Msx1 cooperates with Runx1 for inhibiting myoblast differentiation.

Myogenesis is an important and complicated biological process, especially during the process of embryonic development. The homeoprotein Msx1 is a crucial transcriptional repressor of myogenesis and maintains myogenic precursor cells in an undifferentiated, proliferative state. However, the molecular mechanism through which Msx1 coordinates myogenesis remains to be elucidated. Here, we determine the interacting partner proteins of Msx1 in myoblast cells by a proteomic screening method. Msx1 is found to interact with 55 proteins, among which our data demonstrate that the cooperation of Runt-related transcription factor 1 (Runx1) with Msx1 is required for myoblast cell differentiation. Our findings provide important insights into the mechanistic roles of Msx1 in myoblast cell differentiation, and lays foundation for the myogenic differentiation process.

Phosphorylation of Msx1 promotes cell proliferation through the Fgf9/18-MAPK signaling pathway during embryonic limb development.

Msh homeobox (Msx) is a subclass of homeobox transcriptional regulators that control cell lineage development, including the early stage of vertebrate limb development, although the underlying mechanisms are not clear. Here, we demonstrate that Msx1 promotes the proliferation of myoblasts and mesenchymal stem cells (MSCs) by enhancing mitogen-activated protein kinase (MAPK) signaling. Msx1 directly binds to and upregulates the expression of fibroblast growth factor 9 (Fgf9) and Fgf18. Accordingly, knockdown or antibody neutralization of Fgf9/18 inhibits Msx1-activated extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation. Mechanistically, we determined that the phosphorylation of Msx1 at Ser136 is critical for enhancing Fgf9 and Fgf18 expression and cell proliferation, and cyclin-dependent kinase 1 (CDK1) is apparently responsible for Ser136 phosphorylation. Furthermore, mesenchymal deletion of Msx1/2 results in decreased Fgf9 and Fgf18 expression and Erk1/2 phosphorylation, which leads to serious defects in limb development in mice. Collectively, our findings established an important function of the Msx1-Fgf-MAPK signaling axis in promoting cell proliferation, thus providing a new mechanistic insight into limb development.

The C-terminal region including the MH6 domain of Msx1 regulates skeletal development.

MSX1 is a causative gene for oligodontia in humans. Although conventional Msx1-deficient mice die neonatally, a mutant mouse lacking the C-terminus MH6 domain of MSX1 (Msx1ΔMH6/ΔMH6) showed two different phenotypes; newborn homozygotes with cleft palates died neonatally, whereas those with thin palates remained alive and had craniofacial dysplasia and growth retardation compared with wild-type mice, with most mice dying by the age of 4-5 weeks. In a previously reported case of human oligodontia caused by a heterozygous defect of the Msx1 MH6 domain, a small foramen was observed on the occipital bone. The aim of this study was to test the hypothesis that the Msx1 MH6 domain is involved in bone formation in vivo. In Msx1ΔMH6/ΔMH6 mice, cranial suture fusion was delayed at embryonic day 18.5, and the anteroposterior cranial diameter was smaller and long bone length was decreased at 3 weeks of age. The femoral epiphysis showed no change in the trabecular number, but decreased bone mass, bone density, and trabecular width in Msx1ΔMH6/ΔMH6 mice. In addition, cancellous bone mass was reduced and the cartilage layer in the growth plate was thinner in Msx1ΔMH6/ΔMH6 mice. The mRNA expression levels of major osteoblast and chondrocyte differentiation marker genes were decreased in Msx1ΔMH6/ΔMH6 mice compared with wild-type mice. These findings suggest that the C-terminal region including the MH6 domain of MSX1 plays important roles not only in tooth development and palatal fusion, but also in postnatal bone formation.

Two novel mutations in MSX1 causing oligodontia.

Tooth agenesis is one of the most common developmental anomalies in humans and can affect dental occlusion and speech pronunciation. Research has identified an association between mutations in MSX1, PAX9, EDA, AXIN2, WNT10A, WNT10B and LRP6 and human tooth agenesis. Two unrelated individuals with non-syndromic tooth agenesis and their families were enrolled in this study. Using Sanger sequencing of the candidate genes, we identified two novel mutations: a missense mutation c.572 T>C and a frameshift mutation c.590_594 dup TGTCC, which were both detected in the homeodomain of MSX1. After identifying the mutations, structural modeling and bioinformatics analysis were used to predict the resulting conformational changes in the MSX1 homeodomain. Combined with 3D-structural analysis of other MSX1 mutations, we propose that there is a correlation between the observed phenotypes and alterations in hydrogen bond formation, thereby potentially affecting protein binding.

Temperature dependence of internal motions of protein side-chain NH3(+) groups: insight into energy barriers for transient breakage of hydrogen bonds.

