p63 is within 10kB of the TSS of Nutlin-3A-induced genes, further suggesting that p63 might co-regulate these specific target genes (Supplementary Figure S4G). In order to test whether p63 regulates p53 activity, we created MCF10A cells stably-expressing shRNA against p63 ( Figure 3F, Supplementary Figure S4H). We then performed RNA-seq analysis of MCF10A cells expressing non-targeting (scr), p53, or p63 shRNA to determine their relative contributions to epithelial-specific target genes.
Loss of p53 expression strongly inhibited Nutlin-3A-induced gene expression consistent with this set of genes representing true p53 targets ( Figure 3G). Knockdown of p63 showed more pleiotropic effects. Globally, Nutlin-3A-induced target gene activation is inhibited in p63 knockdown relative to non-targeting controls, suggesting p63 activity is required for a set of epithelial p53 targets ( Figure 3G). Whereas 40% of p53 target genes show defective induction in the absence of p63, the remainder of the genes are either unaffected or display increased p53-dependent transactivation (Supplementary Figure S4I). This observation is consistent with previous reports of direct repression of p53-dependent transactivation through binding site competition (18,19).
Our data suggest that p63 also functions to co-activate a set of p53-dependent genes specifically in epithelial cell types. We then validated these results by qRT-PCR on a panel of epithelial target genes after p53 or p63 knockdown. RIC3, previously demonstrated to be a p53-dependent gene ( Figure 1F), is also derepressed and shows higher p53-dependent transactivation in the absence of p63 ( Figure 3H). Conversely, both IL1A and IL1B are dependent on p53 and p63 for full transactivation, with IL1B showing a strong dependence on p63 for basal expression ( Figure 3H). Knockdown of p63 also led to basal gene expression changes for p53 targets (Supplementary Figure  S4J) and genes related to cell cycle and cell adhesion/extracellular matrix (Supplementary Table S5), as has previously been described (49). In total, these results suggest that p63 functions as both a co-activator and repressor of p53-dependent transcription in MCF10A, but how these opposing mechanisms lead to differential p53 activity is not clear.  Figure S5A). In total, over 20% of H3K4me2+ and 15% of H3K27ac+ p63-bound sites showed at least a 2-fold depletion in MCF10A-p63sh cells relative to scrambled shRNA ( Figure 4D). These enhancer-associated histone modifications were generally unaffected by the loss of p53 ( Figure 4D), suggesting this behavior is specific to p63. This loss of enhancer-associated histone modifications at p63 binding sites suggests that p63 plays a direct role in the maintenance of enhancer activity.
Our observations suggest that p63 activity is required for H3K27ac and H3K4me2 enrichment at a subset of its bound enhancers. p63-bound enhancers dependent on p63 for H3K4me2 enrichment (p63-dependent) were significantly more likely to overlap epithelial-specific DNase hypersensitive sites than those bound by p63, Our data indicates that p63 likely functions to maintain chromatin structure and activity at a set of epithelial-specific enhancers, but not for the entire complement of p63-bound enhancers. A number of studies have demonstrated that p53 has pioneer factor activity as it can directly bind its consensus sequence in the context of a nucleosome (23,54,55). p53 may possess pioneer factor activity, but the large majority of p53 binding sites are constitutively closed or are within previously accessible chromatin (3,22,23). Therefore, we examined whether p53 might serve pioneer or maintenance roles at enhancers as well. Basal H3K4me2 and H3K27ac enrichment at p53 binding sites was unaffected by the loss of p53 (Fig 4G, Supplementary Figure 5D).
p63 knockdown led to a slight loss of H3K27ac and H3K4me2 enrichment at p53 binding sites ( Figure 4G, Supplementary Figure S4D), an expected result as approximately 75% of p53 binding events in MCF10A cells are also p63 binding sites ( Figure 3B). Examination of p53+/p63-binding events displayed a similar trend, with both H3K27ac and H3K4me2 unaffected by p53 loss (Supplementary Figure S4E).
