Deconvolving the recognition of DNA shape from sequence
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE65073 | GEO | 2015/01/21
SECONDARY ACCESSION(S): PRJNA272858
REPOSITORIES: GEO
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Project description:Binding of transcription factors to DNA is mediated by the recognition of the chemical signatures of the DNA bases and the three-dimensional shape of the DNA molecule. The direct contribution of DNA shape to DNA-binding specificity has been difficult to assess, as DNA shape is a consequence of its sequence. Here, we teased apart these two modes of recognition in the context of Hox-DNA binding. We made a series of mutations in Hox residues that, in a co-crystal structure, only recognize DNA shape, and tested the effect on DNA binding preferences using SELEX-seq. Analysis of shape features of selected sequences revealed that these residues are both necessary and sufficient for selection of sequences with distinct shape features. We used statistical machine learning to show that the accuracy of binding specificity predictions improves by adding shape features to a model that only depends on sequence. We conclude that shape readout is a direct and critical component of binding site selection by Hox proteins. Three rounds of SELEX were performed on a series of Hox mutants as described in Slattery et al, Cell, 2011 (PMID 22153072) and Riley et al, Methods in molecular Biology, 2014 (PMID 25151169). Briefly, His-tagged Scr and Antp mutant proteins were incubated with a randomized 16mer oligonucleotide library, and bound DNA was amplified and sequenced as described (PMID 22153072, PMID 25151169).
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Project description:DNA binding protein are generally thought to bind specific DNA sequences through selective interactions with DNA bases. However, it is now becoming more widely appreciated that DNA shape, which may not be specified by a unique base sequence, also contributes to site-specific binding. Here we elucidate how DNA sequence and shape confer site specificity on a genomic scale, and relate this to specificity imparted indirectly through occlusion of sequences by the in vivo environment. For simplicity, we focus on the set of General Regulatory Factors (GRFs) that do not rely on other factors for binding. They also serve a related function in organizing chromatin. Remarkably, we find that GRFs will not bind to their cognate motif if the DNA surrounding that sequence lacks a specific shape. While proper DNA sequence/shape properties tend to be restricted to promoter regions, weaker sites that are still binding-competent reside in gene bodies, but are prevented from binding by resident chromatin. Thus, site-specificity is achieved across a genome in vivo by the combined action of favorable DNA sequence and shape interactions, and occlusion by chromatin.
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