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.

Keywords: Biotinylatable tag; In vivo protein complexes; Knock-in; Sox2.

Fig. 1

Development of biotin tagged Sox2 locus. a Nuclear extracts of transiently transfected HEK cells with control vector (control) or expression constructs for birA and bioSox2 (bioSox2) were incubated with streptavidin coupled dynabeads. Total input (T), unbound fraction (U) and the streptavidin bound fraction (B) analyzed on western blot using HRP coupled streptavidin. Arrow indicates the purified 40 kDa biotinylated Sox2 protein. b Identical experiment as in a, except that the purified samples were incubated with or without TEV protease. Although the bioSox2 was cleaved by the TEV, the protein remained attached to the beads. T is a fraction of the total input material before the beads were added to the extract. U represents the unbound fraction eluted from the beads after incubation with the TEV protease, whereas B represents the bound fraction, which was left on the beads after the TEV protease incubation. The B fraction was subsequently retrieved by boiling the beads. c Construct design to modify the Sox2 locus by homologous recombination. The Neomycin cassette was introduced upstream the transcriptional start site (TS), flanked by two loxP sites (arrow heads). The restriction sites used to isolate the fragment used to electroporate ES cells are indicated (N: NheI; A: Asp718), as well as the EcoRI sites (E) to analyze genomic DNA with the 3′ probe. d Representative Southern blot of ES genomic DNA digested with EcoRI and probed with the indicated 3′ probe, resulting in the wild type band at 15709 bp, and a mutant band 6771. e Schematic overview of the bioTEV-Sox2 protein and the N-terminal amino acids representing the translational start of the Sox2 protein (MA) followed by the Bio tag, a short hinge region (AGL) and the TEV protease cleavage recognition site (TEV). The asterisk indicates the lysine residue that is biotinylated by the BirA protein

Fig. 2

Functional analysis of the bioSox2 mouse. a Immunohistochemistry analysis of esophagus and lungs of adult bioSox2, birA and bioSox2/birA mice showing the expression of Sox2 using an antibody against Sox2 protein (Sox2). Using HRP coupled streptavidin (Strep) shows that the biotinylated Sox2 is expressed in the same cells as the normal Sox2. Moreover, biotinylation only occurs in the mice that carries the bioSox2 allele and the birA transgene. b Total protein extracts isolated from E 17.5 brains of heterozygous Sox2/bioSox2 mice (wt/bio) and homozygous bioSox2 mice (bio/bio) were analyzed by western blot analysis using a Sox2 antibody. This showed that the expression of the Sox2 (34 kDa) and bioSox2 (40 kDa) are comparable (lane wt/bio). c In vivo purification of bioSox2 from nuclear extracts of brain and lung tissue isolated at E18 from bioSox2/birA (bio/birA) and birA only (birA) mice using dynabeads. Arrowheads indicate the band representing the bioSox2, showing specific purification of the bioSox2 in the bioSox2/birA extracts. The western blot is labeled with streptavidine coupled HRP. Indicate lanes are total input (T), unbound fraction (U) and the purified fraction (P)

Fig. 3

In vivo isolation of bioSox2 complexes. a Large scale purification of bioSox2 complexes from E18.5 lungs revealed several putative Sox2 associating proteins. The expression pattern of some of these partners is represented (genepaint). b, c Physical interaction between Wdr5 (b) and Tcf3 (c) with Sox2 was confirmed in co-immunoprecipitations. The myc antibody precipitated the myc-Sox2 (b) or myc-Tcf3 (c), and coprecipitated the FLAG tagged Wdr5 (b) or Sox2 (c). These interactions were confirmed by performing the reciprocal imunoprecipitations