Complexity of transcription factor target search: effect of DNA natural decoys and DNA methylation through interplay with methyl-CpG-binding proteins on transcription factor DNA association

Functions of transcription factors require formation of specific complexes at particular sites in cis-regulatory elements of genes. However, chromosomal DNA contains numerous sites that are similar to the target sequences recognized by transcription factors. These sequences potentially serve as natural decoys that sequester transcription factors. The impact that these decoys have on transcription factor target search is largely unknown, and various factors such as methylation, other DNA binding proteins, and side chain dynamics of individual protein residues may play a role in transcription factor target search during binding and scanning.

First, we quantitatively demonstrate the impact of quasi-specific DNA on the kinetics of the inducible transcription factor Egr-1 using stopped flow assays on a fluorescence spectrometer. We show that the impact of quasi-specific sites depends strongly on their affinity to Egr-1 and on their concentration, and that the presence of these sites are highly probable within genomic DNA. We show that naturally abundant quasi-specific sites(natural decoys) on DNA can considerably impede the target search processes of sequence-specific DNA-binding proteins.

Second, using a stopped-flow fluorescence method, we examined the kinetic impact of DNA methylation of decoys on the search process of the Egr-1 zinc-finger protein. We analyzed its association with an unmethylated target site on fluorescence-labeled DNA in the presence of competitor DNA duplexes, including Egr-1 decoys. The results suggest that methylated decoys attract methyl binding proteins, effectively blocking them and thereby allowing Egr-1 to avoid sequestration in non-functional locations. This effect may occur in vivo for DNA methylation outside CpG islands (CGIs) and facilitate localization of some transcription factors within regulatory CGIs, where DNA methylation is rare.

Lastly, we address the conformational dynamics of Egr-1's arginine side chains' guanidino N_epsilon-H_epsilon moieties when Egr-1 is bound to completely nonspecific and quasi-specific DNA. We compare Egr-1 in complex with both nonspecific and quasi-specific DNA. The resulting data gives a more detailed look at the molecular interactions and dynamics during the scanning process. We demonstrate that there is a significant decrease in mobility of some of the Arg side chains when Egr-1 is non-specifically bound. However, there is a crucial residue which is mobile that may contribute to a conformational switch of the protein from when it is scanning (scanning mode) to when it is bound (recognition mode).

As a whole, these studies suggest that there are significant underlying influences affecting a protein's interaction with DNA. It is possible that DNA is used as a regulatory mechanism of protein interactions through its quasi-specific sequences, methylation, and interaction with other DNA binding proteins.