Andrew R. Gehrke

Sequence preferences of DNA binding proteins are a primary mechanism by which cells interpret the genome. Despite the central importance of these proteins in physiology, development, and evolution, comprehensive DNA binding specificities have been determined experimentally for only a few proteins. Here, we used microarrays containing all 10-base pair sequences to examine the binding specificities of 104 distinct mouse DNA binding proteins representing 22 structural classes. Our results reveal a complex landscape of binding, with virtually every protein analyzed possessing unique preferences. Roughly half of the proteins each recognized multiple distinctly different sequence motifs, challenging our molecular understanding of how proteins interact with their DNA binding sites. This complexity in DNA recognition may be important in gene regulation and in the evolution of transcriptional regulatory networks.

Members of the large ETS family of transcription factors (TFs) have highly similar DNA-binding domains (DBDs)-yet they have diverse functions and activities in physiology and oncogenesis. Some differences in DNA-binding preferences within this family have been described, but they have not been analysed systematically, and their contributions to targeting remain largely uncharacterized. We report here the DNA-binding profiles for all human and mouse ETS factors, which we generated using two different methods: a high-throughput microwell-based TF DNA-binding specificity assay, and protein-binding microarrays (PBMs). Both approaches reveal that the ETS-binding profiles cluster into four distinct classes, and that all ETS factors linked to cancer, ERG, ETV1, ETV4 and FLI1, fall into just one of these classes. We identify amino-acid residues that are critical for the differences in specificity between all the classes, and confirm the specificities in vivo using chromatin immunoprecipitation followed by sequencing (ChIP-seq) for a member of each class. The results indicate that even relatively small differences in in vitro binding specificity of a TF contribute to site selectivity in vivo.