Identification of Nuclear Localization Signals in the Human Homeoprotein, MSX1

MSX1 is one of the homeoproteins with the homeodomain (HD) sequence, which regulates proliferation and differentiation of mesenchymal cells. In this study, we investigated the nuclear localization signal (NLS) in the MSX1 HD by deletion and amino acid substitution analyses. The web-based tool NLStradamus predicted two putative basic motifs in the N- and C-termini of the MSX1 HD. Green fluorescent protein (GFP) chimera studies revealed that NLS1 (161RKHKTNRKPR170) and NLS2 (216NRRAKAKR223) were independently insufficient for robust nuclear localization. However, they can work cooperatively to promote nuclear localization of MSX1, as was shown by the two tandem NLS motifs partially restoring functional NLS, leading to a significant nuclear accumulation of the GFP chimera. These results demonstrate a unique NLS motif in MSX1, which consists of an essential single core motif in helix-I, with weak potency, and an auxiliary subdomain in helix-III, which alone does not have nuclear localization potency. Additionally, other peptide sequences, other than predicted two motifs in the spacer, may be necessary for complete nuclear localization in MSX1 HD.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Characterization of novel MSX1 mutations identified in Japanese patients with nonsyndromic tooth agenesis.

Since MSX1 and PAX9 are linked to the pathogenesis of nonsyndromic tooth agenesis, we performed detailed mutational analysis of these two genes sampled from Japanese patients. We identified two novel MSX1 variants with an amino acid substitution within the homeodomain; Thr174Ile (T174I) from a sporadic hypodontia case and Leu205Arg (L205R) from a familial oligodontia case. Both the Thr174 and Leu205 residues in the MSX1 homeodomain are highly conserved among different species. To define possible roles of mutations at these amino acids in the pathogenesis of nonsyndromic tooth agenesis, we performed several functional analyses. It has been demonstrated that MSX1 plays a pivotal role in hard tissue development as a suppressor for mesenchymal cell differentiation. To evaluate the suppression activity of the variants in mesenchymal cells, we used the myoD-promoter, which is one of convenient reporter assay system for MSX1. Although the gene products of these MSX1 variants are stable and capable of normal nuclear localization, they do not suppress myoD-promoter activity in differentiated C2C12 cells. To clarify the molecular mechanisms underlying our results, we performed further analyses including electrophoretic mobility shift assays, and co-immunoprecipitation assays to survey the molecular interactions between the mutant MSX1 proteins and the oligonucleotide DNA with MSX1 consensus binding motif or EZH2 methyltransferase. Since EZH2 is reported to interact with MSX1 and regulate MSX1 mediated gene suppression, we hypothesized that the T174I and L205R substitutions would impair this interaction. We conclude from the results of our experiments that the DNA binding ability of MSX1 is abolished by these two amino acid substitutions. This illustrates a causative role of the T174I and L205R MSX1 homeodomain mutations in tooth agenesis, and suggests that they may influence cell proliferation and differentiation resulting in lesser tooth germ formation in vivo

Novel frameshift variant of WNT10A in a Japanese patient with hypodontia

Abstract Congenital tooth agenesis is caused by the impairment of crucial genes related to tooth development, such as Wnt signaling pathway genes. Here, we investigated the genetic causes of sporadic congenital tooth agenesis. Exome sequencing, followed by Sanger sequencing, identified a novel single-nucleotide deletion in WNT10A (NC_000002.12(NM_025216.3):c.802del), which was not found in the healthy parents of the patient. Thus, we concluded that the variant was the genetic cause of the patient’s agenesis

An aberrant splice acceptor site due to a novel intronic nucleotide substitution in MSX1 gene is the cause of congenital tooth agenesis in a Japanese family.

Congenital tooth agenesis is caused by mutations in the MSX1, PAX9, WNT10A, or AXIN2 genes. Here, we report a Japanese family with nonsyndromic tooth agenesis caused by a novel nucleotide substitution in the intronic region between exons 1 and 2 of the MSX1 gene. Because the mutation is located 9 bp before exon 2 (c.452-9G>A), we speculated that the nucleotide substitution would generate an abnormal splice site. Using cDNA analysis of an immortalized patient blood cell, we confirmed that an additional 7-nucleotide sequence was inserted at the splice junction between exons 1 and 2 (c.451_452insCCCTCAG). The consequent frameshift generated a homeodomain-truncated MSX1 (p.R151fsX20). We then studied the subcellular localization of truncated MSX1 protein in COS cells, and observed that it had a whole cell distribution more than a nuclear localization, compared to that of wild-type protein. This result suggests a deletion of the nuclear localization signal, which is mapped to the MSX1 homeodomain. These results indicate that this novel intronic nucleotide substitution is the cause of tooth agenesis in this family. To date, most MSX1 variants isolated from patients with tooth agenesis involve single amino acid substitutions in the highly conserved homeodomain or deletion mutants caused by frameshift or nonsense mutations. We here report a rare case of an intronic mutation of the MSX1 gene responsible for human tooth agenesis. In addition, the missing tooth patterns were slightly but significantly different between an affected monozygotic twin pair of this family, showing that epigenetic or environmental factors also affect the phenotypic variations of missing teeth among patients with nonsyndromic tooth agenesis caused by an MSX1 haploinsufficiency

MSX1 variants isolated from tooth agenesis patients.

<p><b>A:</b> 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>B:</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 <i>C. elegans</i> Msx1 (See Additional file 1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102944#pone.0102944-Finnerty1" target="_blank">[43]</a>). <b>C:</b> 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).</p

Nuclear localization of MSX1 variants.

<p>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.</p

Summary of Tooth Agenesis Phenotypes in the L205R and T174I families.

<p>1, central incisor; 2, lateral incisor; 3, canine; 4 and 5, first and second premolars, respectively; and 6–8, first, second, and third molars, respectively.</p