Thermodynamic Additivity for Impacts of Base-Pair Substitutions on Association of the Egr-1 Zinc-Finger Protein with DNA

Abstract

The transcription factor Egr-1 specifically binds as a monomer to its 9-bp target DNA sequence, GCGTGGGCG, via three zinc fingers and plays important roles in the brain and cardiovascular systems. Using fluorescence-based competitive binding assays, we systematically analyzed the impacts of all possible single nucleotide substitutions in the target DNA sequence and determined the change in binding free energy for each. Then, we measured the changes in binding free energy for sequences with multiple substitutions and compared them with the sum of the changes in binding free energy for each constituent single substitution. For the DNA variants with 2 or 3 nucleotide substitutions in the target sequence, we found excellent agreement between the measured and predicted changes in binding free energy. Interestingly, however, we found that this thermodynamic additivity broke down with a larger number of substitutions. For DNA sequences with 4 or more substitutions, the measured changes in binding free energy were significantly larger than predicted. Based on these results, we analyzed the occurrences of high-affinity sequences in the genome and found that the genome contains millions of high-affinity sequences that might functionally sequester Egr-1. Figure S1); comparison of uncertainties in dissociation constants estimated from 4 replicates and those from fitting for single datasets (Table S1); and high-affinity Egr-1-binding sequences and their total numbers in the human genome Graphical abstract

Measuring quantitative effects of methylation on transcription factor–DNA binding affinity

“…For most applications, only the relative affinities to different DNA sequences or, equivalently, the differences in binding free energy are needed, not absolute K D values. Relative binding affinities can be measured using CFA, where the binding to the labeled DNA is in competition with an unlabeled DNA with a different sequence ( 22 ). That approach relieves some of the complications of standard fluorescence anisotropy experiments, but there remain issues about knowing the absolute differences in concentration of the two DNAs and variations in conditions between different reactions.…”

Potential role of DNA methylation as a facilitator of target search processes for transcription factors through interplay with methyl-CpG-binding proteins

“…The DNA-binding domain of the human Egr-1 protein (residues 335–423) comprised three zinc fingers was expressed in Escherichia coli and purified using affinity, size-exclusion and cation-exchange columns, as previously described ( 32 ). This construct was used in our previous nuclear magnetic resonance (NMR) ( 32 – 38 ), X-ray ( 24 ) and fluorescence ( 13 , 15 , 30 , 31 ) studies. For simplicity's sake, this construct is referred to as the Egr-1 zinc-finger protein hereafter.…”

“…The sequence specificity and binding free energy as a function of DNA sequence have been well studied for Egr-1 ( 14 – 16 ). It was shown that the genome contains millions of high-affinity sequences for Egr-1, and even though ∼90% of them are buried in nucleosomes, ∼10 6 sites should remain accessible ( 15 ). Due to the huge number of decoys, it seems difficult for Egr-1 to adequately occupy each functional target because sequestration of Egr-1 molecules may occur in off-target locations.…”

Single-molecule DNA unzipping reveals asymmetric modulation of a transcription factor by its binding site sequence and context

“…Hence, elucidating how the structure and dynamics of the Egr-1 complex is modulated by the binding site sequence and its genomic context is of great interest. Moreover, it is predicted that the mammalian genome harbors ∼10 6 sites that are highly similar, but not identical, to the classical recognition sequence of Egr-1 ( 46 , 47 ). Most of these sites, without a regulatory role, are expected to affect the search process, as they can momentarily trap Egr-1 ( 46 ).…”

“…Unfortunately, since most of the experiments have concentrated on the consensus sequence, it is not clear how Egr-1 binds to the variable ones. Interestingly, a thermodynamic additivity model was proposed to predict the affinity of near-consensus sites, but it was found to fail for sequences with four or more substitutions ( 47 ).…”