RESUMO
A human MusTRD (muscle TFII-I repeat domain (RD)-containing protein) isoform was originally identified in a yeast one-hybrid screen as a protein that binds the slow fibre-specific enhancer of the muscle gene troponin I slow [O'Mahoney, Guven, Lin, Joya, Robinson, Wade and Hardeman (1998) Mol. Cell. Biol. 18, 6641-6652]. MusTRD shares homology with the general transcription factor TFII-I by the presence of diagnostic I-RDs [Roy (2001) Gene 274, 1-13]. The human gene encoding MusTRD, GTF2IRD1 ( WBSCR11 / GTF3 ), was subsequently located on chromosome 7q11.23, a region deleted in the neurodegenerative disease, Williams-Beuren Syndrome [Osborne, Campbell, Daradich, Scherer, Tsui, Franke, Peoples, Francke, Voit, Kramer et al. (1999) Genomics 57, 279-284; Franke, Peoples and Francke (1999) Cytogenet. Cell. Genet. 86, 296-304; Tassabehji, Carette, Wilmot, Donnai, Read and Metcalfe (1999) Eur. J. Hum. Genet. 7, 737-747]. The haploinsufficiency of MusTRD has been implicated in the myopathic aspect of this disease, which manifests itself in symptoms such as lowered resistance to fatigue, kyphoscoliosis, an abnormal gait and joint contractures [Tassabehji, Carette, Wilmot, Donnai, Read and Metcalfe (1999) Eur. J. Hum. Genet. 7, 737-747]. Here, we report the identification of 11 isoforms of MusTRD in mouse skeletal muscles. These isoforms were isolated from a mouse skeletal muscle cDNA library and reverse transcription-PCR on RNA from various adult and embryonic muscles. The variability in these isoforms arises from alternative splicing of a combination of four cassettes and two mutually exclusive exons, all in the 3' region of the primary transcript of Gtf2ird1, the homologous mouse gene. The expression of some of these isoforms is differentially regulated spatially, suggesting individual regulation of the expression of these isoforms. Co-transfection studies in C2C12 muscle cell cultures reveal that isoforms differentially regulate muscle fibre-type-specific promoters. This indicates that the presence of different domains of MusTRD influences the activity exerted by this molecule on multiple promoters active in skeletal muscle.
Assuntos
Processamento Alternativo , Regulação da Expressão Gênica , Fibras Musculares de Contração Lenta/fisiologia , Proteínas Musculares/genética , Proteínas Nucleares , Regiões Promotoras Genéticas , Transativadores , Fatores de Transcrição/genética , Motivos de Aminoácidos , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Elementos Facilitadores Genéticos/genética , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fibras Musculares de Contração Lenta/citologia , Fibras Musculares de Contração Lenta/metabolismo , Proteínas Musculares/metabolismo , Proteínas Musculares/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
Synthetic ligands for the vitamin D receptor (VDR) are potential therapeutic agents for metabolic, neoplastic, and autoimmune disorders. Some of these ligands have similar or more potent antiproliferative, yet reduced hypercalcemic actions, than calcitriol. However, the mechanisms for these differential actions have not been clearly defined. We hypothesized that these gene- and tissue-specific effects may relate to ligand-directed selective recruitment of transcriptional coactivators. To identify key elements in ligand structure that facilitate VDR-coactivator interactions, the current studies assessed the ability of the VDR to recruit the coactivators GRIP1 and RAC3 following activation by a series of 20-R- and 20-S (20-epi)-modified analogues. The strength of VDR-coactivator interactions was ligand-specific and did not always correlate with ligand-receptor binding affinity. In general, the 20-epi analogues enhanced these interactions, whereas the 20-R-modified analogues were less effective than calcitriol. The 16-ene,23-yne modification and fluorinated substituents to the side-chain attenuated interaction with coactivators. The enhanced ability of the VDR to recruit GRIP1 following activation by the 20-epi analogues was consistent with potentiation of 20-epi analogue-induced transactivation of the osteocalcin gene promoter by GRIP1. Overall, the structure of the ligand side-chain as well as its orientation seemed to affect the avidity of coactivator binding. These results suggest that selective recruitment of coactivators may contribute to gene- and tissue-specific effects of vitamin D analogues.
