Transcriptional Analysis of the Human Cardiac Calsequestrin Gene in Cardiac and Skeletal Myocytes*

Calsequestrin is the main calcium-binding protein inside the sarcoplasmic reticulum of striated muscle. In mammals, the cardiac calsequestrin gene (casq2) mainly expresses in cardiac muscle and to a minor extent in slow-twitch skeletal muscle and it is not expressed in non-muscle tissues. This work is the first study on the transcriptional regulation of the casq2 gene in cardiac and skeletal muscle cells. The sequence of the casq2 genes proximal promoter (180 bp) of mammals and avians is highly conserved and contains one TATA box, one CArG box, one E-box, and one myocyte enhancer factor 2 (MEF-2) site. We cloned the human casq2 gene 5′-regulatory region into a luciferase reporter expression vector. By functional assays we showed that a construct containing the first 288 bp of promoter was up-regulated during myogenic differentiation of Sol8 cells and had higher transcriptional activity compared with longer constructs. In neonatal rat cardiac myocytes, the larger construct containing 3.2 kb showed the highest transcriptional activity, demonstrating that the first 288 bp are sufficient to confer muscle specificity, whereas distal sequences may act as a cardiac-specific enhancer. Electrophoretic mobility shift assay studies demonstrated that the proximal MEF-2 and CArG box sequences were capable of binding MEF-2 and serum response factor, respectively, whereas the E-box did not show binding properties. Functional studies demonstrated that site-directed mutagenesis of the proximal MEF-2 and CArG box sites significantly decreased the transcription of the gene in cardiac and skeletal muscle cells, indicating that they are important to drive cardiac and skeletal muscle-specific transcription of the casq2 gene.

Calsequestrin is the main calcium-binding protein inside the sarcoplasmic reticulum of striated muscle. In mammals, the cardiac calsequestrin gene (casq2) mainly expresses in cardiac muscle and to a minor extent in slow-twitch skeletal muscle and it is not expressed in non-muscle tissues. This work is the first study on the transcriptional regulation of the casq2 gene in cardiac and skeletal muscle cells. The sequence of the casq2 genes proximal promoter (180 bp) of mammals and avians is highly conserved and contains one TATA box, one CArG box, one E-box, and one myocyte enhancer factor 2 (MEF-2) site. We cloned the human casq2 gene 5-regulatory region into a luciferase reporter expression vector. By functional assays we showed that a construct containing the first 288 bp of promoter was up-regulated during myogenic differentiation of Sol8 cells and had higher transcriptional activity compared with longer constructs. In neonatal rat cardiac myocytes, the larger construct containing 3.2 kb showed the highest transcriptional activity, demonstrating that the first 288 bp are sufficient to confer muscle specificity, whereas distal sequences may act as a cardiacspecific enhancer. Electrophoretic mobility shift assay studies demonstrated that the proximal MEF-2 and CArG box sequences were capable of binding MEF-2 and serum response factor, respectively, whereas the E-box did not show binding properties. Functional studies demonstrated that site-directed mutagenesis of the proximal MEF-2 and CArG box sites significantly decreased the transcription of the gene in cardiac and skeletal muscle cells, indicating that they are important to drive cardiac and skeletal musclespecific transcription of the casq2 gene.
The sarcoplasmic reticulum (SR) 2 is an intracellular organelle present in striated muscle cells, which has a key role on the regulation of the calcium concentrations during muscle contraction and relaxation (1). The SR is the main storage site of intracellular calcium, storing Ca 2ϩ up to 20 mM while maintaining the free SR Ca 2ϩ concentration at ϳ1 mM (2,3). The SR storage capacity enables the muscle cell to continuously contract without diminishing the Ca 2ϩ available for each contraction-relaxation cycle. Calsequestrin (CASQ) is the most abundant protein in the lumen of the SR; it has a high capacity to bind Ca 2ϩ (40 -50 mol of Ca 2ϩ /mol of CASQ) with a moderate affinity (K d ϳ 1 mM) and prevents the precipitation of Ca 2ϩ inside the SR. It is a highly acidic protein with over 50 Ca 2ϩ binding sites, which are formed by the clustering of two or more negatively charged residues. The molecular weight of the CASQ monomer is 40 kDa; and is capable of polymerizing in response to increasing Ca 2ϩ concentrations (Ͼ1 mM) (3). CASQ locates near the ryanodine receptor and attaches to it, via direct interaction or through anchoring by triadin and junctin. It has been suggested that CASQ has a regulatory role in SR Ca 2ϩ release, by inhibiting the ryanodine receptor through interactions via triadin/junctin at high luminal Ca 2ϩ concentrations (1-2 mM) (4), although the exact mechanism and physiological role of this inhibition has not been established. Thus, in recent years it has become clearer that CASQ has a role beyond its capability to buffer Ca 2ϩ .
