Abstract
Sox2 is a Sry-box containing family member of related transcription factors sharing homology in their DNA binding domain. Sox2 is important during different stages of development, and previously we showed that Sox2 plays an important role in branching morphogenesis and epithelial cell differentiation in lung development. The transcriptional activity of Sox2 depends on its interaction with other proteins, leading to ‘complex-specific’ DNA binding and transcriptional regulation. In this study, we generated a mouse model containing a biotinylatable-tag targeted at the translational start site of the endogenous Sox2 gene (bioSox2). This tag was biotinylated by the bacterial birA protein and the resulting bioSox2 protein was used to identify associating partners of Sox2 at different phases of lung development in vivo (the Sox2 interactome). Homozygous bioSox2 mice are viable and fertile irrespective of the biotinylation of the bio tag, indicating that the bioSox2 gene is normally expressed and the protein is functional in all tissues. This suggests that partners of Sox2 are most likely able to associate with the bioSox2 protein. BioSox2 complexes were isolated with high affinity using streptavidin beads and analysed by MALDI-ToF mass spectrometry analysis. Several of the identified binding partners are already shown to have a respiratory phenotype. Two of these partners, Wdr5 and Tcf3, were validated to confirm their association in Sox2 complexes. This bioSox2 mouse model will be a valuable tool for isolating in vivo Sox2 complexes from different tissues.
Highlights
Sox2 is a Sry-box containing family member of related transcription factors sharing homology in their DNA binding domain
In order to isolate and identify in vivo Sox2 complexes with high specificity, we generated a mouse line expressing Sox2 with a small artificial peptide tag of seventeen amino acids at the N-terminus. This biotag can be biotinylated in vivo by a biotin ligase protein and the protein can subsequently be isolated with high affinity using streptavidin
Western blot analysis showed that the Tobacco Etch Virus (TEV) protease recognized and processed the bioSox2 protein, but the cleaved Sox2 did not elute from the beads (Fig. 1b)
Summary
Sox is a Sry-box containing family member of related transcription factors sharing homology in their DNA binding domain. Expression of Sox is restricted to the anterior ectoderm, from which the neuroectoderm and anterior surface ectoderm will arise (Papanayotou et al 2008; Wood and Episkopou 1999). Together with Oct and Nanog, Sox is part of the core transcription factor network in embryonic stem cells, and Sox is one of the key factors required to induce pluripotent stem cells (iPSC) from in somatic cells (Avilion et al 2003; Masui et al 2007; Takahashi et al 2007; Takahashi and Yamanaka 2006; Yamanaka 2012). Together with Oct and Klf, Sox first co-occupies non-permissive chromatin and the subsequent activation of pluripotency-associated genes by Sox (Smith et al 2016; Xu et al 2016)
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