CAREER: Leveraging Microfluidics for High-Throughput in Vitro Investigations of Transcriptional Regulation

职业：利用微流体进行转录调控的高通量体外研究

• 批准号: 2142336
• 负责人: Polly Fordyce
• 金额: $ 83.84万
• 依托单位: Stanford University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-15 至 2026-12-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2142336&HistoricalAwards=false
• 关键词: CAREER; Leveraging; Microfluidics; Throughput; Vitro

All biological processes depend on the precise regulation of when genes are transcribed and how many RNA transcripts are produced. This regulation is driven primarily by transcription factor (TF) proteins that bind specific DNA sequences in the genome and then recruit additional ‘effector’ proteins to either activate or repress gene transcription. While scientists have ‘cracked’ the DNA code specifying the sequences of RNA and protein molecules produced from protein-coding DNA, it remains unclear how the TF/DNA ‘regulatory’ code that governs the strength and timing of gene expression. It is not known how closely related TFs with apparently similar DNA binding preferences recognize different sites in the genome to regulate distinct transcriptional programs. There is little known about the web of interactions between TFs and the ‘effector’ proteins required to activate transcription. This project will utilize new microfluidic technologies that enable accurate measurement of 1000s of protein/DNA and protein/protein binding interactions simultaneously and at low cost. This project will apply these technologies to better understand how TFs find and bind their DNA targets, how bound TFs recruit ‘effector’ proteins, and the degree to which the ‘regulatory code’ relies on thermodynamics. The PI will expand a hands-on microfluidics device laboratory to provide inquiry-based summer research experiences to community college students, increasing training opportunities for student populations traditionally underrepresented in STEM. Regulated gene expression is central to biology, sculpting the transformation from embryo to animal and enabling cells to respond dynamically to environmental changes. At a molecular level, this regulation is accomplished primarily by transcription factor (TF) proteins that bind DNA regulatory elements and then recruit additional protein cofactors to either activate or repress transcription. Biological diversity is simply too vast for us to ever measure TF binding and transcription in all organisms and all tissues under all conditions of interest. This project will provide in vitro measurements that quantify thermodynamic and kinetic constants of reconstituted macromolecular interactions at unprecedented scale by using multiple novel microfluidic platforms capable of measuring affinities and kinetics for up to one million protein/DNA or protein/protein interactions in parallel that were developed in the PI’s laboratory. In this project, these in vitro technologies will be used to develop quantitative and predictive models of how TFs find and bind their genomic targets and how bound TFs recruit cofactors to regulate gene expression. Using cutting-edge in silico tools, these measurements will then be integrated with existing in vivo data sets to develop quantitative models that, in turn, will be directly tested in vivo by quantifying TF and cofactor binding and gene expression for sequence variants. This project is funded by the Molecular Biophysics and Genetic Mechanisms Clusters in the Division of Molecular and Cellular Biosciences.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.

所有的生物学过程都依赖于基因转录时间和RNA转录产物数量的精确调控。这种调节主要由转录因子（TF）蛋白驱动，转录因子（TF）蛋白结合基因组中的特定DNA序列，然后招募额外的“效应”蛋白来激活或抑制基因转录。虽然科学家们已经“破解”了由编码蛋白质的DNA产生的RNA和蛋白质分子序列的DNA密码，但仍不清楚TF/DNA“调控”密码如何控制基因表达的强度和时间。目前尚不清楚具有明显相似DNA结合偏好的密切相关TF如何识别基因组中的不同位点以调节不同的转录程序。目前对转录因子和激活转录所需的“效应”蛋白之间的相互作用网络知之甚少。该项目将利用新的微流体技术，能够同时以低成本精确测量1000个蛋白质/DNA和蛋白质/蛋白质结合相互作用。该项目将应用这些技术来更好地了解TF如何找到并结合它们的DNA靶点，结合的TF如何招募“效应”蛋白，以及“调控代码”依赖热力学的程度。PI将扩大一个动手微流体设备实验室，为社区大学生提供基于调查的夏季研究经验，为传统上在STEM中代表性不足的学生群体增加培训机会。调控基因表达是生物学的核心，塑造了从胚胎到动物的转变，并使细胞能够对环境变化做出动态反应。在分子水平上，这种调节主要是通过转录因子（TF）蛋白质来完成的，TF蛋白质结合DNA调节元件，然后招募额外的蛋白质辅因子来激活或抑制转录。生物多样性实在是太大了，我们无法在所有感兴趣的条件下测量所有生物体和所有组织中的TF结合和转录。该项目将提供体外测量，通过使用PI实验室开发的多个新型微流体平台，以前所未有的规模量化重构大分子相互作用的热力学和动力学常数，该平台能够并行测量多达一百万个蛋白质/DNA或蛋白质/蛋白质相互作用的亲和力和动力学。在这个项目中，这些体外技术将用于开发定量和预测模型，以了解TF如何找到并结合其基因组靶标，以及结合的TF如何招募辅因子来调节基因表达。使用尖端的计算机工具，这些测量结果将与现有的体内数据集整合，以开发定量模型，反过来，通过定量TF和辅因子结合以及序列变体的基因表达，直接在体内进行测试。该项目由分子和细胞生物科学部的分子生物物理学和遗传机制集群资助。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Short tandem repeats bind transcription factors to tune eukaryotic gene expression

• DOI: 10.1126/science.add1250
• 发表时间: 2023-09-22
• 期刊: SCIENCE
• 影响因子: 56.9
• 作者: Horton，Connor A.;Alexandari，Amr M.;Fordyce，Polly M.
• 通讯作者: Fordyce，Polly M.