In mammals two CASQ isoforms have been described, each one encoded by a different gene. In humans, the casq1 gene is located in chromosome 1q21, and encodes for the CASQ1 isoform; and the casq2 gene, located in chromosome 1p23, which encodes for the CASQ2 isoform. The adult fast-twitch skeletal muscle expresses exclusively the casq1 gene, whereas slowtwitch skeletal muscle expresses mainly the casq1 gene (ϳ75% of total) and to a minor extent the casq2 gene (ϳ25% of total). Cardiac muscle expresses exclusively the casq2 gene. Neither of the CASQ isoforms are present in non-muscle tissues nor in smooth muscle (5). Both human isoforms share a high nucleotide and amino acid homology, 84 and 80%, respectively (5). At this time there are no studies that point to differences in the physiological role of both CASQ isoforms, thus their roles shall be considered equivalent. The rabbit CASQ1 and the dog CASQ2 have been crystallized (6,7); results of crystallization studies shown that the casq monomer was found to be constituted by three almost identical domains (I, II, and III) similar to that of Escherichia coli thioredoxin domain. It was found that CASQ polymerizes in response to rising concentrations of Ca 2ϩ in the lumen of the SR, to form a homotetrameric complex (10 M to 1 mM) and at higher concentrations of polymers, in concentrations higher than 10 mM the CASQ polymer dissociates from the Ca 2ϩ releasing channel (8).
In the heart, the casq2 gene expresses during fetal development and continues to adult life. In fast-twitch skeletal muscle, CASQ2 is the predominant isoform during the fetal period, and during neonatal life. Afterward, there is a switch to CASQ1 gene expression, making the CASQ1 isoform the only one found in adult muscle. Although, in slow-twitch skeletal muscle there is also a switch on CASQ isoform expression during development, in the adult life the casq2 gene is still expressed (9). The CASQ2 mRNA levels have been measured in several heart pathological states such as cardiac hypertrophy, dilated cardiomyopathy, and heart failure, showing no changes on its expression level (10). Interestingly, in transgenic mice that overexpress CASQ2, it was observed that the mice develop cardiac hypertrophy and heart failure, with a typical fetal phenotype, associated with a higher Ca 2ϩ storage capacity of the SR, as well as an impaired Ca 2ϩ release, which leads to a diminished contractility (11). Another pathological state where CASQ2 is involved is the catecholaminergic polymorphic ventricular tachycardia (CPVT), where several mutations in the casq2 gene are present, inserting stop codons in the first exon of the gene, thus nullifying the expression of the casq2 gene. These patients have a morphologically normal heart but are susceptible to develop ventricular arrhythmias when exercise or another stressing event are present (12)(13)(14)(15). Taken together, these findings are suggestive that the transcription of the casq2 gene is finely controlled during skeletal and cardiac muscle development, as well as in cardiac pathologies. Although the casq2 gene of rabbit and mouse have already been cloned (16,17), there are no studies regarding transcriptional regulation of the casq2 gene.
The identification of cardiac-specific transcriptional regulatory elements and transcription factors controlling gene expression have been studied in vitro using the 5Ј-regulatory regions from contractile genes (␣-myosin heavy chain, cardiac troponin I, ventricular myosin light chain 2), atrial natriuretic factor and brain natriuretic peptide (18 -22). The studies have shown that GATA4, MEF-2, Nkx2.5, HAND1, HAND2, and serum response factor (SRF) are major regulatory factors involved in the transcriptional regulation of cardiac genes (23)(24)(25). However, the relative contribution of the transcription factors mentioned above varies in different gene promoters. This has made it difficult to understand the molecular mechanisms responsible for cardiac-specific gene expression.
In this work, we report the first attempt to understand the molecular basis of tissue specificity of the casq2 gene. We cloned 3.2 kb of 5Ј-regulatory region of the human casq2 gene, generated deletion constructs and performed functional analyses. We performed targeted mutagenesis and DNA-protein binding analysis in cardiac myocytes and in the skeletal muscle cell line Sol8. We identified proximal and distal regulatory elements important for the expression of skeletal myotubes and neonatal cardiac myocytes.
Our efforts were directed to a highly conserved region among species located in the first 180 bp of the 5Ј-regulatory region of the casq2 gene, which contains one MEF-2, one E-box, and one CArG box putative binding sites. The results obtained in this work suggest that the proximal promoter is necessary and sufficient for cardiac and skeletal muscle expression, and that MEF-2 and SRF transcription factors participate for the tissuespecific expression of the casq2 gene.

