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

• Published in: Journal of neuroscience methods 2020-07, Vol.341, p.108720-108720, Article 108720
• Main Authors: Sabatucci, A., Berchet, V., Bellia, F., Maccarrone, M., Dainese, E., D’Addario, C., Pucci, M.
• Format: Article
• Language: English
• Subjects: Alphascreen assay; BDNF; Chromatin Immunoprecipitation; CpG methylation; CREB; Cyclic AMP Response Element-Binding Protein - genetics; DNA Methylation; Epigenesis, Genetic; Humans; Promoter Regions, Genetic; Protein Binding; TF binding
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/j.jneumeth.2020.108720

[Display omitted] •Determination of the affinity constant of BDNF/CREB1 binding.•CpG metylation role on DNA/TF binding directly measured by Alphascreen assay.•CpG methylation on CREB1 consensus hampers DNA binding like point mutation. 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.