Regulators of Epidermal Growth and Differentiation

描述（由申请人提供）：表皮内稳态需要在增殖和分化之间保持适当的平衡，当这种平衡被改变时可能导致超增性疾病，如鳞状细胞癌。这一过程的调节因子可能包括RNA降解酶，如由11个亚基组成的外泌体。我们最近表明，EXOSC9、EXOSC7和EXOSC10等外泌体亚基通过促进祖细胞中分化特异性转录因子的mRNA降解，对维持表皮自我更新至关重要。目前尚不清楚外泌体的其他8个亚基及其相关接头蛋白的功能。目的/假设：本提案旨在了解参与维持正常表皮稳态的基因调控机制。我们假设外泌体亚基以不同功能的亚复合物形式存在于表皮细胞中。EXOSC9、EXOSC7和EXOSC10等亚复合物是维持祖细胞功能所必需的，而其他亚复合物则是分化所必需的。我们还假设衔接蛋白招募不同的外泌体亚复合物来靶向rna，以维持自我更新或促进表皮分化。具体目的：(1)确定外泌体亚基在表皮内稳态中的作用；(2)确定外泌体相关适配蛋白在表皮内稳态中的作用。研究设计：为了在更临床相关的环境中研究表皮稳态，我们建立了新的方法，将遗传元件的特定组合引入三维完整的人皮肤，在免疫缺陷小鼠身上再生的人表皮细胞中含有人真皮基质和基底膜。通过使用该模型，我们可以对再生人类皮肤中的外泌体亚基和接头蛋白进行功能丧失实验，以表征它们在表皮生长和分化中的作用。我们还将使用RNA免疫沉淀法，然后进行下一代测序，以确定与外泌体亚基以及衔接蛋白相关的RNA。

DESCRIPTION (provided by applicant): Epidermal homeostasis requires a proper balance between proliferation and differentiation which when altered can lead to hyperproliferative disorders such as squamous cell carcinomas. Regulators of this process may include RNA degradation enzymes such as the exosome which is composed of 11 subunits. We have recently shown that exosome subunits such as EXOSC9, EXOSC7, and EXOSC10 are essential for maintaining epidermal self-renewal by promoting the mRNA degradation of differentiation specific transcription factors in progenitor cells. It is currently unknown the function of the other 8 subunits of the exosome as well as their associated adaptor proteins. Objective/hypothesis: This proposal seeks to understand the gene regulatory mechanisms involved in maintaining normal epidermal homeostasis. We hypothesize that exosome subunits exist in epidermal cells in subcomplexes with distinct functions. Subcomplexes such as EXOSC9, EXOSC7, and EXOSC10 are necessary to maintain progenitor function while other subcomplexes are necessary for differentiation. We also hypothesize that adaptor proteins recruit distinct exosome subcomplexes to target RNAs to either maintain self-renewal or to promote epidermal differentiation. Specific Aims: (1) To determine the role of the exosome subunits in epidermal homeostasis and (2) to determine the role of exosome associated adaptor proteins in epidermal homeostasis. Study Design: To study epidermal homeostasis in a more clinically relevant setting, we established new methods to introduce specific combinations of genetic elements into 3- dimensionally intact human skin, containing human epidermal cells in the context of human dermal stroma and basement membrane, regenerated on immune deficient mice. By using this model, we can perform loss of function experiments on exosome subunits and adaptor proteins in regenerated human skin to characterize their role in epidermal growth and differentiation. We will also use RNA immunoprecipitations followed by next generation sequencing to determine the RNAs associated with exosome subunits as well as adaptor proteins.