Identification of Weight-bearing-responsive Elements in the Skeletal Muscle Sarco(endo)plasmic Reticulum Ca2+ ATPase (SERCA1) Gene
2000; Elsevier BV; Volume: 275; Issue: 30 Linguagem: Inglês
10.1074/jbc.m003678200
ISSN1083-351X
AutoresHeather Mitchell-Felton, R. Bridge Hunter, Eric J. Stevenson, Susan C. Kandarian,
Tópico(s)Adipose Tissue and Metabolism
ResumoThe 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 Ca2+ handling gene by changes in muscle loading condition. 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 Ca2+ handling gene by changes in muscle loading condition. sarco(endo)plasmic reticulum Ca2+ ATPase nuclear factor of activatedT cells polymerase chain reaction relative light units The sarco(endo)plasmic reticulum is the major organelle that regulates the Ca2+ signaling associated with a multitude of cellular processes (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6148) Google Scholar, 2Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1755) Google Scholar). The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA)1 proteins translocate Ca2+ from the cytosol to the sarcoplasmic reticulum or endoplasmic reticulum lumen, thereby reducing intracellular Ca2 and refilling the sarcoplasmic/endoplasmic reticulum Ca2+ stores. The cloning, expression, and functional characterization of three SERCA genes and their splicing variants have been described (3Burk S.E. Lytton J. MacLennan D.H. Shull G. J. Biol. Chem. 1989; 264: 18561-18568Abstract Full Text PDF PubMed Google Scholar, 4Brandl C.J. deLeon S. Martin D.R. MacLennan D.H. J. Biol. Chem. 1987; 262: 3768-3774Abstract Full Text PDF PubMed Google Scholar, 5Korczak B. Zarain-Herzberg A. Brandl C. James Ingles C. Green N.M. MacLennan D.H. J. Biol. Chem. 1988; 263: 4813-4819Abstract Full Text PDF PubMed Google Scholar, 6Gunteski-Hamblin A.-M. Greeb J. Shull G.E. J. Biol. Chem. 1988; 263: 15032-15040Abstract Full Text PDF PubMed Google Scholar, 7Lytton J. Zarain-Herzberg A. Periasamy M. MacLennan D.H. J. Biol. Chem. 1989; 264: 7059-7065Abstract Full Text PDF PubMed Google Scholar, 8Lytton J. MacLennan D.H. J. Biol. Chem. 1988; 263: 15024-15031Abstract Full Text PDF PubMed Google Scholar, 9Wu K.-D. Lytton J. Am. J. Physiol. 1993; 264: C333-C341Crossref PubMed Google Scholar, 10Zarain-Herzberg A. MacLennan D.H. Periasamy M. J. Biol. Chem. 1990; 265: 4670-4677Abstract Full Text PDF PubMed Google Scholar, 11Poch E. Leach S. Snape S. Cacic T. MacLennan D.H. Lytton J. Am. J. Physiol. 1998; 275: C1449-C1458Crossref PubMed Google Scholar, 12Dode L. De Greef C. Mountian I. Attard M. Town M.M. Casteels R. Wuytack F. J. Biol. Chem. 1998; 273: 13982-13994Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). At least one of the SERCA gene products is expressed in every mammalian tissue studied, emphasizing the fundamental role of this protein in cellular function. Products of the SERCA1 gene are the predominant isoforms expressed in skeletal muscle (SERCA1a adult, SERCA1b neonatal) (4Brandl C.J. deLeon S. Martin D.R. MacLennan D.H. J. Biol. Chem. 1987; 262: 3768-3774Abstract Full Text PDF PubMed Google Scholar, 9Wu K.-D. Lytton J. Am. J. Physiol. 1993; 264: C333-C341Crossref PubMed Google Scholar), and one product of the SERCA2 gene is the predominant isoform expressed in cardiac myocytes (SERCA2a (3Burk S.E. Lytton J. MacLennan D.H. Shull G. J. Biol. Chem. 1989; 264: 18561-18568Abstract Full Text PDF PubMed Google Scholar)). The alternative splicing product of SERCA2 (SERCA2b (3Burk S.E. Lytton J. MacLennan D.H. Shull G. J. Biol. Chem. 1989; 264: 18561-18568Abstract Full Text PDF PubMed Google Scholar)) and the products of the SERCA3 gene (3Burk S.E. Lytton J. MacLennan D.H. Shull G. J. Biol. Chem. 1989; 264: 18561-18568Abstract Full Text PDF PubMed Google Scholar, 11Poch E. Leach S. Snape S. Cacic T. MacLennan D.H. Lytton J. Am. J. Physiol. 1998; 275: C1449-C1458Crossref PubMed Google Scholar) are ubiquitously expressed in muscle and non-muscle tissue but in much lower levels. SERCA expression is much higher in striated muscle than in smooth muscle or non-muscle to accommodate the large calcium fluxes associated with excitation-contraction coupling.Contractile activity has a marked effect on SERCA expression in striated muscle. Increased contractile activity leads to decreases in SERCA1 and SERCA2a expression in skeletal (13Kandarian S.C. Peters D.G. Taylor J.A. Williams G.A. Am. J. Physiol. 1994; 266: C1190-C1197Crossref PubMed Google Scholar, 14Hu P. Zhang K.M. Spratt J.A. Wechsler A.S. Briggs F.N. Biochim. Biophys. Acta. 1998; 1395: 121-125Crossref PubMed Scopus (9) Google Scholar) and cardiac (15Arai M. Matsui H. Periasamy M. Circ. Res. 1994; 74: 555-564Crossref PubMed Scopus (323) Google Scholar, 16Cadre B.M. Qi M. Eble D.M. Shannon T.R. Bers D.M. Samarel A.M. J. Mol. Cell Cardiol. 1998; 30: 2247-2259Abstract Full Text PDF PubMed Scopus (34) Google Scholar) muscle, respectively, whereas decreased contractile activity leads to increases in expression (17Schulte L.M. Navarro J. Kandarian S.C. Am. J. Physiol. 1993; 264: C1308-C1315Crossref PubMed Google Scholar, 18Bassani J.W.M. Qi M. Samarel A.M. Bers D.M. Circ. Res. 1994; 74: 991-997Crossref PubMed Scopus (36) Google Scholar, 19Peters D.G. Mitchell-Felton H. Kandarian S.C. Am. J. Physiol. 1999; 276: C1218-C1225Crossref PubMed Google Scholar). SERCA1 expression is particularly sensitive to the reduction of contractile activity due to rat soleus muscle unloading, with significant increases by 2 days and 7-fold increases by 7 days (19Peters D.G. Mitchell-Felton H. Kandarian S.C. Am. J. Physiol. 1999; 276: C1218-C1225Crossref PubMed Google Scholar). Moreover, the increase in expression is due to increases in SERCA1 gene transcription (19Peters D.G. Mitchell-Felton H. Kandarian S.C. Am. J. Physiol. 1999; 276: C1218-C1225Crossref PubMed Google Scholar). The up-regulation of SERCA1 with unloading is counterintuitive as it occurs in a background of overall decreases in total protein and mRNA synthesis (20Thomason D.B. Booth F.W. J. Appl. Physiol. 1990; 68: 1-12Crossref PubMed Scopus (2) Google Scholar). This suggests that the up-regulation of SERCA1 is an active and highly regulated process. However, the functional significance and the molecular mechanisms of selective gene activation are unknown.In previous work we isolated the rat 5′ flank and promoter region of SERCA1 to −3636 base pairs (21Mitchell-Felton H. Kandarian S.C. Am. J. Physiol. 1999; 277: C1269-C1276Crossref PubMed Google Scholar). As a first step in elucidating the transcriptional regulation of SERCA1 by unloading, the sequence from −3636 to +172 was examined using in vivo somatic gene transfer to identify the region(s) both necessary and sufficient for weight-bearing sensitivity. Comprehensive promoter analysis revealed two regions of SERCA1 that were sufficient to confer unloading responsiveness and which were necessary for the unloading sensitivity of the −3636 to +172 construct. Mutagenesis and further deletion analysis of these regions identified a CACC box/E-box interaction, which was required for transcriptional activation with unloading. This is the first demonstration of specific cis-elements required for the in vivo regulation of a Ca2+ handling gene by changes in muscle loading activity.RESULTSPrevious work in this laboratory showed that 75 μg was an optimal amount of SERCA1 plasmid to inject in soleus muscles with respect to the efficiency of plasmid uptake and luciferase activity (21Mitchell-Felton H. Kandarian S.C. Am. J. Physiol. 1999; 277: C1269-C1276Crossref PubMed Google Scholar). Southern blot analysis revealed that neither the size of the constructs (Ref. 21Mitchell-Felton H. Kandarian S.C. Am. J. Physiol. 1999; 277: C1269-C1276Crossref PubMed Google Scholar and data not shown) nor the intervention of hind limb unloading altered plasmid uptake in soleus muscles (control = 378 ± 40 densitometric absorbance units, n = 108; unloaded = 440 ± 47 absorbance units, n = 109; p = 0.32). To determine whether different amounts of injected DNA affected the extent of transactivation with muscle unloading, 50, 75, or 125 μg of SERCA1 (−3636)-pGL3 was examined. Luciferase activity of SERCA1 (−3636)-pGL3 normalized to plasmid uptake was increased 7-fold after 7 days of unloading (Fig.1). Since the extent of increase in luciferase activity was similar at the different quantities tested, the amount of DNA injected did not influence the magnitude of the unloading response. The increase in luciferase activity was the same as the unloading-induced activation of the endogenous SERCA1 gene (19Peters D.G. Mitchell-Felton H. Kandarian S.C. Am. J. Physiol. 1999; 276: C1218-C1225Crossref PubMed Google Scholar), suggesting that elements sufficient for unloading sensitivity were contained in this construct.Initial in vivo deletion analysis of the SERCA1 5′ flank was performed with the constructs depicted in Fig.2. In control muscles, expression of the SERCA1-pGL3 reporter constructs decreased with serial deletions. Unloading responsiveness (i.e. fold activation) was evident in all constructs except SERCA1 (−3174)-pGL3, SERCA1 (−2874)-pGL3, SERCA1 (−1158)-pGL3, SERCA1 (−962)-pGL3, SERCA1 (−612)-pGL3, and SERCA1 (−103)-pGL3. The most obvious loss of unloading responsiveness occurred upon deletion of the region between −1373 and −1158. Therefore, this region was of interest and was selected for further study. Unloading responsiveness was not observed with the deletion of DNA between −1158 and −612. However, injection of the SERCA1 (−330)-pGL3 construct resulted in a return of the unloading response, which was lost again with injection of SERCA1 (−103)-pGL3. The return of unloading responsiveness was unexpected, so the SERCA1 (−330)-pGL3 injection was repeated with newly amplified DNA and a different set of muscles. The same unloading response was observed in the second experiment. Thus, the sequence between −330 and +172 was sufficient in isolation to confer unloading responsiveness, so it was a second region designated for further study. We also observed loss of unloading responsiveness in SERCA1 (−3174)-pGL3 and SERCA1 (−2874)-pGL3 compared with SERCA1 (−3636)-pGL3, although the decrease in fold activation was due to increased control values as opposed to decreased unloading values. These regions were also tested further. Taken together these data suggest that there are multiple regions in the SERCA1 promoter-enhancer that can confer unloading responsiveness. The intricacy of the unloading response in this figure is likely due to the comprehensiveness of the promoter analysis as well as the complexity of the in vivo physiological phenomena under study.Figure 2In vivo deletion analysis of the rat SERCA1 5′ flank. A construct containing the SERCA1 sequence from the indicated nucleotide to +172 upstream of a luciferase reporter gene (Luc) was injected into soleus muscles (75 μg/muscle). Luciferase activity was determined for control (black bars) and 7-day-unloaded (gray bars) muscles. Fold activation is the unloaded divided by the control value. Plasmid uptake was measured in all muscles by Southern blot analysis, which showed no difference in uptake by unloading or plasmid size. For internal consistency, luciferase activity is reported. n = 8–16 muscles per group. Data are the means ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To test regions of SERCA1 for sufficiency in activating transcription with unloading, constructs were created containing SERCA1 fragments upstream of the heterologous SV40 promoter (Fig.3 A). SERCA1 (−1373 to −1158)-SV40 and SERCA1 (−962 to −612)-SV40 resulted in the strongest response with 5.0- and 5.7-fold activation, respectively, over control muscles. The activation of SERCA1 (−962 to −612)-SV40 was unexpected since neither SERCA1 (−962)-pGL3 nor SERCA1 (−612)-pGL3 showed unloading responsiveness (Fig. 2). This finding emphasized the context specificity of the promoter analysis. Other fragments of the SERCA1 5′ flank showed either modest increases or no change in luciferase activity when examined in isolation. Next, we sought to determine whether the regions that were sufficient for unloading responsiveness were required in the context of SERCA1 (−3636)-pGL3. To do this, inverse PCR was used to delete a specific region from the −3636 to +172 parent construct, thereby creating internal deletion constructs (Fig. 3 B). Removal of sequence between −1403 and −1158 abolished the unloading response, indicating that this region is required for unloading induced transcriptional activation. Deletion of other sequences did not result in a loss of unloading sensitivity. These data reveal that one obvious region of the SERCA1 5′ flank containing elements both necessary and sufficient for unloading responsiveness was between −1373 and −1158. The other observation made from this experiment was that sequences involved in high level expression and unloading sensitivity are distinct and, furthermore, that the sequence required for high