SGER: High-throughput Determination of Position Weight Matrices

Duke University, Durham NC

Investigators

Abstract

Transcription factors regulate gene expression by binding in a sequence-specific manner to double-stranded DNA and modulating the initiation of new transcripts. The unique binding specificities of transcription factors are critical components of their ability to regulate gene expression. Yet determining binding specificity is time-consuming and detailed information is available for only a fraction of transcription factors, even in well-studied organisms. The goal of this project is to develop and validate a rapid, simple, high-throughput assay to determine the full spectrum of binding specificities (or position weight matrix) for any transcription factor. The approach uses a microarray containing thousands of different dsDNA hairpin probes to assay relative binding of all permutations of seven base pairs. This method will be tested by comparing results to those obtained using existing, more laborious methods, and then refined, calibrated, and optimized for general use. This high-throughput method should be applicable to understanding the binding specificity of almost any protein:DNA interaction. It is expected that this approach will offer several distinct advantages over existing methods for determining position weight matrices: speed, simplicity, and comprehensive sampling of all potential binding sites. Successful validation of this method should benefit basic research on mechanisms of gene expression by providing detailed information about the binding specificities of many different transcription factors. Other aspects of basic research will also benefit, including automated annotation of genome sequences and evolutionary analyses of gene networks.