EXPERIMENTAL PROCEDURES
Materials-All restriction enzymes were acquired from Invitrogen and New England Biolabs. [␥-32 P]ATP and [␣-32 P]dCTP were acquired from PerkinElmer Life Sciences. All DNA oligonucleotides were synthesized in the Molecular Biology Unit from the Instituto de Fisiología Celular, UNAM, México. Human heart total RNA was acquired from BD Biosciences.
DNA Cloning-A 3276-bp Stu1-Stu1 human genomic fragment from clone RP11-485H8 (BACPAC Resources), which contains 3102 bp of the 5Ј-regulatory region of the human casq2 gene and 176 bp of the 5Ј-nontranslated sequence of exon 1, was subcloned into a pGL3-basic plasmid previously cut with Stu1, using standard techniques. Five more constructs were generated by digestion with restriction enzymes NcoI, AflII, PvuII, XhoI, and BlpI generating fragments of 2322, 1169, 754, 462, and 254 bp, respectively.
Primer Extension Analysis-A synthetic DNA oligonucleotide (300 ng) complementary to the coding sequence of exon 1 (5Ј-GGAAAGACTTACCACTCGGTC-3Ј) was labeled using T4 polynucleotide kinase and [␥-32 P]ATP. Reverse transcription was performed using poly(A) ϩ RNA from human adult heart (BD Biosciences). A sequencing reaction of a known template was used as a ladder for determining the molecular size of the extended DNA fragments.
Cell Culture-Sol8 myoblasts (obtained from ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen), designated as growth media, until they reached a 70% confluence then, were induced to differentiate to myotubes by switching the media to DMEM supplemented with 3% horse serum (HS) (Invitrogen), designated as differentiation media. The C3H10T1/2 cells were maintained in growth media. Primary cultures of neonatal rat cardiomyocytes were maintained with growth media. All the media were supplemented with kanamycin (60 mg/liter), penicillin G (10 units/ml), and streptomycin (10 mg/ml), amphotericin B (0.025 mg/ml), and nystatin (10 units/ml).
Transfection and Reporter Assays-The pGL3-hcasq2 constructs (0.8 g) and the pRL-CMV (0.05 g) were co-transfected into cells grown in 12-well plates using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. After 2 h the transfection mixture was replaced by DMEM with 3% HS to induce differentiation of Sol8 cells to myotubes; or by DMEM with 10% fetal bovine serum to maintain the myoblast phenotype, as well for C3H10T1/2 and neonatal rat cardiomyocytes. The positive control in all experiments was the pGL3promoter, which contains the proximal 202 bp of the SV40 promoter. Following 48 h incubation at 37°C and 5% CO 2 , the cells were lysed and assayed for firefly and Renilla luciferase, using the dual luciferase assay reagent kit (Promega) in a multiwell plate counter Wallac Victor 2 luminometer (PerkinElmer Life Sciences). The results were normalized with the Renilla luciferase activity.
Stably transfected Sol8 cell lines were generated by co-transfecting the pGL3-hcasq2 constructs containing 288 and 3102 bp of the 5Ј-regulatory region of the human casq2 gene with pCDNA3.1, containing the resistance gene to the selecting antibiotic G418. The cells were grown in DMEM supplemented with 10% fetal bovine serum and 500 g/ml G418, and selected after 1 week of growth. Afterward, colonies selected were tested for luciferase activity. The number of copies of the hcasq2/Luc fragment integrated into genomic DNA was verified by dotblot analysis using a DNA probe for luciferase. The colonies selected were grown on 24-well plates until they reached 70% confluence; at that time the cells were induced to differentiate by replacing the media with differentiation media. Cells were lysed on a time course from myoblasts to 5-day myotubes; cell extracts were assayed for luciferase activity. The results were normalized dividing the luciferase activity by the protein concentration.
Real Time RT-PCR-Total RNA was extracted from Sol8 myoblasts and myotubes daily up to day 5 of muscle differentiation, with TRIzol reagent (Invitrogen). RT was performed with 2 g of total RNA using SuperScript III First Strand Synthesis SuperMix for qRT-PCR (Invitrogen) according to the manufacturer's instructions, cDNA was diluted 15 times, and 9.2 l/reaction were used for qPCR with SYBR Green ER qPCR Supermix for iCycler (Invitrogen). Results were analyzed according to the method suggested by Pfaffl (26), using GAPDH as loading control. Primers sequences are indicated on Table 1.