level expression is not confined to the 5′ end of the construct (Fig. 3 B). Data from Fig. 3,A and B were re-plotted as fold activation for comparative purposes, since heterologous and wild type promoters were used to help visualize that −1373 to −1158 was both necessary and sufficient for transcriptional activation with 7 days of muscle unloading (Fig. 3 C).Figure 3Identification of regions of the SERCA1 5′ flank both necessary and sufficient for unloading sensitivity. A, a fragment of the SERCA1 5′ flank (black box) was ligated upstream of the heterologous SV40 promoter to test each fragment for sufficiency of unloading. B, the indicated regions of the SERCA1 5′ flank were removed from the SERCA1 (−3636)-pGL3 parent construct to test their necessity for unloading sensitivity in the context of SERCA1 (−3636)-pGL3. The lack of an exact match between the removed nucleotides in B and their corresponding fragment in A is because the PCR primers used to create deletion constructs had stringent criteria that could not always be met. Control (black bars) and 7-day-unloaded (gray bars) values are as plotted. C, data fromA and B re-plotted as fold activation to emphasize that the region between −1373 and −1158 is both necessary and sufficient for the unloading response. Fold activation is the unloaded divided by the control value. n = 8–16 muscles per group. Data are means ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A more extensive analysis of SERCA1 (−1373 to −1158)-SV40 showed that removal of the sequence from −1373 to −1314 did not alter the magnitude of transcriptional activation, but the removal of the region between −1373 and −1248 decreased the response from 5.0- to 2.2-fold. Thus, sequence between −1314 and −1248 is involved in unloading sensitivity (Fig. 4). However, the sequence from −1373 to −1248 was not sufficient, in isolation, to up-regulate activity, suggesting that an interaction between −1314 to −1248 and −1248 to −1158 exists that confers unloading responsiveness. This interaction may involve the E-box at −1248, which is deleted in the constructs showing only a 2-fold activation. Thus, the E-box (−1248) may be necessary for weight-bearing responsiveness.Figure 4Deletion analysis of the SERCA1 region from −1373 to −1158. The cis-elements present in each construct are as indicated. The E-boxes at −1314 and −1248 were disrupted in the creation of the different deletion constructs. Constructs were injected into control (black bars) or 7-day-unloaded (gray bars) soleus muscles. Fold activation is the unloaded divided by the control value. n = 8–16 muscles/group. Data are the means ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mutagenesis of SERCA1 (−1373 to −1158)-SV40 was then performed to target cis-elements required for unloading induced transactivation (Fig. 5). Consensus cis-elements were identified using MatInspector (Version 2.2). Mutation of the CACC box at position −1262 abolished the unloading response, indicating that this CACC site is involved in the up-regulation of SERCA1 (−1373 to −1158)-SV40. Disruption of the NFAT (−1367) and CACC (−1272) sites did not change transcriptional activation, whereas mutation of CACC (−1305) and CCAAT (−1287) sites show moderate decreases in the unloading response. Surprisingly, with the exception of the E-box at −1248, mutation of the known elements between −1248 and −1158, the NF-I (−1198) and Sp1 (−1171) sites, did not decrease the unloading response. Since an interaction was predicted from the results of Fig.4, we explored the possibility that the E-box (−1248) is interacting with the CACC (−1262) site.Figure 5Mutagenesis of cis-elements in SERCA1 between −1373 and −1158. Individual cis-elements were mutated as indicated (for mutated bases see Table I). Constructs were injected into control (black bars) or 7-day-unloaded (gray bars) soleus muscles. Fold activation is the unloaded divided by the control value. n = 8–16 muscles/group. Data are the means ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To test if a CACC/E-box interaction was sufficient to increase transcription, constructs containing trimerized CACC sites and E-boxes upstream of SV40 were examined in control and 7-day unloaded soleus muscles (Fig. 6). Since weight-bearing sensitivity was abolished with the mutation of the CACC box at −1262, the