RESUMO
M-cadherin is a skeletal muscle-specific transmembrane protein mediating the cell-cell adhesion of myoblasts during myogenesis. It is expressed in the proliferating satellite cells and highly induced by myogenic regulatory factors (MRFs) during terminal myogenic differentiation. Several conserved cis-elements, including 5 E-boxes, 2 GC boxes, and 1 conserved downstream element (CDE) were identified in the M-cadherin proximal promoter. We found that E-box-3 and -4 close to the transcription initiation site (TIS) mediated most of its transactivation by MyoD, the strongest myogenic MRF. Including of any one of the other E-boxes restored the full activation by MyoD, suggesting an essential collaboration between E-boxes. Stronger activation of M-cadherin promoter than that of muscle creatine kinase (MCK) by MyoD was observed regardless of culture conditions and the presence of E47. Furthermore, MyoD/E47 heterodimer and MyoD â¼ E47 fusion protein achieved similar levels of activation in differentiation medium (DM), suggesting high affinity of MyoD/E47 to E-boxes 3/4 under DM. We also found that GC boxes and CDE positively affected MyoD mediated activation. The CDE element was predicted to be the target of the chromatin-modifying factor Meis1/Pbx1 heterodimer. Knockdown of Pbx1 significantly reduced the expression level of M-cadherin, but increased that of N-cadherin. Using ChIP assay, we further found significant reduction in MyoD recruitment to M-cadherin promoter when CDE was deleted. Taken together, these observations suggest that the chromatin-modifying function of Pbx1/Meis1 is critical to M-cadherin promoter activation before MyoD is recruited to E-boxes to trigger transcription.
Assuntos
Caderinas/genética , Elementos E-Box/genética , Regulação da Expressão Gênica/genética , Desenvolvimento Muscular/genética , Regiões Promotoras Genéticas/genética , Animais , Sequência de Bases , Células Cultivadas , Sequência Conservada , Fibroblastos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Proteína Meis1/fisiologia , Proteína MyoD/metabolismo , Mioblastos , Fator de Transcrição 1 de Leucemia de Células Pré-B/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
In Saccharomyces cerevisiae, Sir proteins mediate heterochromatin epigenetic gene silencing. The assembly of silent heterochromatin requires histone deacetylation by Sir2, conformational change of SIR complexes, and followed by spreading of SIR complexes along the chromatin fiber to form extended silent heterochromatin domains. Sir2 couples histone deacetylation and NAD hydrolysis to generate an epigenetic metabolic small molecule, O-acetyl-ADP-ribose (AAR). Here, we demonstrate that AAR physically associates with Sir3 and that polySir3-AAR formation has a specific and essential role in the assembly of silent SIR-nucleosome pre-heterochromatin filaments. Furthermore, we show that AAR is capable of stabilizing binding of the Sir3 BAH domain to the Sir3 carboxyl-terminal region. Our data suggests that for the assembly of SIR-nucleosome pre-heterochromatin filament, the structural rearrangement of SIR-nucleosome is important and result in creating more stable interactions of Sir3, such as the inter-molecule Sir3-Sir3 interaction, and the Sir3-nucleosome interaction within the filaments. In conclusion, our results reveal the importance of AAR, indicating that it not only affects the conformational rearrangement of SIR complexes but also might function as a critical fine-tuning modulatory component of yeast silent SIR-nucleosome pre-heterochromatin by stabilizing the intermolecular interaction between Sir3 N- and C-terminal regions.
Assuntos
Heterocromatina/metabolismo , Nucleossomos/metabolismo , O-Acetil-ADP-Ribose/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Epigênese Genética , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/química , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Sirtuína 2/genética , Sirtuína 2/metabolismoRESUMO
M- and N-cadherin are members of the Ca(2+)-dependent cell-cell adhesion molecule family. M-cadherin is expressed predominantly in developing skeletal muscles and has been implicated in terminal myogenic differentiation, particularly in myoblast fusion. N-cadherin-mediated cell-cell adhesion also plays an important role in skeletal myogenesis. In the present study, we found that both genes were differentially expressed in C2C12 and Sol8 myoblasts during myogenic differentiation and that the expression of M-cadherin was preferentially enhanced in slow-twitch muscle. Interestingly, most MRFs (myogenic regulatory factors) significantly activated the promoter of M-cadherin, but not that of N-cadherin. In line with this, overexpression of MyoD in C3H10T1/2 fibroblasts strongly induced endogenous M-cadherin expression. Promoter analysis in silico and in vitro identified an E-box (from -2 to +4) abutting the transcription initiation site within the M-cadherin promoter that is bound and differentially activated by different MRFs. The activation of the M-cadherin promoter by MRFs was also modulated by Bhlhe40 (basic helix-loop-helix family member e40). Finally, chromatin immunoprecipitation proved that MyoD as well as myogenin binds to the M-cadherin promoter in vivo. Taken together, these observations identify a molecular mechanism by which MRFs regulate M-cadherin expression directly to ensure the terminal differentiation of myoblasts.
Assuntos
Caderinas/genética , Fatores de Regulação Miogênica/fisiologia , Regiões Promotoras Genéticas/genética , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Elementos E-Box/genética , Ensaio de Desvio de Mobilidade Eletroforética , Fatores de Transcrição MEF2 , Camundongos , Proteína MyoD/genética , Proteína MyoD/metabolismo , Proteína MyoD/fisiologia , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Miogenina/genética , Miogenina/metabolismo , Miogenina/fisiologia , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Yeast silent heterochromatin provides an excellent model with which to study epigenetic inheritance. Previously we developed an in vitro assembly system to demonstrate the formation of filament structures with requirements that mirror yeast epigenetic gene silencing in vivo. However, the properties of these filaments were not investigated in detail. Here we show that the assembly system requires Sir2, Sir3, Sir4, nucleosomes, and O-acetyl-ADP-ribose. We also demonstrate that all Sir proteins and nucleosomes are components of these filaments to prove that they are SIR-nucleosome filaments. Furthermore, we show that the individual localization patterns of Sir proteins on the SIR-nucleosome filament reflect those patterns on telomeres in vivo. In addition, we reveal that magnesium exists in the SIR-nucleosome filament, with a role similar to that for chromatin condensation. These results suggest that a small number of proteins and molecules are sufficient to mediate the formation of a minimal yeast silent pre-heterochromatin in vitro.
Assuntos
Inativação Gênica/fisiologia , Nucleossomos/metabolismo , O-Acetil-ADP-Ribose/metabolismo , Sítios de Ligação , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Epigenômica/métodos , Heterocromatina/metabolismo , Histonas/metabolismo , Magnésio , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuínas/metabolismo , Telômero/metabolismoRESUMO
PGC-1α is a transcriptional coactivator promoting oxidative metabolism in many tissues. Its expression in skeletal muscle (SKM) is induced by hypoxia and reactive oxidative species (ROS) generated during exercise, suggesting that PGC-1α might mediate the cross talk between oxidative metabolism and cellular responses to hypoxia and ROS. Here we found that PGC-1α directly interacted with Bhlhe40, a basic helix-loop-helix (bHLH) transcriptional repressor induced by hypoxia, and protects SKM from ROS damage, and they cooccupied PGC-1α-targeted gene promoters/enhancers, which in turn repressed PGC-1α transactivational activity. Bhlhe40 repressed PGC-1α activity through recruiting histone deacetylases (HDACs) and preventing the relief of PGC-1α intramolecular repression caused by its own intrinsic suppressor domain. Knockdown of Bhlhe40 mRNA increased levels of ROS, fatty acid oxidation, mitochondrial DNA, and expression of PGC-1α target genes. Similar effects were also observed when the Bhlhe40-mediated repression was rescued by a dominantly active form of the PGC-1α-interacting domain (PID) from Bhlhe40. We further found that Bhlhe40-mediated repression can be largely relieved by exercise, in which its recruitment to PGC-1α-targeted cis elements was significantly reduced. These observations suggest that Bhlhe40 is a novel regulator of PGC-1α activity repressing oxidative metabolism gene expression and mitochondrion biogenesis in sedentary SKM.