Transcription factors (TFs) play a fundamental role in virtually all cellular processes by dynamically regulating the expression levels of genes across the genome through sequence-specific interactions with genomic DNA. In order to globally map TFs to their target genes and understand the regulatory interactions that govern cellular identity and behavior, precise knowledge of the DNA binding specificities of TFs is necessary. Despite their central importance, however, comprehensive binding site measurements have been obtained for only a small number of TFs.

Protein binding microarray (PBM) technology provides a rapid, high-throughput means of characterizing the in vitro DNA binding specificities of TFs, yet early PBMs were individually tailored to specific TFs and limited in the amount of sequence that could be assayed in any experiment. Here, I describe the development of a new generation of PBMs—first, using all intergenic sequences in the yeast Saccharomyces cerevisiae genome, and second, using synthetic sequence capturing all 10-mer variants—to enable the identification of all binding sites for any TF of interest. Using yeast intergenic PBMs, I identified hundreds of genomic targets for several well-characterized yeast TFs, confirming and expanding upon the set of targets already known from in vivo studies. The development of a universal, species-independent platform enabled the characterization of the binding specificities of a diverse collection of TFs at even higher resolution.

Finally, I present the application of this universal PBM technology on a large scale in a collaborative effort to capture the comprehensive binding specificities of hundreds of mouse TFs. The results of these experiments have revealed an unexpectedly diverse and complex landscape of binding, with closely-related TFs exhibiting unique binding preferences and individual TFs often recognizing multiple distinct classes of sites. A focused analysis of nearly 170 members of the homeodomain family exposed rich patterns of sequence specificity and facilitated the prediction of the DNA binding preferences of homeodomain TFs in dozens of other species. By providing comprehensive binding measurements for all DNA sequence variants, universal PBMs carry great potential to transform our current understanding of transcriptional regulation throughout the genome.