first step was to examine this cis-element. Injection of SERCA1 (CACC)-SV40, a trimerized construct containing the CACC (−1262) site, resulted in no significant change in luciferase activity, supporting the hypothesis that an interaction of CACC (−1262) with a second cis-element is required for transcriptional up-regulation. Examination of SERCA1 (CACC/E-box)-SV40, containing CACC (−1262) and E-box (−1248), resulted in a 9.0-fold increase over control values. Thus, the combination of CACC (−1262) and E-box (−1248) is sufficient to transduce an unloading response, suggesting communication between trans-factors binding these elements. A multimerized construct containing CACC (−1272), CACC (−1262), and E-box (−1248) had a 4.6-fold increase in luciferase activity with unloading. However, the decrease in fold activation was due to a greater increase in the control versus the unloaded luciferase activity, suggesting that the CACC box at −1272 may be important for general high level transcription of SERCA1. This is consistent with the overall decrease in luciferase activity in both control and unloaded muscles with mutagenesis of the CACC box at −1272 base pairs (Fig. 5).Figure 6Identification of cis-elements sufficient for transactivation with 7 days of unloading. Constructs were made which contain trimerized CACC sites and E-boxes (as indicated, see Table II for sequence) and injected into control (black bars) and unloaded (gray bars) soleus muscles. Fold activation is the unloaded divided by the control value.n = 8 muscles/group. Data are the means ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The sequence between −330 to +172 was further examined because 1) SERCA1 (−330)-pGL3 shows a 9.3-fold increase with unloading, 2) the sequence is in close proximity to the basal promoter, and 3) the sequence contains multiple CACC and E-boxes, which were found to be important in −1373 to −1158. Further interest in the −330 region was spurred by the finding that SERCA1 (−3636 to −330)-SV40 could not activate transcription with unloading (Fig.7), suggesting that the SERCA1 (−3636)-pGL3 construct requires the sequence from −330 to +172 for transactivation. Since the sequence between −330 and −103 was not necessary for unloading responsiveness (Figs. 3 B and 7), it was particularly interesting to find that luciferase activity was increased in SERCA1 (−3636 to −103)-SV40 with unloading. In addition, SERCA1 (−330 to −103)-SV40 (Figs. 3 A and 7) and SERCA1 (−103)-pGL3 (Figs. 2 and 7) were not sufficient to activate transcription, whereas SERCA1 (−330)-pGL3 was sufficient (Figs. 2 and7). The results indicate that sequence between −330 and +172 was required for the unloading response seen with SERCA1 (−3636)-pGL3, but that required elements were found in both −330 to −103 and −103 to +172, regions that contain both CACC sites and E-boxes in high numbers. To delineate the minimal required sequence for activation, further deletion analysis was performed. SERCA1 (−330 to +27)-pGL3 was sufficient to up-regulate transcription, whereas SERCA1 (−330 to −21)-SV40 was not, suggesting that the sequence from −21 to +27 contributes to transducing the unloading signal. It is of interest that the spacing of the E-box and CACC site between −21 and +27 is similar to the CACC site and E-box in −1373 to −1158. Given the density of CACC and E-boxes in −330 to +172, the necessity of this sequence for unloading sensitivity of SERCA1 (−3636)-pGL3, and the requirement for the sequence between −21 and +27, which contains a CACC site and an E-box, it is likely that these elements are critical to transducing transactivation in response to the removal of weight-bearing in the soleus muscle.Figure 7Examination of the region between −330 and +172 for elements involved in unloading responsiveness. The position of both CACC sites and E-boxes between −330 and +172 are indicated. Data are presented as fold activation, since both wild type and heterologous (SV40) promoters were used. Fold activation is the unloaded divided by the control value. n = 8–16 muscles/group. Data are the means ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONIntracellular Ca2+ regulates the signaling associated with diverse cellular processes such as excitation-contraction coupling, cell proliferation, differentiation, and death (2Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1755) Google Scholar). SERCA1, by translocating Ca2+ from the cytosol to the lumen of the sarcoplasmic reticulum, is a critical protein in the regulation of skeletal muscle Ca2+ homeostasis. The SERCA1 gene is highly mutable, with mRNA expression and transcription being up-regulated 7-fold by 7 days of unloading in rat soleus muscles (19Peters D.G. Mitchell-Felton H. Kandarian S.C. Am. J. Physiol. 