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1.
Protein Expr Purif ; 179: 105797, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33242573

RESUMO

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.


Assuntos
Diferenciação Celular/fisiologia , Subunidade alfa 2 de Fator de Ligação ao Core , Fator de Transcrição MSX1 , Mioblastos , Animais , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core/química , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Técnicas de Inativação de Genes , Fator de Transcrição MSX1/química , Fator de Transcrição MSX1/genética , Fator de Transcrição MSX1/metabolismo , Fator de Transcrição MSX1/fisiologia , Camundongos , Mioblastos/citologia , Mioblastos/metabolismo
2.
Nucleic Acids Res ; 48(20): 11452-11467, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33080014

RESUMO

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.


Assuntos
Proliferação de Células , Extremidades/embriologia , Fator 9 de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Sistema de Sinalização das MAP Quinases , Fator de Transcrição MSX1/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Linhagem Celular , Fator 9 de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/genética , Fator de Transcrição MSX1/química , Fator de Transcrição MSX1/genética , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Knockout , Mioblastos/citologia , Mioblastos/enzimologia , Mioblastos/metabolismo , Fosforilação , Serina/metabolismo
3.
Biochem Biophys Res Commun ; 526(1): 62-69, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32192766

RESUMO

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.


Assuntos
Desenvolvimento Ósseo , Fator de Transcrição MSX1/química , Fator de Transcrição MSX1/metabolismo , Animais , Animais Recém-Nascidos , Desenvolvimento Ósseo/genética , Diferenciação Celular , Condrócitos/citologia , Regulação da Expressão Gênica , Masculino , Camundongos , Morfogênese , Osteoblastos/citologia , Domínios Proteicos , Deleção de Sequência , Relação Estrutura-Atividade
4.
PLoS One ; 15(1): e0227287, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31914153

RESUMO

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.


Assuntos
Anodontia/genética , Fator de Transcrição MSX1/genética , Adolescente , Análise Mutacional de DNA , Feminino , Mutação da Fase de Leitura , Humanos , Fator de Transcrição MSX1/química , Masculino , Modelos Estruturais , Mutação de Sentido Incorreto , Linhagem , Conformação Proteica em alfa-Hélice/genética , Domínios Proteicos/genética , Adulto Jovem
5.
Biochemistry ; 54(2): 538-45, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25489884

RESUMO

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.


Assuntos
DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Lisina/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Proteínas de Homeodomínio/química , Humanos , Ligação de Hidrogênio , Lisina/química , Fator de Transcrição MSX1/química , Fator de Transcrição MSX1/metabolismo , Camundongos , Modelos Moleculares , Movimento (Física) , Proteínas de Neoplasias/química , Ressonância Magnética Nuclear Biomolecular , Temperatura , Termodinâmica
6.
J Biomol Struct Dyn ; 33(10): 2069-82, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25484111

RESUMO

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.


Assuntos
DNA/química , Glutamina/química , Lisina/química , Fator de Transcrição MSX1/química , Simulação de Dinâmica Molecular , Motivos de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Fator de Transcrição MSX1/genética , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Termodinâmica
7.
Reproduction ; 139(5): 857-70, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20176746

RESUMO

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.


Assuntos
Blastocisto/fisiologia , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Fator de Transcrição MSX1/genética , Supressão Genética , Animais , Bovinos , Feminino , Fertilização in vitro , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição MSX1/química , Fator de Transcrição MSX1/fisiologia , Masculino , Metáfase , Microinjeções , Análise de Sequência com Séries de Oligonucleotídeos , Oócitos/citologia , Oócitos/fisiologia , RNA de Cadeia Dupla , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Homologia de Sequência do Ácido Nucleico , Fatores de Tempo , Zigoto/fisiologia
8.
J Cell Physiol ; 220(2): 303-10, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19334036

RESUMO

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.


Assuntos
Regulação da Expressão Gênica , Fator de Transcrição MSX1/química , Fator de Transcrição MSX1/metabolismo , RNA Antissenso/metabolismo , Transcrição Gênica , Animais , Linhagem Celular , Olho/anatomia & histologia , Olho/metabolismo , Hibridização in Situ Fluorescente , Fator de Transcrição MSX1/genética , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Camundongos Transgênicos , RNA Antissenso/genética
9.
Biochem Biophys Res Commun ; 358(2): 655-60, 2007 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-17499211

RESUMO

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.


Assuntos
Elementos Facilitadores Genéticos/genética , Fator de Transcrição MSX1/química , Fator de Transcrição MSX1/genética , Fatores de Transcrição Otx/química , Fatores de Transcrição Otx/genética , Regiões Promotoras Genéticas/genética , Animais , Sítios de Ligação , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica
10.
Biochem Biophys Res Commun ; 345(1): 74-7, 2006 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-16678795

RESUMO

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.


Assuntos
Fator de Transcrição MSX1/química , Fator de Transcrição MSX1/metabolismo , Organogênese/fisiologia , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Camundongos , Camundongos Endogâmicos C3H , Ligação Proteica
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