Segregated Regulatory Elements Direct β-Myosin Heavy Chain Expression in Response to Altered Muscle Activity

Abstract

Our previous transgenic analyses revealed that a 600-base pair ␤-myosin heavy chain (␤MyHC) promoter conferred mechanical overload (MOV) and non-weightbearing (NWB) responsiveness to a chloramphenicol acetyltransferase reporter gene. Whether the same DNA regulatory element(s) direct ␤MyHC expression following MOV or NWB activity in vivo remains unknown. We now show that a 293-base pair ␤MyHC promoter fused to chloramphenicol acetyltransferase (␤293) responds to MOV, but not NWB activity, indicating a segregation of these two diverse elements. Inclusion of the ␤MyHC negative regulatory element (؊332 to ؊300; ␤NRE) within transgene ␤350 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 ␤NRE sense strand (d␤NRE-S; ؊332 to ؊311). Supershift electrophoretic mobility shift assay revealed that the binding at the distal region of the ␤NRE sense strand was antigenically distinct from cellular nucleic acidbinding protein and Y-box-binding factor 1, two proteins shown to bind this element. Two-dimensional UV crosslinking and shift Southwestern blotting analyses detected two proteins (50 and 52 kDa) that bind to this element. These in vivo results demonstrate that segregated ␤MyHC promoter elements transcriptionally regulate ␤MyHC transgene expression in response to two diverse modes of neuromuscular activity.The sarcomeric myosin heavy chain (MyHC) 1 is a major contractile protein that is encoded by a multigene family constituted by eight members (1). Each member has been shown to be responsive to a complex set of intrinsic and extrinsic signals that collectively regulate their expression in a defined developmental stage-and muscle fiber type-specific pattern. Within the MyHC gene family, the ␤MyHC is the one member whose regulated expression in both cardiac and skeletal muscle has been most extensively studied. Expression of the ␤MyHC gene has been detected in the ventricular myocardium and skeletal muscles comprising the hind limb of fetal mice, whereas in the adult, its expression is primarily restricted to skeletal muscles or muscle regions composed predominately of slow twitch type I fibers (2, 3). In vitro investigations of the control of ␤MyHC gene transcription have led to the identification of a control region located within the proximal promoter (nucleotides Ϫ300 to Ϫ188 of the human ␤MyHC gene) region (4 -7). This control region was found to be composed of three discrete DNA regulatory elements termed MCAT-like, C-rich and ␤e3, which are highly conserved in location and sequence across species (4 -7). The absolute requirement for these elements to obtain high levels of gene expression was demonstrated in transient transfection studies where the independent disruption of any of these conserved elements decreased and/or abolished musclespecific expression of ␤MyHC reporter genes (4 -7).Located just upstream from the ␤MyHC control region is a highly conserved ne...

Skeletal Muscle Adaptations with Age, Inactivity, and Therapeutic Exercise

“…28,54 The reduced protein synthesis appears to be regulated at the molecular level and results from changes in transcription and translation. 54,55 …”

“…18,54,55 In rats, it is well established that inactivity is associated with a selective loss of contractile protein and slow-to-fast transformations in contractile and regulatory proteins. 28,54 The reduced protein synthesis appears to be regulated at the molecular level and results from changes in transcription and translation.…”

Spaceflight results in increase of thick filament but not thin filament proteins in the paramyosin mutant of Caenorhabditis elegans

“…After microgravity exposure, the fibers that express slow type MHCs are decreased and those that express fast type MHCs are increased in soleus slow muscle (Caiozzo et al, 1996;Harrison et al, 2003). At the molecular level, distinct β-MHC promoter sequences mediate β-MHC expression in response to overload and unloading in mice (McCarthy et al, 1999).…”

Functional overload increases β-MHC promoter activity in rodent fast muscle via the proximal MCAT (βe3) site

“…Several highly conserved positive and negative regulatory elements within the proximal region (Ϫ408 bp) promoter have been identified (7,20,29,34). A putative repressor element, ␤e1 (Ϫ321/Ϫ297), appears to play a distinct role in ␤-MHC expression in skeletal and cardiac muscle cells (7,9,10) and has been implicated in conferring the decreased expression of ␤-MHC that occurs in response to soleus unloading (23). It has been suggested that four positive elements [␤e2 or muscle CAT (MCAT)-binding site (Ϫ285/Ϫ269); an A/T-rich site (Ϫ267/Ϫ259); the CCAC box (Ϫ245/Ϫ233); and the ␤e3 element (Ϫ214/Ϫ190)] are essential for muscle-specific gene activation (20,29,34).…”