1999; 276: C1218-C1225Crossref PubMed Google Scholar). Given the central role that SERCA1 plays in maintaining Ca2+homeostasis, it is surprising how little is known about the transcriptional regulation of the gene. Thus, the focus of the present study was to identify elements involved in the transcriptional activation of the SERCA1 gene by muscle inactivity due to unloading. We found that there were specific CACC sites and E-boxes that were required for unloading-induced transactivation.The rat SERCA1 5′ flank contains cis-elements between −3636 and +172 that are known to be important in regulating muscle-specific genes. These include 21 CACC sites (CACCC), 17 myogenic E-boxes (CAGNTG), 2 M-CAT sites (GGAATG), 4 NFAT sites (WGGAAANH), 1 Sp1 site (GGGCGG), 10 Sp1-like sites (GGGAGG), and 12 TRE half-sites (RGGTSA). Interestingly there are no MEF2 sites (YTAWWWWTAR), MEF3 sites (SSTCAGGTTWC), or CArG boxes (CCWWWWWWGG) previously shown to be important for transcription of some muscle genes (22Muscat G.E. Gustafson T.A. Kedes L. Mol. Cell. Biol. 1988; 8: 4120-4133Crossref PubMed Scopus (63) Google Scholar, 23Karns L.R. Kariya K. Simpson P.C. J. Biol. Chem. 1995; 270: 410-417Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 24Grayson J. Bassel-Duby R. Williams R.S. J. Cell. Biochem. 1998; 70: 366-375Crossref PubMed Scopus (38) Google Scholar, 25Parmacek M.S. Ip H.S. Jung F. Shen T. Martin J.F. Vora A.J. Olson E.N. Leiden J.M. Mol. Cell. Biol. 1994; 14: 1870-1885Crossref PubMed Scopus (98) Google Scholar, 26Vincent C.K. Gualberto A. Patel C.V. Walsh K. Mol. Cell. Biol. 1993; 13: 1264-1272Crossref PubMed Scopus (87) Google Scholar, 27Sartorelli V. Webster K.A. Kedes L. Genes Dev. 1990; 4: 1811-1822Crossref PubMed Scopus (231) Google Scholar). Given the relatively high number of CACC sites and E-boxes, it is not surprising that these two elements were found to be necessary for transcriptional regulation of the SERCA1 gene with unloading. There is precedence in the literature for the regulatory pairing of CACC and MEF2 sites necessary for transcription of many muscle genes, including rat myosin light chain 2 slow (28Esser K. Nelson T. Lupa-Kimball V. Blough E. J. Biol. Chem. 1999; 274: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), human myoglobin (24Grayson J. Bassel-Duby R. Williams R.S. J. Cell. Biochem. 1998; 70: 366-375Crossref PubMed Scopus (38) Google Scholar), rat muscle creatine kinase (29Horlick R.A. Benfield P.A. Mol. Cell. Biol. 1989; 9: 2396-2413Crossref PubMed Scopus (63) Google Scholar), mouse troponin C slow (25Parmacek M.S. Ip H.S. Jung F. Shen T. Martin J.F. Vora A.J. Olson E.N. Leiden J.M. Mol. Cell. Biol. 1994; 14: 1870-1885Crossref PubMed Scopus (98) Google Scholar), and human β-enolase (30Feo S. Antona V. Barbieri G. Passantino R. Cali L. Giallongo A. Mol. Cell. Biol. 1995; 15: 5991-6002Crossref PubMed Google Scholar), but the regulatory pairing of CACC sites and E-boxes is less well defined (31Calvo S. Venepally P. Cheng J. Buonanno A. Mol. Cell. Biol. 1999; 19: 515-525Crossref PubMed Scopus (75) Google Scholar, 32Nakayama M. Stauffer J. Cheng J. Banerjee-Basu S. Wawrousek E. Buonanno A. Mol. Cell. Biol. 1996; 16: 2408-2417Crossref PubMed Scopus (72) Google Scholar).To date, the transfactor(s) that binds CACC sites for functional activation has not been definitively determined, although there are a number of candidates including the myocyte nuclear factor, a member of the winged helix family of proteins (33Bassel-Duby R. Hernandez M.D. Yang Q. Rochelle J.M. Seldin M.F. Williams R.S. Mol. Cell. Biol. 1994; 14: 4596-4605Crossref PubMed Scopus (75) Google Scholar), and CBP40 (34Bassel-Duby R. Hernandez M.D. Gonzalez M.A. Krueger J.K. Williams R.S. Mol. Cell. Biol. 1992; 12: 5024-5032Crossref PubMed Scopus (59) Google Scholar). In addition, members of the Sp1/kruppel-like factor family interact with CAC
Referência(s)