ΔNp63 is a pioneer factor that binds inaccessible chromatin and elicits chromatin remodeling

Abstract

Background ΔNp63 is a master transcriptional regulator playing critical roles in epidermal development and other cellular processes. Recent studies suggest that ΔNp63 functions as a pioneer factor that can target its binding sites within inaccessible chromatin and induce chromatin remodeling. Methods In order to examine if ΔNp63 can bind to inaccessible chromatin and to determine if specific histone modifications are required for binding, we induced ΔNp63 expression in two p63-naïve cell lines. ΔNp63 binding was then examined by ChIP-seq and the chromatin at ΔNp63 targets sites was examined before and after binding. Further analysis with competitive nucleosome binding assays was used to determine how ΔNp63 directly interacts with nucleosomes. Results Our results show that before ΔNp63 binding, targeted sites lack histone modifications, indicating ΔNp63’s capability to bind at unmodified chromatin. Moreover, the majority of the sites that are bound by ectopic ΔNp63 expression exist in an inaccessible state. Once bound, ΔNp63 induces acetylation of the histone and the repositioning of nucleosomes at its binding sites. Further analysis with competitive nucleosome binding assays reveal that ΔNp63 can bind directly to nucleosome edges with significant binding inhibition occurring within 50 bp of the nucleosome dyad. Conclusion Overall, our results demonstrate that ΔNp63 is a pioneer factor that binds nucleosome edges at inaccessible and unmodified chromatin sites and induces histone acetylation and nucleosome repositioning.

p63 and p53: Collaborative Partners or Dueling Rivals?

“…SFN was identified as a direct p53 and Np63 target that is FIGURE 2 | (A) p53 and Np63 can act as pioneer factors to evict/remodel nucleosomes and facilitate DNA accessibility, although other pioneer factors are likely responsible for nucleosome eviction at the majority of p53/ Np63 binding sites. (B) Certain nucleosome contexts, such as when the p53/p63 RE motif is near the nucleosome dyad, are recalcitrant to p53 and/or Np63 pioneer factor activity and remain unbound (Yu and Buck, 2019;Yu et al, 2021). (C) In epithelial cell types, p53 DNA binding can be facilitated by Np63 s pioneer factor activity, although specific biochemical mechanisms are still being fully elucidated.…”

“…This rule is broken by pioneer transcription factors, which can bind to nucleosomal DNA either sequence-dependent or independent ( Zaret and Mango, 2016 ). Numerous recent studies suggest that the p53 family of transcription factors are pioneer transcription factors ( Sammons et al, 2015 ; Yu and Buck, 2019 ; Yu et al, 2021 ; Figure 2A ). Indeed, differential pioneer activity of p53 and p63 has the potential to explain some of the observed gene regulatory differences between these two siblings.…”

“…Given the high sequence and functional homology to p53, perhaps it is unsurprising that p63 has also emerged as a pioneer transcription factor with similar nucleosomal constraints ( Yu et al, 2021 ). Control of cell type-specific chromatin accessibility and gene expression is a feature shared by many other pioneer transcription factors ( Iwafuchi-Doi and Zaret, 2014 , 2016 ; Zaret and Mango, 2016 ).…”

ΔNp63 drives dysplastic alveolar remodeling and restricts epithelial plasticity upon severe lung injury

“…ΔNp63 is necessary to maintain basal cell identity and function; without ΔNp63, these cells downregulate basal markers, exit the cell cycle, and undergo a program of precocious differentiation into more committed epithelial cells of their resident tissue (Haas et al, 2019; Romano et al, 2012; Truong et al, 2006; Yang et al, 2018). While ΔNp63 exterts its control over basal cell identity directly by transcriptionally regulating expression of basal cell markers and functional components such as basement membrane-anchoring adhesion molecules (Carroll et al, 2006; Ferone et al, 2013; Ihrie et al, 2005; Kurata et al, 2004; Laurikkala et al, 2006; Romano et al, 2009), recent studies propose an epigenetic role for ΔNp63 as well, in which ΔNp63 cooperates with higher order chromatin remodelers and histone modifiers to rearrange the 3D topology and chromatin accessibility of the genome (Fan et al, 2018; Fessing et al, 2011; Mardaryev et al, 2014; Qu et al, 2019; Sethi et al, 2014), bookmark tissue-specific enhancer regions during differentiation (Kouwenhoven et al, 2015; Lin-Shiao et al, 2019), and acts as a pionner-like factor by binding and opening inaccessible chromatin (Fan et al, 2018; Santos-Pereira et al, 2019; Yu et al, 2021). With its multi-tiered influence over the fundamental basal cell program and critical role in virtually all epithelial tissue contexts in which its been studied, ΔNp63 serves as a promising target to modulate basal cell fate choice in regions of dysplastic remodeling within injured lungs.…”

Lola-I is a developmentally regulated promoter pioneer factor

“…Furthermore, incubation of Lola-I protein with nucleosome-bound sequences in vitro suggested that Lola-I preferentially binds to its motifs near the nucleosome edge when the DNA is partially unwrapped. Although there is no consensus on whether pioneer factors are defined by a certain binding mode in vitro (Klemm et al, 2019), such binding preference has previously been observed for some pioneer factors (Yu and Buck, 2019; Yu et al, 2021; Zhu et al 2018). Like for other pioneer factors, binding of Lola-I may lead to the recruitment of chromatin remodelers that remove the nucleosome (Judd et al, 2021; Zaret, 2020).…”