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. 2012 Sep 7;91(3):435-43.
doi: 10.1016/j.ajhg.2012.07.007. Epub 2012 Aug 23.

Genomic profiling of a human organotypic model of AEC syndrome reveals ZNF750 as an essential downstream target of mutant TP63

Brian J Zarnegar  1 , Dan E Webster, Vanessa Lopez-Pajares, Brook Vander Stoep Hunt, Kun Qu, Karen J Yan, David R Berk, George L Sen, Paul A Khavari
Affiliations

Genomic profiling of a human organotypic model of AEC syndrome reveals ZNF750 as an essential downstream target of mutant TP63

Brian J Zarnegar et al. Am J Hum Genet. .

Abstract

The basis for impaired differentiation in TP63 mutant ankyloblepharon-ectodermal dysplasia-clefting (AEC) syndrome is unknown. Human epidermis harboring AEC TP63 mutants recapitulated this impairment, along with downregulation of differentiation activators, including HOPX, GRHL3, KLF4, PRDM1, and ZNF750. Gene-set enrichment analysis indicated that disrupted expression of epidermal differentiation programs under the control of ZNF750 and KLF4 accounted for the majority of disrupted epidermal differentiation resulting from AEC mutant TP63. Chromatin immunoprecipitation (ChIP) analysis and ChIP-sequencing of TP63 binding in differentiated keratinocytes revealed ZNF750 as a direct target of wild-type and AEC mutant TP63. Restoring ZNF750 to AEC model tissue rescued activator expression and differentiation, indicating that AEC TP63-mediated ZNF750 inhibition contributes to differentiation defects in AEC. Incorporating disease-causing mutants into regenerated human tissue can thus dissect pathomechanisms and identify targets that reverse disease features.

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Figures

Figure 1
Figure 1
Disrupted Epidermal Differentiation by AEC TP63 Mutants in Regenerated Organotypic Human Epidermal Tissue Differentiation markers (A) KRT1 and (B) FLG in control (EV), TP63-ΔNp63α-WT (WT), TP63-ΔNp63α-Cys467Trp, TP63-ΔNp63α-Leu459Phe, TP63-ΔNp63α-Ile482Thr, and TP63-ΔNp63α-Arg500Pro AEC mutant-expressing regenerated organotypic human epidermal tissue (orange = differentiation protein, blue = Hoechst 33342 nuclei, dotted line denotes basement membrane). Differentiation markers (C) KRT1 and (D) FLG in normal skin and in AEC lesional skin from a 1-day-old AEC-affected individual with a confirmed p.Phe458Ser alteration.
Figure 2
Figure 2
Retention of Mutated SAM Domain in TP63 AEC Mutants Is Required for Inhibition of Epidermal Differentiation (A) Immunoblot analysis of TP63-ΔNp63α levels in human KCs transduced with TP63-ΔNp63α-WT, TP63-ΔNp63α-Arg500Pro, and TP63-ΔNp63δ. KCs were treated with control or TP63 targeting siRNA, as indicated. (B) KRT1 in TP63-ΔNp63α-WT, TP63-ΔNp63α-Arg500Pro, and TP63-ΔNp63δ expressing regenerated organotypic human epidermal tissue (orange = differentiation protein, blue = Hoechst 33342, green = basement membrane).
Figure 3
Figure 3
Transcriptional Profiling of AEC Model Tissue (A) Venn diagram illustrating overlap between changes identified in p.Ile482Thr (AEC1) and p.Arg500Pro (AEC2) AEC mutant versus wild-type (WT) control organotypic tissue. Heat map of the 651 genes shared between the two profiles (p value < 10−250, Fisher’s exact test); blue (repressed) and red (induced), log2-based scale. (B) Gene ontology (GO) analysis of the p.Ile482Thr and p.Arg500Pro gene-profile overlap (p values represent a Bonferroni-corrected EASE score).
Figure 4
Figure 4
Identification of TP63-Dependent Candidate Regulators Responsible for the AEC Phenotype (A) Venn diagram illustrating overlap between changes identified in AEC cultures and KCs treated with siRNA targeting total TP63. Heat map of the 206 genes shared between the two profiles (p value < 10−148, Fisher’s exact test); blue (repressed) and red (induced), log2-based scale. (B) Barcode plot showing AEC and TP63i gene-number overlap with indicated epidermal gene set. (C) GSEA enrichment values for top overlapping gene sets.
Figure 5
Figure 5
TP63 ChIP-Seq Identifies TP63 Binding to the ZNF750 Locus during Epidermal Differentiation (A) Heat map showing stable p63 genomic binding in undifferentiated progenitor-containing KC populations and in differentiated KCs. (B) TP63 binding near the TSS of the ZNF750 locus in undifferentiated progenitor-containing KC populations and in differentiated KCs. (C) Immunoblot analysis of TP63-ΔNp63α in KCs infected with pBABE-Empty Vector (EV), pBABE-TP63-ΔNp63α-WT (WT), pBABE-p.Ile482Thr (AEC1), and pBABE-p.Arg500Pro (AEC2) AEC TP63 mutants. Each population of KCs was treated with control or TP63 targeting siRNA, as indicated. (D) ChIP analysis comparing binding capacity of wild-type TP63 versus AEC TP63 mutants at the ZNF750 locus in undifferentiated and differentiated KCs treated with TP63i (specific for endogenous TP63 mRNA). Error bars are ± SD between qPCR triplicate samples.
Figure 6
Figure 6
Rescue of AEC Differentiation Defects by ZNF750 Human epidermal tissue was regenerated from KCs infected with either pBABE-Empty Vector (EV) or pBABE-p.Arg500Pro AEC mutant TP63, then with either pBABE-EV or pBABE-ZNF750. Quantitative mRNA levels of (A) ZNF750 and (B) epidermal differentiation markers and regulators in the indicated day 4 organotypic tissues. Error bars are ± SD between triplicate samples. Differentiation proteins KRT1 (C) and FLG (D) in the indicated organotypic tissues.
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