Genomic and epigenomic investigations of the transcriptional regulatory network of skin keratinocytes in defined genetic models

项目总结 自改变以来，皮肤表皮的发育和分化是一个重要的研究领域 角质形成细胞功能是各种皮肤疾病的潜在原因，包括炎症性皮肤病， 癌症和伤口愈合缺陷。控制发育的转录和表观遗传机制 角质形成细胞的分化还不是很清楚，特别是在活体皮肤的背景下。因此 我们的长期目标是检查角质形成细胞的染色质状态，并识别和表征关键的 转录因子在健康和疾病中作为表皮分化的重要调节因子。它是 已证实转录因子Δnp63在细胞形态发生和分化中起关键作用。 皮肤表皮，尤指在胚胎发育过程中。然而，我们目前对政府的认识 ΔNp63与染色质和转录调控环境相互作用和塑造的原理 对角质形成细胞的研究相当有限，而且是基于主要从细胞培养中生长的角质形成细胞收集的数据。 此外，ΔNp63是否以及如何在成人皮肤成熟后维持皮肤动态平衡方面发挥作用 由于缺乏有针对性的遗传系统，尚未得到充分解决。要解决这些问题 知识空白，我们已经产生了定义良好的ΔNp63转基因和基因敲除小鼠模型，使我们能够 分离纯净的富含碱基的ΔNp63ve角质形成细胞，并进行健壮的、可诱导的缺失 ΔNp63在成人组织中的表达。我们将利用新兴基因组学和表观基因组学工具箱的力量，以及我们的 新开发的鼠标模型解决了两个重要问题。考察……的开拓性作用 在目标1中，我们将进行Δ-SEQ和CHIP-SEQ实验，并比较染色质 Δnp63缺失型角质形成细胞与野生型角质形成细胞的胚胎基底层角质形成细胞的构筑。我们 还将在体内识别ΔNp63靶点，并整合表观基因组和转录数据集以更好地 了解ΔNp63依赖的基因调控机制对胚胎表皮的重要性 成熟。在目标2中，我们将生成ΔNp63条件基因敲除，并研究ΔNp63的丢失如何影响 成人皮肤表皮维持和动态平衡程序的细胞和分子动力学。 此外，我们将描述Δnp63控制的转录调控机制和信号 在成人皮肤中的通路，并确定皮肤表型受缺失阻碍的分子机制 Δnp63。总的来说，这些实验将阐明基因的基本转录机制。 在广泛和动态的范围内调节和具体确定角质形成细胞特有的调节网络。 从长远来看，这些信息对人类患者具有临床和治疗意义。 角质形成细胞分化缺陷引起的毁容和衰弱的皮肤病，尤其是由于 P63信号转导通路的失调。

PROJECT SUMMARY The development and differentiation of the skin epidermis is an important area of research since altered keratinocyte function is an underlying cause for various skin conditions including inflammatory skin diseases, cancer and wound healing defects. The transcriptional and epigenetic mechanisms that control development and differentiation of keratinocytes is not well understood, particularly in the in vivo context of the skin. Hence our long-term goal is to examine the chromatin state of keratinocytes and identify and characterize the crucial transcription factors that function as important regulators of epidermal differentiation in health and disease. It is well established that the transcription factor ΔNp63 plays a critical role in morphogenesis and differentiation of the skin epidermis, particularly during embryogenesis. However, our current knowledge of the governing principles by which ΔNp63 interacts with and shapes the chromatin and transcriptional regulatory environment of keratinocytes is quite limited and based on data amassed primarily from keratinocytes grown in cell culture. Furthermore, if and how ΔNp63 plays a role in maintaining adult skin homeostasis after the matured epidermis is formed, has not been adequately addressed due to lack of targeted genetic systems. To address these knowledge gaps, we have generated well-defined ΔNp63 transgenic and knockout mouse models that allow us to isolate a pure population of basal-enriched ΔNp63+ve keratinocytes, and to perform robust, inducible deletion of ΔNp63 in adult tissues. We will leverage the power of emerging genomics and epigenomics toolbox and our newly developed mouse models to address two important questions. To examine the pioneering function of ΔNp63, in Aim 1, we will perform ATAC-seq and ChIP-seq experiments and compare the chromatin architecture of embryonic basal keratinocytes of ΔNp63-null keratinocytes to their wildtype counterparts. We will also identify ΔNp63 targets in vivo and integrate epigenomic and transcriptomic datasets to better understand the ΔNp63-dependent gene regulatory mechanisms that are important for embryonic epidermal maturation. In Aim 2, we will generate ΔNp63 conditional knockouts and examine how loss of ΔNp63 affects the cellular and molecular dynamics of the maintenance and homeostasis program of the adult skin epidermis. Furthermore, we will characterize the ΔNp63-governed transcriptional control mechanisms and signaling pathways in adult skin and identify the molecular mechanisms of the skin phenotype encumbered by loss of ΔNp63. Collectively, these experiments will shed light on fundamental transcriptional mechanisms of gene regulation and specifically identify keratinocyte-specific regulatory networks on a broad and dynamic scale. Long term, such information has clinical and therapeutic implications for human patients who suffer from disfiguring and debilitating skin diseases resulting from defective keratinocyte differentiation, particularly due to dysregulation in the p63 pathway.