Mutagenesis-Site-directed mutagenesis of the proximal MEF-2, E-box, and CArG box sequences was conducted using the QuikChange site-directed mutagenesis kit (Stratagene) as indicated by manufacturer's instructions, the MEF-2, E-box, and CArG box mutated oligonucleotides used are listed in Table 1. Mutation of the above elements was confirmed by DNA sequencing of the plasmids obtained.
Electrophoretic Mobility Shift Assay Studies-Nuclear extracts from Sol8 myoblasts and myotubes, and from neonatal rat cardiomyocytes were prepared as previously described (21). The sequence of the double-stranded DNA oligonucleotides were the same ones that were used for site-directed mutagenesis; consensus oligonucleotides for MEF-2 and CArG box used as specific competitors were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) (see Table 1). Binding reactions were performed by preincubating at room temperature in 1 ϫ Binding Buffer (Tris-HCl, 50 mM; glycerol, 20%; NaCl, 250 mM; MgCl 2 , 5 mM; EDTA, 2.5 mM; dithiothreitol, 1 mM; phenyl-FIGURE 1. Structure of the human casq2 gene. A, the human casq2 gene is contained in human genomic clones RP11-485H8 and RP5-929G5 (BACPAC Resources). The gene is organized in 11 exons and 10 introns, which span over 68.8 kb of the genome, producing a single mRNA transcript of 2548 nucleotides (nt). B, the transcription initiation site was determined by primer extension of human cardiac mRNA as mentioned under "Experimental Procedures." Two main cDNA extension products of 358 and 348 nucleotides were obtained, locating the transcription initiation sites at 251 and 241 bp upstream from the ATG translation initiation codon and are indicated by arrows. To detect accurately the size of the extended cDNA products a sequencing ladder of a known template was used. The exposure time for the primer extension reaction was 24 h, and for the sequencing ladder 8 h. methylsulfonyl fluoride, 1 mM), 10 g of nuclear extract, and 1 mg of poly(dI-dC), 100-fold molar excess unlabeled competitor when needed, and ␥-32 P-labeled double-stranded DNA oligonucleotides. Supershift antibodies for MEF-2 (sc-313x) and SRF (sc-335x) were purchased from Santa Cruz Biotechnology.
Statistical Analysis-Values are expressed as means of at least 3 independent experiments Ϯ S.E. Mean values were compared by analysis of variance (SPSS 11) applying the Bonferroni method for multiple comparisons. p values Ͻ0.05 were considered significant.

Characterization of the Human casq2 Gene 5Ј-Regulatory
Region-The human genomic clones (RP11-485H8 and RP5-929G5) were analyzed in silico with DNA analysis software (NCBI Blast, Genomatix suite and MacVector) to determine the complete human casq2 gene structure and its 5Ј-regulatory region. The analyses revealed that both clones overlap and contain the complete casq2 gene, integrated with 11 exons and interrupted by 10 introns that span over 68 kb of the genome (Fig. 1A).
To determine the transcription initiation site of the human casq2 gene, primer extension analysis of human CASQ2 mRNA was performed using human heart poly(A) ϩ RNA. Two main extension products (358 and 348 bp) were obtained, mapping two transcription initiation sites at positions 241 and 251 bp upstream from the ATG translation initiation codon (Fig. 1B).