These results suggest that p53 plays a limited role in the specification and maintenance of basal enhancer. The observed p53-independence of basal enhancers is consistent with previous observations that p53 is dispensable for basal eRNA abundance at enhancers (37). Unlike its role in basal enhancer activity, p53 is absolutely required for de novo Nutlin-3A-induced H3K27ac levels at p53-bound enhancers ( Figure 4G-H).
Depletion of p63 results in a substantial increase in Nutlin-3A-induced H3K27ac at p53 binding sites ( Figure 4G). Nearly 85% of p53-bound enhancers gain H3K27ac (> 2-fold relative to DMSO) in the absence of p63, suggesting that p63 binding can locally inhibit p53-dependent chromatin modification enrichment and activity.
Epithelial cell types like MCF10A express multiple p53 family members that might redundantly regulate enhancer activity, making it difficult to assess the direct role of p53 in modulating enhancer activity. We therefore extended our analysis to isogenic WT or TP53-/-HCT116 human colon carcinoma cell lines and WT or Trp53-/-mouse embryonic fibroblasts (MEF). Both cell models lack endogenous p63 expression and thus should allow us to define the direct role of p53 in enhancer maintenance and activity. Similar to our observations in MCF10A cells, basal enhancer-associated H3K4me1/2 methylation at p53-bound enhancers is unaffected by the loss of p53 ( Figure 4I-J). These data suggest that p53 is not an enhancer maintenance factor and contributes primarily to de novo histone acetylation and transcriptional activation at already established enhancers.

DISCUSSION
In this report, we focused on identifying mechanisms that control differential expression of p53 target genes across cell types. Our results demonstrate that differential cis-regulatory activity and p53 binding serve as critical determinants of cell type-specific, p53-dependent transcriptomes. The acute p53 response in foreskin fibroblasts and breast epithelial cells (MCF10A) leads to distinct transcriptional programs that appear to reflect cell type-specific functions. In the case of MCF10A, p53 activates a series of genes involved directly in epithelial cell identity, such as epithelial cornification genes and ZNF750, a key regulator of epithelial cell differentiation (56, 57).
Knockout studies in mice suggest p53's role in development is limited, although neural tube and germ cell defects are observed at low penetrance (58-60). Therefore, our observations that p53 regulates key epithelial development and differentiation genes is surprising. Loss of p53 activity is a strong predictor of epithelial-to-mesenchymal transition (EMT) (61,62). One potential rationale for p53-dependent regulation of these key epithelial lineage genes is a mechanism to protect against EMT by supporting transcription of epithelial identity genes. Of note, canonical p53 transcriptional pathways appear to be dispensable for tumor suppression (4). Recent work demonstrated that the p53 transcriptional network is highly distributed as to allow loss of multiple target genes/pathways without loss of p53-dependent tumor suppression (3). Certainly, additional investigations into cell type-dependent p53 transcriptional targets, and their roles in context-dependent tumor suppression are warranted given these findings.
Our data suggest that transcriptional activation by p53 requires both geneproximal p53 binding and pre-establishment of cis-regulatory elements. Fibroblastspecific p53 target genes have active promoters, characterized by canonical H3K4me3 enrichment, and p53 engagement with distal enhancer regions. Conversely, the same enhancers were active and engaged by p53 in MCF10A, but the gene promoters had chromatin-associated hallmarks of inactivity. It is currently unclear whether the lack of activity in MCF10A is driven by lack of promoter-bound cofactors, through repressive chromatin-associated mechanisms, or both. Repressive DNA and histone modifications have been implicated in differential control of p53 activity in cancer cell lines (63)(64)(65).
These observations suggest that promoter competence, independent of p53 binding, potently controls cell specific p53 transcription responses.
