(Received for publication, October 17, 1995; and in revised form, December 18, 1995)
From the
Expression of the muscle creatine kinase (MCK) gene in skeletal and heart muscle is controlled in part by a 5` tissue-specific enhancer. In order to identify new regulatory elements, we designed mutations in a previously untested conserved portion of this enhancer. Transfection analysis of these mutations delineated a new control element, named Trex (Transcriptional regulatory element x), which is required for full transcriptional activity of the MCK enhancer in skeletal but not cardiac muscle cells. Gel mobility shift assays demonstrate that myocyte, myoblast, and fibroblast nuclear extracts but not primary cardiomyocyte nuclear extracts contain a trans-acting factor that binds specifically to Trex. The Trex sequence is similar (7/8 bases) to the TEF-1 consensus DNA-binding site involved in regulating other muscle genes. To determine if TEF-1 interacts with Trex, selected TEF-1 binding sites such as GTIIc and M-CAT and two anti-TEF-1 antisera were used in gel shift assays. These experiments strongly suggest that a factor distinct from TEF-1 binds specifically to Trex. Thus it appears that MCK transcription is regulated in skeletal muscles through a Trex-dependent pathway while Trex is not required for MCK expression in heart. This distinction could account partially for the difference in levels of muscle creatine kinase in these tissues.
The differentiation of embryonic mesodermal cells into skeletal
or cardiac muscle cells requires the controlled expression of numerous
muscle-specific
genes(1, 2, 3, 4, 5) .
Because the muscle creatine kinase (MCK) ()gene is expressed
at different levels in mature skeletal and cardiac muscle
cells(6) , it is an instructive model for comparing the
mechanisms that regulate expression of the same muscle-specific gene in
both striated muscle tissues. The current study identifies a new
regulatory element within the MCK enhancer, which exhibits such
differential activity in skeletal and cardiac muscle cells.
Expression of the MCK gene in differentiated muscle cells is controlled by an array of cis-elements and trans-acting factors. Particular attention has been given to the analysis of a 206-bp upstream enhancer, which confers muscle-specific expression both in cultured cells and in transgenic mice(7, 8, 9) . Mutational analysis, gel mobility shift studies, and footprinting assays have identified six transcriptional regulatory elements in the MCK enhancer (from 5` to 3`): the CArG, AP2, A/T-rich, left and right E boxes, and MEF-2 sites (6, 10, 11) . Each of these MCK gene control elements has been well conserved during mammalian evolution(7, 12, 13, 14) , and similar nucleotide motifs have been implicated in the transcriptional control of numerous other muscle-specific genes(15) .
Mutating these six MCK enhancer control elements causes different relative effects in cultured cardiomyocytes and skeletal muscle cells (6) . Mutation of the MCK right E box site decreases enhancer activity more dramatically in skeletal than in cardiac muscle cells, while mutations of the CArG site or the A/T-rich site seem more deleterious in cardiac than in skeletal muscle cells. In contrast to these differential effects, mutations of the MEF-2 site or the left E box site decrease enhancer activity to about the same extent in both cell types. Unlike the other MCK enhancer control elements, the AP2 site seems to repress transcription in cultured skeletal and cardiac muscle cells as its mutation leads to increased expression in both cell types.
These six enhancer regulatory elements are the target sites
for an array of muscle and non-muscle-specific DNA binding factors. The
left and right E boxes contain the consensus core sequence CANNTG,
which is the target for skeletal muscle-specific determination factors
of the MyoD family of transcription factors(16) . These two E
boxes, which differ in their flanking regions, exhibit noticeably
different transcriptional activities and protein binding
properties(17, 18) . Although E boxes are involved in
the cardiac regulation of MCK as well as a number of other cardiac
muscle genes (19, 20, 21, 22, 23) , ()MyoD family members are not detected in the heart at any
time during development(4) . The MEF-2 site and the A/T-rich
site are both rich in adenine and thymidine(25, 26) .
The MEF-2 site is the target of MEF2 proteins, which are also known as
the Related to Serum Response Factor (RSRF)
family(27, 28) , and BBF-1, a serum-inducible factor
identified in cardiac but not in skeletal muscle(29) . MEF2
proteins have been shown to cooperate in transactivation with members
of the MyoD family of transcription factors (30, 31, 32, 33) and bind with lower
affinity to the MCK A/T-rich site (34) . The mouse MCK A/T-rich
element is also a DNA-binding site for both the homeoprotein MHox and
the ubiquitous Oct-1 factor(26, 34) . The CArG motifs
have been shown to bind serum response factors (28) .
The
MCK enhancer does not contain all of the regulatory elements identified
in other muscle genes. For example it does not contain functional Sp1
and CCAC sites found in regulatory regions of the actin (22) and myoglobin genes(35) . The MCK enhancer also
appears to contain no perfect match to the M-CAT sites, key elements in
regulating the cardiac troponin T(36) ,
- and
-myosin
heavy
chain(21, 37, 38, 39, 40) ,
and skeletal
-actin genes(41) , and targets for MCBF
(M-CAT binding factors) and the ubiquitous TEF-1 factor(42) .
M-CAT sites were of particular interest to the present study because a newly identified MCK enhancer control element called Trex (Transcriptional regulatory element x) bears close similarity to the consensus MCBF/TEF-1-binding sites in other genes. We show here that the MCK enhancer Trex site is required for full transcriptional activity of the enhancer in MM14 skeletal muscle cell cultures. However, mutations in the Trex site that greatly reduce MCK reporter gene expression in these cultures exhibit virtually no effect in heart cell cultures. Furthermore, despite its similarities to the consensus TEF-1-binding site, we show that the Trex site is not a target for TEF-1, nor do previously identified TEF-1-binding sites (such as M-CAT or the SV40 enhancer GTIIc site) compete with the Trex sequence for specific Trex binding. A Trex-specific binding complex has been identified in MM14 skeletal myocyte, MM14 myoblast, NIH-3T3 fibroblast, and hybridoma cell nuclear protein extracts. However, no such complex was identified in nuclear protein extracts from neonatal cardiomyocytes. Trex may thus be partially responsible for the differential expression of MCK and potentially other genes in skeletal versus cardiac muscle cells.
Cardiac muscle gene regulation was studied in primary newborn Sprague-Dawley rat ventricular cardiomyocytes prepared as described previously (6, 46) and maintained in Dulbecco's modified Eagle's medium/M199 (4:1), 10% horse serum, 5% fetal bovine serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. These cultures contained about 95% cardiomyocytes as assessed by immunohistochemistry with myosin antibody MF-20(47) . After transfection, cardiomyocytes were switched to serum-free Dulbecco's modified Eagle's medium/M199 (4:1) supplemented with 6 µg/ml insulin and Fungizone.
Skeletal and cardiac muscle cells were maintained in differentiation medium at least 24 h before harvesting. In both transient and stable cultures, at least 95% of the cells were differentiated as assessed by myosin immunostaining(47) . The cells were analyzed either for chloramphenicol acetyltransferase (CAT) and placental alkaline phosphatase gene expression in the case of transient transfections (6) or for CAT and protein concentration (Bradford assay) in the case of stable transfections(48) . For each MCKCAT construct tested, at least two plasmid preparations were used. The effect of each mutation was evaluated as the relative amount of CAT/placental alkaline phosphatase or CAT/[protein] and was compared with the wild-type enhancer-CAT construct whose activity was set to 100. The results of independent transfections are shown as the average of at least three transient transfections of three plates each or three plates of stably transfected cells.
Figure 1: Muscle creatine kinase gene enhancer. A, partial nucleotide sequence comparison of the mouse(7) , rat(12) , human(13) , and rabbit (14) MCK enhancers. Residues are compared with the mouse sequence, and identical nucleotides are indicated by a dot. Known regulatory sites are labeled in boldface. Non-conserved residues are indicated in lowercase letters. Gaps have been introduced to optimize the alignment. The Trex region is underlined. B, mutations introduced into the mouse MCK enhancer Trex region are indicated in boldface. The 22-bp oligonucleotide TrexG-C containing a single underlined base pair change is also indicated. C, constructs tested in mutational analysis. The enhancer-containing plasmids are shown schematically with known regulatory sites in stippled boxes and introduced mutations in black boxes. E indicates E box motifs. Each mutation (M1, M2, and M3) was tested separately. 1256MCKCAT is composed of the 206-bp enhancer with the -1050 through +7 MCK promoter fused to a CAT gene. (+enh)80MCKCAT and (-enh)80MCKCAT are composed of the 206-bp enhancer in its native, (+), or reversed(-) orientation linked to the -80-bp basal MCK promoter and fused to CAT.
Figure 2: Effects of Trex site mutations on MCK-CAT reporter gene expression. For each cell type and construct tested, the wild-type reporter gene activity is set at 100. A, transcriptional activity of wild-type (WT) and mutant (mut) M1 1256MCKCAT constructs in either transiently or stably transfected MM14 skeletal myocyte cultures and in transiently transfected rat primary cardiomyocyte cultures. The relative activity of 1256MCKCAT is about 12-fold higher in skeletal myocytes than in cardiomyocytes. B, transcriptional activity of wild-type or mutated MCK enhancers carrying mutations M1, M2, or M3 in MM14 skeletal muscle cells. Each construct was tested in the positive (+enh)80MCKCAT or the negative (-enh)80MCKCAT orientation. Data are plotted as the mean value and standard deviation of CAT/placental alkaline phosphatase for the transient transfections and as CAT/[protein] for the stable transfections.
In the context of the full-length promoter 1256MCKCAT, mutation M1 decreased enhancer activity 2-4-fold in both transiently and stably transfected MM14 myocyte cultures (Fig. 2A). In contrast, the 1256MCKCAT enhancer construct bearing the M1 mutation was expressed at a similar level to the wild-type enhancer in rat primary cardiomyocyte cultures (Fig. 2A). These results suggest that mutation M1 disrupts a positive regulatory site that is required for full transcriptional activity of the enhancer in skeletal myocytes but not in cardiomyocytes. A more dramatic effect of mutation M1 was observed in constructs containing the enhancer, in either its normal (+) or reversed(-) orientation, fused to a truncated promoter ((+enh)80MCKCAT and (-enh)80MCKCAT). In these contexts the M1 mutation caused nearly a 10-fold decrease in enhancer activity in both transient (Fig. 2B) and stable transfection assays of MM14 myocytes (data not shown). The greater relative effects of Trex site mutations when tested in (+enh)80MCKCAT versus 1256MCKCAT contexts may be due to compensatory effects of sequences in the -1050- to -81-bp region contained in the later construct; however, no Trex site has been identified in this region.
To determine if Trex is involved in silencing MCK gene expression in cells that do not express MCK, NIH-3T3 fibroblasts and MM14 myoblasts were transfected with 1256MCKCAT constructs. As expected, the wild-type MCK enhancer construct was expressed at a low background level (data not shown), consistent with the lack of endogenous MCK expression in these cell types. Mutation M1 did not result in elevated levels of reporter gene expression, indicating that Trex is not responsible for silencing the MCK gene in fibroblasts and myoblasts.
To further delineate the Trex site the sequence between the AP2 and A/T-rich sites was examined via two smaller mutations, M2 and M3 (Fig. 1B). Mutation of bases in the 5`-region of the M1 mutation (mut M3) had no effect on expression of the (enh)80MCKCAT reporter gene when tested in either the positive or negative enhancer orientation (Fig. 2B). These results indicate that none of the residues modified by M3 are critical parts of Trex. In contrast, disruption of bases within the more 3`-sequence by mutation M2 impaired expression severely, reducing the enhancer function about 8-fold (Fig. 2B). Since the nucleotide substitutions made in mutation M2 differ markedly from those made in mutation M1, it seems very unlikely that both mutations would create negative regulatory sites with similar repressor activity. The most straightforward interpretation is that the M1 and M2 mutations both disrupt the same positive control element.
Transfections of MM14
myocytes or 3T3 fibroblasts with either -80MCKCAT or a
multimerized Trex sequence ligated to the basal MCK promoter,
(Trex)ATCAT, result in background levels of CAT gene
expression (data not shown), indicating that four copies of the Trex
site are not sufficient to promote transcription. Therefore, the Trex
site appears to require additional regulatory elements in order to
confer transcriptional activity to skeletal muscle genes.
Figure 3: Binding of nuclear factors from skeletal muscle cells and fibroblasts to the MCK enhancer Trex site. The 22-bp, 5`-labeled Trex probe was mixed with nuclear extracts from MM14 myocytes (lanes 2-4), MM14 myoblasts (lanes 5-7), or NIH-3T3 fibroblasts (lanes 8-10) and analyzed via gel mobility shift assay. Lane 1 shows the mobility of the free probe, indicated by an arrow at the bottom of the gel. A slowly migrating DNA-protein complex of the same apparent mobility was formed with the Trex probe and nuclear proteins from each cell type. The Trex binding complex, indicated by an arrow, is abolished by addition of a 100-fold molar excess unlabeled Trex probe (lanes 3, 6, and 9) but not by addition of an equivalent amount of a similar 22-bp oligonucleotide carrying the mutation M1 (lanes 4, 7, and 10, respectively).
Figure 4: Sequence specificity of the Trex binding complex. The 22-bp, 5`-labeled Trex probe was mixed with MM14 myocyte nuclear extracts. A and B, lane 8 shows the mobility of the free probe. Binding of the Trex-specific complex (lanes 1 and 9) is competed away by addition of an excess of unlabeled Trex oligonucleotide (lanes 10-13) but not by unlabeled oligonucleotides carrying the mutation M1 (lanes 14-16), the single base pair mutation TrexG-C (lanes 17-19), the closely related GTIIc oligonucleotide (lanes 2-4), or the M-CAT oligonucleotide (lanes 5-7).
Gel mobility shift assays showed that the specific complex formed by Trex and nuclear proteins from MM14 myocytes was not abolished by competition with unlabeled GTIIc site or M-CAT site oligonucleotides (Fig. 4A, lanes 2-4 and lanes 5-7). Similarly, this complex persisted upon addition of a 22-mer oligonucleotide (Fig. 1B, Trex G-C) containing a 1-base pair mutation that converts the Trex site into a sequence equivalent to the TEF-1 consensus sequence (Fig. 4B, lanes 17-19). Cross-competition studies showed that binding to GTIIc, TrexG-C, or to M-CAT probes was not altered by addition of unlabeled Trex oligonucleotide (data not shown). Since binding to Trex is not altered by increasing amounts of known TEF-1-binding sites, it appears that TrexBF is not TEF-1.
Further evidence that the TrexBF differs from TEF-1 was obtained by immunological studies using anti-TEF-1 sera that recognize both human and mouse TEF-1(40) . Addition of either N-terminal or C-terminal anti-TEF-1 polyclonal antiserum to MM14 nuclear extracts produced no change in migration of the TrexBF complex (Fig. 5, lanes 6 and 7). However, both antisera caused a supershift when the same skeletal muscle extracts were combined with GTIIc oligonucleotides (Fig. 5, lanes 2 and 3). As a control, no supershift was observed upon addition of nonimmune serum (Fig. 5, lanes 1 and 5). This result indicates that MM14 nuclear extracts contain TEF-1 or a TEF-1-related protein that can participate as part of a GTIIc binding complex. However, the DNA binding activity defined as TrexBF is not TEF-1.
Figure 5: Immunological analysis of the skeletal muscle GTIIc and Trex binding complexes. GTIIc probe (lanes 1-4) and MCK Trex probe (lanes 5-8) were mixed with MM14 myocyte nuclear extracts. DNA-protein complexes are shown in lanes 4 and 8 for GTIIc and Trex, respectively. Upon addition of antiserum (N) against the N-terminal domain of TEF-1 and antiserum (C) against the C-terminal domain of TEF-1, the GTIIc probe is supershifted (lanes 2 and 3). In contrast, no supershift is observed with the Trex probe (lanes 6 and 7). Control assays with non-immune sera (P) are shown in lanes 1 and 5, respectively.
Taken together these results indicate that both TEF-1 and a previously unrecognized factor, TrexBF, are present in MM14 nuclear extracts. However, despite the close sequence similarity between the MCK Trex site and the TEF-1-binding sites, TrexBF is not TEF-1.
Figure 6: Trex-specific binding activity is undetectable in cardiac myocyte nuclear extracts. Nuclear extracts from primary rat cardiomyocytes were mixed with 5`-labeled Trex oligonucleotide (lanes 1-3) or TrexG-C, a single base pair mutant Trex oligonucleotide that conforms to the TEF-1 consensus binding sequence (lanes 4-7). No nuclear proteins from the cardiomyocyte extract form a shifted complex with the Trex probe (lanes 1-3). In contrast, two DNA-protein complexes formed between cardiac proteins and the TrexG-C labeled probe (lane 4). Both complexes are competed by addition of a 100-fold molar excess unlabeled Trex G-C sequence (lane 5), as well as by excess GTIIc sequence (lane 6). Neither complex is abolished by addition of excess unlabeled wild-type Trex sequence (lane 7).
To examine the possibility that cardiomyocyte extracts contain an inhibitor that prevents binding to Trex, up to a 3-fold excess of cardiomyocyte nuclear extract was added to a skeletal extract and tested in gel mobility shift assays. In no case was there a specific reduction of skeletal muscle extract binding to Trex (data not shown), indicating that no inhibitor of Trex binding exists in these extracts.
Although TrexBF is present in various cell extracts, its activity has not been defined. TrexBF's activity could be regulated in these cell types through several mechanisms, for example by post-translational modification (e.g. phosphorylation) or by interaction with ubiquitous or cell-type specific accessory factors. Considering the fact that TrexBF activity is found in both replicating and postmitotic MM14 cells, mitogen-regulated phosphorylation is probably not involved in TrexBF activity. The alternative mechanism, involving protein-protein interactions, has been proposed for regulation of the cardiac troponin T gene. In this gene the ubiquitous TEF-1 factor binds to M-CAT control elements(42) , but its full transcriptional activity requires intermediary factors, termed TIFs(50) . Understanding the mechanisms involved in regulating TrexBF function awaits its cloning and characterization.
Five TEF-1 isoforms have been characterized(59) . Although we cannot rule out the possibility that TrexBF is a novel TEF-1 isoform, this seems unlikely since neither TEF-1 antiserum supershifted the Trex-TrexBF complex (Fig. 5). In addition, a single base pair mutation that converts the MCK enhancer Trex site to a consensus TEF-1-binding site does not compete for Trex binding (Fig. 4B). This suggests that association of TrexBF with the Trex site is very sequence-specific. Since a single amino acid substitution in a helix-loop-helix protein can change the protein specificity for bases flanking the core E box motif(60) , it is possible that TrexBF and TEF-1 may have very similar structures but that slight changes are sufficient to alter their binding specificity. An additional distinction between TEF-1 and TrexBF is that TEF-1 is enriched in skeletal muscles as well as in heart tissue(40, 59) , whereas TrexBF is undetected in heart. Moreover, knocking out the TEF-1 gene affects cardiac muscle but not skeletal muscle development(61) . When considered as a whole, these observations strongly support the hypothesis that TrexBF is not TEF-1.
The activation and steady state expression of the same
genes in both skeletal and cardiac muscles could imply transcription
factors that are common to both striated muscle types. Indeed since
combinatorial interactions of multiple cis-acting elements underlie the
regulation of muscle-specific genes, the relative ratios of common
factors could be responsible for the quantitative differences in a
gene's expression level in each type of
muscle(35, 38) . However, in many cases heart and
skeletal muscle cells use alternative transcriptional strategies to
regulate tissue-specific expression of the same
gene(3, 62) . For instance, distinct enhancer
fragments control expression of the cardiac troponin C, cardiac
troponin T, myosin light chain 2, and -myosin heavy chain genes in
cardiac versus skeletal muscle cells (24, 56, 63, 64) . Here we present
evidence that a novel MCK enhancer regulatory element, Trex, is
required for skeletal muscle but not for cardiac muscle expression of
the MCK gene. Thus it appears that MCK is expressed in skeletal muscle
through a Trex-dependent pathway and in heart through a
Trex-independent pathway. It will be instructive to determine both how
TrexBF is activated in skeletal muscles and how this factor is
restricted in cardiomyocytes. It will also be important to extend our
observations to transgenic mice as well as to evaluate the role of Trex
in other muscle genes.