©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Role of Transcription Enhancer Factor-1 (TEF-1) Related Proteins in the Formation of M-CAT Binding Complexes in Muscle and Non-muscle Tissues (*)

(Received for publication, November 10, 1995; and in revised form, January 16, 1996)

Iain K. G. Farrance (§) Charles P. Ordahl

From the Department of Anatomy and Cardiovascular Research Institute, University of California, San Francisco, California 94143

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

M-CAT sites are required for the activity of many promoters in cardiac and skeletal muscle. M-CAT binding activity is muscle-enriched, but is found in many tissues and is immunologically related to the HeLa transcription enhancer factor-1 (TEF-1). TEF-1-related cDNAs (RTEF-1) have been cloned from chick heart. RTEF-1 mRNA is muscle-enriched, consistent with a role for RTEF-1 in the regulation of muscle-specific gene expression. Here, we have examined the tissue distribution of TEF-1-related proteins and of M-CAT binding activity by Western analysis and mobility shift polyacrylamide gel electrophoresis. TEF-1-related proteins of 57, 54 and 52 kDa were found in most tissues with the highest levels in muscle tissues. All of these TEF-1-related proteins bound M-CAT DNA and the 57- and 54-kDa TEF-1-related polypeptides were phosphorylated. Proteolytic digestion mapping showed that the 54-kDa TEF-1-related polypeptide is encoded by a different gene than the 52- and 57-kDa TEF-1-related polypeptides. A comparison of the migration and proteolytic digestion of the 54-kDa TEF-1-related polypeptide with proteins encoded by the cloned RTEF-1 cDNAs showed that the 54-kDa TEF-1-related polypeptide is encoded by RTEF-1A. High resolution mobility shift polyacrylamide gel electrophoresis showed multiple M-CAT binding activities in tissues. All of these activities contained TEF-1-related proteins. One protein-M-CAT DNA complex was muscle-enriched and was up-regulated upon differentiation of a skeletal muscle cell line. This complex contained the 54-kDa TEF-1-related polypeptide. Therefore, RTEF1-A protein is a component of a muscle-enriched transcription complex that forms on M-CAT sites and may play a key role in the regulation of transcription in muscle.


INTRODUCTION

Development of cardiac and skeletal muscle requires the coordinate regulation of a large number of contractile protein genes in a stage- and cell type-specific manner. Coregulated genes share cis regulatory elements that are targets for common factors which activate and/or repress groups of genes. In skeletal muscle, these regulatory factors include the MyoD family of helix-loop-helix proteins (MDFs), the MEF-2 family, and SRF(1, 2, 3, 4) . Regulatory factors in cardiac muscle also include the MEF-2 family and SRF(1, 2, 3, 4) , while some factors, such as GATA-4 (5, 6) and USF(7) , are required in cardiac but have not been shown to be required in skeletal muscle. The MDF proteins are not involved in the regulation of any genes in cardiac muscle since they are not expressed in heart(4) , and the activity of some promoters in cardiac muscle is E-box-independent (cTNT(8) ; cMLC2(9, 10) ; betaMHC (11, 12) ; cTNC(13, 14) ; skeletal alpha-actin(15, 16) ).

The cTNT gene is activated early in cardiac and skeletal muscle development and becomes restricted to cardiac muscle later in development(17, 18) . A conserved sequence motif (CATTCCT termed ``M-CAT''), present in two copies in the cTNT promoter region, is necessary for the transcription of this promoter in cardiac and embryonic skeletal muscle(8, 19) . M-CAT elements are required for the activity of other promoters in cardiac muscle cells (skeletal alpha-actin (15, 16) ; beta-myosin heavy chain(11, 12, 20, 21) ; alpha-myosin heavy chain(22, 23) ; vascular smooth muscle alpha-actin(24, 25) ; beta-acetylcholine receptor(26) ). DNase I footprinting and mobility shift PAGE have identified nuclear factors that bind M-CAT motifs(27, 28) . In previous work, we have shown that this M-CAT binding activity is found in many tissues, but is muscle-enriched and is biochemically and immunologically related to the HeLa transcription enhancer factor-1 (TEF-1)(^1)(27, 28) . HeLa cell TEF-1 binds to the M-CAT-related GTIIC and Sph motifs of the SV40 enhancer(29, 30) . These TEF-1 binding sites are required for activation of the SV40 late promoter(31, 32, 33) .

TEF-1 was the first isolated member of a mutigene family. TEF-1-related genes fall into four classes (detailed in Azakie et al.(55) ). cDNAs most closely related to the original HeLa cell TEF-1 (30) are herein referred to as NTEF-1(34, 35, 55) . (^2)The RTEF-1 (for Related to TEF-1) class contains the cloned TEF-1-related cDNAs from chicken tissues (the predominant cDNAs are termed here RTEF-1A and -1B)(^3)(36) . The other two classes are termed DTEF-1 (for Divergent TEF-1) (55) and ETEF-1 (for Embryonic TEF-1)(37) . (^4)Generally, mRNA for TEF-1 family members are muscle enriched but are expressed in multiple tissues(34, 35, 36, 55) . Expression of recently cloned TEF-1 family members is more restricted. Chicken DTEF-1 is expressed many tissues including cardiac muscle but is notably absent from skeletal muscle (55) and murine ETF-1 is found in almost exclusively in neuronal tissue(37) .

In addition to the basal transcriptional machinery, many transcription factors, including TEF-1, require cofactors for full transcriptional activation. In HeLa cells, which contain TEF-1, the activity of TEF-1-dependent promoters was inhibited by low levels of exogenous TEF-1. Also, no transactivation was seen in cell lines that do not contain TEF-1(30) . This led to the proposal that TEF-1 requires cell-specific cofactors for activity. Some cofactors have been partially purified and are found associated with and also separate from TBP(38, 39) . Although found at varying levels in different cell lines (40) , it is not known whether these cofactors differ between tissue types. TEF-1 can also interact with viral proteins to activate transcription. Efficient transactivation of the SV40 late promoter requires direct interaction of TEF-1 with TBP and T-antigen (41) .

