RESUMEN
To examine the role of the beta-myosin heavy chain (betaMyHC) distal muscle CAT (MCAT) element in muscle fiber type-specific expression and mechanical overload (MOV) responsiveness, we conducted transgenic and in vitro experiments. In adult transgenic mice, mutation of the distal MCAT element led to significant reductions in chloramphenicol acetyltransferase (CAT) specific activity measured in control soleus and plantaris muscles when compared with wild type transgene beta293WT but did not abolish MOV-induced CAT specific activity. Electrophoretic mobility shift assay revealed the formation of a specific low migrating nuclear protein complex (LMC) at the betaMyHC MCAT element that was highly enriched only when using either MOV plantaris or control soleus nuclear extract. Scanning mutagenesis of the betaMyHC distal MCAT element revealed that only the nucleotides comprising the core MCAT element were essential for LMC formation. The proteins within the LMC when using either MOV plantaris or control soleus nuclear extracts were antigenically related to nominal transcription enhancer factor 1 (NTEF-1), poly(ADP-ribose) polymerase (PARP), and Max. Only in vitro translated TEF-1 protein bound to the distal MCAT element, suggesting that this multiprotein complex is tethered to the DNA via TEF-1. Protein-protein interaction assays revealed interactions between nominal TEF-1, PARP, and Max. Our studies show that for transgene beta293 the distal MCAT element is not required for MOV responsiveness but suggest that a multiprotein complex likely comprised of nominal TEF-1, PARP, and Max forms at this element to contribute to basal slow fiber expression.
Asunto(s)
Regulación de la Expresión Génica , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/fisiología , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Proteínas Nucleares , Animales , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Sitios de Unión , Fenómenos Biomecánicos , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Sustancias Macromoleculares , Ratones , Ratones Transgénicos , Complejos Multiproteicos , Mutagénesis Sitio-Dirigida , Sondas de Oligonucleótidos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Proteínas Recombinantes/metabolismo , Factores de Transcripción de Dominio TEA , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Our previous transgenic analyses revealed that a 600-base pair beta-myosin heavy chain (betaMyHC) promoter conferred mechanical overload (MOV) and non-weight-bearing (NWB) responsiveness to a chloramphenicol acetyltransferase reporter gene. Whether the same DNA regulatory element(s) direct betaMyHC expression following MOV or NWB activity in vivo remains unknown. We now show that a 293-base pair betaMyHC promoter fused to chloramphenicol acetyltransferase (beta293) responds to MOV, but not NWB activity, indicating a segregation of these two diverse elements. Inclusion of the betaMyHC negative regulatory element (-332 to -300; betaNRE) within transgene beta350 repressed expression in all transgenic lines. Electrophoretic mobility shift assays showed highly enriched binding activity only in NWB soleus nuclear extracts that was specific to the distal region of the betaNRE sense strand (dbetaNRE-S; -332 to -311). Supershift electrophoretic mobility shift assay revealed that the binding at the distal region of the betaNRE sense strand was antigenically distinct from cellular nucleic acid-binding protein and Y-box-binding factor 1, two proteins shown to bind this element. Two-dimensional UV cross-linking and shift Southwestern blotting analyses detected two proteins (50 and 52 kDa) that bind to this element. These in vivo results demonstrate that segregated betaMyHC promoter elements transcriptionally regulate betaMyHC transgene expression in response to two diverse modes of neuromuscular activity.
Asunto(s)
Regulación de la Expresión Génica , Músculos/fisiología , Cadenas Pesadas de Miosina/genética , Soporte de Peso/fisiología , Animales , Northern Blotting , Mapeo Cromosómico , ADN/metabolismo , Genes Reguladores , Humanos , Ratones , Ratones Transgénicos , Fenotipo , Estrés MecánicoRESUMEN
Non-weight-bearing (NWB) activity [space flight and hindlimb suspension (HS)] results in the loss of soleus muscle mass, a slow-to-fast fiber-type conversion, and decreased beta-myosin heavy chain (beta-MHC) protein and mRNA expression. To identify beta-MHC promoter sequences required for decreased beta-MHC expression in response to HS, we have modified an existing noninvasive hindlimb unweighting model to accommodate the use of (transgenic) mice. After 2 wk of HS, body and muscle (soleus > gastrocnemius > plantaris) weights were decreased as was the proportion of histochemically classified type I fibers in HS soleus muscle. Northern blot analysis revealed decreases in endogenous mRNA representing beta-MHC, slow myosin light chain 1 and 2, and cardiac/slow troponin C, whereas those representing skeletal troponin C, muscle creatine kinase, and glyceraldehyde-3-phosphate dehydrogenase increased. Protein extracts prepared from HS soleus (SS) muscle of mice harboring transgenes comprised of 5.6 or 0.6 kilobase of wild type (wt) mouse beta-MHC promoter (beta 5.6 wt, beta 0.6wt) and those carrying the simultaneous mutation (mut) of the MCAT, C-rich, and beta e3 subregions (beta 5.6mut3, beta 0.6mut3) revealed decreases in chloramphenicol acetyltransferase (CAT) specific activity relative to respective controls. Decreased CAT mRNA was observed for transgene beta 5.6mut3, line 85. Two weeks of the simultaneous imposition of mechanical overload (synergist ablation) and HS (MOV/HS) countermanded the loss in absolute and normalized SS weight but did not decrease beta 0.6wt transgene expression. These transgenic results demonstrate that regulatory sequences within a 600-base pair beta-MHC promoter are sufficient to direct decreased transcription of beta-MHC transgenes after 2 wk of HS.