Although charged side chains play important roles in protein function, their dynamic properties are not well understood. Nuclear magnetic resonance methods for investigating the dynamics of lysine side-chain NH3(+) groups were established recently. Using this methodology, we have studied the temperature dependence of the internal motions of the lysine side-chain NH3(+) groups that form ion pairs with DNA phosphate groups in the HoxD9 homeodomain-DNA complex. For these NH3(+) groups, we determined order parameters and correlation times for bond rotations and reorientations at 15, 22, 28, and 35 °C. The order parameters were found to be virtually constant in this temperature range. In contrast, the bond-rotation correlation times of the NH3(+) groups were found to depend strongly on temperature. On the basis of transition state theory, the energy barriers for NH3(+) rotations were analyzed and compared to those for CH3 rotations. Enthalpies of activation for NH3(+) rotations were found to be significantly higher than those for CH3 rotations, which can be attributed to the requirement of hydrogen bond breakage. However, entropies of activation substantially reduce the overall free energies of activation for NH3(+) rotations to a level comparable to those for CH3 rotations. This entropic reduction in energy barriers may accelerate molecular processes requiring hydrogen bond breakage and play a kinetically important role in protein function.

Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes.

In most of homeodomain-DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1-DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein-DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein-DNA complexes. The order of stability of protein-DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein-DNA complexes. Among specific protein-DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.

Suppression of the transcription factor MSX1 gene delays bovine preimplantation embryo development in vitro.

This study was conducted to investigate the effect of suppressing transcription factor gene MSX1 on the development of in vitro produced bovine oocytes and embryos, and identify its potential target genes regulated by this gene. Injection of long double-stranded RNA (LdsRNA) and small interfering RNA (siRNA) at germinal vesicle stage oocyte reduced MSX1 mRNA expression by 73 and 37% respectively at metaphase II stage compared with non-injected controls. Similarly, injection of the same anti-sense oligomers at zygote stage reduced MSX1 mRNA expression by 52 and 33% at 8-cell stage compared with non-injected controls. Protein expression was also reduced in LdsRNA- and siRNA-injected groups compared with non-injected controls at both stages. Blastocysts rates were 33, 28, 20 and 18% in non-injected control, scrambled RNA (scRNA), LdsRNA- and siRNA-injected groups respectively. Cleavage rates were also significantly reduced in Smartpool siRNA (SpsiRNA)-injected group (53.76%) compared with scRNA-injected group (57.76%) and non-injected control group (61%). Large-scale gene expression analysis showed that 135 genes were differentially regulated in SpsiRNA-injected group compared with non-injected controls, of which 54 and 81 were down- and up-regulated respectively due to suppression of MSX1. Additionally, sequence homology mapping and gene enrichment analysis with known human pathway information identified several functional modules that were affected due to suppression of MSX1. In conclusion, suppression of MSX1 affects oocyte maturation, embryo cleavage rate and the expression of several genes, suggesting its potential role in the development of bovine preimplantation embryos.

Autoregulatory loop of Msx1 expression involving its antisense transcripts.

The Msx1 homeogene plays an important role in epithelial-mesenchymal interactions leading organogenesis. Msx1 gene is submitted to bidirectional transcription generating a long non-coding antisense (AS) RNA potentially involved in Msx1 expression regulation. RT-Q-PCR and RNA-FISH studies indicated that transient overexpression of the Msx1 AS transcript in 705IC5 mouse odontoblasts decreased the abundance of endogenous Msx1 S mRNA at the post-transcriptional level. Conversely, Msx1 overexpression increased the AS RNA level probably by activating AS transcription. In vivo mapping by RT-PCR evidenced both Msx1 RNAs in all adult mouse tissues tested raising the issue of Msx1 function during adulthood. The expression patterns of the two RNAs were similar, confirming the tight S/AS relationship. In particular, both Msx1 mRNAs and Msx1 protein were similarly distributed in eyes, and were found in regions with a common ectodermic origin and in cells potentially involved in regeneration. In conclusion, we report that Msx1 S RNA is negatively controlled by its AS RNA at a post-transcriptional level, and that the AS RNA is retrocontrolled positively by Msx1. The tight link between Msx1 S and AS RNAs constitutes a regulatory loop resulting in a fine-tuned expression of Msx1 which appears to be significant for adult homeostasis.

Otx2 is a putative candidate to activate mice Msx1 gene from distal enhancer.

A comparative analysis between sequences of Msx1 promoter gene from human, mouse, and fugu allowed us to identify sequences highly conserved among these animals. One of the regions of great homology is localized between the positions -4622 and -4572, including the region described as distal enhancer. In this region putative transcription factors binding sites for Nkx2.5, CTF-CBP, Bicoid, Brn2, and Oct were found. To evaluate the functionality of these sites we performed EMSA analysis using two different regions from the distal enhancer and nuclear protein extracts from embryos. The results showed that in the presence of a Bicoid consensus binding site a DNA-protein complex can be formed. The identification of the proteins involved in this complex by mass spectrometry and Western blotting identified OTX2, a Bicoid-like protein. This protein was shown to be present in nuclear extracts of the embryonic stages analyzed by Western blot. Altogether these results suggest that OTX2 is a putative candidate to activate mice Msx1 gene from distal enhancer.

Modification of Msx1 by SUMO-1.

The small ubiquitin-related modifier SUMO reversibly modifies many proteins, including promoter-specific transcription factors. Genetic studies in both humans and mice indicate that the Msx1 transcription factor is associated with specific disorders, including cleft palate. We show that Msx1 conjugation to SUMO-1 in vivo is enhanced by an E3 SUMO ligase, PIAS1, suggesting that sumoylation of Msx1 is a new mechanism for modulating the molecular function of Msx1 during organogenesis.