Identification of weight-bearing-responsive elements in the skeletal muscle sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1) gene.
The skeletal muscle sarco(endo)plasmic reticulum calcium ATPase (SERCA1) gene is transactivated as early as 2 days after the removal of weight-bearing (Peters, D. G., Mitchell-Felton, H., and Kandarian, S. C. (1999) Am. J. Physiol. 276, C1218-C1225), but the transcriptional mechanisms are elusive. Here, the rat SERCA1 5' flank and promoter region (-3636 to +172 base pairs) was comprehensively examined using in vivo somatic gene transfer into rat soleus muscles (n = 804) to identify region(s) that are both necessary and sufficient for sensitivity to weight-bearing. In all, 40 different SERCA1 reporter plasmids were constructed and tested. Several different regions of the SERCA1 5' flank were sufficient to confer a transcriptional response to 7 days of muscle unloading when placed upstream of a heterologous promoter. Two of these regions were analyzed further because they were necessary for the unloading response of -3636 to +172, as demonstrated using internal deletion constructs. Deletion analysis of these regions (-1373 to -1158 and -330 to +172) suggested that unloading responsiveness corresponded to CACC sites and E-boxes. Mutagenesis of cis-elements in the first region showed that a specific CACC box (-1262) was involved in SERCA1 transactivation and a nearby E-box (-1248) was also implicated. Constructs containing trimerized CACC sites and E-boxes showed that the presence of both elements is required to activate transcription. This is the first identification of specific cis-elements required for the regulation of a Ca(2+) handling gene by changes in muscle loading condition.
Mitchell-Felton
H
H
Department of Health Sciences, Boston University, Boston, Massachusetts 02215, USA.
Hunter
R B
RB
Stevenson
E J
EJ
Kandarian
S C
SC
D000818:Animals; D001483:Base Sequence; D000252:Calcium-Transporting ATPases / Q000235:genetics*; D017931:DNA Primers; D005260:Female; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D051381:Rats; D017208:Rats, Wistar; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D015533:Transcriptional Activation
C506945:Atp2a1 protein, rat; D017931:DNA Primers; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases