Generation of a biotinylatable Sox2 mouse model to identify Sox2 complexes in vivo

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.

Identification of SOX2 Interacting Proteins in the Developing Mouse Lung With Potential Implications for Congenital Diaphragmatic Hernia

“…The transcription factor SOX2 is expressed early in the developing lung and is one of the first molecular signs of epithelial cells that are committed to the proximal airway fate. To identify molecular switches that hallmark the differentiation of the proximal cell toward the different cell lineages eventually covering the upper airways, such as ciliated cells, club cells and basal cells, we generated a mouse line expressing a biotinylatable tagged SOX2 ( 26 ). An additional mouse strain ubiquitously expressing the bacterial biotin birA ligase was used to biotinylate the bioSOX2 protein in vivo ( 27 ).…”

“…Comparable immunoprecipitations were performed to validate the interaction between SOX2 and CHD4 using a FLAG-tagged CHD4 and a myc-tagged SOX2 protein, indicating that SOX2 and CHD4 also interact ( Figure 3C ). The interactions between SOX2 and TCF3 or WDR5 were used as positive controls, as we previously showed that these proteins interact with SOX2 ( 26 ).…”

“…BioSOX2 and birA mice were previously generated and maintained on C57/Bl6 background under standard conditions and experiments were performed following guidelines of the ethics committee of the Erasmus Medical Center ( 26 , 27 ).…”

“…Therefore, we used a specific mouse model expressing a biotinylatable SOX2, the bioSOX2 mouse, to isolate SOX2 complexes in vivo during airway epithelium differentiation ( 26 ). We identified several interacting proteins and showed that CHD4, FOXP2, FOXP4, and CUX1 act as SOX2 interacting partners during lung development.…”

Distinct roles for SOX2 and SOX21 in differentiation, distribution and maturation of pulmonary neuroendocrine cells

“…Mice were kept under standard conditions. Mouse strains bioSOX2flox (Schilders, Eenjes et al 2018) and SOX21-KO (gift of Stavros Malas) were used. Wild-type animals were C57BL/6.…”