Proteomics

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Genomic regions flanking E-box sites influence DNA binding specificity of bHLH transcription factors through DNA shape (validation)


ABSTRACT: DNA sequence is a major determinant of the binding specificity of transcription factors (TFs) for their genomic targets. However, eukaryotic cells often express, at the same time, TFs with highly similar DNA binding motifs but distinct in vivo targets. Currently, it is not well understood how TFs with seemingly identical DNA motifs achieve unique specificities in vivo. Here, we used custom protein binding microarrays to analyze TF specificity for putative binding sites in their genomic sequence context. Using yeast TFs Cbf1 and Tye7 as our case study, we found that binding sites of these bHLH TFs (i.e., E-boxes) are bound differently in vitro and in vivo, depending on their genomic context. Computational analyses suggest that nucleotides outside E-box binding sites contribute to specificity by influencing the 3D structure of DNA binding sites. Thus, local shape of target sites might play a widespread role in achieving regulatory specificity within TF families. Two protein binding microarray (PBM) experiments of Saccharomyces cerevisiae transcription factors were performed. Briefly, the PBMs involved binding GST-tagged yeast transcription factors Cbf1 and Tye7 to double-stranded 44K Agilent microarrays in order to determine the accuracy of our regression models for TF-DNA binding specificity. Briefly, this array contains 30-bp genomic sequences from our initial custom array (Gordan et al 2013, submitted), with 0 through 4 mutations designed at various positions in the genomic sequences. Each sequence in represented in 6 replicate spots. We report the PBM signal intensity for each spot. The PBM protocol is described in Berger et al., Nature Biotechnology 2006 (PMID 16998473).

ORGANISM(S): Saccharomyces cerevisiae  

SUBMITTER: John Horton   Iris Dror  Martha L Bulyk  Ning Shen  Tianyin Zhou  Raluca Gordan  Remo Rohs 

PROVIDER: E-GEOD-44437 | ArrayExpress | 2013-03-29

SECONDARY ACCESSION(S): GSE44437PRJNA190630

REPOSITORIES: GEO, ArrayExpress

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