The 5Ј-regulatory region was analyzed with the MacVector program and Genomatix suite, to identify possible binding sites for transcription factors. In this analysis we identified a highly conserved DNA homology region among several species including the chimp (100%), mouse (95%), rat (98%), dog (98%), cow (93%), and chicken (87%), located between Ϫ30 to Ϫ140 bp that contains one imperfect TATA box, one MEF-2 site, one E-box, and one CArG box (Fig. 2). FIGURE 2. DNA sequence analysis of the proximal 5-regulatory region of the casq2 genes among species. Nucleotide sequence analysis comparison was performed using the MacVector 6.5.3 program and BLAST algorithm from NCBI. The proximal 5Ј-regulatory region of the casq2 gene revealed that the first 180 bp are conserved in human, chimp, mouse, rat, dog, cow, and chicken genomic sequences with just a few mismatches. In this region, we found a putative TATA box, one CArG box, one E-box, and one MEF-2 site. The two transcription initiation sites are indicated with arrows. The BlpI, StuI, and XhoI restriction sites used to generate the Ϫ71 and Ϫ288 bp constructs are underlined. The ATG translation initiation codon is underlined. DECEMBER 7, 2007 • VOLUME 282 • NUMBER 49

JOURNAL OF BIOLOGICAL CHEMISTRY 35557
Transcriptional Activity of the hcasq2 Gene 5Ј-Regulatory Region in Cardiac and Skeletal Muscle Cells-Six deletion constructs in pGL3-basic containing 3102, 2148, 1095, 580, 288, and 71 bp of the human casq2 gene 5Ј-regulatory region and 176 bp of 5Ј-nontranslated sequence of exon 1 were generated and used for transfection experiments, to identify the basal pro-moter region and upstream 5Ј-regulatory sequences of the human casq2 gene in neonatal rat cardiac myocytes and Sol8 skeletal muscle cells. The transcriptional activity of the different constructs was determined by transiently transfecting them into neonatal rat cardiomyocytes, the skeletal muscle cell line Sol8, and C3H10T1/2 cells. The results obtained by transient transfection in neonatal rat cardiac myocytes showed that the Ϫ288-bp construct had a transcriptional activity similar to the one observed with the pGL3-promoter construct (202 bp of the SV40 proximal promoter), which has been described as a strong promoter in muscle. The transcriptional activity observed with the Ϫ288-bp construct was 2-fold higher of that observed with the Ϫ71-bp construct, which contains only a TATA box, but no other regulatory elements. The Ϫ580 and Ϫ1095-bp constructs exhibited ϳ2-fold higher activity compared with the Ϫ288-bp construct, the Ϫ2148 bp was 3.5-fold higher and the Ϫ3102-bp construct had the highest activity, increasing over 3.7fold relative to the Ϫ288-bp construct (Fig. 3A). The promoter-less pGL3-basic plasmid showed only background firefly luciferase activity (Fig. 3A).
In 2-day Sol8 myotubes, the highest transcriptional activity was observed with the Ϫ288-bp construct, showed 12-fold more activity than the Ϫ71-bp construct, and was similar to that observed with the SV40 promoter construct. The Ϫ3102-bp construct had 65% less activity compared with the Ϫ288-bp construct. The other intermediate length constructs showed similar activity than the Ϫ3102-bp construct. The Ϫ71-bp construct activity in myotubes was almost equal to the activity observed on undifferentiated myoblasts with all of the constructs, reflecting basal promoter activity. The pGL3-basic construct had only background activity. (Fig. 3B). In C3H10T1/2 fibroblasts, all the casq2 gene constructs showed only basal transcriptional activity, similar to the activity of the promoter less construct, whereas the SV40 promoter showed strong transcriptional activity (Fig. 3C). To analyze the transcriptional activity of the casq2 gene regulatory region constructs during the course of myogenic differentiation, we generated stable transfectant Sol8 cell lines, which integrated into two cell genome of the chimeric casq2/Luc constructs, of which, one contains Ϫ288 bp and the other contains Ϫ3.1 kb of the 5Ј-regulatory region. The resulting stable transfectant cell lines were induced to differentiate by serum withdrawal, and the luciferase activity was determined daily during 5 days of myogenic differentiation. The results indicated that both constructs have a very similar pattern of activation, showing increases in the activity level since day 1 of differentiation (Fig. 4A). The pattern of activation of the casq2 gene constructs was very similar to the pattern of increase of the endogenous level of casq2 mRNA expression determined by real time RT-PCR (Fig.  4B). The myogenin, MEF2C, and SRF mRNA levels were also quantified by real time RT-PCR. Myogenin mRNA increased since day 1, whereas MEF2C and SRF mRNA increased after 3 days of myogenic differentiation (Fig. 4C).

The Proximal MEF-2 and CArG Box Sites Are Functional in Cardiac
Myocytes-Due to the results obtained by transcriptional activation of the chimeric casq2 gene constructs, which suggest an important role played by the first 288 bp of the 5Ј-regulatory region in cardiac and skeletal muscle myocytes, we determined the DNA-protein binding capabilities of the putative binding sites (MEF-2, E-box, and CArG box) present in this region. Because MEF-2, basic helix-loop-helix, and SRF transcription factors are present in Sol8 myotubes and have been demonstrated to participate in the regulation of several muscle genes, we decided to perform electrophoretic mobility shift assay studies using synthetic double-stranded DNA oligonucleotides and specific antibodies for MEF2 and SRF for supershift assays (see "Experimental Procedures").
The results showed that the proximal casq2 gene MEF-2 site was capable of specifically binding a protein present in nuclear extracts of neonatal rat cardiomyocytes, which was of the same size to the one observed with a previously tested MEF-2 consensus oligonucleotide (Fig. 5A). The complex was not competed by the addition of 50 -100-fold molar excess of the mutated casq2 MEF-2 oligonucleotide or the mutated consensus MEF-2 oligonucleotide. The protein contained in the complex was supershifted using a MEF-2 antibody (Fig. 5A). Nuclear extracts of Sol8 myoblasts and myotubes were also tested; both were capable of binding specifically the casq2 gene MEF-2 oligonucleotide, the signal obtained with myoblasts had  DECEMBER 7, 2007 • VOLUME 282 • NUMBER 49 a lesser intensity than the one observed with myotubes (Fig. 5B). The complex formed was of similar apparent size to the one observed in cardiac cells. The complex observed with Sol8 nuclear extracts was also supershifted in the presence of the MEF-2 antibody (Fig. 5B).

Transcriptional Analysis of the Human Cardiac Calsequestrin Gene
The proximal casq2 gene CArG box was also assayed to test its capabilities of binding the transcription factors present in nuclear extracts of cardiomyocytes as well as Sol8 myoblasts and myotubes. The CArG box oligonucleotide formed a complex when incubated with cardiomyocytes nuclear extracts (Fig. 5C). This complex was specifically competed with the addition of the wild type CArG box casq2 (50 -100-fold molar excess) unlabeled probe, or a previously tested consensus CArG box oligonucleotide, whereas it did not change by the addition of the mutated casq2 gene CArG box oligonucleotide. The protein present in this complex was identified by supershift assay, using a SRF-specific antibody (Fig. 5C). We also assayed the binding capabilities with Sol8 myoblasts and myotube nuclear extracts. The results showed a complex of similar apparent size to the one observed in cardiomyocytes; it also was a specific complex and was supershifted with a SRF antibody. The complex produced by myoblast nuclear extracts was of lesser intensity than the one observed with myotubes (Fig. 5D).
The proximal casq2 E-box site was also assayed to determine its binding properties. When incubated with cardiomyocytes or Sol8 myoblasts and myotubes nuclear extracts, no DNA-protein complex was observed. The same nuclear extracts were assayed with a consensus E-box sequence (5Ј-CANNTG-3Ј) and a specific complex was observed (data not shown).
"Experimental Procedures" of the sites previously tested by electrophoretic mobility shift assay.
The four mutated constructs containing the mutations of the MEF-2 site (mMEF-2), E-box (mE-box), CArG box (mCArG box), and MEF-2 and CArG box (mMEF2 ϩ mCArG-box), were transiently transfected into neonate rat cardiomyocytes, and assayed for luciferase activity 48 h later (Fig. 6A). The results showed that the mutation of the MEF-2 site was associated with a 70% reduction of transcriptional activity compared with the wild type construct. The mutation of the E-box showed a nonsignificant reduction of the transcriptional activity. The mutation of the CArG box was associated with a reduction of 40% of transcriptional activity. The construct containing both the MEF-2 and CArG box mutations was associated with a further reduction of the activity up to 80% to that observed with the wild type construct, which was an activity equivalent to that observed with the Ϫ71-bp construct lacking the MEF-2 and CArG box elements and showed only basal promoter activity in all the cell types analyzed.
We performed the same experiment in Sol8 myoblasts (white bars) and myotubes (black bars) (Fig. 6B). The results showed that in myoblasts the level of transcriptional activity of all the casq2 gene constructs was low and minimally affected by the mutation of the MEF2 CArG box sites. In myotubes the activity of the Ϫ288-bp construct is high, we observed a similar behavior to the one seen in cardiomyocytes, with a reduction of 70% in activity with mMEF-2 construct, a 60% reduction with the mCArG box construct, and a 80% reduction when both the double mutated mMEF-2 and mCArG box construct were assayed. Both cardiomyocytes and Sol8 myotubes, when transfected with the Ϫ288-bp construct containing the mutated MEF-2 and CArG box sites, exhibited very low transcriptional activity, quite similar to that observed with the Ϫ71-bp construct, which has only basal transcriptional activity.

DISCUSSION
Calsequestrin has a major role to maintain the calcium homeostasis in the striated muscle cells, also the data regarding its specific tissue expression and absence of changes on its expression associated with pathological conditions, evidences the existence of a fine regulation of its expression. The results presented in this work are the first attempt to understand the transcriptional regulation of the expression of the casq2 gene.
