In Vitro Screening for Regulated Transcription Factors with Differential Display of DNA-Binding Proteins (DDDP)

Abstract

INTRODUCTIONStrict regulation of transcription factor activity is essential to establish and maintain gene expression. Eukaryotic cells control transcription factors at many different levels. Post-translational regulatory mechanisms (e.g., phosphorylation, nuclear translocation, multimerization, regulated degradation, etc.) play particularly important roles because they enable cells to respond to various intra- and extracellular stimuli quickly and without prior protein synthesis. However, extensive post-translational changes can make it difficult to identify differentially regulated transcription factors. Common genomic screening techniques such as DNA microarray analysis are unable to detect any mode of regulation beyond that of mRNA stability. This protocol describes the differential display of DNA-binding proteins (DDDP), which is based on the electrophoretic mobility shift assay (EMSA) and detects DNA-binding transcription factors, independent of the number or nature of regulatory steps required for activation. DDDP is an unbiased screening technique that can be used in any experimental system that uses concentrated protein extracts. A plasmid library containing random DNA sequences is constructed. This library is then used to generate radioactive DNA probes to test protein extracts from different sources in parallel for differentially regulated DNA-binding proteins. Plasmids corresponding to probes that display differential DNA-binding activity can be sequenced, and the binding sequence can be narrowed down in a two-step procedure. The corresponding transcription factors can then be identified by bioinformatic and/or biochemical methods.