A new methodological approach for in vitro determination of the role of DNA methylation on transcription factor binding using AlphaScreen® analysis: Focus on CREB1 binding at hBDNF promoter IV.
Alphascreen assay
BDNF
CREB
CpG methylation
TF binding
Journal
Journal of neuroscience methods
ISSN: 1872-678X
Titre abrégé: J Neurosci Methods
Pays: Netherlands
ID NLM: 7905558
Informations de publication
Date de publication:
15 07 2020
15 07 2020
Historique:
received:
17
12
2019
revised:
09
03
2020
accepted:
01
04
2020
pubmed:
18
5
2020
medline:
22
6
2021
entrez:
17
5
2020
Statut:
ppublish
Résumé
DNA methylation plays a relevant role in the regulation of gene transcription, but currently the exact quantification of transcription factors binding to methylated DNA is not being determined. The binding of the transcription factor cAMP response element-binding protein-1 to its cognate CpG containing motif is known to be impaired upon methylation. It thus represents a paradigmatic system to experimentally verify the validity of a new in vitro method to measure the role of methylation on DNA/transcription factors binding. An AlphaScreen® assay was developed to quantitatively measure the contribution of DNA CpG methylation on the interaction with transcription factors. The method was validated measuring the variation in affinity of cAMP response element-binding protein-1 and its recognition motif in human Brain-derived neurotrophic factor gene exon IV promoter as a function of CpG methylation. For the first time, a quantitative direct correlation between DNA methylation and transcription factors binding is reported showing a dramatic reduction in binding affinity between fully methylated and non-methylated DNA. This methodology allows to directly measure DNA/transcription factors binding ability as a function of DNA methylation levels thus improving not quantitative methods available today. Moreover, it allows to work with purified proteins and oligonucleotides without need of chromatin. The present methodology is suggested as a new analytical tool for the quantitative determination of the effect of CpG methylation on the interaction of gene promoters with transcription factors regulating gene expression, a key epigenetic mechanism implicated in many physiological and pathological conditions.
Sections du résumé
BACKGROUND
DNA methylation plays a relevant role in the regulation of gene transcription, but currently the exact quantification of transcription factors binding to methylated DNA is not being determined. The binding of the transcription factor cAMP response element-binding protein-1 to its cognate CpG containing motif is known to be impaired upon methylation. It thus represents a paradigmatic system to experimentally verify the validity of a new in vitro method to measure the role of methylation on DNA/transcription factors binding.
METHOD
An AlphaScreen® assay was developed to quantitatively measure the contribution of DNA CpG methylation on the interaction with transcription factors. The method was validated measuring the variation in affinity of cAMP response element-binding protein-1 and its recognition motif in human Brain-derived neurotrophic factor gene exon IV promoter as a function of CpG methylation.
RESULTS
For the first time, a quantitative direct correlation between DNA methylation and transcription factors binding is reported showing a dramatic reduction in binding affinity between fully methylated and non-methylated DNA.
COMPARISON WITH EXISTING METHODS
This methodology allows to directly measure DNA/transcription factors binding ability as a function of DNA methylation levels thus improving not quantitative methods available today. Moreover, it allows to work with purified proteins and oligonucleotides without need of chromatin.
CONCLUSIONS
The present methodology is suggested as a new analytical tool for the quantitative determination of the effect of CpG methylation on the interaction of gene promoters with transcription factors regulating gene expression, a key epigenetic mechanism implicated in many physiological and pathological conditions.
Identifiants
pubmed: 32416472
pii: S0165-0270(20)30143-6
doi: 10.1016/j.jneumeth.2020.108720
pii:
doi:
Substances chimiques
CREB1 protein, human
0
Cyclic AMP Response Element-Binding Protein
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Review
Langues
eng
Sous-ensembles de citation
IM
Pagination
108720Informations de copyright
Copyright © 2020 Elsevier B.V. All rights reserved.