Targeted p63 isoform modulation corrects dominant mutations in AEC syndrome without disrupting epidermal homeostasis [ChIP-seq]

The transcription factor p63 is a master regulator of stratified epithelial development. Its disruption leads to severe congenital defects affecting the skin, limbs, and craniofacial structures. Among p63-related disorders, Ankyloblepharon-Ectodermal Defects-Cleft Lip/Palate (AEC) syndrome is caused by dominant mutations predominantly in the SAM/TID domains of the TP63 gene, affecting the p63a isoform. These mutations promote protein aggregation and transcriptional dysregulation, resulting in debilitating skin erosions. To explore a therapeutic strategy based on isoform switching, we developed a conditional mouse model in which exon 13 was deleted, replacing p63a with the shorter p63ß isoform, which is expressed in the skin, albeit at lower levels . Despite the essential role of p63a in limb and palate formation, we found that p63ß is sufficient to sustain epidermal development, postnatal skin homeostasis, and wound healing. At the molecular level, the switch from p63a to p63ß preserved chromatin binding and global transcriptional programs in keratinocytes. We next applied CRISPR/Cas9-mediated exon 13 deletion in human primary keratinocytes. This isoform conversion maintained proliferation and gene expression. Importantly, in AEC patient-derived keratinocytes, p63ß expression rescued protein aggregation, restored mechanical integrity, and normalized key epidermal gene expression. Together, these findings demonstrate that p63ß can functionally compensate for p63a in the skin and establish isoform switch as a promising therapeutic strategy for AEC syndrome. Overall design: Mouse dorsal skin was harvested P0, and the epidermis was separated from the dermis by overnight incubation in Dispase solution at 4°C and single-cell suspensions were obtained by incubation in Accutase for 20 minutes at RT. Cells were cross-linked in 1% formaldehyde for 10 minutes at 37°C. After quenching with 125mM glycine for 5 minutes at RT, cells were collected by centrifugation and washed twice with ice-cold PBS containing 1mM PMSF and a protease inhibitor cocktail (Roche). Pellets containing 4×106 cells were resuspended in 500µL of SDS lysis buffer (50mM Tris-HCl pH8.1, 1% SDS, 10mM EDTA) supplemented with protease inhibitors, incubated on ice for 10 minutes. Samples were sonicated in 250µL aliquots using a Bioruptor® Plus (Diagenode) at high power (60s ON, 90s OFF) for 45 minutes at 4°C to obtain chromatin fragments of approximately 300bp. Chromatin was diluted in ChIP dilution buffer (16.7mM Tris-HCl at pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS) and incubated overnight at 4°C with p63-specific antibodies (EPR570, Abcam) conjugated with Dynabeads Protein A and G (ThermoFisher Scientific). Immunoprecipitated samples were washed with Low salt buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-Hcl pH 8.1, 150 mM NaCl) High salt buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-Hcl pH 8.1, 500mM NaCl) and LiCl Immune complex wash buffer (10mM Tris-Hcl pH 8.1, 0.25 M LiCl, 1 mM EDTA, 1% Na deoxycholate, 1% NP-40) and twice with TE buffer. After immunoprecipitation, DNA was eluted in 1% SDS plus 0.1 M NaHCO3, purified with QiaQuick columns (ThermoFisher Scientific)

转录因子（transcription factor）p63是复层上皮发育的核心主调控因子。其功能缺失会引发累及皮肤、四肢及颅面结构的严重先天性缺陷。在p63相关疾病中，睑缘粘连-外胚层发育不良-唇腭裂（Ankyloblepharon-Ectodermal Defects-Cleft Lip/Palate, AEC）综合征主要由TP63基因SAM/TID结构域的显性突变所致，该突变会影响p63α亚型。此类突变会促进蛋白质聚集与转录失调，进而导致衰弱性皮肤糜烂。为探索基于亚型转换的治疗策略，我们构建了条件性小鼠模型：该模型中外显子13被敲除，以皮肤中低水平表达的较短p63β亚型替换p63α。尽管p63α在四肢与腭部发育中发挥必需作用，但我们发现p63β足以维持表皮发育、出生后皮肤稳态及伤口愈合。在分子层面，从p63α到p63β的转换保留了角质形成细胞中的染色质结合能力与全局转录程序。我们随后在人类原代角质形成细胞中应用CRISPR/Cas9介导的外显子13敲除，该亚型转换维持了细胞增殖与基因表达谱。重要的是，在AEC患者来源的角质形成细胞中，p63β的表达挽救了蛋白质聚集现象，恢复了细胞机械完整性，并使关键表皮基因表达回归正常水平。综上，这些研究结果表明p63β可在皮肤中功能性补偿p63α的功能，并确立亚型转换作为AEC综合征极具前景的治疗策略。

实验整体设计：于新生第0天（P0）采集小鼠背侧皮肤，通过4℃下在Dispase酶溶液中过夜孵育将表皮与真皮分离，随后在室温下用Accutase消化20分钟制备单细胞悬液。将细胞置于1%甲醛中于37℃交联10分钟，室温下用125mM甘氨酸淬灭交联反应5分钟后，通过离心收集细胞，并用含1mM苯甲基磺酰氟（PMSF）与蛋白酶抑制剂混合物（Roche）的冰预冷PBS洗涤两次。将含4×10⁶个细胞的沉淀重悬于500μL添加了蛋白酶抑制剂的SDS裂解缓冲液（50mM Tris-HCl pH8.1、1% SDS、10mM EDTA）中，冰浴10分钟。将样品以250μL分装，使用Bioruptor® Plus超声破碎仪（Diagenode）以高功率（超声60秒，暂停90秒）于4℃超声破碎45分钟，以获得约300bp的染色质片段。将染色质用ChIP稀释缓冲液（16.7mM Tris-HCl pH8.1、167mM NaCl、1.2mM EDTA、1.1% Triton X-100、0.01% SDS）稀释后，于4℃下与结合了Dynabeads蛋白A/G磁珠（ThermoFisher Scientific）的p63特异性抗体（EPR570，Abcam）孵育过夜。免疫沉淀后的样品依次经低盐缓冲液（0.1% SDS、1% Triton X-100、2mM EDTA、20mM Tris-HCl pH8.1、150mM NaCl）、高盐缓冲液（0.1% SDS、1% Triton X-100、2mM EDTA、20mM Tris-HCl pH8.1、500mM NaCl）、氯化锂免疫复合物洗涤缓冲液（10mM Tris-HCl pH8.1、0.25M LiCl、1mM EDTA、1%脱氧胆酸钠、1% NP-40）洗涤，再用TE缓冲液洗涤两次。免疫沉淀完成后，用1% SDS与0.1M NaHCO₃洗脱DNA，随后通过QiaQuick柱（ThermoFisher Scientific）纯化DNA。