Role of Histone H3K9 Methyltransferase Eset in Intestinal Stem Cells

 描述（由申请人提供）：肠干细胞（ISC）是上皮稳态和再生的关键驱动因素。关于不同ISC群体的身份以及表观遗传调节因子在ISC自我更新和多能性中的作用，人们的知识存在重大差距。缺乏这样的知识，显着阻碍了了解的功能，以及参与肠道内稳态和再生的调控网络的ISCs。申请人研究的长期目标是利用表观遗传修饰的可逆性，包括组蛋白的翻译后修饰，以促进组织 再生和修复。本申请的目的是确定Eset（也称为Setdb 1和KMT 1 E）（一种在一部分ISC中表达的组蛋白H3 K9甲基转移酶）在ISC功能（如肠内稳态和再生）中的作用。核心假设是Eset特异性标记活跃的ISCs，并作为转录因子的关键辅阻遏物，调节对这些细胞的维持或功能至关重要的基因程序。这一假设是根据申请人实验室产生的初步数据提出的。拟议研究的基本原理是，定义活性ISCs的身份和功能并了解它们是如何调节的，有可能为肠道损伤提供预防和治疗价值，这通常与化疗和放疗，药物介导的毒性和各种炎症性疾病有关。在强有力的初步数据的指导下，这一假设将在小鼠模型中通过追求三个特定目标进行测试：1）Eset作为活性肠干细胞的特异性标志物; 2）确定Eset在肠干细胞功能中的作用; 3）阐明Eset在肠干细胞中发挥功能的分子机制。具体而言，申请人提出比较Eset阳性和阴性ISCs的增殖速率并鉴定其他关键特性（放射敏感性，转录谱）（目的1），以确定Eset缺陷和过表达对体内肠稳态和再生以及离体ISC活力、自我更新和分化的影响（目的2），并鉴定Eset上游调节子和下游效应子，阐明Eset与关键转录因子在ISCs基因表达调控中的相互作用机制（目的3）。申请人认为，所提出的研究是创新的，因为Eset特异性标记活性ISC的概念是新颖的，并且将使用创新的方法（例如，体内谱系追踪、离体ISC培养、组蛋白肽阵列）。该项目意义重大，因为拟议研究的结果有望通过进一步定义活性ISCs的身份和功能，并为ISC功能的表观遗传调控提供新的见解，从而垂直推进肠干细胞和上皮生物学领域。这些结果也具有潜在的翻译影响。Eset在ISCs中的有限表达使其成为肠损伤预防和治疗干预的有希望的靶点。

 DESCRIPTION (provided by applicant): Intestinal stem cells (ISCs) are critical drivers of epithelial homeostasis and regeneration. There are major gaps in knowledge concerning the identities of distinct ISC populations and the roles of epigenetic regulators in ISC self-renewal and multipotency. Lack of such knowledge has significantly hindered the understanding of the functions of ISCs, as well as the regulatory networks involved, in intestinal homeostasis and regeneration. The long-term goal of the applicant's research is to harness the reversibility of epigenetic modifications, including post-translational modifications of histones, to promote tissue regeneration and repair. The objective of this application is to determine the role of Eset (also known as Setdb1 and KMT1E), a histone H3K9 methyltransferase expressed in a fraction of ISCs, in ISC functions such as intestinal homeostasis and regeneration. The central hypothesis is that Eset specifically marks active ISCs and acts as a key corepressor of transcription factors in regulating a gene program that is essential for the maintenance or functioning of these cells. This hypothesis has been formulated on the basis of preliminary data generated in the applicant's laboratory. The rationale for the proposed research is that defining the identity and functions of active ISCs and understanding how they are regulated have the potential to deliver preventive and therapeutic value for intestinal damage, which is frequently associated with chemo- and radiotherapy, drug-mediated toxicity, and various inflammatory disorders. Guided by strong preliminary data, this hypothesis will be tested in mouse models by pursuing three specific aims: 1) Validate Eset as a specific marker of active intestinal stem cells; 2) Determine the role of Eset in intestinal stem cell functions; and 3) Elucidate the molecular mechanisms by which Eset exerts its function in intestinal stem cells. Specifically, the applicant proposes to compare the proliferation rates of Eset-positive and -negative ISCs and identify other key properties (radiosensitivity, transcriptional profile) of Eset-positive ISCs (aim 1), to determine the effects of Eset deficiency and overexpression on intestinal homeostasis and regeneration in vivo and on ISC viability, self-renewal, and differentiation ex vivo (aim 2), and to identify Eset upstream regulators and downstream effectors and to elucidate the mechanistic interactions between Eset and key transcription factors in the regulation of gene expression in ISCs (aim 3). The proposed research is innovative, in the applicant's opinion, because the concept that Eset specifically marks active ISCs is novel and innovative approaches (e.g. in vivo lineage tracing, ex vivo ISC culture, histone peptide arrays) will be used. The project is significant, because results from the proposed studies are expected to vertically advance the fields of intestinal stem cells and epithelial biology by further defining the identity and functions of active ISCs and providing novel insights into epigenetic regulation of ISC functions. These results also have potential translational impact. The restricted expression of Eset in ISCs makes it a promising target for preventive and therapeutic interventions for intestinal damage.