Stem cell-specific transcriptional control by dyskerin ribonucleoprotein complex

项目总结/摘要。转录因子OCT 4和SOX 2是转录调控的关键因子。 干细胞维持和多能性，但它们如何调节基因表达知之甚少。这 知识差距使我们无法理解OCT 4和SOX 2如何控制多能性。长期 本项目的目标是了解调控干细胞自我更新的转录机制， 分化转录辅因子被激活因子募集是基因激活的关键步骤。 然而，OCT 4和SOX 2的这些辅因子的身份和功能尚不清楚。我们开发了一个 完全重构的体外转录测定，以检测和分离OCT 4和SOX 2的新型辅因子。一 在这些辅因子中，dyskerin核糖核蛋白（RNP）复合物在遗传干细胞中发生突变， 疾病先天性角化不良（DC），并且是本申请的焦点。本报告的总体目标 我们的建议是了解干细胞特异性转录和多能性是如何被dyskerin调节的。 RNP。中心假设是dyskerin RNP是OCT 4/SOX 2的重要组分 转录机制和多能性和干细胞维持的新调节剂。的 这项工作的基本原理是破译dyskerin RNP如何调节多能性基因转录 将产生一个新的理解如何转录辅因子控制干细胞多能性。中央 将通过追求三个具体目标来测试假设：（1）通过以下方式确定转录机制： 其中OCT 4/SOX 2/dyskerin核糖核蛋白复合物调节干细胞维持， （2）识别dyskerin相关的非编码RNA及其机制， 用OCT 4和SOX 2激活转录，和（3）确定dyskerin中的致病性突变如何 损害RNA结合和转录共激活。在第一个目标下，全基因组分析 将用于定义转录靶点、基因表达程序和干细胞分化 由dyskerin RNP控制的途径。在第二个目标下，通过鉴定与细胞凋亡相关的RNA， dyskerin复合物，对非编码RNA调节辅因子的机制的基本见解 活动将被调查。根据第三个目标，患者来源的诱导多能干细胞（iPSC）将 用于鉴定致病性突变引起的RNA组成和转录活性的变化 在dyskerin基因（DKC 1），并建立DKC 1突变和 我们在DKC 1突变型iPSC中发现了受损的多能性基因网络。拟议的研究是 创新，因为它采用体外重建和转录测定来揭示新的转录 人类多能干细胞的机制。这一贡献是重要的，因为预计它将产生一个 对多能性如何由OCT 4和SOX 2控制的基本理解，从而增强我们的 控制干细胞命运的能力，用于治疗和治疗疾病，如DC。

PROJECT SUMMARY/ABSTRACT. The transcription factors OCT4 and SOX2 are critical regulators of stem cell maintenance and pluripotency, but how they regulate gene expressions is poorly understood. This knowledge gap prevents us from understanding how OCT4 and SOX2 control pluripotency. The long-term goal of this project is to understand transcriptional mechanisms governing stem cell self-renewal and differentiation. Recruitment of transcriptional cofactors by activators is a key step in gene activation. However, the identity and function of these cofactors for OCT4 and SOX2 are not known. We developed a fully reconstituted in vitro transcription assay to detect and isolate novel cofactors for OCT4 and SOX2. One of these cofactors, the dyskerin ribonucleoprotein (RNP) complex, is mutated in the inherited stem cell disorder dyskeratosis congenita (DC), and is the focus of this application. The overall objective of this proposal is to understand how stem cell-specific transcription and pluripotency are regulated by the dyskerin RNP. The central hypothesis is that dyskerin RNP is an essential component of OCT4/SOX2 transcriptional machinery and a novel regulator of pluripotency and stem cell maintenance. The rationale for this work is that deciphering how the dyskerin RNP regulates pluripotency gene transcription will yield a new understanding of how transcriptional cofactors control stem cell pluripotency. The central hypothesis will be tested by pursuing three Specific Aims: (1) Identify transcriptional mechanisms by which the OCT4/SOX2/dyskerin ribonucleoprotein complex regulates stem cell maintenance and pluripotency, (2) Identify dyskerin-associated non-coding RNAs and the mechanism by which they activate transcription with OCT4 and SOX2, and (3) Determine how pathogenic mutations in dyskerin compromise RNA binding and transcriptional co-activation. Under the first aim, genome-wide analyses will be used to define transcriptional targets, gene expression programs, and stem cell differentiation pathways controlled by the dyskerin RNP. Under the second aim, by identifying RNAs associated with the dyskerin complex, fundamental insight into the mechanisms by which non-coding RNAs regulate cofactor activity will be investigated. Under the third aim, patient-derived induced pluripotent stem cells (iPSCs) will be used to identify changes in the RNA composition and transcriptional activity due to pathogenic mutations in the dyskerin gene (DKC1), and establish mechanistic connection between DKC1 mutations and a compromised pluripotency gene network that we found in DKC1-mutant iPSCs. The proposed research is innovative, because it employs in vitro reconstitution and transcription assay to uncover novel transcriptional mechanims in human pluripotent stem cells. This contribution is significant, because it is expected to yield a foundational understanding of how pluripotency is controlled by OCT4 and SOX2, thereby enhancing our ability to control stem cell fate for therapies and the treatment of diseases such as DC.