The DNA sequence analysis of the human casq2 gene 5Ј-regulatory region, compared with the regulatory region of this gene in other species (chimp, mouse, rat, cow, dog, and chicken), reveals that the only region that has a high homology among them is contained within the Ϫ30 to Ϫ140-bp region upstream from the main transcription initiation site. Upstream from this region only a few small isolated sequences (20 -30-bp long) display homology among mammalian casq2 genes, but not with the chicken casq gene (data not shown). This observation is also relevant when one considers that avians only have one casq gene, which is expressed in fast-and slow-twitch skeletal and cardiac muscles. Also, this highly conserved proximal promoter region contains one MEF2, one E-box, and one CArG box putative DNA that are of relevance for gene expression in skeletal and cardiac muscle cells.
The functional results we report demonstrate that the first 288 bp of the human casq2 gene 5Ј-regulatory region are capable of directing the expression of the casq2/Luc constructs in neonatal rat cardiomyocytes and myotubes of the mouse Sol8 cell line; but not in C3H10T1/2 fibroblasts. The results also showed that the Ϫ288-bp casq2 gene construct drive a similar expression in Sol8 myotubes and in neonatal rat cardiomyocytes. The construct containing 3.1 kb of 5Ј-regulatory region increased transcriptional activity relative to the 288-bp construct (4-fold) in cardiac myocytes. In contrast, in Sol8 myotubes the 288 bp had the higher transcriptional activity, which is similar to the activity of the SV40 promoter construct, but constructs containing longer sequences showed 50 -60% less transcriptional activity. The above results agree with the casq2 mRNA and protein tissue expression pattern, where CASQ2 has been shown to be abundantly expressed in cardiac muscle, but only to a minor extent in slow-twitch skeletal muscle (17,27). These results also demonstrate that the first 288 bp are necessary and sufficient to confer a striated muscle-specific  DECEMBER 7, 2007 • VOLUME 282 • NUMBER 49 expression, and that DNA elements located between 1.1 and 2.14 kb may function as an enhancer with specific activity in cardiac myocytes, and may have a negative role in the regulation of transcription in skeletal muscle. The results obtained with the stable transfected Sol8 cell lines showed a similar pattern of induction of transcriptional activity of the Ϫ288and Ϫ3102-bp casq2 gene constructs during skeletal muscle differentiation, which also followed a similar pattern to the level of expression of endogenous casq2 mRNA. Interestingly, the expression of MEF-2C and SRF mRNA was induced during Sol8 muscle differentiation increasing from myoblasts to myotubes 3-and 1.5-fold, respectively.

Transcriptional Analysis of the Human Cardiac Calsequestrin Gene
By DNA sequence analysis of the highly conserved casq2 gene proximal 5Ј-regulatory region, we identify three putative binding sites for transcription factors relevant in the regulation of cardiac and skeletal muscle-specific genes (MEF-2, E-box, and CArG box). The proximal putative MEF-2 and E-box elements share high homology with the consensus sequences for these sites (17). Although, the CArG box sequence present in the casq2 gene (CC(A/T) 7 GG), is not a canonical site (CC(A/T) 6 GG), previously a CArG box site with the same configuration of the one present in the casq2 gene regulatory region has been described and demonstrated to be functional in the cardiac ␣-myosin heavy chain gene (21). Therefore, we further analyzed the role of these muscle regulatory elements on the regulation of the casq2 gene. The results demonstrate that the MEF-2 and CArG box sites are capable of binding the MEF-2 and SRF, respectively; whereas the E-box site did not bind any protein present in the nuclear extracts of cardiomyocytes, C3H10T1/2 cells, as well in Sol8 myoblasts and myotubes.
Because of these results, we decided to focus our work on analyzing the function of the proximal MEF-2, E-box, and CArG box sites, to understand their role in the tissue-specific regulation of the casq2 gene. We generated mutants for these three sites by site-directed mutagenesis. The functional studies demonstrate that the MEF-2 and CArG box mutations decrease the transcriptional activity of the Ϫ288-bp human casq2 gene construct, 70 and 40%, respectively. The DNA protein binding studies confirmed that the mutated sites were unable to bind MEF-2 and SRF. However, mutation of the putative E-box site did not change the transcriptional activity confirming the results showing the absence of protein binding. The mutation of both the MEF-2 site and CArG box on the same construct decreases the transcriptional activity to basal level on both cell types (cardiac, skeletal), similar to that observed with the Ϫ71-bp construct, which is devoid of both elements.
