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

所有的生物过程都依赖于基因何时转录以及产生多少RNA转录物的精确调控。这种调节主要是由转录因子（TF）蛋白驱动的，它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”密码是如何控制基因表达的强度和时间的。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来调节不同的转录程序。人们对tf和激活转录所需的“效应”蛋白之间的相互作用知之甚少。该项目将利用新的微流体技术，能够以低成本同时精确测量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靶标，结合的tf如何招募“效应”蛋白，以及“调节代码”在多大程度上依赖热力学。PI将扩展一个动手微流体设备实验室，为社区大学生提供基于探究的夏季研究经验，增加传统上在STEM中代表性不足的学生群体的培训机会。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，使细胞能够对环境变化做出动态反应。在分子水平上，这种调节主要是由转录因子（TF）蛋白完成的，转录因子结合DNA调节元件，然后招募额外的蛋白质辅助因子来激活或抑制转录。生物多样性太大了，我们无法测量所有生物和组织在所有条件下的TF结合和转录。该项目将提供体外测量，通过使用多个新型微流控平台，在前所未有的规模上量化重建大分子相互作用的热力学和动力学常数，这些平台能够测量多达一百万蛋白质/DNA或蛋白质/蛋白质相互作用的亲和和动力学，这些平台是在PI实验室开发的。在本项目中，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点，以及结合的tf如何招募辅助因子来调节基因表达。使用尖端的硅工具，这些测量结果将与现有的体内数据集相结合，以开发定量模型，反过来，将通过定量TF和辅因子结合以及序列变异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小组资助。该奖项反映了美国国家科学基金会的法定使命，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。

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.