(Received for publication, November 10, 1995)
From the
The rabbit cardiac/slow twitch muscle sarcoplasmic reticulum
Ca-ATPase (SERCA2) gene encodes a Ca
transport pump whose expression is regulated during skeletal and
cardiac muscle development and in response to various
pathophysiological and hormonal states. Employing transient
transfection analyses in Sol8 muscle cells, we have identified two
positive regulatory regions, one distal (-1810 base pair (bp) to
-1110 bp) and one proximal (-284 bp to -72 bp),
within the SERCA2 promoter. The proximal promoter region from
-284 bp to -80 bp was shown to confer muscle-specific
expression to a heterologous promoter in Sol8 cells. This region is
highly GC-rich containing the consensus sequence for four Sp1 elements
(GGGCGG) and three Sp1-like elements (GGGAGG). DNase I footprint
analysis with Sol8 nuclear extracts and purified Sp1 protein showed the
protection of the seven Sp1 binding sites. In addition, site-directed
mutagenesis of the Sp1 consensus sites demonstrated that Sp1 sites are
essential for the muscle-specific expression of the SERCA2 promoter.
Furthermore, we demonstrate that cotransfection of an Sp1 expression
vector together with SERCA2-CAT constructs can up-regulate SERCA2
promoter activity. These results imply that the Sp1 transcription
factor plays an important role in the transcriptional regulation of
SERCA2 within muscle cells.
The sarcoplasmic reticulum (SR) ()Ca
-ATPase (SERCA) is a calcium transport
pump and plays an essential role in the contraction-relaxation cycle of
muscle. During excitation-contraction coupling, the SR
Ca
-ATPase is responsible for the active transport of
cytosolic calcium into SR vesicles producing muscle relaxation. The SR
Ca
-ATPase is localized in the longitudinal SR and
represents approximately 35-40% of the total SR protein in
cardiac muscle(1, 2) . Five distinct
sarco(endo)plasmic reticulum Ca
-ATPase (SERCA)
isoforms have been described which are encoded by three distinct genes:
SERCA1, SERCA2, and SERCA3 (reviewed in (3, 4, 5) ). SERCA2 encodes two alternatively
spliced Ca
-ATPase isoforms, SERCA2a(6, 7) and SERCA2b(8, 9) , which diverge in the
COOH-terminal region (SERCA2a = 4 amino acids; SERCA2b =
49 amino acids). These isoforms differ in Ca
affinity, Ca
turnover rate, and ATP
hydrolysis(10) . Functional differences between the two
isoforms have been attributed to 12 amino acids located within the
carboxyl-terminal region of SERCA2b(11) .
SERCA2a, the
cardiac/slow twitch Ca-ATPase, is the primary isoform
expressed in cardiac muscle, both in the atrium and the
ventricle(12, 13, 14) . The earliest
expression of SERCA2a mRNA can be traced to the heart tube of 10-day
post-coitum rat embryos(15, 16) . During heart
development the SERCA2a mRNA level increases gradually from fetal to
adult stages(15, 17) .
SERCA2a mRNA is also expressed in developing skeletal muscle cells in fetal and neonatal stages(17, 18) . However, in fast twitch skeletal muscle the SERCA2a isoform is replaced by the SERCA1 isoform in the adult stage(17) . In contrast, the SERCA2a isoform predominates in both fetal and adult slow twitch skeletal muscle tissue. SERCA2b, on the other hand, is found primarily in smooth muscle and non-muscle cells (9, 14, 19, 20) .
Our
previous studies with the rabbit SERCA2 promoter showed that the
upstream region extending to -1110 bp from the transcriptional
start site is capable of promoting reporter activity in differentiating
CC
muscle cells(21) . In particular,
these studies demonstrated that the proximal promoter region -284
bp to +350 bp is highly active in skeletal muscle cells.
The goal of this study is to perform a detailed analysis of SERCA2 proximal promoter elements important for high level expression in muscle cells. In this study, we use a slow twitch muscle cell line, Sol8 (22) (derived from mouse soleus), which expresses high levels of SERCA2a (23) for promoter analyses. Our DNA transfection analyses reveal two positive regulatory regions, one proximal (-284 bp to -72 bp) and one distal (-1810 bp to -1110 bp). We demonstrate that the proximal promoter region (-284 bp to -80 bp) functions like an enhancer element in muscle cells when transferred to a heterologous promoter-reporter system (TK-CAT). This region is highly GC-rich and contains seven Sp1 binding sites. Using DNase I footprint analyses and site-directed mutagenesis of the Sp1 sites, we demonstrate that the Sp1 elements are critical for SERCA2 promoter activity in muscle cells. Furthermore, we show that overexpression of Sp1 can up-regulate SERCA2 promoter activity in Sol8 muscle cells.
Mutagenic oligonucleotides were annealed to denatured double-stranded pALTER-284 template along with appropriate ampicillin and tetracycline knockout and recovery oligonucleotides. Synthesis of a mutant strand was performed with T4 DNA polymerase and T4 DNA ligase. Competent ES1301mutS (restriction +) cells were co-transformed with the extended product along with R408 helper phage DNA, which allows the production of phagemid DNA from the pALTER vector. JM109 cells were infected with phagemid containing the mutant DNA and selected with the appropriate antibiotic. Isolates were monitored for appropriate antibiotic resistance, and mutations were confirmed by DNA sequencing before cloning into the pBLCAT3 vector. Serial mutations of the Sp1 sites were made progressively following the same protocol.
Figure 1: SERCA2 promoter analysis in Sol8 muscle cells. A, schematic representation of the serial deletions created within the SERCA2 promoter region from positions -1810 bp to +350 bp relative to the transcription initiation site. SERCA2 promoter deletions were fused to the chloramphenicol acetyltransferase reporter gene in the pBLCAT3 vector. Known consensus elements for Sp1, Ap2, E-box, CarG, MCAT, and GATA1 are indicated. B, expression of SERCA2-CAT deletion constructs in Sol8 muscle cells. CAT assay from a typical transient transfection experiment performed in Sol8 myotubes. C, comparison of CAT activity in NIH 3T3 fibroblasts, Sol8 myoblasts, and Sol8 myotubes. Data represent the averages of three or more transfection experiments. CAT activity is represented as the -fold expression of SERCA2-CAT construct over background (pBLCAT3) expression relative to the activity of the -1810-CAT construct.
To determine whether SERCA2 promoter activity is restricted to differentiating myotubes, the promoter constructs were transfected into Sol8 myoblasts (undifferentiated) and a fibroblast cell line (NIH 3T3), and CAT activity was determined (Fig. 1C). SERCA2 promoter activity was significantly lower in myoblasts and is negligible in NIH 3T3 fibroblasts (Fig. 1C), suggesting that SERCA2 promoter activity may require additional muscle-specific regulatory factors that are induced during myogenesis.
Figure 2: The SERCA2 proximal promoter (-284 bp to -80 bp) up-regulates the TK-CAT reporter in Sol8 muscle cells. A, schema showing the -284 bp to -80 bp region linked in both 5` (sense and antisense) and 3` (antisense) to the TK-CAT reporter. The locations of Ap2 sites, Sp1 sites, CCAAT box, and GATAA box are illustrated. B, the SERCA2/TK-CAT activity was determined in Sol8 myotubes and NIH 3T3 fibroblasts. The CAT data represent the averages of at least three transient transfection experiments in Sol8 myotubes. The CAT data are represented as the -fold activity over the pBLCAT2 construct activity.
Figure 3: Nucleotide sequence of the SERCA2 proximal promoter (-284 bp to -72 bp). The consensus binding sequences for the Sp1 transcription factor (sites I, III, VI, and VII) are indicated. Sp1-like (GGGAGG) sequences (sites II, IV, and V) are denoted by an asterisk, and Ap2 consensus sites are also boxed. A 17-bp region from -284 bp to -267 bp responsible for high level expression of the proximal promoter in Sol8 cells is also indicated.
Figure 4: DNase I footprint analysis of the -284 bp to -80 bp SERCA2 promoter region with Sol8 myotube nuclear extract. The -284 bp to -80 bp DNA fragment of the SERCA2 proximal promoter was used to perform DNase I footprint analysis on both the sense and antisense strand in the presence of Sol8 myotube nuclear extracts. Lanes 2 and 3 and lanes 6 and 7 correspond to increasing concentrations of nuclear extract in the presence of DNase I. Lanes 4 and 5 were incubated with DNase I (D) alone. A Maxam and Gilbert A+G DNA sequence ladder of the same region is shown in lanes 1 and 8. Black boxes indicate protected regions corresponding to Sp1 consensus or Sp1-like sites. The hatched box represents an additional protected region designated CATP1. Lanes are designated 1-8, starting from left to right.
To determine the authenticity of Sp1 protein binding to the Sp1 consensus sequences, DNase I footprint analysis was carried out with purified Sp1 protein. Purified Sp1 protein protected all seven Sp1 binding sites (Fig. 5). Interestingly, the Sp1 sites which are in tandem, II-III and V-VI (Fig. 3), produced an extended footprint, revealing that Sp1 can occupy both sites simultaneously. Taken together, these results demonstrate that Sp1 has the ability to bind to all seven Sp1 sites within the proximal promoter. The variations observed in Sp1 binding patterns may be due to differences in the concentration of Sp1 present in nuclear extracts as compared to purified Sp1 protein. It is also possible that nuclear protein binding to certain sites could affect SP1 protein binding to sites I and VII.
Figure 5: DNase I footprint analysis of the -284 bp to -80 bp SERCA2 promoter region using purified Sp1. Labeled probe was incubated with increasing concentrations of purified human Sp1 protein (lanes 3-5 and 9-11) or in the absence of Sp1 (lanes 2 and 8). A Maxam and Gilbert A+G DNA sequence ladder of the same region is shown in lanes 1 and 6 and lanes 7 and 12. Black boxes indicate protected regions that correspond to Sp1 consensus (GGGCGG) sequences or to Sp1-like (GGGAGG) sequences. Lanes are designated 1-12, starting from left to right.
Figure 6: Effects of mutations to Sp1 consensus sites on SERCA2 promoter activity. Left panel, schematic representation of mutations to individual Sp1 consensus sites I, III, VI, and VII and in combinations I+III and I+III+VI. The Sp1 consensus sequences were mutated from the consensus GGGCGG to GTTCGG by site-directed mutagenesis of the -284 bp to +350 bp SERCA2 promoter fragment. Right panel, promoter activity of Sp1 mutant and wild type constructs determined in Sol8 myotubes. CAT activity is represented as the -fold SERCA2-CAT activity over pBLCAT3 relative to the wild type -284-CAT construct.
Figure 7: Overexpression of Sp1 in Sol8 muscle cells. The SP1 expression vector pPACSP1 was cotransfected with SERCA2 promoter constructs (-284-CAT, -267-CAT, and -72-CAT) and pBLCAT3 in Sol8 muscle cells. CAT activity is represented relative to the control -284-CAT (no pPACSP1). Data represent the average of at least three experiments.
Figure 8: Sp1 protein binding activity in nuclear extracts of Sol8 myoblasts and myotubes. A double-stranded Sp1 consensus oligonucleotide was end-labeled and incubated without nuclear extract (lane 1), with Sol8 myoblast nuclear extract (lane 2), or with Sol8 myotube nuclear extract (lanes 3 and 4).
In this study we show that the SERCA2 promoter (-1810
bp to +350 bp) contains multiple cis regulatory elements
responsible for the complex regulation of this gene in muscle cells. We
have identified two positive regulatory regions, a proximal region
(-284 bp to -72 bp) and a distal region (-1810 bp to
-1110 bp), as important for high level SERCA2 gene expression. In
addition, sequences within the -490 bp to -562 bp region
had a negative effect on SERCA2 promoter activity. The SERCA2 proximal
promoter region (-284 bp to +350 bp) proved to be sufficient
for high level expression in Sol8 muscle cells. Interestingly, a short
deletion (17 bp) between -284 bp and -267 bp produced a
significant decrease (69%) in promoter activity, suggesting that this
region may contain an important regulatory element. This region (17-bp
UPE) was previously shown to bind a 43-kDa protein from
CC
muscle nuclear extracts(21) .
However, when the 17-bp UPE was linked to a heterologous promoter, it
was unable to promote high level expression, (
)suggesting
that other regulatory elements within the proximal promoter are
required.
Here, we demonstrate that the SERCA2 proximal promoter region (-284 bp to -80 bp) is capable of activating a heterologous (TK) promoter in muscle cells. Interestingly, this DNA fragment functions in a position- and orientation-independent manner, characteristic of a classical enhancer. The proximal promoter region is highly GC-rich and contains seven Sp1 binding sites, which are conserved between the rat and rabbit SERCA2 genes(32) .
The results presented in this paper demonstrate that Sp1 plays an important role in the transcriptional regulation of the SERCA2 gene. DNase I footprinting with purified Sp1 protein revealed that all seven Sp1 sites within the proximal promoter can bind Sp1. Nuclear extracts from Sol8 myotubes showed strong protection of Sp1 sites II-VI and weaker protection of sites I and VII. Moreover, site-directed mutagenesis demonstrated that Sp1 binding sites were required for high level expression of the SERCA2 proximal promoter. Furthermore, overexpression of Sp1 in Sol8 muscle cells clearly established that Sp1 can up-regulate SERCA2 promoter activity. Our results can be interpreted to suggest that alterations in Sp1 concentrations can modulate SERCA2 gene transcription.
Although Sp1 is generally regarded as an ubiquitous transcription factor, levels of Sp1 have been shown to vary among different tissues (33) . In mouse, Sp1 was shown to be expressed at high levels in tissues undergoing differentiation, and at much lower levels in terminally differentiated cells(33) . Our results show that Sp1 binding is increased during Sol8 myogenesis (Fig. 8). Such increases in Sp1 binding could be due to an increase in availability of Sp1 protein or post-translational modifications of the Sp1 protein by phosphorylation or differential glycosylations(34, 35) .
It is not, however, surprising that Sp1 overexpression could produce an increase in SERCA2 promoter activity since this promoter contains seven adjacent Sp1 binding sites. Sp1 has been well established as an activator of native(36) , as well as artificial (25) promoters that contain multiple Sp1 consensus sites. Sp1, which binds to adjacent sites as seen in the SERCA2 promoter, can interact synergistically to form higher order complexes and activate transcription(30) . Our DNase I footprinting data demonstrate that sequences containing adjacent Sp1 sites produce regions of extended protection, suggesting that Sp1 protein can interact to form a higher order complex over this region. Formation of Sp1 complexes are believed to generate an activation environment that allows for more efficient interactions of the transcription apparatus, thus activating transcription at a higher level(30) . Transactivation of SERCA2 proximal promoter by Sp1 overexpression provides strong evidence for this type of mechanism.
Although the SERCA2 promoter is expressed at higher levels in Sol8
muscle cells than in NIH 3T3 cells, it is unlikely that muscle
specificity is due to the Sp1 transcription factor alone. We propose
that the Sp1 protein interacts with other trans acting factors to
produce maximal expression of the SERCA2 gene in muscle cells. The
-284 bp to -267 bp region, which binds a 43-kDa protein,
appears to be essential for the expression of the SERCA2 gene ( (21) and this report). Our DNase I footprinting analysis also
revealed a new protein binding site, CATP1, indicating additional trans
acting factors might bind within the SERCA2 proximal promoter. However,
the relevance of this site in the regulation of the SERCA2 gene remains
to be determined. In addition, there are three Ap2 consensus sequences
within the proximal promoter that may contribute to the regulation of
the SERCA2 gene. Furthermore, the distal promoter region, -1810
bp to -1110 bp, contains consensus sequences for CarG box, E box,
and MCAT elements, which have all been implicated in muscle-specific
gene regulation. Our preliminary data indicate that the distal promoter
region -1810 bp to -1110 bp can also up-regulate
heterologous promoter (TK) expression in muscle cells.
Interactions between proximal and distal promoter elements may form the basis for the tissue-specific expression of the SERCA2 gene. Sp1 has been shown to be involved in the interactions of proximal and distal enhancers through DNA looping(37) . Thus, Sp1 can act as a physical link between proximal and distal promoter activation elements. Sp1 binding in the SERCA2 proximal promoter may play a role in linking distal promoter elements (located in the -1810 bp to -1110 bp) via DNA bending (38) or looping(37) , thus providing a better activation environment for the SERCA2 promoter.
Recent studies have also shown that thyroid-responsive elements are located within the proximal promoter of the SERCA2 gene(32, 39, 40) . In the rat gene three TRE elements were identified between nucleotides -485 bp and -190 bp(39) . In the rabbit gene TRE elements are located between -254 bp and -72 bp(40) . Sp1 binding sites were included in the SERCA2 constructs, which gave maximal induction by thyroid hormone. Deletion of the Sp1 binding region decreased T3 inducibility of the promoter(39) . Therefore, Sp1 binding sites might be important for thyroid hormone-induced activation of the SERCA2 gene. However, it remains to be determined whether TRE inducibility is dependent on Sp1 interactions with the T3 receptor.
The role of Sp1
in muscle-specific gene expression has been widely established.
Muscle-specific genes including cardiac and skeletal -actin (41, 42) and
MHC (43) require Sp1 for
efficient gene expression. Furthermore, the Sp1 transcription factor
has been shown to play an important role in muscle-specific gene
expression and gene induction by interacting with additional factors
that are highly tissue-specific. For example, the cardiac
-actin
gene has been shown to require Sp1 in addition to MyoD1 and an SRF-like
factor for muscle-specific gene expression(41) . Additionally,
a Sp1 site was shown to be required along with M-CAT and CarG elements
for
-adrenergic induction of the skeletal
-actin
gene (44) . The
-adrenergic inducibility of
the ANF gene also appears to be dependent on Sp1 and CarG
elements(45) . Finally, Sp1 has been shown to regulate gene
expression through competition with other factors for binding sites.
Sp1 sites within mouse acetylcholinesterase gene proximal promoter
region have been shown to overlap with Egr-1 sites resulting in
competition between Sp1 and Egr-1 for binding(46) . Occupation
of the overlapping site by Sp1 up-regulates the acetylcholinesterase
gene expression in muscle cells.
In summary, the data presented here suggest that Sp1 plays an essential role in regulating SERCA2 gene expression in muscle cells. We propose that the Sp1 protein may interact with other transcription factors in controlling the efficient expression of this gene in muscle cells. Future experiments will attempt to characterize how the Sp1 binding sites interact with other regulatory elements that are present in the proximal and distal region.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) M33834[GenBank].