Figure 1.
Pedigree analysis of tooth agenesis.
A: Plaster cast of the maxillary (upper panel), and mandibular (lower panel) of the T174I proband exhibiting five missing teeth. B: Panoramic radiograph of the L205R proband with nine missing teeth. C and D: Pedigree for the tooth agenesis family of both the T174I (C), and L205R (D) probands. Darkened symbols represent affected, clear symbols indicate normal unaffected, hatched symbols indicate unable to diagnose caused by acquired tooth loss, squares indicate males, and circles indicate females. A type of CA-repeat polymorphism in the MSX1 intron is indicated i.e. CA1 (12×CAs), CA2 (11×CAs), CA3 (10×CAs), CA4 (9×CAs), CA5 (8×CAs). Asterisks indicate family members that were not analyzed.
Figure 2.
MSX1 variants isolated from tooth agenesis patients.
A: DNA sequences of MSX1 exon 2 in unaffected (a and b) and affected (c and d) individuals. The C-T transition at position 521 (T174I), and T-G transition in position 614 (L205R) in the mutant sequences cause amino acid substitutions within the MH4/homeodomain of MSX1. B: Schematic representation of the human MSX1 protein. Multiple sequence alignments of the MSX1 MH4/homeodomain sequences sorted by species and homology are shown. Sequences were obtained from the SwissProt database and thereby aligned. Conservation of the key Thr174 and Leu205 amino acids (black arrows) across species is indicated by gray shading. The leucine at position 205 is universally conserved at that position but the threonine at position 174 is replaced by serine, which has a similar polarity, in C. elegans Msx1 (See Additional file 1 in [43]). C: 3D models of mutant and wild-type MSX1 based on the crystal structures of the Msx1 MH4/homeodomain in complex with DNA (PDB ID 1IG7).
Table 1.
Summary of Tooth Agenesis Phenotypes in the L205R and T174I families.
Figure 3.
Functional analysis of tooth agenesis-causing MSX1 mutations.
A: myoD-promoter repression activity of T174I and L205R variants in C2C12 cells. The C2C12 cells were cotransfected with an MSX1 expression vector, the firefly luciferase reporter gene driven by the myoD-promoter and sea pansy luciferase reporter gene driven by the thymidine kinase promoter. After differentiation treatments, firefly luciferase activity was measured and normalized with respect to the sea pansy luciferase activity. Open bars indicate human wild-type MSX1 transfectant data (3–15 ng/well); hatched bars denote data from experiments utilizing transfectants of the T174I (a) and L205R (b) variants of MSX1 (3–20 ng/well), respectively. The relative luciferase activity is expressed as a percentage of the negative control (gray bars). Each transfection point was carried out in triplicate in a gelatin-coated 24-well plate. The experiments were performed three times and representative data are shown. Error bars indicate standard deviation. Asterisks indicate no significant differences against the control (P-value >0.1). B: Stability and expression level of wild-type and mutant proteins. Western blot analysis was performed using extracts prepared from HEK293 cells transfected with FLAG-wild-type-MSX1 (lane 1), FLAG-T174I-MSX1 (lane 2) or FLAG-L205R-MSX1 (lane 3). These whole-cell extracts were immunoblotted with an anti-FLAG antibody (upper panel) and anti-beta actin antibody as an internal standard (lower panel). C: Electrophoretic mobility shift assay of MSX1. a: A double-stranded oligonucleotide probe containing a consensus binding-site for MSX1 interacts with proteins in the wild-type MSX1 transfectant cell lysate (lane 1, arrow and small arrowhead). The DNA binding ability of MSX1 is diminished by the T174I and L205R substitutions. Wild-type-MSX1 protein and oligo DNA complex observed in lane 1 (arrow) was not detected in the MSX1 variant transfectant lysates (lane 2, T174I-MSX1; lane 3, L205R-MSX1), or HEK293 parental cells (lane 4, negative control). b: Supershift experiments using an anti-FLAG antibody. The binding specificity was confirmed by a super-shift induction with anti-FLAG antibody (lane 2). A protein-DNA-antibody complex formed (large arrowhead), and the band indicated by the arrow disappeared. This indicates that this molecular complex denoted by the arrow contained FLAG-tagged wild-type MSX1 protein, and that the protein indicated by the small arrowhead is an unknown product that bound to the probe. D: Molecular interaction between MSX1 and EZH2 histone methyltransferase. Myc-EZH2 protein was coimmunoprecipitated with FLAG-MSX1 (lane 1). EZH2 protein was not detected in the MSX1-null EZH2 transfectant HEK293 cell lysate (lane 2). The MSX1-EZH2 interaction was observed in the FLAG-T174I-MSX1 sample (lane 3), and in the FLAG-L205R-MSX1 sample (lane 4).
Figure 4.
Nuclear localization of MSX1 variants.
Immunolocalizations of FLAG-tagged wild-type, T174I, and L205R MSX1 products were similar when these variants were expressed in HEK293 cells. MSX1 (FLAG, red signal); nuclei (DAPI, blue signal). Cell morphologies were visualized by F-actin staining (Phalloidin, green signal). Neither the T174I nor L205R mutation affects the nuclear localization of MSX1 in vitro. Scale bar 20 µm.