Many M-CAT-dependent cellular promoters that have been identified to date are muscle-specific. However, the distribution of M-CAT binding activity and TEF-1 mRNA (27, 28, 34, 35, 36) raises the question of how muscle-specific gene expression is generated using a factor that itself is expressed in muscle and non-muscle tissues. We have investigated the tissue distribution of TEF-1 proteins and M-CAT binding activities using a TEF-1 antiserum and high resolution mobility shift PAGE. We found multiple TEF-1 proteins and M-CAT binding activities in tissues. We have isolated proteins from these complexes and have shown that RTEF-1A encodes the sole TEF-1 protein recognized by the antiserum used here in a muscle-enriched M-CAT binding activity.


EXPERIMENTAL PROCEDURES

Materials

Oligonucleotides were synthesized by Operon Technologies Inc. (Alameda, CA). DNA oligonucleotides were labeled at their 5`-ends using [-P]ATP and T4 polynucleotide kinase. V8 protease (sequencing grade), chymotrypsin (sequencing grade) and subtilisin were purchased from Boehringer Mannheim.

Crude Nuclear Extracts

Crude nuclear extracts were made from embryonic day 12 chicken leg and breast muscle, heart and gizzard using the previously published procedure(28) . Tissues for extracts from embryonic day 12 chicken lung, kidney, liver and brain, were collected and washed in 25 mM HEPES, 130 mM NaCl, pH 7.8, prior to homogenization. For cultured cell extracts, plates were washed twice with 25 mM HEPES, 130 mM NaCl, pH 7.8, twice for 5 min each with relaxation buffer 1(28) , twice for 5 min each with relaxation buffer 2, and once briefly with low salt buffer (25 mM Tris, MES, pH 7.6, 1 mM dithiothreitol, 0.1% Triton X-100). Cells were soaked in low salt buffer for 10 min, scraped, allowed to swell for 10 min, and lysed by 5 to 20 strokes with the tight pestle in a Dounce homogenizer. Nuclei were pelleted at 300 times g and resuspended in 20 mM HEPES, pH 7.6, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM dithiothreitol, 25% glycerol. The NaCl concentration was raised to 0.45 M and nuclear proteins were extracted for 30 min prior to removal of the chromatin by centrifugation at 12,000 times g. All steps were at 4 °C and all buffers contained 0.5 mM phenylmethylsulfonyl fluoride and 2 µg/ml aprotinin and leupeptin.

Mobility Shift Polyacrylamide Gel Electrophoresis

Mobility shift PAGE reactions (containing 750 ng poly(dI-dC), 0.5 ng (0.4 fmol) M-CAT-1 DNA, and 4 µg of nuclear extract) were as described previously(27) . Reactions were loaded at the bottom of 1.2-cm wide wells in a 1.5-mm thick polyacrylamide gel (6% acrylamide; 44:1, acrylamide:bis) in 45 mM Tris, 45 mM boric acid, 1 mM EDTA (42) and electrophoresed at 10 V/cm until bromphenol blue in a separate well had migrated 20 cm.

Antibodies

The TEF-1 antiserum was raised against pooled synthetic peptides deduced from the chicken RTEF-1A cDNA sequence (36) (RQIYDKFPEKKG, amino acids 259-270; LQVVTNRDTQETL, amino acids 387-399; and the carboxyl-terminal peptide of RTEF-1A HGAQHHIRLVKD, amino acids 413-425). The peptides were cross-linked to keyhole limpet hemocyanin and injected into rabbits using standard protocols. IgG was purified using protein A agarose(43) . Affinity-purified antibody was isolated using a RSET:RTEF-1A fusion protein, produced as recommended by the manufacturer (Invitrogen), lacking the two internal peptides but containing the carboxyl-terminal peptide. Fusion protein was separated by SDS-PAGE, transferred to nitrocellulose, and blotted as described below for Western analysis without treatment of the blot with secondary antibody. Specific antiserum were eluted from the fusion protein with basic and acidic buffers as described(43) .

Western Analysis

Nuclear proteins (10 µg) were separated by SDS-PAGE on a 10% gel and transferred to nitrocellulose. Filters were blocked with 5% bovine serum albumin in 25 mM Tris, pH 7.5, 130 mM NaCl (TBS) for 16 h at 4 °C, washed with TBS, 0.05% Tween-20 (TBST), incubated with TEF-1 antibody (0.5 to 1.0 µg IgG/ml in TBST). After 2 h at 25 °C, the blot was washed with TBST, incubated with goat anti-rabbit IgG peroxidase (1/5000, Vector Laboratory) in TBST with 2% goat serum for 1 h at 25 °C, and washed with TBST. Bands were visualized by chemiluminescence. Southwestern analysis was done as described previously(27) .

For immunodetection of TEF-1 proteins in mobility shift complexes, mobility shift PAGE reactions were as above except the reactions were scaled up 5-fold in a final volume of 20 µl. After electrophoresis, gels were soaked twice for a total of 3 min in 62.5 mM Tris (pH 6.8), 2% SDS, 25 mM dithiothreitol and dried. Protein-M-CAT DNA complexes were localized by autoradiography, excised from the gel, and loaded onto a 10% SDS-PAGE gel as described below for proteolytic digestions but without the addition of protease(43) . After electrophoresis and transfer to poly(vinylidene fluoride) membrane, TEF-1 proteins were detected by Western analysis.

Immunoprecipitation

Primary chick muscle cultures were grown as described elsewhere(44) . Forty-eight hours after plating, cells were fed with complete medium 2 h prior to labeling. Cells were labeled in 2 ml per 100-mm plate of 90% Dulbeccco's modified Eagle's medium (Cys/Met-free), 5% complete minimal essential medium, 5% fetal calf serum containing S-Met/Cys (TransLabel, 125 µCi/ml, 1000 Ci/mmol) or in 2 ml per 100-mm plate of minimal essential medium (PO(4)-free), 5% fetal calf serum containing PO(4) (0.5 mCi/ml, 9000 Ci/mmol) for 4 h. Nuclear proteins were prepared from labeled cells as above with the addition of 1 mM sodium orthovanadate, 2 mM NaF, and 2 mM sodium pyrophosphate to the low salt buffer and of 1 mM sodium orthovanadate, 10 mM NaF, and 10 mM sodium pyrophosphate to the nuclear extraction buffer.

Nuclear extract from a single 100-mm plate per treatment (50 µl; 2 times 10^6 cpm for S-labeled extracts and 4.5 times 10^6 cpm for P-labeled extracts) was used in each immunoprecipitation reaction. SDS was added to 2%, and the extract was heated to 100 °C for 2 min and centrifuged in a microcentrifuge for 10 min. The SDS concentration was diluted to 0.1% with 25 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate (radioimmune precipitation buffer). The reactions were precleared with 20 µl of protein A-agarose for 30 min at room temperature. TEF-1 antibody (10 µg) was added and incubated at 4 °C for 2 h. Protein A-agarose (20 µl) was added to remove immune complexes from solution. After 2 h at 4 °C, immune complexes were centrifuged briefly and washed three or four times with radioimmune precipitation buffer with 0.1% SDS. For phosphatase treatments, immune complexes were washed an additional two times with incubation buffer (bacterial alkaline phosphatase, 100 mM Tris, pH 8.0, 50 mM MgCl(2), 100 µg/ml aprotinin; potato acid phosphatase, 100 mM MES, pH 6.0, 20 µg/ml aprotinin and leupeptin, 1 mM phenylmethylsulfonyl fluoride; calf intestinal alkaline phosphatase, 50 mM Tris, pH 8.5, 1 mM EDTA, 2 µg/ml aprotinin and leupeptin, 1 mM phenylmethylsulfonyl fluoride) and incubated with enzymes (bacterial alkaline phosphatase, 2.8 units over 1 h at 30 °C; potato acid phosphatase, 4 units over 1 h at 37 °C; calf intestinal alkaline phosphatase, 200 units over 1 h at 37 °C). Proteins were eluted from the complexes with SDS sample buffer, subjected to SDS-PAGE, and visualized by fluorography.

Proteolytic Mapping

Overexpressed RTEF-1A and -1B were produced by cloning full-length cDNAs into the expression vector pXJ40 (30) and transfecting QM7 cells (45) using calcium phosphate (46) . Nuclear extracts from ED12 skeletal muscle (50 µg for each 2-cm wide well) and from QM7 cells overexpressing RTEF-1A (10 µg/well) were subjected to SDS-PAGE on a 10% gel. Each 2-cm wide well from the first dimension SDS-PAGE gel was used for four proteolytic digestion wells. A strip of the gel was analyzed by Western blot analysis to localize the TEF-1 proteins; the remainder of the gel was dried. The portion of the gel containing TEF-1 proteins was excised, rotated 90°, placed in the well of a second SDS gel (15%), and subjected to proteolytic digestion in the stacking gel(43) . The gel was transferred to poly(vinylidene fluoride) membrane and proteolytic fragments containing TEF-1 peptides were detected by Western analysis as above.


RESULTS

TEF-1 Protein Diversity

An antiserum was raised against peptides derived from the cloned TEF-1-related cDNA RTEF-1A(36) . The peptides chosen as immunogens were derived from conserved regions of the TEF-1 gene family to maximize detection of diverse TEF-1-related gene products. The antiserum obtained has broad specificity. It recognizes TEF-1 proteins overproduced from cloned TEF-1 cDNAs (human NTEF-1 and chicken RTEF-1A, see below) and endogenous TEF-1 proteins in extracts from avian (see below) and mammalian (22, 23, 47, 48) cells and tissues.

To determine the diversity of TEF-1 proteins, nuclear extracts from various chick embryo tissues were subjected to SDS-PAGE, blotted, and probed with the TEF-1 antiserum. Proteins of 57, 54, and 52 kDa were detected in nuclear extracts from a range of tissues (Fig. 1A, see legend). These proteins were enriched in extracts of muscle-containing tissues (gizzard, lane 3; cardiac muscle, lane 4; skeletal muscle, lane 5) as compared to tissues containing little or no muscle (kidney, lane 6; lung, lane 7). Within most tissues, the polypeptides are present at similar relative levels. Notably, however, liver (lane 1) and brain (lane 2) nuclear extracts contain the 57- and 52-kDa polypeptides but very low levels of the 54-kDa polypeptide. In nuclear extracts of the muscle cell line QM7, the 57-, 54-, and 52-kDa polypeptides are present at similar levels before and after differentiation (Fig. 1A, compare lanes 8 and 9; see legend). These results show that, although TEF-1 proteins are enriched in muscle tissues, none of the TEF-1 proteins shows a high degree of muscle specificity.


