RUI: Investigating enhancer grammar that underlies naive-state pluripotency

RUI：研究朴素态多能性基础的增强子语法

• 批准号: 2335201
• 负责人: Sharon Torigoe
• 金额: $ 70万
• 依托单位: Lewis and Clark College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-03-15 至 2027-02-28
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2335201&HistoricalAwards=false
• 关键词: RUI; Investigating; enhancer; grammar; underlies

Despite decades of research, it remains a challenge to predict enhancer functions from genetic information. Enhancers are sequences in genomes that control when and where genes are expressed and have profound roles during development of multicellular organisms. While they do not code for proteins, the information that enhancers contain is encoded as binding sites for transcription factors (TFs), which are key proteins in gene regulation. It is thought that a key contributor to enhancer function is grammar, or the characteristics of TF binding sites, such as type, number, binding affinities, and arrangement. However, there are still many questions about the principles of enhancer grammar and how those underlie mechanisms for gene regulation. A deeper understanding of enhancers will help us predict phenotypes from genetic information and can be applied in other areas, such as medicine and agriculture. This project will study enhancer grammar in naïve-state pluripotent stem cells, which represent the earliest stages of mammalian development. In addition to advancing knowledge about how gene regulation is encoded into the genome, this work will engage 25-50 undergraduate students per year in mentored, investigative, and original research in preparation for science careers.To investigate grammatical constraints that underlie naïve-state specific enhancer function, this project will focus on the enhancers for the naïve-state gene Klf4, which contain binding sites for the TFs OCT4, SOX2, ESRRB and STAT3. Preliminary work has indicated that low-affinity TF binding sites are critical for the naïve-state specific function of one Klf4 enhancer. To illuminate the role of this binding affinity constraint in transcription activation mechanisms, this project will utilize molecular approaches to measure the effects of optimizing TF binding sites in this enhancer. Prior studies also have shown that TFs bind to the Klf4 enhancers in a hierarchical order, suggesting that the arrangement of TF binding sites is key to facilitate enhanceosome assembly, particularly interactions among TFs at the Klf4 enhancers. To address these questions, this research will utilize a combination of reporter assays, gene editing, and molecular techniques. Lastly, this project will apply knowledge from these and prior studies of the Klf4 enhancers to other naïve-state specific enhancers. A combination of bioinformatics and molecular biology techniques will be employed to identify and analyze putative naïve-state specific enhancers.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

尽管经过了数十年的研究，从遗传信息预测增强子功能仍然是一个挑战，增强子是基因组中控制基因表达的时间和地点的序列，并且在多细胞生物的发育过程中发挥着深远的作用，虽然它们不编码蛋白质，但信息。增强子所含的编码为转录因子 (TF) 的结合位点，转录因子是基因调控中的关键蛋白质，人们认为增强子功能的关键因素是语法或 TF 结合位点的特征，例如类型、数量、结合亲和力，和然而，关于增强子语法的原理以及这些原理如何构成基因调控机制仍然存在许多问题，对增强子的深入了解将有助于我们从遗传信息中预测表型，并可应用于其他领域，例如医学和农业。该项目将研究幼稚状态多能干细胞的增强子语法，这代表了哺乳动物发育的最早阶段。除了增进有关基因调控如何编码到基因组中的知识之外，这项工作每年还将吸引 25-50 名本科生参与。在为科学职业做准备的指导性、调查性和原创性研究。为了研究幼稚状态特定增强子功能背后的语法限制，该项目将重点关注幼稚状态基因 Klf4 的增强子，其中包含 TF OCT4 的结合位点， SOX2、ESRRB 和 STAT3 表明，低亲和力 TF 结合位点对于 Klf4 增强子的初始状态特异性功能至关重要。为了了解这种结合亲和力约束在转录激活机制中的作用，该项目将利用分子方法来测量优化该增强子中 TF 结合位点的效果，之前的研究也表明 TF 以分层顺序与 Klf4 增强子结合。 TF 结合位点的排列是促进增强体组装的关键，特别是 Klf4 增强子上 TF 之间的相互作用。为了解决这些问题，本研究将结合报告基因检测、基因编辑和分子技术。将这些和先前 Klf4 增强子研究的知识应用于其他幼稚态特异性增强子将采用生物信息学和分子生物学技术的组合来识别和分析假定的幼稚态特异性增强子。该奖项是 NSF 的法定使命，并已被视为。值得通过使用基金会的智力优点和更广泛的影响审查标准进行评估来支持。