RUI: Investigating enhancer grammar that underlies naive-state pluripotency

尽管经过了几十年的研究，但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时间和地点的序列，在多细胞生物体的发育过程中具有深远的作用。虽然它们不编码蛋白质，但增强子包含的信息编码为转录因子(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，语法或TF结合位点的特征，如类型、数量、结合亲和力和排列，是增强子功能的关键因素。然而，关于增强子语法的原理以及这些原理如何作为基因调控机制的基础，仍然存在许多问题。对增强子的深入了解将有助于我们根据遗传信息预测表型，并可应用于其他领域，如医学和农业。这个项目将研究幼稚状态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。除了提高基因调控如何编码到基因组中的知识，这项工作每年将有25-50名本科生参与指导、调查和原创研究，为科学生涯做准备。为了调查天真状态特定增强子功能背后的语法限制，该项目将重点放在天真状态基因Klf4的增强子上，它包含TFOCT4、SOX2、Esrrb和STAT3的结合位点。初步工作表明，低亲和力的Tf结合位点对于KLF4增强子的幼稚状态特异性功能至关重要。为了阐明这种结合亲和力限制在转录激活机制中的作用，本项目将利用分子方法来测量优化这种增强子中TF结合位点的效果。先前的研究还表明，TF与KLF4增强子的结合具有层次性，这表明TF结合位点的排列是促进增强体组装的关键，特别是在KLF4增强子上TF之间的相互作用。为了解决这些问题，这项研究将利用记者分析、基因编辑和分子技术的组合。最后，该项目将把来自这些和先前对KLF4增强剂的研究的知识应用于其他幼稚状态特定的增强剂。生物信息学和分子生物学技术的结合将被用来识别和分析假定的幼稚状态的特定增强剂。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.