Figure 1: A, Multiple TEF-1 proteins are expressed in tissues and cell lines. Western blot analyses of ED12 tissues and from cultured cells using the TEF-1 antibody. 57- and 52-kDa TEF-1 polypeptides are detected in liver (Lv, lane 1) and brain (Br, lane 2) extracts, while polypeptides of 57, 54, and 52 kDa are detected in gizzard (G, lane 3), cardiac muscle (H, lane 4), skeletal muscle (Sk, lane 5), kidney (K, lane 6), and lung (Lu, lane 7). Growing (G, lane 8) and differentiated (D, lane 9) QM7 cells contain the 57-, 54-, and 52-kDa TEF-1 polypeptides. The thickness and appearance in this blot and others of the 57-kDa band in brain, gizzard, and heart suggests that this band may contain more than one polypeptide. In this blot it appears that the 54-kDa TEF-1 polypeptide increased upon differentiation of QM7 cells (compare lanes 8 to 9). This difference was not seen with other blots of this extract nor with other QM7 extracts. Preimmune serum did not recognize any protein in the nuclear extracts (data not shown). Asterisks mark bands that are not found on every immunoblot. The position of molecular mass standards is indicated on the left. B, all TEF-1 proteins bind M-CAT DNA. An M-CAT DNA probe (OGT2-56)(27, 29) recognizes 57- and 52-kDa polypeptides in extracts from ED12 liver (Lv, lane 1) and brain (Br, lane 2). 57-, 54-, and 52-kDa polypeptides are detected in extracts from gizzard (G, lane 3), skeletal muscle (Sk, lane 4), and cardiac muscle (H, lane 5). The molecular mass of the M-CAT binding polypeptides are indicated. It is possible that a protein that binds M-CAT DNA but unrelated to TEF-1 comigrates with the TEF-1 proteins. We could not resolve this possibility here. C, some TEF-1 proteins are phosphorylated. Primary chick skeletal muscle cultures were metabolically labeled with S-Cys/Met (lanes 1-4) or PO(4) (lanes 5-8). Nuclear extracts were subjected to immunoprecipitation with TEF-1 antiserum. Immunoprecipitates were treated with no phosphatase (-, lanes 1 and 5), calf intestinal alkaline phosphatase (C, lanes 2 and 6), bacterial alkaline phosphatase (B, lanes 3 and 7), or potato acid phosphatase (P, lanes 4 and 8). Proteins were subjected to SDS-PAGE and detected by fluorography. The reduction of the intensity of the bands in lane 4 is likely due to the acidic incubation buffer for potato acid phosphatase. Positions of molecular mass standards are indicated on the left. The 57- and 54-kDa TEF-1 polypeptides are phosphorylated in skeletal muscle.



All TEF-1 Polypeptides Bind M-CAT DNA

Southwestern blot analysis was used to determine if the proteins that bind M-CAT DNA in tissue extracts are TEF-1-related. Fig. 1B shows that M-CAT DNA binds bands of 57, 54, and 52 kDa in extracts from ED12 liver (lane 1), brain (lane 2), gizzard (lane 3), skeletal muscle (lane 4), and cardiac muscle (lane 5). Western analysis of parallel lanes shows that the M-CAT-binding proteins comigrate with the proteins recognized by the TEF-1 antiserum (data not shown). Therefore, all proteins in the extracts that bind M-CAT DNA are recognized by the TEF-1 antiserum and vice versa (see legend to Fig. 1B).

Based upon intensity of the labeled bands, the 54-kDa band appears to bind more M-CAT DNA than the 52- and 57-kDa bands in gizzard, skeletal muscle, and cardiac muscle. In liver and brain, the 54-kDa TEF-1 band binds an equal amount of M-CAT DNA as the 52- and 57-kDa bands (Fig. 1B). The Western blot analysis reported above showed that the TEF-1 proteins are present at similar levels in gizzard, skeletal muscle, and cardiac muscle and that the 54-kDa polypeptide is present at much lower levels in liver and brain (see above). Therefore, we tentatively conclude that the 54-kDa polypeptide binds M-CAT DNA with higher affinity than the 52- and 57-kDa polypeptides.

TEF-1 Proteins Are Differentially Phosphorylated

TEF-1 proteins are likely to be phosphorylated since M-CAT sites are required for the transcriptional induction of the beta-MHC and skeletal alpha-actin promoters in cardiac muscle cultures by the alpha(1)-adrenergic stimulation and by protein kinase C(11, 16) . To determine if differential phosphorylation accounts for the three TEF-1 bands detected by Western and Southwestern analysis, chick primary muscle cultures were metabolically labeled with S or P, and TEF-1 was immunoprecipitated from nuclear extracts.

Fig. 1C shows that 57-, 54-, and 52-kDa polypeptides were immunoprecipitated from S-labeled extracts (lane 1). Immunoprecipitation of P-labeled extracts showed that only the 57- and 54-kDa bands are phosphorylated, while the 52-kDa band is not (lane 5). Identical results were obtained after metabolic labeling of primary cardiac muscle and liver fibroblasts (data not shown). Treatment of immunoprecipitates with phosphatases removes all the P from the TEF-1 proteins (lanes 6-8). However, treatment of S-labeled immunoprecipitates with phosphatases does not change the mobility of the 57-, 54-, and 52-kDa polypeptides (lanes 2-4). These results indicate that the TEF-1 proteins are not related by differential phosphorylation of a single polypeptide backbone. Therefore, the 57-, 54-, and 52-kDa polypeptides are likely to represent distinct translation products.

Proteolytic Mapping of TEF-1 Proteins

The primary sequence relationship between the TEF-1 proteins was analyzed by partial proteolytic mapping. Skeletal muscle extracts were subjected to SDS-PAGE and the region of the gel containing TEF-1 proteins was applied to a second SDS gel and partially digested with various amounts of V8 protease within the stacking gel. Partial proteolytic fragments were resolved in the second gel and fragments containing immunogenic peptides were detected by Western blot with the TEF-1 antiserum. The proteolytic cleavage pattern of the 52- and 57-kDa polypeptides was identical (Fig. 2A and diagrammed in Fig. 2B; see also Fig. 3B for other proteases). Digestion of the 57- or 52-kDa TEF-1 polypeptide yields the same three major products: fragment 1 of 49 kDa, fragment 2 of 36 kDa, and fragment 3 of 12.5 kDa (Fig. 2B and Table 1). Affinity-purified TEF-1 antibody specific for the carboxyl-terminal peptide of RTEF-1A reacted with the undigested 52- and 57-kDa TEF-1 proteins (U, Fig. 2, B and C) and with fragments 1 and 3 (data not shown). This shows that the 5-kDa difference between the 52- and 57-kDa TEF-1 polypeptides is located within the amino-terminal portion of the polypeptides.


Figure 2: TEF-1 proteins are encoded by at least two genes. A, ED12 skeletal muscle extract was subjected to SDS-PAGE on a 10% gel. The region of the gel containing TEF-1 proteins was isolated and rotated 90°, and the protein was digested in the stacking gel of a second 15% SDS gel with 80 ng (lane 1) or 200 ng (lane 2) of V8 protease (Lys-C). Proteolytic fragments containing TEF-1 peptides were detected by Western blot using the TEF-1 antiserum. The migration position of the TEF-1 proteins in the first dimension SDS gel is indicated above each lane. B, schematic representation of the proteolytic digestion pattern of TEF-1 proteins. Peptides derived from the 52- and 57-kDa TEF-1 polypeptides, as indicated above each series, are represented by solid black spots. Peptides derived from the 54-kDa TEF-1 polypeptide are represented by unfilled spots. Fragments are identified by numbers for molecular mass comparison (see Table 1) and for mapping using a larger font for major peptides than for minor peptides. U indicates undigested TEF-1 proteins. C, proteolytic cleavage maps of TEF-1 polypeptides. Maps of the cleavage sites for V8 protease in the TEF-1 proteins were generated using molecular masses of the peptides and reactivity of the peptides with antibody specific to the carboxyl-terminal peptide of RTEF-1A (see Table 1). Unfilled boxes represent intact TEF-1 proteins. Small filled boxes in the 54-kDa TEF-1 protein represent the position of immunogenic peptides used to raise TEF-1 antibody (see ``Experimental Procedures''). Filled triangles indicate major cleavage sites in the proteins while unfilled triangles below the TEF-1 proteins indicate minor cleavage sites in the 54-kDa TEF-1 polypeptide. The position of the DNA binding domain of TEF-1 (TEA domain) (30, 36, 53, 54) is shown in its approximate location. Vertical lines marked with asterisks indicate positions of known alternative splicing in cloned chick RTEF-1. The map positions of the peptides numbered in B are shown by lines next to the peptide number. Two lines next to the peptide number indicate ambiguity in the assignment of map positions.




Figure 3: RTEF-1A encodes the 54-kDa TEF-1 protein. A, RTEF-1A comigrates with the 54-kDa TEF-1 polypeptide. Nuclear extracts from cardiac muscle and QM7 cells expressing RTEF-1A and -1B were analyzed by Western blot with the TEF-1 antibody. Lane 1, extract from QM7 cells overexpressing RTEF-1A (3 µg); lane 2, extract from QM7 cells overexpressing RTEF-1A (3 µg) mixed with cardiac muscle extract (10 µg); lane 3, cardiac muscle extract (10 µg); lane 4, extract from QM7 cells overexpressing RTEF-1B (3 µg) mixed with cardiac muscle extract (10 µg); lane 5, extract from QM7 cells overexpressing RTEF-1B (3 µg). The minor products of RTEF-1A and -1B cDNAs (55.5 and 58 kDa, respectively) result from heterogeneity at the amino terminus of the proteins (lanes 1, 2, 4, and 5; see below). B, proteolytic digestion pattern of RTEF-1A is identical to the 54-kDa TEF-1 polypeptide. Nuclear extracts from skeletal muscle (lanes 1, 4, and 6) and QM7 cells overexpressing RTEF-1A (lanes 2, 3, 5, and 7) were separated by 10% PAGE. As in Fig. 2, regions of the gel containing TEF-1 proteins were applied to a second SDS gel and subjected to proteolytic digestion with V8 protease (lane 1, 80 ng; lane 2, 20 ng; lane 3, 100 ng), subtilisin (lane 4, 20 ng; lane 5, 20 ng), or chymotrypsin (lane 6, 800 ng; lane 7, 500 ng). Proteolytic fragments containing the immunogenic peptides were detected with the TEF-1 antiserum. Arrows indicate proteolytic fragments that are common between RTEF-1A and the 54-kDa TEF-1 protein. Slight differences in mobility of the proteolytic fragments are the result of uneven heating of the gel during electrophoreseis. Removal by all proteases of 7 kDa from the major RTEF-1A translation product causes the major and minor forms of RTEF-1A to comigrate (see lanes 2 and 3). Since the antibody specific for the carboxyl-terminal peptide of TEF-1 reacts with these products, the multiple forms of RTEF-1A (and presumably RTEF-1B) are due to to heteorgeneity in the amino-terminal portion of RTEF-1A.





Using these data a map of the proteolytic digestion sites was determined (Fig. 2C). Two cleavage sites are found in both the 52- and 57-kDa TEF-1 proteins: one 12.5 kDa from the carboxyl terminus of both proteins and a second 8 and 3 kDa from the amino terminus of the 57- and 52-kDa TEF-1 proteins, respectively. The amino-terminal difference between the 52- and 57-kDa TEF-1 proteins could result from alternative splicing in regions of their pre-mRNAs encoding their amino termini because TEF-1 and TEF-1-related genes have been shown to be subject to alternative splicing in this region(30, 36) ^2 Also, this difference may result from multiple sites of translational initiation (30, 36) (see ``Discussion''). Alternatively, the 52- and 57-kDa TEF-1 proteins may be encoded by two genes whose primary sequence divergence cannot be resolved with the proteases and antiserum used here.

The proteolytic pattern of the 54-kDa TEF-1 polypeptide showed an overall similarity to that of the 52/57-kDa polypeptides (Fig. 2A and diagrammed in Fig. 2B). There are two protease-sensitive regions yielded three major fragments (fragment 1, 47 kDa; fragment 2, 34 kDa; fragment 3, 11.7 kDa; Fig. 2B and Table 1) and five minor fragments (fragment 4, 37.5 kDa; fragment 5, 32 kDa; fragment 6, 30 kDa; fragment 7, 13 kDa, and fragment 8, 9.8 kDa). Affinity purified TEF-1 antibody specific for the carboxyl-terminal peptide of RTEF-1A reacted with the undigested 54-kDa TEF-1 polypeptide (U) and with peptides 1, 3, 7, and 8 (Fig. 2B, data not shown). A proteolytic cleavage map generated from these data show two major cleavage sites and multiple minor cleavage sites (Fig. 2C). One major site is 7 kDa from the amino terminus of this protein and the other major cleavage site is 11.7 kDa from the carboxyl terminus.

These proteolytic cleavage studies show that the 54-kDa TEF-1 protein is encoded by a different TEF-1 family member than the 52- and 57-kDa TEF-1 proteins. First, the carboxyl-terminal-most major cleavage site of the 54-kDa TEF-1 polypeptide is found at a different position (11.8 kDa from the end of the protein) than the carboxyl-terminal most major cleavage site in the 57 and 52 TEF-1 polypeptides (12.5 kDa from the end of the proteins). This difference likely results from a primary sequence difference between the TEF-1 proteins and not from alternative splicing since no alternative splicing has been found in the carboxyl-terminal region of any cloned TEF-1 cDNAs(30, 34, 35, 36, 37, 55) . Second, the minor cleavage sites, yielding peptides 4-8, in the 54-kDa TEF-1 protein are not found in the 52- or 57-kDa TEF-1 proteins. This shows that the 54-kDa TEF-1 polypeptide has many cleavage sites not found or not exposed in the 52- or 57-kDa TEF-1 proteins. Finally, an antibody (kindly provided by M.-H. Disatnik and P. C. Simpson) directed against the amino terminus of rat NTEF-1, a region divergent between TEF-1 family members, recognizes the NTEF-1 class of TEF-1 proteins but not RTEF-1A (data not shown). This NTEF-1 antiserum recognized the 57 kDa TEF-1 polypeptide but not the 54- or 52-kDa TEF-1 polypeptides (data not shown). This supports the amino-terminal difference between the 57- and 52-kDa TEF-1 proteins and that the 54-kDa TEF-1 protein is encoded by a different gene than the 52- and 57-kDa TEF-1 proteins. Therefore, the TEF-1 proteins analyzed here are encoded by at least two genes. One gene encodes the 54-kDa polypeptide, while one or more other gene(s) encodes the 52- and 57-kDa polypeptides. This conclusion is further supported by the recent cloning of multiple TEF-1-related cDNAs from chicken, mouse, and rat(37, 55) (^5)(see ``Discussion'').

The 54-kDa TEF-1 Protein Is Encoded by RTEF-1A mRNA

The majority of RTEF-1 cDNAs isolated from embryonic chick represent two mRNAs, RTEF-1A and RTEF-1B, that are identical except for the presence of a 13-amino acid exon in RTEF-1B(36) . RTEF-1B is present in cardiac cells at 10-20% of the level of RTEF-1A. (^6)To determine how these RTEF-1 cDNAs are related to the TEF-1 proteins characterized above, RTEF-1A and -1B were expressed in QM7 cells and subjected to Western analysis and partial proteolytic mapping as described above (Fig. 3).

Extracts from cardiac muscle and QM7 cells overexpressing RTEF-1A or -1B were subjected to SDS-PAGE and analyzed by Western blot with the TEF-1 antiserum (Fig. 3A). The major product produced by the RTEF-1A cDNA is 54 kDa (see legend to Fig. 3) and comigrates with the 54-kDa band detected by the TEF-1 antibody in cardiac muscle extracts (compare lanes 1 and 3). The major product produced by the TEF-1B cDNA is 55.5 kDa and does not comigrate with any of the bands detected by the TEF-1 antibody in cardiac muscle extracts (compare lanes 3 and 5). Mixing of the QM7 extracts with cardiac muscle extract confirms the comigration of the proteins (lanes 2 and 4).

Comigration of RTEF-1A polypeptide and the natural 54-kDa TEF-1 polypeptide on SDS gels suggests that they are products of the same gene. To test this hypothesis, TEF-1 proteins from skeletal muscle (Fig. 3B, lanes 1, 4, and 6) and RTEF-1A overproduced in QM7 cells (Fig. 3B, lanes 2, 3, 5, and 7) were digested with V8 protease (lanes 1-3), subtilisin (lanes 4 and 5), and chymotrypsin (lanes 6 and 7). All peptides detected in digests of RTEF-1A by each protease comigrate with those detected in digests of the 54-kDa TEF-1 protein (compare arrow pairs in lane 1 with lanes 2 and 3, lane 4 with lane 5, and lane 6 with lane 7). Taken together, the comigration of the 54-kDa TEF-1 polypeptide with overexpressed RTEF-1A (Fig. 3A) and the results of these proteolytic digests (Fig. 3B) indicate that the 54-kDa TEF-1 protein is encoded by RTEF-1A mRNA.

A Muscle Enriched M-CAT Binding Activity

Using previously published methods M-CAT binding activities migrate as a broad band on mobility shift PAGE gels(27, 28) . To determine the precise distribution and potential muscle-specificity of individual M-CAT binding activities, we developed a high resolution mobility shift assay (see ``Experimental Procedures''). Using this technique, the broad gel shift band seen in other studies could be resolved into as many as three protein-M-CAT DNA complexes, depending upon the tissue analyzed (Fig. 4).


