Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities

Abstract

Transcription factors (TFs) regulate the expression of genes involved in myriad cellular processes through sequence-specific interactions with DNA. In order to predict DNA regulatory elements and the TFs targeting them with greater accuracy, detailed knowledge of the binding preferences of TFs is needed. Protein binding microarray (PBM) technology permits rapid, high-throughput characterization of the in vitro DNA binding specificities of proteins 1 . Here, we present a novel, maximally compact, synthetic DNA sequence design that represents all possible DNA sequence variants of a given length k (i.e., all "k-mers") on a single, universal microarray. We constructed such all k-mer microarrays covering all 10 base pair (bp) binding sites by converting high-density single-stranded oligonucleotide arrays to double-stranded DNA arrays. Using these microarrays, we comprehensively determined the binding specificities over a full range of affinities for five TFs of diverse structural classes from yeast, worm, mouse, and human. Importantly, the unbiased coverage of all k-mers permits an interrogation of binding site preferences, including nucleotide interdependencies, at unprecedented resolution.

Polycomb-like proteins link the PRC2 complex to CpG islands

“…Both PHF1 and MTF2 showed higher binding affinity for the (G/T)CpGG containing sequences. To further validate the DNA motifs recognized by PCL proteins, we performed unbiased protein binding microarray experiments using universal “all 10mer” arrays 23 , which confirmed that PHF1 and MTF2 preferentially bind to DNAs containing the (T/G)CpGG motifs, with guanines slightly preferred as the flanking bases on each side of the motifs (Fig. 2f).…”

“…Subsequently, custom-designed “all-10mer” universal oligonucleotide arrays in 8 x 60K GSE array format (Agilent Technologies; AMADID #030236) were double-stranded and duplicate protein binding microarray experiments were performed essentially as described 23,28 . MTF2 was assayed at a final concentration of either 500 nM or 900 nM, while PHF1 was assayed at a final concentration of 900 nM, in binding reactions containing 50 μM zinc acetate, on either a fresh slide or a slide that had been stripped exactly once.…”

“…Scans were acquired using a GenePix 4400A (Molecular Devices) microarray scanner. Microarray data quantification, normalization, and motif derivation were performed essentially as described previously using the Universal PBM Analysis Suite and the Seed-and-Wobble motif-derivation algorithm 23,28 .…”

Predicting the sequence specificities of DNA- and RNA-binding proteins by deep learning

“…Weirauch et al 17 20 and Seed-and-Wobble 21 . For each individual algorithm, they optimized the data preprocessing steps to attain best test performance.…”

“…3). E-score correlation is robust to outliers and array biases 21 . Correlations of 7-mer scores can be computed using measured data, providing upper bounds on performance.…”