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.

尽管进行了数十年的研究，但从遗传信息中预测增强子功能仍然是一个挑战。增强子是控制基因何时何地表达基因并在多细胞生物发育中具有重要作用的基因组序列。尽管它们不为蛋白质进行编码，但增强子包含的信息被编码为转录因子（TFS）的结合位点，这是基因调节中的关键蛋白质。据认为，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，数字，结合亲和力和布置。但是，关于增强剂语法原理以及基因调节机制的基础仍然存在许多问题。对增强剂的更深入了解将有助于我们从遗传信息中预测表型，并可以在其他领域（例如医学和农业）应用。该项目将研究幼稚的多能干细胞中的增强子语法，这代表了哺乳动物发育的最早阶段。除了促进有关基因调节如何编码中的知识外，这项工作还将与25至50名本科生参与，以准备进行科学职业准备，调查，调查和原始研究。为了调查基于幼稚的特定增强功能的语法约束，该项目将集中在naïvevevevevevestate kene knee knee knee knee klfffff的情况下，该项目将集中在4个，该项目均包含iex seee kie k klfffffff so e。 ESRRB和STAT3。初步工作表明，低亲和力TF结合位点对于一个KLF4增强子的幼稚特异性功能至关重要。为了阐明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衡量优化该增强子中TF结合位点的效果。先前的研究还表明，TFS以层次顺序结合了KLF4增强子，这表明TF结合位点的排列是促进增强体组装的关键，尤其是KLF4增强器的TFS相互作用。为了解决这些问题，这项研究将利用记者评估，基因编辑和分子技术的结合。最后，该项目将将KLF4增强剂的这些知识应用于其他幼稚的特定增强剂。将聘请生物信息学和分子生物学技术的结合，以识别和分析推定的幼稚态特异性增强剂。该奖项反映了NSF的法定任务，并被认为是通过基金会的智力优点和更广泛的影响来评估来获得的支持。