The role of p53 family members in epithelial lineage establishment and maintenance

P53家族成员在上皮细胞谱系建立和维持中的作用 项目总结 P53是一种转录因子，在肿瘤抑制中发挥重要作用，经常在 癌症。另一方面，p63只在上皮细胞中表达，在癌症中很少发生突变。 虽然TrP53(编码P53蛋白)的缺失在出生时没有表现出表型，但Trp63(编码 P63蛋白)导致小鼠鳞状上皮明显的发育和形态缺陷。 表皮。在人类中，p63基因的胚系突变会导致外胚层发育不良综合征，从而导致裂隙 腭部和四肢畸形。为了改进和开发治疗上皮癌和p63的新方法 相关的遗传性疾病，我们对它们在上皮谱系建立和维持中的作用的理解 需要在很大程度上扩大。特别是，P53家族成员在新生上皮细胞谱系中的作用 承诺及其维持上皮细胞特性的分子和表观遗传机制 虽然增强子的调节和先锋因子的活性仍有待阐明。我们的实验室此前显示， 成纤维细胞系中40%的P53结合位点位于染色质构象封闭的部位 缺乏启动子或增强子特异性的组蛋白修饰。进一步的分析强烈表明，这些 近2000个地点采用开放的染色质构象，由增强子特定标记(H3K4me)装饰 和H3K27ac)在上皮细胞中特异表达。此外，p53和p63都直接与这些位点结合在一起。 上皮谱系。根据我们实验室以前的发现和专业知识，我建议：(1)剖析 P53家族成员在上皮性增强子建立和上皮性谱系承诺中的作用。为此，我 将利用一种新的时间控制的成纤维细胞角质形成细胞重编程系统。这套多功能的系统 再加上基因组和生化工具，我也可以研究p63亚型和突变体的作用。 作为P53基因的S在上皮细胞特性的从头建立中的作用。(2)确定p63如何维持上皮细胞 身份。在这里，我将利用CRISPR Cas9在正常人表皮角质形成细胞上引入突变 P63和在p63反应元件中位于特定的基因组座位。这将使我能够直接观察到 P63的突变和p63结合的丢失会导致上皮细胞特性的丧失。这些目标加在一起将 对P53家族成员在上皮细胞谱系建立和维持中的作用提供新的见解， 以及与疾病相关的分子机制。 第1条

The role of p53 family members in epithelial lineage establishment and maintenance Project summary p53 is a transcription factor that plays a fundamental role in tumor suppression and is often mutated in cancer. p63, on the other hand, is exclusively expressed in epithelial cells and is rarely mutated in cancer. While deletion of Trp53 (encoding the p53 protein) shows no phenotype at birth, deletion of Trp63 (encoding the p63 protein) in mice leads to clear developmental and morphological defects in the squamous epithelia and epidermis. In humans, germline mutations in p63 cause ectodermal dysplastic syndromes, leading to cleft palate and limb malformations. In order to improve and develop novel treatments for epithelial cancers and p63 related genetic disorders, our understanding of their role in epithelial lineage establishment and maintenance needs to be largely expanded. In particular, the role of p53 family members in de novo epithelial lineage commitment and the molecular and epigenetic mechanisms by which they maintain epithelial cell identity though enhancer regulation and pioneer factor activity remain to be elucidated. Our lab previously showed that 40% of p53 binding sites in the fibroblast lineage are located at sites with a closed chromatin conformation and devoid of promoter or enhancer specific histone modifications. Further analysis strongly suggested that these nearly 2000 locations adopt an open chromatin conformation, decorated by enhancer specific marks (H3K4me and H3K27ac) specifically in epithelial cells. Furthermore, both p53 and p63 bind directly to these sites in epithelial lineages. Based on our lab's previous discoveries and expertise I propose to: (1) Dissect the role of p53 family members in epithelial enhancer establishment and epithelial lineage commitment. To this end, I will utilize a novel temporally-controlled fibroblast to keratinocyte reprogramming system. This versatile system coupled with genomic and biochemical tools will allow me to study the role p63 isoforms and mutants, as well as p53's role in de novo establishment of epithelial identity. (2) Determine how p63 maintains epithelial identity. Here, I will utilize CRISPR Cas9 on normal human epidermal keratinocytes to introduce mutations in p63 and in p63 response elements at specific genomic loci. This will allow me to directly observe how mutations in p63 and loss of p63 binding contribute to loss of epithelial identity. Together these aims will provide novel insight into the roles of p53 family members in epithelial lineage establishment and maintenance, as well as molecular mechanisms related to disease.   1