Single molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation.
Nucleic Acids Res
; 49(19): 10975-10987, 2021 11 08.
Article
em En
| MEDLINE
| ID: mdl-34606618
ABSTRACT
The interaction of transcription factors with their response elements in DNA is emerging as a highly complex process, whose characterization requires measuring the full distribution of binding and dissociation times in a well-controlled assay. Here, we present a single-molecule assay that exploits the thermal fluctuations of a DNA hairpin to detect the association and dissociation of individual, unlabeled transcription factors. We demonstrate this new approach by following the binding of Egr1 to its consensus motif and the three binding sites found in the promoter of the Lhb gene, and find that both association and dissociation are modulated by the 9 bp core motif and the sequences around it. In addition, CpG methylation modulates the dissociation kinetics in a sequence and position-dependent manner, which can both stabilize or destabilize the complex. Together, our findings show how variations in sequence and methylation patterns synergistically extend the spectrum of a protein's binding properties, and demonstrate how the proposed approach can provide new insights on the function of transcription factors.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
DNA
/
Metilação de DNA
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Elementos de Resposta
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Hormônio Luteinizante Subunidade beta
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Proteína 1 de Resposta de Crescimento Precoce
Limite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Israel