Asunto(s)
Músculo Esquelético/fisiología , Cadenas Pesadas de Miosina/genética , Animales , Peso Corporal , Regulación de la Expresión Génica , Ratones , Ratones Transgénicos , Músculo Esquelético/anatomía & histología , Miosinas/metabolismo , Tamaño de los Órganos , Regiones Promotoras Genéticas , ARN Mensajero/genética , Estrés Mecánico , Transcripción Genética , TransgenesRESUMEN
The DNA regulatory element(s) involved in beta-myosin heavy chain (beta-MHC) induction by the physiological stimulus of mechanical overload have not been identified as yet. To delineate regulatory sequences that are required for mechanical overload induction of the beta-MHC gene, transgenic mouse lines were generated that harbor transgenes containing serial deletions of the human beta-MHC promoter to nucleotides -293 (beta 293), -201 (beta 201), and -141 (beta 141) from the transcription start site (+1). Mechanically overloaded adult plantaris and soleus muscles contained 11- and 1.9-fold increases, respectively, in endogenous beta-MHC-specific mRNA transcripts (Northern blot) compared with sham-operated controls. Expression assays (chloramphenicol acetyltransferase specific activity) revealed that only transgene beta 293 expression was muscle specific in both fetal and adult mice and was induced in the plantaris (10- to 27-fold) and soleus (2- to 2.5-fold) muscles by mechanical overload. Histochemical staining for myosin adenosinetriphosphatase activity revealed a fiber-type transition of type II to type I in the overloaded plantaris and soleus muscles. These transgenic data suggest that sequences located between nucleotides -293 and +120 may be sufficient to regulate the endogenous beta-MHC gene in response to developmental signals and to the physiological signals generated by mechanical overload in fast- and slow-twitch muscles.
Asunto(s)
Regulación de la Expresión Génica , Ratones Transgénicos/genética , Músculos/enzimología , Cadenas Pesadas de Miosina/genética , Regiones Promotoras Genéticas , Adenosina Trifosfatasas/metabolismo , Envejecimiento/metabolismo , Animales , Secuencia de Bases , Fenómenos Biomecánicos , Peso Corporal , Feto/metabolismo , Dosificación de Gen , Humanos , Ratones , Músculo Esquelético/enzimología , Músculos/anatomía & histología , Músculos/fisiología , Miocardio/enzimología , Tamaño de los Órganos , ARN Mensajero/metabolismo , Especificidad por SustratoRESUMEN
Mechanical overload leads to hypertrophy, increased type I fiber composition, and beta-myosin heavy chain (beta-MHC) induction in the fast-twitch plantaris muscle. To better understand the mechanism(s) involved in beta-MHC induction, we have examined inducible expression of transgenes carrying the simultaneous mutation of three DNA regulatory subregions [muscle CAT (MCAT), C-rich, and beta e3] in the context of either 5,600-base pair (bp; beta 5.6mut3) or 600-bp (beta 0.6mut3) beta-MHC promoter in overloaded plantaris muscles of transgenic mice. Protein extract from mechanically overloaded plantaris muscle of mice, harboring either mutant transgene beta 5.6mut3 or beta 0.6mut3, showed an unexpected 2.8- to 4.5-fold increase in chloramphenicol acetyltransferase (CAT) specific activity relative to their respective controls. Similar results were obtained with wild-type (wt) beta-MHC transgenes (beta 5.6wt, beta 0.6wt). Histochemical staining for both myofibrillar ATPase and CAT activity and CAT immunohistochemistry revealed a striking increase in type I fibers and that CAT expression was restricted to these fibers in overloaded plantaris muscle of beta 5.6mut3 transgenic mice. Our transgenic data suggest that beta-MHC transgenes, and perhaps the endogenous beta-MHC gene, are induced by mechanical overload via a mechanism(s) that does not exclusively require the MCAT, C-rich, or beta e3 subregions.