Figure 4: Multiple M-CAT binding activities are found in tissues and cells. 5`-end-labeled M-CAT-1 DNA was mixed with extracts from muscle and non-muscle tissues or QM7 cells and analyzed by mobility shift PAGE. Skeletal muscle (Sk, lane 1), cardiac muscle (H, lane 2), gizzard (G, lane 3), brain (Br, lane 4), kidney (K, lane 5), lung (Lu, lane 6), and liver (Lv, lane 7). Extracts from a quail myogenic cell line (QM7) from growing (G, lane 8) and differentiated muscle cells (D, lane 9) were also assayed. Complexes 1, 2, and 3 are indicated. In brain extracts, complex 1 was formed in reactions containing low levels of nonspecific competitor DNA (200 ng). Under these conditions complex 1 was not observed with liver, lung, or kidney extracts.



The slowest mobility protein-M-CAT DNA complex (complex 1) was found in nuclear extracts from skeletal muscle (lane 1), cardiac muscle (lane 2), and gizzard (lane 3) and was present at extremely low levels in brain (lane 4, see legend). Complex 1 was absent in kidney (lane 5), lung (lane 6), and liver (lane 7). Therefore, complex 1 was highly enriched in muscle tissues. The intermediate mobility protein-M-CAT DNA complex (complex 2) was found in all tissues examined. Complex 2 in gizzard, kidney, and lung is a broader band than complex 2 in skeletal and cardiac muscle and brain. Upon extensive electrophoresis this band can be resolved into two complexes in gizzard, lung and kidney (data not shown). The fastest mobility protein-M-CAT DNA complex (complex 3) was enriched in gizzard (lane 3), kidney (lane 5), and lung (lane 6), but was found at lower levels in skeletal muscle (lane 1), cardiac muscle (lane 2), and liver (lane 7), and was absent in brain (lane 4).

QM7 cells gave a similar pattern of protein-M-CAT DNA complexes as that found in muscle tissue (Fig. 4, lanes 8 and 9). Complex 1 is up-regulated upon differentiation while complexes 2 and 3 are not (compare lanes 8 to 9). Quantitation of the levels of complex 1 relative to complexes 2 and 3, in three independent sets of QM7 extracts, showed that formation of complex 1 increases 2.5-5-fold upon differentiation (data not shown). Similar results were obtained using extracts from primary cultures of growing and differentiating quail skeletal muscle cells (data not shown). These experiments show that complex 1 is muscle-enriched and preferentially increases upon differentiation.

All M-CAT Binding Activities Contain TEF-1 Proteins

Experiments described above (see Fig. 1) suggest that all M-CAT-binding proteins in all tissues are related to TEF-1 because all M-CAT-binding proteins detected by Southwestern blot analysis were also detected with the TEF-1 antiserum. However, previous experiments with antiserum against a human NTEF-1 peptide (antiserum P2) (30) and the antibody reported here (data not shown) failed to quantitatively supershift chick M-CAT binding activity in mobility shift PAGE assays, indicating that some M-CAT binding activities may not be related to TEF-1(27) . To test this notion more directly, we asked whether each of the three protein-M-CAT DNA complexes contained polypeptides antigenically related to TEF-1. Briefly, protein-M-CAT DNA complexes were separated by high resolution mobility shift PAGE and the position of each complex localized by autoradiography. Each complex was cut from the gel and the proteins in the complexes subjected to SDS-PAGE and Western blot analysis with TEF-1 antiserum.

Fig. 5shows that all protein-M-CAT DNA complexes contain TEF-1 proteins. Complex 1 contains the 54-kDa TEF-1 polypeptide (skeletal muscle, lane 1; gizzard, lane 4; and cardiac muscle, data not shown). A long exposure of lane 7 shows that, even though complex 1 is of low abundance in brain, this complex contains the 54-kDa TEF-1 polypeptide in brain. Complex 2 in skeletal muscle (lane 2) and cardiac muscle (data not shown), and brain (lane 8) contains both the 57- and 52-kDa TEF-1 polypeptide. In gizzard (lane 5) and lung (lane 9), complex 2 contains all three of the TEF-1 polypeptides. However, when complex 2 from gizzard, lung, and kidney is further separated into its two distinct subcomplexes (see above), the upper portion contained the 54-kDa TEF-1 protein while the lower portion contained both the 52- and 57-kDa TEF-1 polypeptides (data not shown). Complex 3, which is found in all tissues except brain, contains both the 57- and 52-kDa TEF-1 polypeptides (skeletal and cardiac muscle, lane 3, and data not shown; gizzard, lane 6; lung, lane 10).


Figure 5: All protein-M-CAT complexes contain TEF-1 proteins. Protein-M-CAT complexes from skeletal muscle (lanes 1-3), gizzard (lanes 4-6), brain (lanes 7 and 8), and lung (lanes 9 and 10) were resolved by mobility shift PAGE. Proteins from complex 1 (lanes 1, 4, and 7), complex 2 (lanes 2, 5, 8, and 9), and complex 3 (lanes 3, 6, and 10) were subjected to Western blot analysis with the TEF-1 antiserum. When no M-CAT DNA was included in mobility shift PAGE reactions, no TEF-1 proteins were detected in the region of the gel containing complexes 1, 2, or 3. The molecular mass of the TEF-1 proteins is indicated on the left.




DISCUSSION

A Muscle-enriched M-CAT Binding Activity Is Encoded by RTEF-1A cDNA

It is a paradox that while many M-CAT dependent promoters are strictly muscle-specific, M-CAT binding activity and TEF-1 mRNA, although enriched in muscle, are expressed in many tissues (11, 21, 23, 27, 28, 34-36). The tissue specificity of M-CAT-dependent promoters could be generated by the specific expression of a TEF-1 protein in muscle or from the presence of a muscle-specific M-CAT binding activity. Therefore, we have examined the tissue distribution of TEF-1 proteins by Western blot and that of M-CAT binding activity by high resolution mobility shift PAGE and Southwestern analysis. Relatively equal levels of TEF-1 proteins of 57, 54, and 52 kDa were found within most tissues tested (skeletal muscle, heart, gizzard, lung, and kidney). In brain and liver the 54-kDa polypeptide was found at lower levels than the 52- and 57-kDa polypeptides (Fig. 1A). Since none of these TEF-1 proteins are muscle-specific, a simple model of tissue-specific expression of a TEF-1-related gene cannot account for the muscle specificity of M-CAT-dependent promoters. Using modifications of previously published mobility shift PAGE methods(27, 28, 42) , at least three protein-M-CAT DNA complexes were resolved (Fig. 4, complexes 1, 2, and 3). The slowest migrating complex (termed complex 1) was enriched in tissues containing striated (skeletal muscle and heart) and smooth (gizzard) muscle. Complex 1 was up-regulated upon differentiation of a cultured skeletal muscle cell line. Thus complex 1 represents a muscle-enriched protein-M-CAT DNA complex.

