RESUMO
Eukaryotic genomes contain numerous non-functional high-affinity sequences for transcription factors. These sequences potentially serve as natural decoys that sequester transcription factors. We have previously shown that the presence of sequences similar to the target sequence could substantially impede association of the transcription factor Egr-1 with its targets. In this study, 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. DNA methylation of decoys alone did not affect target search kinetics. In the presence of the MeCP2 methyl-CpG-binding domain (MBD), however, DNA methylation of decoys substantially (â¼10-30-fold) accelerated the target search process of the Egr-1 zinc-finger protein. This acceleration did not occur when the target was also methylated. These results suggest that when decoys are methylated, MBD proteins can block them and thereby allow Egr-1 to avoid sequestration in non-functional locations. This effect may occur in vivo for DNA methylation outside CpG islands (CGIs) and could facilitate localization of some transcription factors within regulatory CGIs, where DNA methylation is rare.
Assuntos
Metilação de DNA , Proteína 2 de Ligação a Metil-CpG/metabolismo , Sítios de Ligação , Ilhas de CpG , DNA/química , DNA/genética , DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Humanos , Cinética , Proteína 2 de Ligação a Metil-CpG/química , Proteína 2 de Ligação a Metil-CpG/genética , Modelos Biológicos , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ativação Transcricional , Dedos de Zinco/genéticaRESUMO
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 two or three 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 four or more substitutions, the measured changes in binding free energy were significantly larger than predicted. On the basis of these results, we analyzed the occurrences of high-affinity sequences in the genome and found that the genome contains millions of such sequences that might functionally sequester Egr-1.
Assuntos
DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Termodinâmica , Dedos de Zinco , Algoritmos , Sequência de Bases , Sítios de Ligação/genética , Ligação Competitiva , DNA/química , DNA/genética , Proteína 1 de Resposta de Crescimento Precoce/química , Proteína 1 de Resposta de Crescimento Precoce/genética , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Mutação Puntual , Ligação Proteica , Domínios ProteicosRESUMO
Due to chemical exchange, the mobility of histidine (His) side chains of proteins is typically difficult to analyze by NMR spectroscopy. Using an NMR approach that is uninfluenced by chemical exchange, we investigated internal motions of the His imidazole NH groups that directly interact with DNA phosphates in the Egr-1 zinc-finger-DNA complex. In this approach, the transverse and longitudinal cross-correlation rates for 15N chemical shift anisotropy and 15N-1H dipole-dipole relaxation interference were analyzed together with 15N longitudinal relaxation rates and heteronuclear Overhauser effect data at two magnetic field strengths. We found that the zinc-coordinating His side chains directly interacting with DNA phosphates are strongly restricted in mobility. This makes a contrast to the arginine and lysine side chains that retain high mobility despite their interactions with DNA phosphates in the same complex. The entropic effects of side-chain mobility on the molecular association are discussed.