Skeletal muscle is a highly organized tissue that performs the specialized function of force development. In the adult mouse, four major myosin heavy chain (MyHC) isoforms [fast IIb, IIx/d, and IIa and slow type I (β)] are expressed in a manner that defines four primary fiber types, termed fast type IIb, IIx/d, and IIa and slow type I (37). Each of these distinct fiber types displays unique functional properties with respect to size, metabolism, fatigability, and intrinsic contractile properties. For example, slow type I fibers primarily populate slow-twitch muscles such as the soleus, rely on oxidative metabolism, have increased resistance to fatigue, and express high levels of the slow type β-isoform of MyHC (βMyHC) which is particularly efficient in energy utilization while maintaining tension. Thus, slow-twitch muscles like the soleus are primarily used in chronic activities such as postural maintenance and for sustained low-force locomotor activities. However, under non-weight-bearing (NWB) conditions, as would be encountered in the microgravity environment of space flight, an inactive lifestyle, injury, or disease, slow-twitch muscles undergo a marked degree of muscle atrophy accompanied by a slow-to-fast phenotypic change characterized by decreased βMyHC mRNA and protein expression (2, 5, 27, 28, 37, 41, 42). Because MyHC is a major determinant of the maximum unloaded shortening velocity (Vmax) of skeletal muscle fibers, the documented decrease in βMyHC protein has important physiological implications for skeletal muscles exposed to NWB conditions (2). While the phenotypic adaptations that occur in response to NWB have been well documented, identification of the precise transcriptional control mechanism(s) that mediates decreased βMyHC gene expression has been elusive since adult-stage muscle phenotypes, fiber-type transitions, and the effects of altered muscle loading conditions cannot be adequately duplicated in cultured muscle cells. Skeletal muscles composed primarily of slow type I fibers, such as the soleus muscle, are more susceptible to atrophy during periods of reduced muscle activity (5). Our previous work has utilized the soleus muscle as a model system to understand the mechanistic basis underlying decreased βMyHC gene expression in response to NWB conditions (27, 28, 42). Our transgenic expression analysis of both the mouse and human βMyHC promoters delineated a 600-bp region of the βMyHC promoter that was sufficient to mimic endogenous βMyHC down-regulation which occurs in response to NWB (27). Further deletion of this 600-bp promoter region identified a strong negative regulatory element (dβNRE-S; −332/−311) and multiple positive-acting elements, including a distal MCAT (dMCAT; −290/−284), proximal MCAT (pMCAT; −210/−203), and an E-box/nuclear factor of activated T cells (E-box/NFAT; −183/−172) (28, 42). MCAT and NFAT elements are frequently found in the control region of muscle-specific genes and have been reported to function as inducible, muscle- or fiber-specific response elements (9, 17, 22, 24, 33, 35, 44, 45). However, when we examined each of these elements in detail, none was found to be solely responsible for NWB responsiveness, indicating the presence of additional elements required for down-regulation of the βMyHC under NWB conditions (42). A computer-assisted analysis of the sequence located downstream from the dMCAT element identified three closely spaced GC-rich (GT/CACC) elements, termed C-richA (−248/−225), C-richB (−160/−140), and C-richC (−61/−41) in this paper. These three elements are highly conserved in sequence and location across species: an arrangement suggesting an important role in βMyHC regulation (Fig. (Fig.1).1). GC/GT elements are frequently found in the transcriptional control region of genes encoding proteins critical to a broad range of subcellular systems, including striated muscle. These elements are known to interact with members of the Sp family of transcription factors. In mammals, there are eight structurally similar Sp protein family members that are expressed in an overlapping fashion. Each contains a nearly identical DNA-binding domain consisting of three Cys2His2 zinc fingers, which likely accounts for their binding to the same GC/GT elements with variable affinity. Ubiquitously expressed Sp1 was the first family member identified, followed by Sp2, Sp3, and Sp4 (6, 30, 39). Targeted mutation of either Sp1, SP3, or SP4 results in early lethality due to a variety of different cellular defects indicating that each Sp protein can serve a distinct physiological role (7, 11, 12, 26, 32, 38). Recently, a subgroup of Sp protein family members comprising Sp5 to -8 has been isolated, and mice carrying targeted mutations of either Sp5, -7, or -8 also display distinct phenotypes (4, 16, 31). FIG. 1. βMyHC GC-rich elements are highly conserved in sequence and position across species. The nucleotide sequence comparison of the βMyHC proximal promoter of various species reveals high conservation of the GC-rich elements (shading). See ... A recent study has implicated multiple CACC elements in regulation of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1) gene under NWB conditions (29). However the proteins which bound to the CACC elements were not identified. In this study, we have identified and characterized the biochemical and functional properties of three βMyHC proximal promoter GC-rich elements. Mutagenesis analysis revealed the importance of each the GC-rich element for βMyHC reporter gene expression in C2C12 myotubes. Methylation interference footprinting delineated a binding site involving strong and weak protein-DNA interactions at the C-richA element that were identical for Sp1, Sp3, and Sp4. Electrophoretic mobility shift assay (EMSA) analysis revealed enriched binding of Sp3 proteins (115, 78, and 80 kDa) to these elements when nuclear extracts from soleus muscle exposed to NWB conditions were used. Binding of Sp3 to these elements was decreased while Sp1 binding increased with nuclear extracts from plantaris muscle following mechanical overload (MOV), a stimulus that induces a fast-to-slow phenotypic transition and increased βMyHC gene expression. Transient expression assays showed that Sp3 functioned as a competitive inhibitor of Sp1-mediated transactivation of a 293-bp βMyHC reporter gene in Drosophila SL-2 cells and C2C12 myotubes. In addition, a protein-DNA complex containing Sp4 was specifically detected when nuclear extracts from adult skeletal muscle were used. This Sp4-containing complex could not be detected with nuclear extracts isolated from a variety of cell lines, including HeLa, C2C12, and SOL8. These results provide clear evidence that Sp3 proteins play a functional role in mediating adult-stage skeletal muscle phenotype transitions in response to altered neuromuscular conditions. We suggest that the differential expression of Sp proteins will contribute to phenotypic adaptations that occur in all tissues affected by loading conditions imposed by disease, an inactive lifestyle, or space travel.