The results indicate that MEF-2 and SRF have a very important role in regulating the expression of the casq2 gene in skeletal and cardiac muscle. Mutation of both sites leaves only basal promoter activity, which may be directed by the TATA-like box present in this region. We must also consider that DNA elements present upstream from Ϫ288 to Ϫ580 bp and from Ϫ1095 to Ϫ3.1 kb have a significant role in regulating the expression of the casq2 gene in cardiac muscle, where these regions show an increase of the activity compared with the Ϫ288-bp construct. DNA sequence analysis of the Ϫ288 to Ϫ3.1 kb region shows the presence of several putative MEF-2, E-box, NFAT, and GATA-4 binding sites (data not shown); the transcription factors that bind to these sites have been mentioned in the regulation of several cardiac and skeletal muscle genes. The presence of several E-box sites in this region may play an important role, because previously it has been reported by DNA microarray analysis that the casq2 gene is regulated in skeletal myotubes by myogenin (28).
In this study, we show that the transcription factors MEF-2 and SRF play a significant role in the regulation of the casq2 gene. However, it is difficult to assume that these are the only factors involved in its regulation because it is well documented that both MEF-2 and SRF are activated via Ca 2ϩ -regulated pathways, like calcineurin for MEF-2 (29 -31) and CaM kinase II for SRF (32,33). When this data is considered along with previous reports that show no changes on the expression of CASQ2 in cardiac pathological states, which imply abnormal intracellular Ca 2ϩ concentrations, it suggests that non-Ca 2ϩregulated pathways may be involved on the regulation of the expression of the casq2 gene (2,10). Therefore, we believe that other transcriptional mechanisms must be involved in the regulation of the expression of casq2 gene, and have to be studied in more detail.
The transcriptional regulation of the casq2 gene is similar to previous publications on cardiac-specific genes like the cardiac troponin C, cardiac ␣-actin, and cardiac ␣-myosin heavy chain genes. The regulatory mechanisms for these genes consist of a basal promoter with the proximal regulatory region activating the transcription in the adult slow-twitch skeletal muscle and cardiac muscle, being sufficient to have striated muscle-specific expression (21). Also in genes with cardiac-specific expression, in the distal regulatory region are enhancers that confer cardiac expression (21,34,35). Recently, it was proposed that a larger enhanceosome complex mediates the cardiac-specific expression, where several transcription factors like GATA4, SRF, MEF-2, NFAT, and HAND2, interact with each other through indirect mechanisms that involve transcriptional scaffolding molecules. The co-activator p300/CBP has been proposed as one of the molecules involved in the formation of the cardiacspecific enhanceosome (29,36,37).
The MEF-2 and SRF factors belong to the MADS family of transcription factors, and are capable of interacting with a large number of transcription factors and cofactors through the MADS DNA binding domain. MEF-2 has been mentioned several times as regulating the transcription of cardiac, skeletal, and smooth muscle genes (38,39). The SRF factor has been particularly involved in the regulation of smooth muscle genes, although it has also been associated with some cardiac and slow-twitch skeletal muscle genes (40 -42). Both factors have the possibility of acting as positive or negative regulators when they bind to DNA, which is dependent of the recruitment of positive or negative transcriptional co-regulators. In recent years the myocardin family of transcription factors, especially myocardin-related transcription factors A/B, have been associated with the regulation of cardiac genes (43)(44)(45)(46), by interactions with the SRF, potentiating the effect of these factors. Thus, it is possible that some of these cofactors contribute with SRF to regulate expression of the casq2 gene. The MEF-2 factor has been mentioned to interact with basic helix-loop-helix transcription factors (47,48), even when the E-box site present on the first 140 bp did not show binding capabilities, it is still possible that basic helix-loop-helix factors acting as cofactors of MEF-2 and are involved in regulating the casq2 gene expression.
In summary, in this work we have demonstrated that the 5Ј-regulatory region of the human casq2 gene has a high homology with other species on the first 180 bp, where only one MEF-2 and one CArG box binding sites are present. These sites are functional and interact with MEF-2 and SRF transcription factors present in cardiac myocytes and Sol8 myotubes. Mutation of the MEF2 or CArG box sites leads to a diminished transcriptional activity in neonatal rat cardiomyocytes and Sol8 myotubes in culture. Remarkably, when both sites are mutated simultaneously the transcriptional activity of the Ϫ288-bp construct decreases to basal level, indicating that the MEF2 and CArG box sites are necessary for transcription of the casq2 gene. We demonstrated that the first 288 bp of the 5Ј-regulatory region of the casq2 gene are necessary and sufficient to regulate the expression in skeletal myotubes and cardiomyocytes, but not in non-muscle cells. The results suggest that, the regions between Ϫ288 and Ϫ580 bp and from Ϫ1095 to Ϫ3102 bp have a role as cardiac-specific enhancers, whereas in skeletal muscle do not play a positive role, but possibly a negative regulatory role. Further studies are still needed on the distal regulatory elements to have a more complete understanding of the muscle-specific transcriptional regulation of the casq2 gene.