RESUMO
The transcription factor Myocyte enhancer factor 2C (MEF2C) plays a key role in the late differentiation of skeletal muscle progenitor cells, the so-called myoblasts. During myoblast differentiation, both MEF2C expression and transcriptional activity are regulated. We have reported that nTRIP6, the nuclear isoform of the focal adhesion LIM domain protein TRIP6, acts as an adaptor transcriptional co-activator for several transcription factors. It interacts with the promoter-bound transcription factors and consequently mediates the recruitment of other co-activators. Based on a described interaction between MEF2C and TRIP6 in a yeast-two-hybrid screen, we hypothesised a co-regulatory function of nTRIP6 for MEF2C. In proliferating myoblasts, nTRIP6 interacted with MEF2C and was recruited together with MEF2C to the MEF2-binding regions of the MEF2C target genes Myom2, Mb, Tnni2 and Des. Silencing nTRIP6 or preventing its interaction with MEF2C increased MEF2C transcriptional activity and increased the expression of these MEF2C target genes. Thus, nTRIP6 acts as a co-repressor for MEF2C. Mechanistically, nTRIP6 mediated the recruitment of the class IIa histone deacetylase HDAC5 to the MEF2C-bound promoters. In conclusion, our results unravel a transcriptional co-repressor function for nTRIP6. This adaptor co-regulator can thus exert either co-activator or co-repressor functions, depending on the transcription factor it interacts with.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas com Domínio LIM/química , Proteínas com Domínio LIM/metabolismo , Mioblastos/citologia , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Diferenciação Celular , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células , Conectina/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Mioblastos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , Troponina I/genéticaRESUMO
Several LIM domain proteins regulate transcription. They are thought to act through their LIM protein-protein interaction domains as adaptors for the recruitment of transcriptional co-regulators. An intriguing example is nTRIP6, the nuclear isoform of the focal adhesion protein TRIP6. nTRIP6 interacts with AP-1 and enhances its transcriptional activity. nTRIP6 is also essential for the transrepression of AP-1 by the glucocorticoid receptor (GR), by mediating GR tethering to promoter-bound AP-1. Here we report on the molecular mechanism by which nTRIP6 exerts these effects. Both the LIM domains and the pre-LIM region of nTRIP6 are necessary for its co-activator function for AP-1. Discrete domains within the pre-LIM region mediate the dimerization of nTRIP6 at the promoter, which enables the recruitment of the Mediator complex subunits THRAP3 and Med1. This recruitment is blocked by GR, through a competition between GR and THRAP3 for the interaction with the LIM domains of nTRIP6. Thus, nTRIP6 both positively and negatively regulates transcription by orchestrating the recruitment of the Mediator complex to AP-1-regulated promoters.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas com Domínio LIM/metabolismo , Complexo Mediador/metabolismo , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas com Domínio LIM/química , Camundongos , Regiões Promotoras Genéticas/genética , Complexo de Endopeptidases do Proteassoma , Multimerização Proteica , Estrutura Quaternária de Proteína , Transporte Proteico , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/química , Transcrição GênicaRESUMO
BACKGROUND: The unconventional motor protein, myosin Va, is crucial for the development of the mouse neuromuscular junction (NMJ) in the early postnatal phase. Furthermore, the cooperative action of protein kinase A (PKA) and myosin Va is essential to maintain the adult NMJ. We here assessed the involvement of myosin Va and PKA in NMJ recovery during muscle regeneration. METHODOLOGY/PRINCIPAL FINDINGS: To address a putative role of myosin Va and PKA in the process of muscle regeneration, we used two experimental models the dystrophic mdx mouse and Notexin-induced muscle degeneration/regeneration. We found that in both systems myosin Va and PKA type I accumulate beneath the NMJs in a fiber maturation-dependent manner. Morphologically intact NMJs were found to express stable nicotinic acetylcholine receptors and to accumulate myosin Va and PKA type I in the subsynaptic region. Subsynaptic cAMP signaling was strongly altered in dystrophic muscle, particularly in fibers with severely subverted NMJ morphology. CONCLUSIONS/SIGNIFICANCE: Our data show a correlation between the subsynaptic accumulation of myosin Va and PKA type I on the one hand and NMJ regeneration status and morphology, AChR stability and specificity of subsynaptic cAMP handling on the other hand. This suggests an important role of myosin Va and PKA type I for the maturation of NMJs in regenerating muscle.