Abstract
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.
Highlights
Protein–protein interactions (PPIs) play a fundamental role in many physiological processes, such as the cell division cycle or cell signaling in health and disease
We chose Tap54beta [35,36] and coexpressed biotin acceptor peptide (BAP)-Tap54beta and BirA-Oct4. This protein was present in comparable amounts in the nuclus and cytoplasm as shown by anti-His-HRP blots, weak biotinylation of the BAP-Tap54beta protein was observed in the nuclear fraction, and no noticeable biotinylation signal was found in the supernatant (Supplementary material, Figure S1). These results indicate that BirA-Oct4 is mainly localized in the nucleus and, in principle, it could biotinylate BAP-Tap54beta as a result of random collision; the biotinylation level of BAP-Tap54beta was much lower in comparison to that obtained when coexpressing the pair BAP-Sox2 and BirA-Oct4
Despite the fact that this method was developed as part of a joint Kazakh–French project during the internship of A.K. at the Gustave Roussy Institute (2007–2011), the work presented is the result of a transfer of technology and was completely carried out at the Kazakhstan National Center for Biotechnology
Summary
Protein–protein interactions (PPIs) play a fundamental role in many physiological processes, such as the cell division cycle or cell signaling in health and disease. The direct interaction between these key pluripotency transcription factors is DNA-dependent [18,19] and involves DNA-binding domains, such as the POU-specific domain (POUS) and a homeodomain (POUHD) of OCT4 and the high-mobility group (HMG) domain of Sox. The direct interaction between these key pluripotency transcription factors is DNA-dependent [18,19] and involves DNA-binding domains, such as the POU-specific domain (POUS) and a homeodomain (POUHD) of OCT4 and the high-mobility group (HMG) domain of Sox2 These proteins often bind to closely localized genomic sites, and their balanced expression as well as their precise mode of interaction with DNA at different time points are crucial for the pluripotency gene regulatory network (PGRN). How the expression levels of Oct of and Sox change over time in naive ES cells, and whether these fluctuations bias germ layer cell fate commitment remain unknown
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