Impact of iASPP knockout on p63 genome occupancy in mouse keratinocytes. Impact of iASPP knockout on p63 genome occupancy in mouse keratinocytes

iASPP (PPP1R13L) is an evolutionarily conserved regulator of p53 family members. In mice, iASPP deletion in keratin 14-expressing keratinocytes is sufficient to cause detrimental phenotypes with squamous epithelium abnormalities. In human skin, wild-type nuclear iASPP colocalizes with the key squamous differentiation regulatory factor, TP63, and iASPP mutations are associated with abnormalities in skin development. To clarify how iASPP modulates p63 genome occupancy, p63 ChIP-seq data from iASPP WT and iASPP KO primary mouse keratinocytes (MKC) were compared. Overall design: Mouse keratinocytes (MKC) were isolated from mice with or without a Krt14-Cre+;Ppp1r13l-/- genotype, which causes a deletion of iASPP exon 8 in Krt14-expressing epithelial cells. ChIP-seq was performed on isolated keratinocytes from Krt14-Cre+;Ppp1r13l-/- (iASPP KO) or iASPP WT mice, using the H129 p63 antibody (Santa Cruz). KO and WT ChIP-seq profiles were then compared using a quantitative method for ChIP-seq sample comparison (MAnorm).

iASPP（PPP1R13L）是p53家族成员的进化保守调控因子。在小鼠模型中，于表达角蛋白14的角质形成细胞内敲除iASPP，即可诱发有害表型并伴随鳞状上皮异常。在人类皮肤组织中，野生型核定位iASPP与关键鳞状分化调控因子TP63存在共定位现象，且iASPP突变与皮肤发育异常存在关联。为阐明iASPP调控p63基因组结合位点的具体机制，研究人员对来自iASPP野生型（WT）与iASPP敲除型（KO）的原代小鼠角质形成细胞（MKC）的p63染色质免疫共沉淀测序（ChIP-seq）数据进行了对比分析。
整体实验设计：从携带Krt14-Cre+;Ppp1r13l-/-基因型（可在表达Krt14的上皮细胞中敲除iASPP第8号外显子）与野生型的小鼠中分离原代小鼠角质形成细胞（MKC）。采用H129 p63抗体（Santa Cruz）对分离得到的角质形成细胞开展ChIP-seq实验。随后利用用于ChIP-seq样本比较的定量分析方法（MAnorm），对敲除组与野生组的ChIP-seq图谱进行对比分析。