Assuntos
Receptores de Calcitriol/efeitos dos fármacos , Receptores de Calcitriol/metabolismo , Vitamina D/análogos & derivados , Animais , Dimerização , Humanos , Ligantes , Coativador 2 de Receptor Nuclear , Coativador 3 de Receptor Nuclear , Conformação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Ratos , Receptores de Calcitriol/química , Receptores de Calcitriol/genética , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Estereoisomerismo , Transativadores/química , Transativadores/metabolismo , Transativadores/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Vitamina D/química , Vitamina D/metabolismo , Vitamina D/farmacologiaRESUMO
Human MusTRD1alpha1 was isolated as a result of its ability to bind a critical element within the Troponin I slow upstream enhancer (TnIslow USE) and was predicted to be a regulator of slow fiber-specific genes. To test this hypothesis in vivo, we generated transgenic mice expressing hMusTRD1alpha1 in skeletal muscle. Adult transgenic mice show a complete loss of slow fibers and a concomitant replacement by fast IIA fibers, resulting in postural muscle weakness. However, developmental analysis demonstrates that transgene expression has no impact on embryonic patterning of slow fibers but causes a gradual postnatal slow to fast fiber conversion. This conversion was underpinned by a demonstrable repression of many slow fiber-specific genes, whereas fast fiber-specific gene expression was either unchanged or enhanced. These data are consistent with our initial predictions for hMusTRD1alpha1 and suggest that slow fiber genes contain a specific common regulatory element that can be targeted by MusTRD proteins.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Proteínas Musculares/fisiologia , Proteínas Nucleares/fisiologia , Transativadores/fisiologia , Animais , Membro Posterior/citologia , Membro Posterior/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Musculares/genética , Proteínas Nucleares/genética , Transativadores/genéticaRESUMO
The vitamin D receptor (VDR) is a ligand-dependent transcription factor that heterodimerizes with retinoid X receptor (RXR) and interacts with the basal transcription machinery and transcriptional cofactors to regulate target gene activity. The p160 coactivator GRIP1 and the distinct coregulator Ski-interacting protein (SKIP)/NCoA-62 synergistically enhance ligand-dependent VDR transcriptional activity. Both coregulators bind directly to and form a ternary complex with VDR, with GRIP1 contacting the activation function-2 (AF-2) domain and SKIP/NCoA-62 interacting through an AF-2 independent interface. It was previously reported that SKIP/NCoA-62 interaction with VDR was independent of the heterodimerization interface (specifically, helices H10/H11). In contrast, the present study defines specific residues within a conserved and surface-exposed region of VDR helix H10 that are required for interaction with SKIP/NCoA-62 and for full ligand-dependent transactivation activity. SKIP/NCoA-62, the basal transcription factor TFIIB, and RXR all interacted with VDR helix H10 mutants at reduced levels compared with wild type in the absence of ligand and exhibited different degrees of increased interaction upon ligand addition. Thus, SKIP/NCoA-62 interacts with VDR at a highly conserved region not previously associated with coregulator binding to regulate transactivation by a molecular mechanism distinct from that of p160 coactivators.
Assuntos
Proteínas Nucleares/metabolismo , Receptores de Calcitriol/metabolismo , Receptores do Ácido Retinoico/metabolismo , Fator de Transcrição TFIIB/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Células COS , Camundongos , Modelos Moleculares , Mutagênese , Ligação Proteica , Receptores de Calcitriol/química , Receptores de Calcitriol/genética , Receptores X de RetinoidesRESUMO
Ski-interacting protein (SKIP), a vitamin D receptor (VDR) coactivator, also functions as a repressor in Notch signalling in association with the corepressor SMRT. Here we show that SKIP bifunctionally modulates (activates or represses) Retinoid-X receptor (RXR)- and VDR-dependent gene transcription in a cell line-specific manner, with activation in CV-1 and repression in P19 cells. The coactivator function of SKIP in these cells appeared to correlate with the relative level and ratio of expression of N-CoR and p300, with greater SKIP activation in higher p300-expressing and lower N-CoR-expressing cell-lines. C-terminal deletion of SKIP (delta334-536 aa) was associated with strong activation in both CV-1 and P19 cells. The corepressors N-CoR and SMRT and the coregulator p300 interacted with SKIP through the same N-terminal region (1-200 aa). Overall these results suggest that transcriptional action of SKIP may depend on distinct functional domains and cell line-specific interactions with both corepressors and coactivators.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Expressão Gênica , Histona Acetiltransferases , Camundongos , Células NIH 3T3 , Proteínas Nucleares/genética , Correpressor 2 de Receptor Nuclear , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Receptores X de Retinoides , Deleção de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Fatores de Transcrição de p300-CBPRESUMO
The novel transcription factor hMusTRD1alpha1 (human muscle TFII-I repeat domain-containing protein 1alpha1; previously named MusTRD1; O'Mahoney, J. V., Guven, K. L., Lin, J., Joya, J. E., Robinson, C. S., Wade, R. P., and Hardeman, E. C. (1998) Mol. Cell. Biol. 18, 6641-6652) was identified in a yeast one-hybrid screen as a protein that binds within an upstream enhancer-containing region of the skeletal muscle-specific gene, TNNI1 (human troponin I slow; hTnIslow). It has been proposed that hMusTRD1alpha1 may play an important role in fiber-specific muscle gene expression by virtue of its ability to bind to an Inr-like element (nucleotides -977 to -960) within the hTnIslow upstream enhancer-containing region that is necessary for slow fiber-specific expression. In this study we demonstrate that both MEF2C, a known regulator of slow fiber-specific genes, and hMusTRD1alpha1 regulate hTnIslow through the Inr-like element. Co-transfection assays in C2C12 cells and Cos-7 cells demonstrate that hMusTRD1alpha1 represses hTnIslow transcription and prevents MEF2C-mediated activation of hTnIslow transcription. Gel shift analysis shows that hMusTRD1alpha1 can abrogate MEF2C binding to its cognate site in the hTnIslow enhancer. Glutathione S-transferase pull-down assays demonstrate that hMusTRD1alpha1 can interact with both MEF2C and the nuclear receptor co-repressor. The data support the role of hMusTRD1alpha1 as a repressor of slow fiber-specific transcription through mechanisms involving direct interactions with MEF2C and the nuclear receptor co-repressor.