Sequence preferences of DNA-binding proteins are a primary mechanism by which cells interpret the genome. Despite these proteins' central importance in physiology, development, and evolution, comprehensive DNA-binding specificities have been determined experimentally for 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 evolution of transcriptional regulatory networks.The interactions between transcription factors (TFs) and their DNA binding sites are an integral part of the gene regulatory networks that control development, core cellular processes, and responses to environmental perturbations. However, only a handful of sequence-specific TFs have been characterized well enough to identify all the sequences that they can and, just as importantly, can not bind. Computational analysis of microarray readout of chromatin immunoprecipitation experiments (ChIP-chip) suggests extensive use of low affinity binding sites in yeast (1), and computational models of gene expression during fly embryonic development suggest that low affinity binding sites contribute as much as high affinity sites (2).The availability of TF binding data spanning the full affinity range would improve our understanding of the biophysical phenomena underlying protein-DNA recognition, and would improve accuracy in analyzing cis regulatory elements. Here we report the comprehensive determination of the DNA binding specificities of 104 known and predicted mouse TFs using the universal protein binding microarray (PBM) technology (3). These TFs represent 22 different DNA binding domain (DBD) structural classes that are the major DBD classes found in metazoan TFs.We created (4) N-terminal GST fusion constructs of the DBDs of 104 known and predicted mouse TFs (Fig. S1 and Table S1). Five of these proteins -Max, Bhlhb2, Gata3, Rfx3, and Sox7 -were also represented as full-length fusions to N-terminal GST, yielding a total set of 109 non-redundant proteins represented by 115 samples (5). Each protein was used in two PBM experiments (6,7) (Figs. S2, S3, S4 and Table S2). DNA binding site motifs initially were derived using the Seed-and-Wobble algorithm (3,8); Seed-and-Wobble first identifies the single 8-mer (ungapped or gapped) with the greatest PBM enrichment score (E-score) (3), and then systematically tests the relative preference of each nucleotide variant at each position both within and outside the seed (5). Later analyses incorporated additional motif finding algorithms, including RankMotif++ (9) and Kafal (5).Beyond simpl...

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before the teleost genome duplication (TGD). The slowly evolving gar genome conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization, and development (e.g., Hox, ParaHox, and miRNA genes). Numerous conserved non-coding elements (CNEs, often cis-regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles of such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses revealed that the sum of expression domains and levels from duplicated teleost genes often approximate patterns and levels of gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes, and the function of human regulatory sequences.

Most homeodomains are unique within a genome, yet many are highly conserved across vast evolutionary distances, implying strong selection on their precise DNA-binding specificities. We determined the binding preferences of the majority (168) of mouse homeodomains to all possible 8-base sequences, revealing rich and complex patterns of sequence specificity and showing that there are at least 65 distinct homeodomain DNA-binding activities. We developed a computational system that successfully predicts binding sites for homeodomain proteins as distant from mouse as Drosophila and C. elegans, and we infer full 8-mer binding profiles for the majority of known animal homeodomains. Our results provide an unprecedented level of resolution in the analysis of this simple domain structure and suggest that variation in sequence recognition may be a factor in its functional diversity and evolutionary success.