Role of Histone H3K9 Methyltransferase Eset in Intestinal Stem Cells

 描述(申请人提供)：肠道干细胞(ISCs)是上皮细胞动态平衡和再生的关键驱动力。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，目前存在着重大的知识空白。缺乏这样的知识已经严重阻碍了对ISCs的功能以及所涉及的调节网络在肠道内稳态和再生方面的了解。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。这项应用的目的是确定Eset(也称为Setdb1和KMT1E)，一种在部分ISCs中表达的组蛋白H3K9甲基转移酶，在肠道内稳态和再生等ISC功能中所起的作用。中心假说是，Eset特异性地标记活跃的ISCs，并在调节对这些细胞的维持或功能至关重要的基因程序中扮演关键的转录因子辅助抑制因子的角色。这一假设是根据申请人实验室产生的初步数据提出的。这项拟议研究的基本原理是，定义活跃的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，肠道损伤经常与化疗和放射治疗、药物介导的毒性和各种炎症性疾病有关。在强大的初步数据的指导下，这一假说将在小鼠模型中进行验证，以追求三个特定的目标：1)验证Eset作为活跃的肠道干细胞的特异性标志物；2)确定Eset在肠道干细胞功能中的作用；3)阐明Eset在肠道干细胞中发挥作用的分子机制。具体地说，申请人建议比较ESET阳性和阴性ISCs的增殖率，并鉴定ESET阳性ISCs的其他关键特性(辐射敏感性、转录图谱)(目标1)，确定ESET缺乏和过度表达对体内肠道稳态和再生以及对ISC活性、自我更新和体外分化的影响(目标2)，并确定ESET的上游调节因子和下游效应因子，并阐明Eset和关键转录因子在调节ISCs基因表达方面的相互作用机制(目标3)。申请人认为，拟议的研究是创新的，因为ESET专门标记活跃的ISCs的概念是新颖的，将使用创新的方法(例如，体内谱系追踪、体外ISC培养、组蛋白多肽阵列)。该项目意义重大，因为拟议的研究结果有望通过进一步定义活跃的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，从而垂直推动肠道干细胞和上皮生物学领域的发展。这些结果也有潜在的翻译影响。Eset在ISCs中的有限表达使其成为肠道损伤预防和治疗干预的一个有前途的靶点。

 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.