Multiprotein Complex Formation at the beta  Myosin Heavy Chain Distal Muscle CAT Element Correlates with Slow Muscle Expression but Not Mechanical Overload Responsiveness*

Dharmesh R. VyasDagger , John J. McCarthyDagger , Gretchen L. TsikaDagger , and Richard W. TsikaDagger §||

From the § Department of Biochemistry, School of Medicine, the Dagger  Department of Biomedical Sciences, School of Veterinary Medicine, and the  Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211

Received for publication, August 24, 2000, and in revised form, September 26, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To examine the role of the beta -myosin heavy chain (beta MyHC) distal muscle CAT (MCAT) element in muscle fiber type-specific expression and mechanical overload (MOV) responsiveness, we conducted transgenic and in vitro experiments. In adult transgenic mice, mutation of the distal MCAT element led to significant reductions in chloramphenicol acetyltransferase (CAT) specific activity measured in control soleus and plantaris muscles when compared with wild type transgene beta 293WT but did not abolish MOV-induced CAT specific activity. Electrophoretic mobility shift assay revealed the formation of a specific low migrating nuclear protein complex (LMC) at the beta MyHC MCAT element that was highly enriched only when using either MOV plantaris or control soleus nuclear extract. Scanning mutagenesis of the beta MyHC distal MCAT element revealed that only the nucleotides comprising the core MCAT element were essential for LMC formation. The proteins within the LMC when using either MOV plantaris or control soleus nuclear extracts were antigenically related to nominal transcription enhancer factor 1 (NTEF-1), poly(ADP-ribose) polymerase (PARP), and Max. Only in vitro translated TEF-1 protein bound to the distal MCAT element, suggesting that this multiprotein complex is tethered to the DNA via TEF-1. Protein-protein interaction assays revealed interactions between nominal TEF-1, PARP, and Max. Our studies show that for transgene beta 293 the distal MCAT element is not required for MOV responsiveness but suggest that a multiprotein complex likely comprised of nominal TEF-1, PARP, and Max forms at this element to contribute to basal slow fiber expression.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Regulation at the level of gene transcription represents a critical control point whereby a cell-specific phenotype can be specified and modulated in response to intrinsic and extrinsic stimuli throughout development. For example, in the day 17 post-coitus mouse fetus, beta -myosin heavy chain (beta MyHC)1 expression is detected in the heart, and subsequently its expression becomes primarily restricted to adult stage type I skeletal muscle fibers (1-4). Once myofibers have differentiated and acquired a distinct adult phenotype, the molecular properties of the fiber are not static but instead are remarkably malleable in response to a broad spectrum of physiological signals (5). A well documented example of this phenotypic plasticity is the induction of beta MyHC expression in fast type II fibers in response to increased neuromuscular activity imposed by mechanical overload (MOV) (5, 6). The regulated control of beta MyHC expression has been shown to involve the summation of inputs from multiple cis-acting modules located within both the beta MyHC proximal promoter and more distal regulatory regions (3, 6-11). However, the precise contribution of each discrete element comprising these regions has not been systematically analyzed in the in vivo context. In addition, the identity of the cell-specific and/or ubiquitous transcription factors that direct beta MyHC slow fiber restricted expression or MOV responsiveness in adult stage skeletal muscle have not been determined as yet.

To investigate the mechanistic basis controlling both slow fiber restricted expression and MOV responsiveness of the beta MyHC gene, we have conducted an extensive in vivo deletion analysis of the human beta MyHC promoter in the context of the transgenic mouse (4, 6, 8-11). These studies defined a minimal promoter region comprised of 293 bp of the beta MyHC promoter (transgene beta 293WT) that closely mimics the expression pattern of the endogenous beta MyHC gene at all developmental stages, and in response to MOV (4, 9). Most striking was the finding that the deletion of an 89-bp region (-293 to -205) resulted in the loss of transgene beta 293WT expression and MOV responsiveness, thus indicating that this 89-bp region contains regulatory elements important for both basal slow fiber expression and MOV responsiveness of the beta MyHC gene (9). Further analysis of this 89-bp region led to the identification of a putative MOV element (beta A/T-rich element: -269 to -258) that formed an enriched multiprotein binding complex only when MOV-plantaris nuclear extract was used (8). In addition to this A/T-rich element, the 89-bp region also contains a muscle CAT (MCAT) site. Whether this regulatory element plays a functional role in conferring beta MyHC basal slow fiber expression and/or MOV responsiveness has not been investigated as yet.

MCAT regulatory elements (5'-CATTCCT-3') are similar in nucleotide sequence to the SV40 enhancer GT-IIC (5'-CATTCCA-3'), SphI (5'-CATGCCT-3', and SphII (5'-CATACCT-3') elements (12, 13). Earlier studies using protein purification and cDNA cloning identified transcription enhancer factor 1 (TEF-1) as the GT-IIC protein binding activity within HeLa cell extracts (14, 15). Subsequent studies have established the presence of a multigene TEF-1 family in vertebrates (nominal TEF-1 (NTEF-1), related TEF-1 (RTEF-1), divergent TEF-1, and embryonic TEF-1). Further diversity within the TEF-1 family of transcription factors is accomplished by alternative splicing (12, 13, 16). In addition to existing as multiple isoforms, distinct TEF-1 isoproteins can modulate gene transcription as a result of post-transcriptional modification via different intracellular signaling pathways (17, 18) or by interacting with various cobinding and/or coactivator proteins that may be cell-specific or ubiquitous (12, 13, 19).

MCAT elements are commonly located in the control region of many striated (skeletal and cardiac) muscle-specific genes (Ref. 13 and references within). A detailed molecular analysis of the cTnT promoter has elucidated the mechanistic basis by which two closely positioned MCAT elements participate in directing embryonic skeletal muscle-specific gene transcription of the cTnT gene (20, 21). These two MCAT elements (MCAT1 and MCAT2) bind several protein complexes that display high, intermediate, and low migrating mobility in electrophoretic mobility shift assays (EMSA). All binding complexes contain TEF-1 protein; however, the low mobility complex (LMC) formed at the MCAT1 element contains an additional protein identified as poly(ADP-ribose) polymerase (PARP) (20-22). Mutation of the 5'-flanking nucleotides of either the MCAT1 or MCAT2 elements revealed their integral requirement for achieving both skeletal muscle-specific expression of cTnT reporter genes, and the formation of a LMC at these MCAT elements when using embryonic chicken skeletal muscle nuclear extract (20). Likewise, Gupta et al. (23) have reported that the positive regulation of alpha MyHC gene expression in cardiomyocytes requires an E-box/MCAT composite element that involves the cobinding of NTEF-1 and the basic helix-loop-helix leucine zipper transcription factor Max. Additionally, Ojamaa et al. (24) have demonstrated that the alpha MyHC E-box/MCAT element can serve as a contractile/mechanical responsive element in cardiomyocytes and under conditions of increased contractile activity binds the basic helix-loop-helix leucine zipper protein, upstream stimulatory factor (USF).

The beta MyHC gene is regulated by mechanical stimuli and contains within its proximal promoter two closely spaced MCAT elements; a distal MCAT (-290/-284) and a proximal MCAT (-210/-203). These two distinct MCAT elements are highly conserved and located within a region required for both slow fiber expression and MOV responsiveness of a 293-bp human beta MyHC transgene (beta 293) in adult skeletal muscle (4). Our previous EMSA analysis revealed that only the distal MCAT element formed a series of protein complexes displaying low, intermediate, and high migrating mobility similar to those formed at the cTnT MCAT element, and in addition, the low mobility complex was enriched only when using MOV-P nuclear extract (25). On the basis of these data obvious questions arise. First, does the distal MCAT element (-293/-284) bind both TEF-1 and PARP? Second, does this element contribute to both basal slow fiber expression and MOV responsiveness of the minimal 293-bp human beta MyHC transgene?

In the current study we have generated multiple independent transgenic mouse lines harboring either a wild type 293-bp human beta MyHC transgene (beta 293WT) or a 293-bp MCAT element mutant transgene (beta 293Mm). These mice were used in studies aimed at determining whether the distal MCAT element is required for: 1) basal slow fiber expression, 2) muscle-specific expression, and 3) MOV responsiveness. We also conducted a detailed molecular analysis to determine what nuclear protein(s) comprise the low mobility binding complex formed at the distal MCAT element. Our findings show that the beta MyHC distal MCAT element contributes to basal slow fiber expression of transgene beta 293 but is not required for MOV-responsive expression of transgene beta 293 in adult transgenic mice. The multiprotein complex formed at the beta MyHC distal MCAT element is comprised of NTEF-1, PARP, and Max. DNA binding studies indicate that PARP and Max are bound to the distal MCAT element via TEF-1, whereas protein-protein interaction assays revealed that these three proteins physically interact in vitro. Collectively, these data support the notion that the assembly of TEF-1, PARP, and Max at the beta MyHC distal MCAT element (LMC) contributes to basal slow fiber expression of transgene beta 293 in adult stage skeletal muscle.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transgenes and Site-directed Mutagenesis-- The wild type beta MyHC 293-bp transgene (beta 293) used in this study has been described previously (4, 9). Briefly, beta MyHC transgenes consist of 293 bp of human beta MyHC 5'-promoter sequence and 120 bp of 5'-untranslated region (includes exon 1), fused to the 5'-end of the bacterial CAT gene (see Fig. 1). The transgenes were released free of pSVOCAT vector sequence by NdeI/BamHI restriction digest, and the digest products were fractionated by agarose gel electrophoresis. The transgene insert DNA was electroeluted from the gel, concentrated by ethanol precipitation, and dialyzed against microinjection buffer (10 mM Tris-HCl, pH 7.4, 0.15 mM EDTA) for 48 h at 4 °C. As a final step in transgene construct purification, the dialyzed DNA was passed through a 0.2-µm filter.

The distal MCAT site in the human beta 293 promoter was mutated within the plasmid pbeta 293CAT using the QuikChangeTM site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Complementary oligonucleotide primers harboring mutations within the MCAT site were designed to meet the length and melting temperature requirements specified by the manufacturer (mutated bases are underlined): 5'-GCATAGTTAAGCCAGCCAAGCGCGTCTTAGGAGGCCTGGCCTGGG-3'. Base pair substitutions were incorporated at nucleotides determined by our previous diethylpyrocarbonate interference footprinting to be crucial DNA-protein contact sites (25). Unintentional transcription factor recognition sites were not created by these mutations as assessed by cross-referencing the mutated primers against the eukaryotic transcription factor data base available on the Wisconsin Package, version 10.0, Genetics Computer Group (Madison, WI). The PCR-mediated incorporation of mutant sequence was performed using 5 ng of double-stranded pbeta 293CAT DNA template utilizing the manufacturer's recommended conditions. The PCR product was transformed into Epicurian coli XL1-Blue supercompetant cells (Stratagene), and the resulting plasmid DNA was isolated using anion exchange columns (Qiagen EndoFreeTM). Successful incorporation of the mutation was verified via automated sequencing of both strands (Applied Biosystems model 377). The distal MCAT mutant transgene (beta 293Mm) was isolated and purified as described above.

Transgenic Mice-- Transgenic mice were generated by microinjection of purified transgene DNA into pronuclei of single cell fertilized embryos as described previously (26). Transgenic founder mice were identified by Southern blot analysis. Subsequent transgene-positive offspring were identified by PCR amplification of genomic DNA using primers specific for the CAT gene. In the present study, multiple independent transgenic lines representing each transgene (wild type beta 293WT and mutant beta 293Mm) were analyzed. All lines were maintained in a heterozygous state by continual outbreeding to nontransgenic FVB/n mice.

The in vivo presence of the MCAT mutant sequence was verified by PCR amplification of beta 293WT and beta 293Mm mouse genomic DNA. PCR conditions were as described above, and the reactions performed were with both a sense strand primer harboring the mutated MCAT sequence on the 3' terminus (5'-GCATAGTTAAGCCAGCCAAGCGCGTCTTAG-3') and an antisense strand primer specific to sequences within the CAT gene (5'-GGATATATCAACGGTGGTAT-3'). Under these conditions, only genomic DNA harboring the transgene beta 293Mm sequence was amplified resulting in a 450-bp PCR product (see Fig. 1D).

Transgene copy number was estimated as described previously (26). Briefly, 10 µg of transgenic mouse genomic DNA was digested with EcoRI, followed by fractionation through a 1% agarose gel and transfer onto a nylon membrane. The membrane was hybridized with a 32P-labeled CAT cDNA probe used to identify each transgene. The intensity of each signal was quantified with the use of a PhosphorImager (Storm860 with IMAGEQUANT version 5.1 software, Molecular Dynamics) and was compared with that of a previously reported single-copy transgenic line (human beta 1285, line 41) (10). The membrane was stripped and rehybridized with a 32P-labeled c-myc (single copy gene) cDNA probe (350 bp of exon 2) to verify that each lane contained equal amounts of DNA.

Animal Care and MOV Surgical Procedure-- The ablation surgery used in this study was performed as described previously (27). All procedures were conducted using adult female transgenic mice (22-26 g) and were approved by the Animal Care Committee for the University of Missouri-Columbia. MOV was imposed on the hindlimb fast twitch plantaris muscle by surgical removal of the gastrocnemius and soleus muscles. All mice demonstrated normal mobility shortly after recovering from anesthesia. The post-surgical experimental period lasted 8 weeks after which time the overloaded plantaris muscle (MOV-P) was removed. Sham operated mice served as controls for the plantaris (CP) and soleus (CS) muscles.

CAT Assays-- CAT assays were performed as described previously (9, 27) and the percent conversion of [14C]chloramphenicol to the acetylated form was quantified using a PhosphorImager (Storm860) with IMAGEQUANT version 5.1 software.

Preparation of Nuclear Protein Extract from Adult Skeletal Muscle-- Nuclear extracts were isolated from adult rat CP, MOV-P, and CS muscle as described previously (8). Protein concentration was determined according to Bradford (28).

Electrophoretic Mobility Shift Assay-- All oligonucleotide probes used in this study are listed in Table I. EMSAs were carried out as described previously (8). Binding reactions were performed using either 3.5 µg of CP, MOV-P, or CS nuclear extract and 20,000 cpm of labeled probe for 20 min at room temperature in a 25-µl total volume. Where indicated, either in vitro transcribed/translated (TnT) NTEF-1, RTEF-1, embryonic TEF-1, divergent TEF-1, PARP, or Max protein were used in place of muscle nuclear extract (see figure legends for specific quantities). The binding reactions were resolved on a 5% nondenaturing polyacrylamide gel at 220 volts for 2.5 h at 4 °C. Supershift EMSAs were performed by first preincubating skeletal muscle nuclear extract with 2 µl of the corresponding antibody for 30 min at room temperature followed by the addition of the 32P-labeled DNA probe. Following electrophoresis, the gels were dried, and DNA-protein complexes were visualized by autoradiography at -80 °C.


                              
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Table I
Oligonucleotide probes and competitors
Core MCAT-binding elements within the oligonucleotides are delineated in boldface type. cTnT, cardiac troponin T. Cohesive termini on cTnT MCAT oligonucleotide are italicized. MCK Trex, muscle creatine kinase transcriptional regulatory element x. Nucleotides similar to the TEF-1 consensus binding site in MCK Trex oligonucleotides are underlined. MCK E-box, MEF-1 or enhancer right E-box (high affinity). Italicized bases depict sticky ends of non-MCK sequence incorporated for cloning purposes. CM-1 oligonucleotide is derived from the MCK right E-box and randomized with nucleotide substitutions that facilitate binding of c-Myc protein to this sequence. Double underlining represents E-box elements.

In Vitro Transcription and Translation-- In vitro coupled TnT was performed using 1 µg of TEF-1, PARP, or Max expression plasmids in the T7 TnT rabbit reticulocyte lysate kit according to the manufacturer's instructions (Promega). The expression plasmids corresponded to either NTEF-1 (pXJ40-TEF-1A, ORF of human TEF-1 (15)), RTEF-1 (pXJ41-TEF-3, ORF of human TEF-3 (29)), embryonic TEF-1 (pXJ41-TEF-4, ORF of mouse TEF-4 (29)), and divergent TEF-1 (pXJ41-TEF-5, ORF of human TEF-5 (30)), PARP (pBS-II SK+ PARP, full-length human PARP (31)), or Max (pVZ1 p21-Max, full-length human Max (23)). Parallel TnT reactions were performed in the presence of [35S]methionine (PerkinElmer Life Sciences). The integrity and expected molecular weights of the protein products were verified by resolving the radiolabeled reaction products on a SDS-12% polyacrylamide gel. Parallel reactions of lysate not programmed with plasmid DNA were used as negative controls (unprogrammed lysate (UL)).

Antibodies-- The antibodies used in this study were as follows: NTEF-1, mouse polyclonal antibody raised against amino acids 86-199 of human TEF-1 (BD Transduction Laboratories); PARP, rabbit polyclonal antibody raised against full-length human PARP (Roche Molecular Biochemicals); and Max, rabbit polyclonal antibody raised against full-length mouse Max (Upstate Biotechnology). All antibodies listed hereafter were purchased from Santa Cruz Biotechnology, Inc. and include: MyoD, rabbit polyclonal antibody raised against full-length, (amino acids 1-318) mouse MyoD; Myogenin, rabbit polyclonal antibody raised against full-length (amino acids 1-225) of rat myogenin; USF-1, rabbit polyclonal antibody raised against carboxyl terminus amino acids 291-310 of human USF-1; E2A.E12, rabbit polyclonal raised a peptide in the carboxyl terminus of human E47 and corresponds to amino acids 422-439 of human E12; and HEB, rabbit polyclonal antibody raised against a peptide in the carboxyl-terminal domain of human HEB (HTF 4).

Production and Purification of GST Fusion Proteins-- The GST-TEF-1 expression plasmid has been described elsewhere (23). Briefly, full-length rat TEF-1 cDNA was subcloned into the XbaI and XhoI sites of the bacterial expression vector pGEX-KG. Full-length Max cDNA was amplified from pVZ1-p21-Max (23) in PCR reactions using the following primers: sense strand, 5'-CCGCTCCCTGGGCGGATCCAAATGAGCGATACC-3' and antisense strand, 5'-TGGCCTGCCCCGCTCGAGTTAGCTGGCCTC-3'. The amplified cDNA was subcloned into the BamHI and XhoI sites of pGEX-5X-1 (Amersham Pharmacia Biotech). The GST fusion proteins were expressed in Escherichia coli and purified according to the manufacturer's (Amersham Pharmacia Biotech) instructions with modifications. Briefly, bacteria (DH5alpha ) containing the pGEX-KG-TEF-1 and PGEX-5X-1-Max plasmids were grown for 15 h at 37 °C in LB-Amp medium. Subsequently, the cells were diluted 1:100 in fresh medium, grown for 4 h (A600 ~0.7), and then induced to express the fusion protein at 37 °C for 4 h, using 0.4 mM isopropyl-beta -D-thiogalactopyranoside. Following induction, the bacteria were pelleted by centrifugation at 5,000 × g for 10 min at 4 °C, and resuspended in 1× phosphate-buffered saline supplemented with protease inhibitors. The cells were lysed by sonication for two 40-s pulses (while on ice). The protein was solubilized (30 min, 4 °C) by the addition of Triton X-100 to a final concentration of 1% in the sonicated lysate and then centrifuged at 20,000 × g for 10 min at 4 °C to remove insoluble material. Fusion proteins were immobilized by the addition of glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) to the supernatant, and the binding reaction was allowed to proceed at 4 °C for 30 min. The beads were pelleted at 500 × g for 5 min at 4 °C and washed five times with 1× phosphate-buffered saline. The proteins were quantified by SDS-PAGE using known concentrations of bovine serum albumin as a standard and stained with Coomassie Blue.

In Vitro Protein-Protein Interactions-- [35S]Methionine labeled TnT proteins corresponding to NTEF-1, PARP, and Max were incubated with 2 µg of immobilized GST, GST-TEF-1, or GST-Max with continuous rocking for 3 h at 4 °C in a modified 1× protein interaction buffer described by Gupta et al. (23); 20 mM HEPES, pH 7.5, 75 mM KCl, 1 mM EDTA, 2 mM MgCl2, 2 mM dithiothreitol, and 1% Triton X-100. Unprogrammed lysate that was also transcribed and translated in the presence of [35S]methionine was used as the negative control. After incubation the glutathione-Sepharose beads were pelleted by centrifugation at 500 × g and washed five times with ice-cold 1× protein interaction buffer. The fusion proteins were eluted off the beads by heating at 95 °C for 3 min in the presence of 1× SDS sample buffer and analyzed on a 4-20% gradient SDS-PAGE gel. The gel was dried and exposed to film for 15 h at room temperature.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mutation of the Distal MCAT Element Decreased Slow Fiber Expression of Transgene beta 293Mm-- We initiated our investigation into the in vivo functional role served by the beta MyHC distal MCAT element in adult stage skeletal muscle by generating multiple independent lines of two classes of transgenic mice (Fig. 1, A-C). The first class of transgenic mouse carries a wild type transgene comprised of 293 bp of human beta MyHC promoter fused to the CAT reporter gene (termed transgene beta 293WT), whereas the second class carries the same transgene except that the highly conserved distal MCAT element has been mutated (termed transgene beta 293Mm) (Fig. 1, A-C). Nucleotide mutations were introduced into the core distal MCAT element by base pair substitution and reflected sites of strong DNA-protein interaction as assessed by our previous diethylpyrocarbonate interference footprint analysis (25).



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Fig. 1.   beta MyHC distal MCAT sequence and mutation. A, nucleotide sequence comparison showing conservation of the distal MCAT element (shading) within the beta MyHC proximal promoter of various species. See Vyas et al. (8) for the accession number and reference of each sequence. A gap (dot) was inserted in the pig sequence to optimize the alignment. B, illustration of the 293-bp beta MyHC wild type and MCAT mutant transgenes analyzed in this study. Transgenes consist of 293 bp of human beta MyHC 5'-promoter sequence and 120 bp of 5'-untranslated region linked to the CAT reporter cDNA. The open box within the 5'-untranslated region represents the first untranslated exon. C, mutation of the distal MCAT binding site (shading) with base pair alterations shown in lowercase and boldface type. D, in vivo verification of MCAT mutant sequence by PCR amplification of beta 293Mm transgenic mouse genomic DNA on a 1% agarose gel stained with ethidium bromide. Under the described PCR conditions (see "Experimental Procedures" for details), only genomic DNA harboring the MCAT mutant transgene sequence (beta 293Mm lines 4, 6, 7, and 9, lanes 3-6) produced a 450-bp PCR product (arrow). Because of the 7-bp mismatch (overlapping the distal MCAT core sequence) at the 3' terminus of the sense strand primer, no PCR product was amplified in genomic DNA of beta 293 wild type transgenic mice (lanes 7-9). +, positive control, amplified product from purified beta 293 MCAT mutant transgene DNA construct (lane 2). MW, DNA size marker (in base pairs) is shown in lane 1.

To assess whether the distal MCAT element was solely responsible for directing both muscle-specific and high levels of slow fiber expression, we measured the CAT specific activity (pmol/µg of protein/min) in muscle and nonmuscle tissues obtained from transgenic mice carrying mutant transgene beta 293Mm. The CAT specific activity measured in the CS and CP muscles of mice representing each of the four independent beta 293Mm lines was significantly decreased as compared with CAT specific activities measured in the CS and CP muscles obtained from mice representing each of the three independent beta 293WT lines (Fig. 2, A and B, and Table II). Our analysis of the beta 293Mm transgenic lines did not reveal measurable levels of CAT specific activity in any nonmuscle tissue examined even under conditions where excess protein and extended incubation times were used (50 µg protein/overnight incubation) (data not shown). These data indicate that the distal MCAT element is not required to direct muscle specific expression of transgene beta 293 in the chromosomal context. Because this result was observed for all four independent transgenic lines each carrying different transgene copy numbers, it is unlikely that chromosomal position or transgene copy number accounts for these results.



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Fig. 2.   Effects of MCAT element mutation on beta 293 CAT specific activity. A, representative CAT assay demonstrating the expression levels in CS extracts of beta 293 wild type and MCAT mutant transgenic lines. All samples were incubated at 30 µg for 17 h to illustrate the low expression levels of the MCAT mutant lines relative to the wild type lines. Extracts obtained from nontransgenic (Ntg) mouse soleus muscle served as the negative control, and purified CAT protein (70 ng) was used as a positive control. B, bar graph represents comparison of wild type (black bars) and MCAT mutant (gray bars) beta 293 transgene expression levels in soleus muscle. Error bars correspond to the S.E. for six or more independent animals for each line.


                              
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Table II
Response of wild-type beta 293 (beta 293wt) and MCAT mutant beta 293 (beta 293Mm) transgene activity
Values are expressed as means ± S.E. CAT specific activity (pmol/µg of protein/min) were measured by incubation of protein extracts with 20 mM acetyl-CoA and [14C]chloramphenicol at 37 °C. Incubation of muscle protein extracts representing each transgenic line was performed as follows: beta 293wt, line 2, CP/MOV-P, 20 µg/60 min; CS, 10 µg/60 min; line 96, CP/MOV-P/CS 7.5 µg/30 min; Line 99, CP/MOV-P/CS 7.5 µg/30 min. beta 293Mm, CP/MOV-P for lines 4, 6, 7, and 9, 30 µg/17 h; CS, lines 4 and 7, 30 µg/60 min; line 6, 15 µg/60 min; line 9, 10 µg/17 h. Note that CAT-specific activity for beta 293 wild-type lines 96 and 99 have been previously reported by McCarthy et al. (9).

Mutation of the Distal MCAT Element Does Not Eliminate MOV Responsiveness of Transgene beta 293Mm-- Our previous transgenic analysis has revealed that the beta MyHC distal MCAT element (-290/-284) is located within an 89-bp region (-293/-205) that is required for MOV responsiveness of transgene beta 293WT (4). Therefore, to determine whether the distal MCAT element functions as an beta MyHC MOV-responsive element, CAT specific activity was measured in sham operated CP and MOV-P muscles of transgenic mice carrying either transgene beta 293WT or beta 293Mm following an 8-week period of MOV. For mice representing each of the three independent lines carrying transgene beta 293WT, expression assays revealed that the CAT specific activity measured in MOV-P muscle extract was 2.9-7.5-fold higher than that measured in sham operated CP muscle extract (Table II). Interestingly, following the 8-week MOV period, the CAT specific activity measured in the MOV-P muscle obtained from mice representing each of the four independent beta 293Mm transgenic lines was also 1.5-7.1-fold higher than that measured in corresponding CP muscles (Table II).

Collectively, our transgenic analysis demonstrates that the beta MyHC distal MCAT element is required for basal slow muscle fiber expression levels of transgene beta 293 in both soleus and plantaris muscles. However, this element is not required for MOV responsiveness because its mutation did not alter the relative fold induction of transgene beta 293Mm in the MOV-P muscle.

Multiple Nuclear Protein Binding Complexes Form at the beta MyHC Distal MCAT Element-- To determine whether the nuclear protein binding properties of the distal MCAT element differed between muscles comprised of different proportions of histochemically classified slow type I fibers, we performed an EMSA analysis using nuclear extracts isolated from rat CS (78-90% type I fibers), CP (4-8% type I fibers), and MOV-P (30% type I fibers) muscles. EMSA analysis of binding reactions containing the 21-bp double-stranded 32P-labeled wild type distal MCAT probe (Table I) and 3.5 µg of either CP, MOV-P, or CS nuclear extract revealed the formation of multiple binding complexes (Fig. 3). These binding complexes are referred to as LMC, intermediate mobility complex (IMC), and high mobility complex (HMC). The LMC appeared as a single band that varied in quantity concordant to the proportion of slow type I fibers populating the muscles from which the nuclear extract was isolated, that is, CS > MOV-P > CP. The IMC consisted of two distinct bands whose intensity appeared to vary inversely to the amount of LMC that was formed. The high mobility complex consisted of multiple bands; however, only one band was easily distinguishable in our experiments regardless of the amount of nuclear extract used or the length of time the gel was exposed to film.



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Fig. 3.   EMSA analysis of skeletal muscle nuclear extract binding at the distal MCAT element. 32P-Labeled distal MCAT probe was incubated with 3.5 µg of either CP, MOV-P, or CS nuclear extract. Binding complexes were resolved by PAGE as described under "Experimental Procedures." Note the enriched DNA-protein interactions at the LMC for the MOV-P (lane 2) and CS (lane 3) nuclear extract but not for the CP (lane 1) nuclear extract. Free probe represents excess unreacted radiolabeled oligonucleotide.

The LMC Formed at the beta MyHC and cTnT Distal MCAT Elements Are Similar-- Interestingly, Larkin et al. (20) have recently reported a similar binding pattern (LMC, IMC, HMC) as assessed by EMSA analysis using the cardiac troponin T (cTnT) MCAT elements (MCAT1 and MCAT2) and chicken embryonic skeletal muscle nuclear extract. Therefore, to address the possibility that the same proteins bind to the beta MyHC distal MCAT and cTnT MCAT elements, we performed competition EMSA using as competitor the cTnT and alpha MyHC MCAT elements (Fig. 4). The addition of 100-fold molar excess cold wild type distal beta MyHC MCAT probe to binding reactions containing either CP (Fig. 4, lane 1 versus lane 2), MOV-P (lane 6 versus lane 7), or CS (lane 11 versus 12) nuclear extract completely abolished complex formation, indicating that these complexes are specific. Interestingly, the addition of 100-fold molar excess cold cTnT MCAT1 probe to binding reactions containing either CP (Fig. 4, lane 1 versus lane 3), MOV-P (lane 6 versus lane 8), or CS (lane 11 versus lane 13) prevented IMC and HMC formation and competed away most of the LMC formation. Similarly, competition using the alpha MyHC E-box/MCAT composite element completely abolished the formation of the IMC and HMC and abolished nearly all of the LMC (data not shown). As an additional competitor, we used the muscle creatine kinase transcriptional regulatory factor x element that has previously been shown not to bind TEF-1 when using TEF-1-containing MM14 nuclear extract, despite its relative sequence homology to MCAT consensus elements (32). The addition of 100-fold molar excess cold transcriptional regulatory factor x element to binding reactions containing either CP (Fig. 4, lane 1 versus lane 4), MOV-P (lane 6 versus lane 9), or CS (lane 11 versus lane 14) nuclear extract did not effectively compete for the formation of the LMC, IMC, or HMC.



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Fig. 4.   Competition EMSA assessment of sequence-specific DNA-protein binding at the distal MCAT element. 3.5 µg of either CP (lanes 1-5), MOV-P (lanes 6-10), or CS (lanes 11-15) nuclear extract was incubated in the presence of the 32P-labeled distal MCAT oligonucleotide. For analysis of sequence specificity of binding at this MCAT element, the following cold (nonradioactive) competitors were added to the reaction mixture at a 100-fold molar excess prior to the addition of the probe: distal MCAT (lanes 2, 7, and 12), cTnT MCAT (lanes 3, 8, and 13), MCK transcriptional regulatory factor x (lanes 4, 9, and 14), and the MCK high affinity right E-box (lanes 5, 10, and 15).

Because adjacent E-box elements have been shown to be important for nuclear protein binding at the alpha MyHC and cTnT MCAT elements, we used the high affinity MCK E-box as competitor to determine whether the E-box in the immediate 5'-flanking region of the beta MyHC distal MCAT element is required for complex formation. Addition of 100-fold molar excess cold MCK E-box element to binding reactions containing either CP (Fig. 4, lane 1 versus lane 5), MOV-P (lane 6 versus lane 10), or CS (lane 11 versus lane 15) nuclear extract slightly abolished formation of the LMC only. Collectively, our direct and competition EMSA data provide evidence that: 1) nuclear protein binding at the beta MyHC distal MCAT element is specific, 2) distinct patterns of nuclear protein binding complexes were formed when using either CP, MOV-P, or CS nuclear extracts, and 3) the nuclear proteins forming the LMC preferentially bind the beta MyHC distal MCAT element versus the cTnT and alpha MyHC MCAT elements. This difference is likely due to sequence specific differences between these three MCAT elements (Table I).

Nucleotides Comprising the beta MyHC Distal Core MCAT Element Are Essential for Skeletal Muscle Nuclear DNA-Protein Binding Complex Formation-- To identify nucleotides within the 21-bp beta MyHC distal MCAT oligonucleotide probe that interact with MOV-P and CS nuclear protein to form the LMC, IMC, and HMC, we performed competition EMSA using scanning mutagenesis. In these experiments, we introduced nucleotide substitutions two base pairs at a time starting within the immediate 5'-flanking region (E-box) and extending throughout the core MCAT element and its immediate 3'-flanking region (Fig. 5A). The resulting unlabeled oligonucleotides were then added in 100-fold molar excess to binding reactions containing either MOV-P or CS nuclear extract and the 32P-labeled wild type distal MCAT probe (Fig. 5B). As shown previously, the addition of excess wild type beta MyHC distal MCAT probe to binding reactions containing either MOV-P (Fig. 5B, lane 1 versus lane 2) or CS (lane 11 versus lane 12) nuclear extract completely abolished the formation of all binding complexes. Similarly, all binding complexes were effectively competed away by the addition of either distal MCAT mut-1, mut-2 (contain mutations within E-box), or mut-7 (3'-flanking region) to binding reactions containing either MOV-P (Fig. 5B, lane 1 versus lanes 3, 4, and 9) or CS (lane 11 versus lanes 13, 14, and 19) nuclear extract. Importantly, mutant MCAT probes carrying nucleotide substitutions within the core MCAT element (mut-3, mut-4, mut-5, and mut-8) did not act as effective competitors of complex formation when added to binding reactions containing MOV-P (Fig. 5B, lane 1 versus lanes 5-7 and 10) or CS (lane 11 versus lanes 15-17 and 20) nuclear extract. Interestingly, the addition of distal MCAT mut-6 probe to binding reactions containing MOV-P (Fig. 5B, lane 1 versus lane 8) or CS (lane 11 versus lane 18) nuclear extract completely abolished LMC formation but not IMC or HMC formation. These data confirm our previous findings using diethylpyrocarbonate interference footprinting (25) by showing that nucleotides comprising the core beta MyHC distal MCAT element are critical for the formation of all binding complexes when using either MOV-P or CS nuclear extract (Fig. 5C).



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Fig. 5.   Competition EMSA determination of specific nucleotides involved in DNA-protein interactions at the distal MCAT element. A, summary of MCAT mutant oligonucleotides used in competition EMSA shown in B. wt, wild type or nonmutated distal MCAT sequence. The core MCAT sequence is indicated with shading. Mutated bases are lowercase and in boldface type. +++, competition was as effective as distal MCAT wild type sequence; ++, partially effective competition; -, no detectable competition. B, 32P-labeled distal MCAT oligonucleotide was incubated in the presence of either MOV-P (lanes 1-10) or CS (lanes 11-20). Where indicated, 100-fold molar excess of either distal MCAT oligonucleotide (lanes 2 and 12) or various distal MCAT mutant oligonucleotides (harboring base pair mutations shown in A) were added to the reaction prior to the addition of the probe. C, distal MCAT oligonucleotide with MCAT core sequence is shaded, and E-box sequence is in boldface type. Two base pair numbering above the oligo sequence represents specific nucleotide mutation. The black box delineates nucleotides whose mutation renders the distal MCAT an ineffective competitor (especially of the LMC), suggesting this region as a putative binding site for the LMC. Diethylpyrocarbonate interference footprint (25) is shown here to illustrate the DNA-protein binding profile. Open circles denote partial interference; closed circles depict complete interference. Sequence numbering begins at the 5'-end of the sense strand.

NTEF-1, PARP, and Max Comprise the LMC Formed at the beta MyHC Distal MCAT Element-- Previous work has shown that TEF-1 and Max interact at the alpha MyHC E-box/MCAT composite element, and that TEF-1 and PARP associate at the cTnT MCAT1 elements (22, 23). In addition, Max has been identified as a component of the cTnT MCAT binding complex formed when using neonatal cardiomyocyte nuclear extracts (23). Thus, to assess whether these nuclear proteins were components of the LMC, IMC, and HMC formed at the beta MyHC distal MCAT element, we performed antibody EMSAs using polyclonal antibodies that recognize each of the aforementioned proteins. The specific binding complexes formed at the 32P-labeled human beta MyHC distal MCAT element when reacted with CP (Fig. 6, lane 1 versus lane 2), MOV-P (lane 4 versus lane 5), or CS (lane 13 versus lane 14) nuclear extract were not altered by preincubation with preimmune serum. The addition of polyclonal NTEF-1 antibody to binding reactions containing CP (lane 1 versus lane 3), MOV-P (lane 4 versus lane 6), or CS (lane 13 versus lane 15) nuclear extract supershifted the top band of the IMC doublet and the prominent HMC band. In addition, the enriched LMC observed when using MOV-P nuclear extract appeared to be nearly immunodepleted, whereas the highly enriched LMC formed when using CS nuclear extract was partially abolished. Interestingly, the addition of either polyclonal PARP or Max antibody to binding reactions using MOV-P nuclear extract essentially immunodepleted the LMC, which resulted in an embellishment of the top band of the IMC, which is comprised entirely of NTEF-1 protein (Fig. 6, lane 4 versus lanes 7 and 8). Identical results were obtained when CS nuclear extract was used in binding reactions with the exception that the highly enriched LMC was again only partially abolished (Fig. 6, lane 13 versus lanes 16 and 17). When all combinations of the three polyclonal antibodies were added to binding reactions using MOV-P (Fig. 6, lane 4 versus lanes 9-12) or CS (lane 13 versus lanes 18-21) nuclear extract, the LMC was immunodepleted. The preincubation of binding reactions containing either CS or MOV-P nuclear extract with antibodies recognizing other E-box binding basic helix-loop-helix and basic helix-loop-helix leucine zipper proteins (MyoD, myogenin, E2A, HEB, and USF) did not alter beta MyHC distal MCAT element binding complex formation, or mobility (data not shown). These data support three notable conclusions: 1) the LMC formed at the human beta MyHC distal MCAT element is comprised of at least three proteins antigenically related to NTEF-1, PARP and Max, 2) the prominent HMC band and the top band of the IMC doublet are comprised of protein antigenically related to NTEF-1 or possibly TEF-1 isoprotein(s) recognized by the NTEF-1 polyclonal antibody used in these experiments, and 3) the bottom band of the IMC doublet was not supershifted/immunodepleted by the addition of any antibodies used herein and thus may be comprised by a TEF-1 isoform not recognized by the polyclonal NTEF-1 antibody or an as yet unidentified nuclear protein(s).



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Fig. 6.   Antibody supershift EMSA analysis of muscle nuclear extract binding complexes at the distal MCAT element. Supershift EMSAs were performed by preincubation of CP (TEF-1 Ab only, lane 3), MOV-P, or CS nuclear extract with 1 µl of anti-Max, anti-PARP, or anti-TEF-1 Ab for 30 min at room temperature prior to the addition of the labeled distal MCAT probe. Various combinations of antibodies were used as indicated by the plus sign above each lane. TEF-1 SS marks the supershifted TEF-1 components of the IMC and HMC. Autoradiographic exposure time for the CS complexes (lanes 13-21) was reduced to visualize the effects of the various antibodies on the LMC without interference from the supershifted TEF-1 complex. Control reactions were performed with preimmune serum (PI; lanes 2, 5, and 14). Ab, antibody.

TEF-1, but Not PARP or Max, Specifically Binds to the beta MyHC Distal MCAT Element in Vitro-- Because our antibody supershift assays indicate that proteins antigenically related to Max, NTEF-1, and PARP comprise the LMC formed at the beta MyHC distal MCAT element, it was important for us to determine the ability of these three factors to independently bind to the 32P-labeled human beta MyHC distal MCAT element. EMSA analysis of binding reactions containing the 32P-labeled human beta MyHC distal MCAT element and rabbit reticulocyte lysate-generated in vitro translated TEF-1 isoproteins (Fig. 7A, inset shows expected 54-kDa TEF-1 proteins) revealed the formation of specific binding complexes that were different from the nonspecific complex formed when unprogrammed lysate was used (Fig. 7A, lane 1 versus lanes 2-5). In contrast, a binding complex was not formed when either [35S]methionine-labeled in vitro translated PARP (Fig. 7B, inset shows expected 120-kDa PARP protein) or Max (Fig. 7C, inset shows expected 22-kDa Max protein) were reacted with the beta MyHC distal MCAT element (Fig. 7B, lanes 1-4, Max, and data not shown). The inability of PARP or Max to form a binding complex with the beta MyHC distal MCAT element remained, even when using 10-100 ng of either purified recombinant baculovirus expressed PARP (data not shown) or Max (Fig. 7C, lanes 1-3). Further, we were unable to reconstitute the formation of a beta MyHC distal MCAT LMC in our in vitro binding assays despite the simultaneous use of TEF-1, PARP, and Max, in binding reactions (data not shown). We were, however, able to detect DNA-protein binding when adding either 5 or 10 ng of purified Max to binding reaction containing the CM-1 oligonucleotide (contains (33) previously shown Myc/Max E-box binding site CACGTG (23, 34)) (Fig. 7C, lanes 4-6), indicating that the purified Max protein used in our LMC reconstitution experiments possessed DNA binding capabilities.



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Fig. 7.   EMSA analysis of recombinant TEF-1, PARP, and Max binding at the distal MCAT element. A, inset, reveals the correct size of [35S]methionine-labeled TEF-1. The rabbit reticulocyte lysate system was programmed with 1 µg of circular TEF-1 expression plasmids in the presence of [35S]methionine. The TnT product was resolved by 12% SDS-PAGE and exposed to film. Molecular mass markers (in kDa) are shown on the left. Main panel, 32P-labeled distal MCAT probe was incubated with rabbit reticulocyte lysate corresponding to various TEF-1 isoforms as indicated (lanes 2-5). ---, labeled distal MCAT electrophoresed in the absence of protein extract (lane 6). NS, nonspecific binding endogenous to the rabbit reticulocyte lysate system. B, inset, reveals the correct size of [35S]methionine-labeled PARP. Main panel, distal MCAT probe was incubated in the presence of UL (lane 1) or increasing amounts (1, 5, and 10 µl) of PARP cDNA programmed rabbit reticulocyte lysate. No difference was observed in sequence-specific DNA-protein binding between the UL and PARP programmed reactions. NS, nonspecific binding endogenous to the UL. C, left inset, reveals correct size of [35S]methionine-labeled Max. Right inset, Coomassie-stained SDS-PAGE shows correct size of recombinant baculovirus expressed Max (200 ng). Main panel, radiolabeled distal MCAT (lanes 1-3) or CM-1 (lanes 4-6) were incubated with purified baculovirus expressed Max in the amounts indicated.

NTEF-1, PARP, and Max Stably Interact-- Our DNA-protein binding assays revealed that of the three proteins tested, only NTEF-1 binds the beta MyHC distal MCAT element; therefore, the protein-protein interactions within our LMC remained to be identified. Importantly, previous work has shown a stable protein-protein interaction between RTEF-1 and PARP (22) and between NTEF-1 and Max (23), and, therefore, it was logical to test for protein-protein interactions between NTEF1, PARP, and Max. To this end, we performed in vitro pairwise protein-protein interaction assays wherein [35S]methionine-labeled in vitro translated Max, NTEF-1, or PARP protein were individually reacted with either bacterially expressed GST-NTEF-1 or GST-Max fusion proteins that were bound to glutathione-Sepharose beads (Fig. 8). Following incubation, the pelleted glutathione-Sepharose beads/fusion protein complex was extensively washed, and this complex was analyzed for bound [35S]methionine-labeled in vitro translated protein by using SDS-PAGE (Fig. 8). Importantly, [35S]methionine-labeled in vitro translated PARP protein was found to bind both GST-Max (Fig. 8A, lane 3) and GST-NTEF-1 (Fig. 8C, lane 3) but not the 26-kDa GST protein (Fig. 8, A and C, lane 2), revealing that this interaction was specific between each respective fusion protein and in vitro translated PARP protein. Similarly, [35S]methionine-labeled in vitro translated NTEF-1 and Max were found to bind specifically to GST-Max (Fig. 8B, lane 2 versus lane 3) and GST-NTEF-1 (Fig. 8D, lane 2 versus lane 3), respectively. In all analyses described above, the [35S]methionine-labeled in vitro translated protein, bound by the GST fusion protein, migrated to a position in the SDS-polyacrylamide gel that aligned with the [35S]methionine-labeled in vitro translated protein that was not reacted with GST fusion protein (Fig. 8, A-D, lanes 1 versus lanes 3). When unprogrammed rabbit reticulocyte lysate was reacted with either GST, GST-NTEF-1, or GST-Max, a [35S]methionine-labeled in vitro translated protein product was not observed, further demonstrating specificity of the interactions between the fusion protein and the in vitro translated product (Fig. 8, A-D, lanes 4-6). Collectively, these data indicate that the assembly of the beta MyHC distal MCAT multiprotein LMC involves DNA binding of TEF-1, and that TEF-1, PARP, and Max are capable of protein-protein interactions between each other. However, it remains to be determined conclusively whether the LMC involves a ternary complex comprised of these proteins or alternatively two subsets of paired protein complexes (see "Discussion").



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Fig. 8.   Protein-protein interactions between TEF-1, PARP, and Max. Full-length Max and NTEF-1 were prepared as GST fusion proteins and immobilized on Sepharose beads as described under "Experimental Procedures." Immobilized GST, GST-Max, or GST-TEF-1 was incubated with [35S]methionine-labeled PARP TnT (A and C, lanes 2 and 3), NTEF-1 TnT (B, lanes 2 and 3), or Max TnT (D, lanes 2 and 3). The labeled proteins bound to the beads were analyzed by 4-20% SDS-PAGE. Positive control labeled TnT reactions are shown for each panel in lane 1. Molecular mass markers in kDa are shown to the left of each panel.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MCAT elements are known to participate in directing muscle-specific expression. Our transgenic findings herein show that the control region distal MCAT element (-290/-284) contributes significantly to basal slow muscle expression of chromosomally integrated transgene beta 293 but is not absolutely required for MOV responsiveness. Furthermore, our in vivo protein-protein interaction studies elucidate the possibility that in adult skeletal muscle NTEF-1, PARP, and Max may physically interact to form a ternary binding complex at the beta MyHC distal MCAT element.

Transgenic Mouse Analyses of the in Vivo Function of the Human beta MyHC Distal MCAT Element Reveals That It Contributes to Basal Slow Fiber Expression but Is Not Absolutely Required for MOV Responsiveness-- In adult mice, basal expression levels of the endogenous beta MyHC gene is high in the slow twitch soleus muscle, whereas it is not expressed to any appreciable degree in the fast twitch plantaris muscle, an expression pattern mimicked by wild type transgene beta 293WT (4, 9). Our analysis of mutant transgene beta 293Mm basal expression revealed that mutation of the distal MCAT element significantly decreased but did not abolish expression in the slow twitch soleus muscle (Fig. 2, A and B, and Table II). In fact, even though the magnitude of mutant transgene beta 293Mm expression was decreased, its expression pattern (soleus > plantaris) still mirrored that of wild type transgene beta 293WT. These results showing low, yet persistent expression of mutant transgene beta 293Mm strongly suggest that other cis-acting elements within the 293-bp human beta MyHC basal promoter must be acting in concert to preserve wild type levels of slow muscle expression. Candidate sites within the beta MyHC proximal control region (-293/-170) that may act in combination with the distal MCAT element to direct high levels of slow muscle expression are an A/T-rich (-269/-258), CCAC/Sp1 (C-rich, -242/-231), and NFAT (-179/-171). In this regard, our results from EMSA and transgenic mouse analysis indicate that the beta MyHC NFAT and beta A/T-rich elements contribute to slow muscle expression because their independent mutation in the context of transgene beta 293 (beta 293Nm, beta 293A/Tm) significantly decreased slow muscle expression.2 Our data provide evidence that the combined inputs from the distal MCAT, beta A/T-rich, and beta NFAT elements underlie transgene beta 293WT slow muscle gene expression in adult transgenic mice. Indirect support for this notion comes from recent findings implicating NFAT and MEF2 proteins as downstream mediators of calcium-activated intracellular signaling pathways that have been proposed to regulate the slow gene program (Ref. 39 and references within). Additional work is underway to clarify whether these three elements collaborate to direct slow muscle specific expression.

Because MCAT elements have been shown to function as inducible promoter elements in response to mechanical stimuli (13)y and the distal MCAT element resides within the 89-bp region required for MOV responsiveness of transgene beta 293WT, we investigated its role in MOV-inducible expression. Our analysis revealed that the range of MOV-induced CAT specific activity measured in the MOV-P muscle of mice harboring mutant transgene beta 293Mm was almost identical to those carrying wild type transgene beta 293WT, indicating that the distal MCAT element is not absolutely required for MOV responsiveness of transgene beta 293 (Table II). Although MOV induction could still be achieved when the distal MCAT element was mutated (beta 293Mm), this element still appears to be necessary to achieve high levels of transgene beta 293 induction in response to MOV (Table II), implicating the requirement for additional element(s). In support of this notion, we have recently identified by EMSA analysis a putative MOV element (A/T-rich element (-269/-258)) that demonstrates enriched binding of two unidentified nuclear proteins (44 and 48 kDa) only under MOV conditions (8). Thus the beta A/T-rich and distal MCAT elements may participate collaboratively to confer MOV responsiveness to transgene beta 293, a study currently under investigation.

In addition to our mutant MCAT transgenic findings, our EMSA analysis provides supportive evidence that the distal MCAT element contributes to slow fiber expression in the control soleus and MOV-P muscle. In direct and competition EMSA experiments we observed the formation of a specific LMC that varied in intensity according to the proportion of slow type I fibers within the adult stage rat skeletal muscles from which nuclear extract was isolated (Fig. 3). This EMSA binding pattern (CP < MOV-P < CS) is relevant to the in vivo expression pattern of our transgenes because the mouse and rat display a qualitatively similar pattern of slow type I fibers within these muscles.

Mutant Transgene beta 293Mm Does Not Display a Loss of Muscle Specific Expression-- Previous studies by Rindt et al. (7) reported that mutation of the distal MCAT element within a 600-bp mouse beta MyHC promoter (beta 0.6MCAT) led to the loss of muscle specific expression (7). Regardless of the aforementioned findings, we did not detect measurable levels of transgene beta 293Mm in any nonmuscle tissue examined (data not shown). Several explanations may account for these different observations. First, the loss of mouse beta MyHC transgene beta 0.6MCAT muscle specific expression was examined in only one transgenic line; therefore, this result may reflect chromosomal integration site effects as opposed to the loss of promoter regulation. Second, the levels of human transgene beta 293Mm studied herein are on average lower than those of mouse transgene beta 0.6MCAT, which may have resulted in our missing possible aberrant (nonmuscle) expression. Last, our mutation was restricted to a 5-nucleotide substitution within the core MCAT element, whereas the mutation within mouse transgene beta 0.6MCAT involved substitution of 19 nucleotides that spanned the core MCAT as well as both 5' and 3' regions (7). Based on the findings of Larkin et al. (20) concerning the requirement of the MCAT 5'-flanking nucleotides to achieve cTnT muscle specific gene expression, it is reasonable to suggest that mutation of the immediate 5'-flanking region may have altered muscle specific expression.

The beta MyHC Distal MCAT LMC Is a Multiprotein Complex and Is Likely Comprised of TEF-1, PARP, and Max-- Because of the relevance of previous findings showing physical interactions between NTEF-1 and Max (23) as well as RTEF-1 and PARP (22), it was important that we determine whether these same proteins comprise the LMC that forms at the beta MyHC distal MCAT element when using adult stage MOV-P or soleus nuclear extract. Our EMSA and protein-protein interaction assays provide multiple lines of evidence that TEF-1, PARP, and Max most likely comprise a ternary protein complex that binds the beta MyHC distal MCAT element to form the LMC. First, EMSA analysis revealed the formation of a LMC with a mobility that resembled the LMC formed at the cTnT element (Fig. 3). Second, in competition EMSAs, oligonucleotides containing either the alpha MyHC E-box/MCAT site or the cTnT MCAT1 element abolished the formation of most but importantly not all of the LMC formed at the beta MyHC distal MCAT element. This is most apparent when using CS nuclear extract (Fig. 4, lane 13). Alternatively, these findings might also suggest that the proteins comprising the LMC at these three different MCAT elements may be the same but reflect differences in binding affinity. Third, the use of polyclonal anti-NTEF-1, PARP and Max, antibodies revealed that the LMC contained proteins antigenically related to TEF-1, PARP, and Max (Fig. 6). Fourth, our in vitro protein-protein interaction assays show that NTEF-1, PARP, and Max all physically interact with each other (Fig. 7). Alternatively, when considering the third and fourth issues above, it remains possible that two different populations of LMC exists in the form of TEF-1/PARP and TEF-1/Max. Although this is possible, it seems unlikely because there would be a difference of approximately 100-kDa between these two distinct LMCs. For example, a PARP (120-140 kDa)/TEF-1 (54 kDa) protein complex would have a mass of 174-194 kDa, whereas a TEF-1/Max (22 kDa) protein complex would have a mass of 76 kDa. This 100-120 kDa difference in mass would likely be resolved in our EMSA assays. Last, in DNA binding assays, our experiments reveal that of the three proteins tested, only in vitro synthesized TEF-1 isoproteins bind to the beta MyHC distal MCAT element (Fig. 8A), indicating that the putative ternary protein complex is tethered to this element via TEF-1 DNA-protein binding.

Despite the fact that some of the nuclear proteins that interact at the beta MyHC, cTnT, and alpha MyHC MCAT elements are the same, our experiments show that the DNA binding properties of the beta MyHC distal MCAT element differ, possibly explaining why Max and PARP did not bind this element. This important distinction is supported by differences in footprint patterns obtained for the beta MyHC, cTnT, and alpha MyHC MCAT elements. Our footprint analysis (25) in conjunction with our current scanning mutagenesis results provide evidence that strong DNA-protein interactions occurred throughout the core MCAT element with weaker interactions in the flanking regions (Fig. 5). In contrast, similar analysis of the cTnT MCAT1 element revealed strong DNA-protein interactions occurring at the 5'-flanking nucleotides, the proposed site (5'-TGTTG-3') of PARP binding that is conspicuously absent from the beta MyHC distal MCAT oligonucleotide (Refs. 20 and 22 and Table I). Likewise, analysis of the alpha MyHC E-box/MCAT hybrid element revealed strong DNA-protein interactions spanning the 5'-flanking nucleotides containing the high affinity Max target binding site (CACGTG) that is also absent from the beta MyHC distal MCAT oligonucleotide (Ref. 40 and Table I). The lack of Max DNA binding to our MCAT element was not completely unexpected because Max was identified as a neonatal cardiomyocyte nuclear protein component of a binding complex that formed at a cTnT core MCAT oligonucleotide devoid of an E-box element (23). Consequently, this same oligonucleotide was shown to be incapable of binding purified Max protein, showing that Max can participate in transcriptional regulation without binding DNA.

Although not an absolute certainty, we suggest that the conditions of our in vitro binding assays do not adequately emulate the in vivo biological conditions reflected in our adult stage skeletal muscle nuclear extract, thereby precluding independent binding by both Max and PARP, as well as the reconstitution of a LMC. For example, it is possible that something in the rabbit reticulocyte lysate may have inhibited DNA binding by both PARP and Max. Alternatively, these proteins may require a post-translational modification that occurs only in the context of intact adult stage skeletal muscle. Nevertheless, our data provide evidence that the beta MyHC distal MCAT element LMC is primarily bound by TEF-1 protein and that PARP and Max may weakly interact with flanking nucleotide.

In summary, our results provide convincing evidence that formation of the beta MyHC distal MCAT LMC when using either MOV-P or soleus nuclear extract is comprised of TEF-1, PARP, and Max and that this interaction correlates with slow muscle expression but is not required for MOV responsiveness of wild type transgene beta 293WT. Mechanistically, our findings suggest that TEF-1 binds to the core MCAT nucleotides where it serves to activate transgene beta 293WT transcription. Because Max does not contain a transactivation domain, its presence in the LMC may serve to mediate favorable interactions between the transcription initiation complex, as well as transcription factors bound at adjacent cis-acting elements via protein-protein interactions. PARP, on the other hand, has been shown to participate in diverse processes that include DNA repair, chromatin remodeling, and gene transcription (41). Thus, it is reasonable to speculate that nuclear PARP is recruited to the beta MyHC distal MCAT element to activate beta MyHC gene expression in slow muscle by altering local chromatin structure, or by modulating transcription factor activity via poly(ADP)-ribosylation. Support for the later notion comes from recent evidence showing the involvement of calcium in the activation of nuclear PARP in neuronal cells (42) and calcium-activated intracellular signaling pathways in regulating, in part, the slow skeletal muscle gene program (39). In our studies, calcium-activated mechanisms should be carefully considered because slow type I skeletal muscle fibers have been reported to have 2-6-fold higher basal levels of intracellular calcium than fast type II fibers (see Ref. 39 and references within), and MOV is associated with an increased proportion of slow type I fibers (6). Thus, it is plausible that the higher basal levels of intracellular calcium within the soleus muscle and within the induced slow type I fibers populating the plantaris following MOV resulted in elevated levels of activated nuclear PARP and thus enhanced LMC formation (Figs. 3 and 4). The physiological importance of the formation of the beta MyHC distal MCAT LMC is underscored by our transgenic mouse analysis wherein mutation of the distal MCAT element led to a significant decrease in slow muscle expression of chromosomally located transgene beta 293Mm. The investigation into the involvement of calcium-activated pathways in the MOV induced increased proportion of slow type I fibers in adult skeletal muscle is currently underway.


    ACKNOWLEDGEMENTS

We thank Dr. Irwin Davidson for TEF cDNAs, Dr. Mahesh Gupta for GST-NTEF-1, Drs. Simbulan-Rosenthal and Smulson for pBS-II SK+ PARP, Dr. R. Eisenman for the Max expression vector, Dr. D. Ayer for purified baculovirus expressed Max protein and the CM-I oligonucleotide, and Dr Hannink for pGEX-5X-1 and pGEX-KG. Also, we thank Drs. Ordahl, Larkin, Butler, and Farrance for oligonucleotides and helpful discussions. Dedicated to the memory of George W. Smith and Marcia Smith.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants R01-AR41464 and R01-AR47197 (to R. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: University of Missouri-Columbia, Dept. of Veterinary Biomedical Sciences and Department of Biochemistry, 1600 E. Rollins Ave., W112 VET Medicine Bldg., Columbia, MO 65211. Tel.: 573-884-4547; Fax: 573-884-6890; E-mail: tsikar@missouri.edu.

Published, JBC Papers in Press, September 28, 2000, DOI 10.1074/jbc.M007750200

2 G. L. Tsika and R. W. Tsika, unpublished observation.


    ABBREVIATIONS

The abbreviations used are: beta MyHC, beta -myosin heavy chain; MOV, mechanical overload; MOV-P, overloaded plantaris muscle; bp, base pair(s); CAT, chloramphenicol acetyltransferase; MCAT, muscle CAT; TEF-1, transcription enhancer factor 1; NTEF-1, nominal TEF-1; RTEF-1, related TEF-1; EMSA, electrophoretic mobility shift assay; LMC, low mobility complex; IMC, intermediate mobility complex; HMC, high mobility complex; PARP, poly(ADP-ribose) polymerase; USF, upstream stimulatory factor; PCR, polymerase chain reaction; CP, control plantaris; CS, control soleus; TnT, troponin T; cTnT, cardiac TnT; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; UL, unprogrammed lysate; GST, glutathione S-transferase.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Lyons, G. E., Ontell, M., Cox, R., Sassoon, D., and Buckingham, M. (1990) J. Cell Biol. 111, 1465-1476[Abstract]
2. Lyons, G. E., Schiaffino, S., Sassoon, D., Barton, P., and Buckingham, M. (1990) J. Cell Biol. 111, 2427-2436[Abstract]
3. Knotts, S., Rindt, H., Neumann, J., and Robbins, J. (1994) J. Biol. Chem. 269, 31275-31282[Abstract/Free Full Text]
4. Wiedenman, J. L., Tsika, G. L., Gao, L., McCarthy, J. J., Rivera-Rivera, I. D., Vyas, D., Sheriff-Carter, K., and Tsika, R. W. (1996) Am. J. Physiol. 271, R688-R695[Abstract/Free Full Text]
5. Booth, F. W., and Baldwin, K. M. (1996) in Handbook of Physiology (Rowell, L. B , and Shepherd, J. T., eds) , pp. 1075-1123, Oxford University Press, New York
6. Tsika, G. L., Wiedenman, J. L., Gao, L., McCarthy, J. J., Sheriff-Carter, K., Rivera-Rivera, I. D., and Tsika, R. W. (1996) Am. J. Physiol. 271, C690-C699[Abstract/Free Full Text]
7. Rindt, H., Gulick, J., and Robbins, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1540-1544[Abstract]
8. Vyas, D. R., McCarthy, J. J., and Tsika, R. W. (1999) J. Biol. Chem. 274, 30832-30842[Abstract/Free Full Text]
9. McCarthy, J. J., Vyas, D. R., Tsika, G. L., and Tsika, R. W. (1999) J. Biol. Chem. 274, 14270-14279[Abstract/Free Full Text]
10. Wiedenman, J. L., Rivera-Rivera, I., Vyas, D., Tsika, G., Gao, L., Sheriff-Carter, K., Wang, X., Kwan, L. Y., and Tsika, R. W. (1996) Am. J. Physiol. 270, C1111-C1121[Abstract/Free Full Text]
11. McCarthy, J. J., Fox, A. M., Tsika, G. L., Gao, L., and Tsika, R. W. (1997) Am. J. Physiol. 272, R1552-R1561[Abstract/Free Full Text]
12. Jacquemin, P., and Davidson, I. (1997) Trends Cardiovasc. Med. 7, 192-197[CrossRef]
13. Larkin, S. B., and Ordahl, C. P. (1999) in Heart Development (Rosenthal, N. , and Harvey, R. P., eds) , pp. 307-329, Academic Press, San Diego, CA
14. Davidson, I., Xiao, J. H., Rosales, R., Staub, A., and Chambon, P. (1988) Cell 54, 931-942[Medline] [Order article via Infotrieve]
15. Xiao, J. H., Davidson, I., Matthes, H., Garnier, J. M., and Chambon, P. (1991) Cell 65, 551-568[Medline] [Order article via Infotrieve]
16. Jiang, S. W., Trujillo, M. A., Sakagashira, M., Wilke, R. A., and Eberhardt, N. L. (2000) Biochemistry 39, 3505-3513[CrossRef][Medline] [Order article via Infotrieve]
17. Ueyama, T., Zhu, C., Valenzuela, Y. M., Suzow, J. G., and Stewart, A. F. (2000) J. Biol. Chem. 275, 17476-17480[Abstract/Free Full Text]
18. Gupta, M. P., Kogut, P., and Gupta, M. (2000) Nucleic Acids Res. 28, 3168-3177[Abstract/Free Full Text]
19. Belandia, B., and Parker, M. G. (2000) J. Biol. Chem. 275, P30801-P30805
20. Larkin, S. B., Farrance, I. K., and Ordahl, C. P. (1996) Mol. Cell. Biol. 16, 3742-3755[Abstract]
21. Farrance, I. K., and Ordahl, C. P. (1996) J. Biol. Chem. 271, 8266-8274[Abstract/Free Full Text]
22. Butler, A. J., and Ordahl, C. P. (1999) Mol. Cell. Biol. 19, 296-306[Abstract/Free Full Text]
23. Gupta, M. P., Amin, C. S., Gupta, M., Hay, N., and Zak, R. (1997) Mol. Cell. Biol. 17, 3924-3936[Abstract]
24. Ojamaa, K., Samarel, A. M., and Klein, I. (1995) J. Biol. Chem. 270, 31276-31281[Abstract/Free Full Text]
25. Vyas, D. R., McCarthy, J. J., Tsika, G. L., and Tsika, R. W. (2000) Basic Appl. Myol. 10, 5-16
26. Tsika, R. W. (1994) Exercise Sport Sci. Rev. 22, 361-388[Medline] [Order article via Infotrieve]
27. Tsika, R. W., Hauschka, S. D., and Gao, L. (1995) Am. J. Physiol. 269, C665-C674[Abstract]
28. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
29. Jacquemin, P., Hwang, J. J., Martial, J. A., Dolle, P., and Davidson, I. (1996) J. Biol. Chem. 271, 21775-21785[Abstract/Free Full Text]
30. Jacquemin, P., Martial, J. A., and Davidson, I. (1997) J. Biol. Chem. 272, 12928-12937[Abstract/Free Full Text]
31. Simbulan-Rosenthal, C. M., Rosenthal, D. S., Iyer, S., Boulares, A. H., and Smulson, M. E. (1998) J. Biol. Chem. 273, 13703-13712[Abstract/Free Full Text]
32. Fabre-Suver, C., and Hauschka, S. D. (1996) J. Biol. Chem. 271, 4646-4652[Abstract/Free Full Text]
33. Ayer, D. E., Kretzner, L., and Eisenman, R. N. (1993) Cell 72, 211-222[Medline] [Order article via Infotrieve]
34. Blackwood, E. M., and Eisenman, R. N. (1991) Science 251, 1211-1217[Medline] [Order article via Infotrieve]
35. Deleted in proof
36. Deleted in proof
37. Deleted in proof
38. Deleted in proof
39. Olson, E. N., and Williams, R. S. (2000) Cell 101, 689-692[Medline] [Order article via Infotrieve]
40. Gupta, M. P., Gupta, M., and Zak, R. (1994) J. Biol. Chem. 269, 29677-29687[Abstract/Free Full Text]
41. D'Amours, D., Desnoyers, S., D'Silva, I., and Poirier, G. G. (1999) Biochem. J. 342, 249-268[CrossRef][Medline] [Order article via Infotrieve]
42. Homburg, S., Visochek, L., Moran, N., Dantzer, F., Priel, E., Asculai, E., Schwartz, D., Rotter, V., Dekel, N., and Cohen-Armon, M. (2000) J. Cell Biol. 150, 293-308[Abstract/Free Full Text]


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