Although p53 is broadly expressed in all cells, distal, p53-bound enhancers display significant differences in chromatin structure across cell types (23). Our results suggest that p53 is not required for establishment or maintenance of chromatin modification patterns at these enhancers. p53 is a pioneer transcription factor capable of binding its response element within nucleosomal DNA (50, 54). Indeed our results demonstrate that the majority of p53 binding events occur within nucleosome rich regions lacking active histone modifications in agreement with previous work (23). We propose that p53, which can bind to closed chromatin, does not broadly control de novo enhancer accessibility. A recent ATAC-seq study in primary human fibroblasts demonstrated increased chromatin accessibility at a limited number of p53 binding sites (22). Taken together, these data and our observations suggest that p53's role in mediating enhancer accessibility may be context-dependent and depend on the  (66,67). Indeed, our data suggests that p63 binds to many locations that appear to be nucleosome rich, although whether p63 is directly engaging nucleosomal DNA is unclear. The context-specific nucleosomal DNA binding activity of the p53 family, therefore, may be important for both facilitating robust transcriptional activation and for locally regulating p53 family transcription factor activity at chromatin. These data support a model whereby the p53-dependent transcriptome is licensed by the enhancer regulatory abilities of other transcription factors. This model is a particularly attractive avenue for further inquiry and provides a straightforward mechanism for cell type-dependent tumor suppressor and homeostatic activities of p53. p53 family members have highly conserved DNA binding domains that allow them to engage with highly similar DNA sequences. Therefore, the extent to which p53 family member competition for DNA binding affects their function has been a longstanding question. Initial observations suggested that p63 primarily represses p53 activity in a dominant negative fashion (18,19). We observed p63-dependent repression of nearly 15% of p53 targets in MCF10A epithelial cells, supporting a dominant negative model. However, our results also demonstrated that p63 is required for p53-dependent activation of approximately 40% of epithelial target genes. The precise mechanisms by which p63 supports p53 activity are unclear, but our data demonstrating that p63 likely functions as an enhancer licensing or maintenance factor provides a straightforward possibility. Our model proposes that p63 first establishes a permissive chromatin environment which then allows additional transcription factors, including p53, to bind. This putative mechanism supports both p63-dependent p53 activity and p63-mediated repression. Whereas p63 activity may be required to facilitate a suitable binding environment, ∆Np63 could also repress p53 through local competition for the same binding site. We note that loss of p63 led to substantial gains in p53dependent H3K27ac enrichment at enhancers, supporting a local inhibition of p53 enhancer activity by p63. Due to the population-based nature of ChIP-seq, it is uncertain whether p53 and p63 truly compete for binding sites in the same cell, or whether p53 and p63 binding occurs in mutually exclusive cells populations. Therefore, our data confirm p53 and p63 have their previously reported antagonistic relationship and reveal a more broadly cooperative partnership than expected (8,9).
Our data demonstrate that p63 is required for enhancer identity in breast basal epithelial cells. Knockdown of p63 in MCF10A cells leads to a site-specific loss of enhancer-associated histone modifications and linked gene expression. p63 is required for lineage commitment and self-renewal of basal epithelial cells (45), and p53 family motifs are highly and specifically enriched in epithelial specific enhancers (44). The specific molecular events linking initial p63 DNA binding to establishment of an active enhancer state are becoming more clear. p63 interacts with the histone H3K4 monomethylase KMT2D in normal epithelial keratinocytes and reduced KMT2D activity leads to preferential reduction of H3K4me1 at p63-bound enhancers (48). Similarly, inactivation of the BAF chromatin remodeling complex is correlated with reduced chromatin accessibility at p63 binding sites (47). Our data, therefore, place p63 in line with other lineage determination factors that directly act as pioneer transcription factors to license cell type-specific enhancers (68,69). Further investigation into p63 control of epithelial enhancer identity is required to determine precisely how p63 pioneer activity controls epithelial lineage determination and self-renewal.

ACCESSION NUMBERS
All raw data associated with this study are deposited at NCBI Gene Expression Omnibus under accession number GSE111009.   Rel. Exp. Rel. Exp.
Supplemental Figure 5