A Microfluidics-Based Platform For Identification and Detailed Characterization of Transcription Factor Binding Sites

Abstract

Transcriptional regulation of gene expression is mediated by transcription factors that bind DNA sequence-specifically within gene promoters to activate or repress transcription. However, biochemical data linking transcription factors to their consensus binding sites has traditionally been difficult to obtain, complicating reconstruction of cellular pathways contributing to a transcriptional response.We have developed a versatile and sensitive microfluidics-based technique for de novo identification and subsequent detailed characterization of transcription factor consensus motifs and binding energy landscapes. Our technique offers several advantages over current methods. First, our technique mechanically traps all complexes at equilibrium prior to measurement, allowing detection of weak or transient interactions and providing direct, quantitative measurements of reaction parameters. Second, our technique requires extremely small amounts of reagents, permitting protein production via cell-free transcription/translation of PCR-generated templates and eliminating laborious and time-consuming cloning steps. Finally, our technique allows high-throughput screening of transcription factor binding to all possible DNA 8mers in a single experiment.To evaluate the performance of our new technique, we probed DNA binding patterns for 30 yeast transcription factors from various families and used a statistical-mechanical model of transcription factor binding to determine preferred consensus motifs. In all cases, the core consensus obtained agreed with previous literature results, validating the utility of our technique for de novo identification of transcription factor binding sites. This quantitative data set provides critical information that can be used to revise and refine current models of transcription factor binding interactions.