Next Article in Journal
Synthetically Lethal Interactions of Heme Oxygenase-1 and Fumarate Hydratase Genes
Previous Article in Journal
Simultaneous Suppression of Two Distinct Serotonin N-Acetyltransferase Isogenes by RNA Interference Leads to Severe Decreases in Melatonin and Accelerated Seed Deterioration in Rice
Open AccessArticle

In Vivo Quantitative Estimation of DNA-Dependent Interaction of Sox2 and Oct4 Using BirA-Catalyzed Site-Specific Biotinylation

1
Republican State Enterprise “National Center for Biotechnology” under the Science Committee of Ministry of Education and Science of Republic of Kazakhstan, 13/5, Kurgalzhynskoye road, Nur-Sultan 010000, Kazakhstan
2
Institut de Cancerologie Gustave Roussy, UMR8126, Universite Paris-Sud 11, CNRS, 94805 Villejuif, France
*
Author to whom correspondence should be addressed.
Biomolecules 2020, 10(1), 142; https://doi.org/10.3390/biom10010142
Received: 12 December 2019 / Revised: 3 January 2020 / Accepted: 9 January 2020 / Published: 16 January 2020
(This article belongs to the Section Molecular Biology)
Protein–protein interactions of core pluripotency transcription factors play an important role during cell reprogramming. Cell identity is controlled by a trio of transcription factors: Sox2, Oct4, and Nanog. Thus, methods that help to quantify protein–protein interactions may be useful for understanding the mechanisms of pluripotency at the molecular level. Here, a detailed protocol for the detection and quantitative analysis of in vivo protein–protein proximity of Sox2 and Oct4 using the proximity-utilizing biotinylation (PUB) method is described. The method is based on the coexpression of two proteins of interest fused to a biotin acceptor peptide (BAP)in one case and a biotin ligase enzyme (BirA) in the other. The proximity between the two proteins leads to more efficient biotinylation of the BAP, which can be either detected by Western blotting or quantified using proteomics approaches, such as a multiple reaction monitoring (MRM) analysis. Coexpression of the fusion proteins BAP-X and BirA-Y revealed strong biotinylation of the target proteins when X and Y were, alternatively, the pluripotency transcription factors Sox2 and Oct4, compared with the negative control where X or Y was green fluorescent protein (GFP), which strongly suggests that Sox2 and Oct4 come in close proximity to each other and interact. View Full-Text
Keywords: protein–protein interactions (PPI); proximity-utilizing biotinylation (PUB); biotin acceptor peptide (BAP); BirA; proteomics; liquid chromatography–tandem mass spectrometry (LC–MS/MS); multiple reaction monitoring (MRM); in vivo DNA-dependent protein–protein interaction; pluripotency transcription factors Sox2 and Oct4 protein–protein interactions (PPI); proximity-utilizing biotinylation (PUB); biotin acceptor peptide (BAP); BirA; proteomics; liquid chromatography–tandem mass spectrometry (LC–MS/MS); multiple reaction monitoring (MRM); in vivo DNA-dependent protein–protein interaction; pluripotency transcription factors Sox2 and Oct4
Show Figures

Graphical abstract

MDPI and ACS Style

Kulyyassov, A.; Ogryzko, V. In Vivo Quantitative Estimation of DNA-Dependent Interaction of Sox2 and Oct4 Using BirA-Catalyzed Site-Specific Biotinylation. Biomolecules 2020, 10, 142.

Show more citation formats Show less citations formats
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop