Stem cell-specific transcriptional control by dyskerin ribonucleoprotein complex

项目摘要/摘要。转录因子OCT4和SOX2是关键的调节因子 干细胞的维持和多能性，但它们如何调控基因表达却知之甚少。这 知识鸿沟阻碍了我们理解OCT4和SOX2是如何控制多能性的。长期的 该项目的目标是了解调控干细胞自我更新和转录的机制 差异化。激活因子对转录辅因子的募集是基因激活的关键步骤。 然而，OCT4和SOX2的这些辅因子的身份和功能尚不清楚。我们开发了一种 完全重组的体外转录实验，以检测和分离OCT4和SOX2的新辅助因子。一 在这些辅因子中，dyskerin核糖核蛋白(Rnp)复合体在遗传干细胞中发生突变。 遗传性角化不良(DC)，是本应用的重点。这样做的总体目标是 建议是了解干细胞特异性转录和多能性是如何受dyskerin调节的 RNP。中心假设是dyskerin RNP是OCT4/SOX2的重要组成部分 转录机制和多能性和干细胞维持的新调节器。这个 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 将对转录辅助因子如何控制干细胞的多能性产生新的理解。中环 假说将通过追求三个具体目标来检验：(1)通过以下方式确定转录机制 其中OCT4/SOX2/dyskerin核糖核蛋白复合体调节干细胞维持和 多能性，(2)识别与dyskerin相关的非编码RNA及其机制 用OCT4和SOX2激活转录，以及(3)确定dyskerin的致病突变 折衷RNA结合和转录共激活。在第一个目标下，全基因组分析 将用于确定转录靶点、基因表达程序和干细胞分化 受dyskerin RNP控制的通路。在第二个目标下，通过识别与 Dyskerin复合体，对非编码RNA调节辅因子机制的基本见解 将对活动进行调查。在第三个目标下，患者来源的诱导多能干细胞(IPSCs)将 用于识别由于致病突变引起的RNA组成和转录活性的变化 在dyskerin基因(DKc1)中，并建立DKc1突变与 我们在DKC1突变的ipscs中发现的多能性基因网络受损。拟议的研究是 创新，因为它使用了体外重组和转录测试来发现新的转录 人类多能干细胞的机制。这一贡献是巨大的，因为它预计将产生 对OCT4和SOX2如何控制多能性的基础理解，从而增强我们的 控制干细胞命运的能力，用于治疗和治疗DC等疾病。

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.