In previous mobility shift PAGE experiments, neither an antibody against human NTEF-1 nor the antiserum described here quantitatively supershifted M-CAT binding activity from skeletal muscle (27) (data not shown). It was possible, therefore, that not all of the M-CAT binding activities (complexes 1, 2, and 3) contained TEF-1 proteins. Here, a comparison of Southwestern and Western analyses show that all proteins that bind M-CAT DNA are TEF-1-related (Fig. 1B). Also, elution of proteins from mobility shift complexes showed that all M-CAT binding activities contain TEF-1 proteins (Fig. 5). These two experimental results indicate that TEF-1 proteins can account for all M-CAT binding activities found in muscle and non-muscle cells.

The 54-kDa TEF-1 polypeptide was the only TEF-1 protein detected in the muscle-enriched complex 1. Two lines of evidence show that the 54-kDa TEF-1 polypeptide is encoded by the previously cloned RTEF-1A cDNA. First, the predominant protein produced from RTEF-1A comigrated with the 54-kDa TEF-1 polypeptide. Second, the proteolytic digestion pattern of the 54-kDa TEF-1 polypeptide and overexpressed RTEF-1A are identical. Therefore, RTEF1-A is a component of a muscle-enriched transcription complex that forms a sequence-specific complex with M-CAT sites. This is the first indication that any isoprotein encoded by a member of the TEF-1-related gene family, recently shown to exist in higher vertebrates(55) , may have a cell-specific transcriptional function.

Generation of Muscle Specificity by Protein-M-CAT DNA Complex 1

Since the 54-kDa TEF-1 protein and RTEF-1A mRNA are present in non-muscle tissues the muscle enrichment of complex 1 must require something in addition to RTEF-1A protein. Several explanations for the muscle enrichment of complex 1 are possible. First, since the RTEF-1A in complex 1 was detected by Western analysis, a muscle-enriched non-TEF-1 protein(s) may also be present in this complex. Mobility shift PAGE with in vitro produced RTEF-1A (36) and Southwestern blots (27) (Fig. 1B) show that any cofactor is not required for M-CAT binding. Nevertheless, the DNA binding mechanism for TEF-1 family members has not been determined so a contribution by a non-TEF-1 protein moiety cannot be ruled out.

Second, RTEF-1A may be modified differentially in muscle cells as compared to non-muscle cells. Both endogenous and overproduced RTEF-1A are phosphorylated in skeletal muscle cells (Fig. 1C and data not shown). Since phosphorylation does not alter the mobility of TEF-1 proteins in SDS gels (Fig. 1C), we could not determine if RTEF-1A has different levels or different specific sites of phosphorylation in differentiated muscle cells than in other cell types. Other types of post-translational modifications, such as glycosylation, could also play a similar role in regulation of RTEF-1 activity. Such modifications could result in altered migration of RTEF-1A-containing complexes in mobility shift gels and may contribute to the transcriptional activation of muscle specific genes. Alternatively, since TEF-1 requires cofactors for transcriptional activation(40) , differential modification of RTEF-1A could be required for the interaction of RTEF-1A with muscle-enriched cofactors or could make the interactions with more generally expressed cofactors or with the basal transcriptional machinery more efficient. In either case, gizzard must represent an intermediate situation. In gizzard, since the RTEF-1A protein is found in both complexes 1 and 2 (Fig. 5), subsaturating amounts of the cofactor are present or the RTEF-1A is incompletely modified in this tissue.

Other M-CAT Binding Activities

Other investigators have shown TEF-1 to be a key component of M-CAT binding activities in tissues and cell lines. The protein-M-CAT complex from rat heart contains TEF-1 (22, 23, 47) . Indeed, the antibody described here was able to quantitatively remove M-CAT binding activity from cardiac nuclear extracts(23) , indicating that, in rat as in chicken, TEF-1 proteins are present in all M-CAT binding activities. Using M-CAT sites from the betaMHC and the cTNT genes, Shimizu and co-workers (34, 42) have found a protein-M-CAT complex (termed A1) that, like complex 1 in chicken, is up-regulated in differentiated skeletal muscle cell lines (C2C12, sol8, and L6). A second protein-M-CAT complex (termed A2) was found in myoblasts, myotubes and in non-muscle cells (HeLa, HepG2, GC). An antibody against human NTEF-1, antibody P2(30) , quantitatively supershifted the A2 complex but did not recognize the differentiation-specific A1 complex. This antibody was raised against a region of NTEF-1 that is not well conserved between different TEF-1 family members(36, 37, 55) . We believe that the A1 complex would be recognized by the TEF-1 antibody used here since this antibody recognizes multiple TEF-1 family members, and that the TEF-1 protein in this complex is encoded by the mammalian homologue of chicken RTEF-1A (37, 55) .^5

We have not yet identified unequivocally which TEF-1 family member encodes the 52- and 57-kDa TEF-1 proteins that are present in mobility shift complexes 2 and 3. The 57-kDa, and probably the 52-kDa, TEF-1 protein is likely to be encoded by avian NTEF-1, since this protein was recognized by the rat NTEF-1 antiserum. None of the data presented precludes complexes 2 and 3 from transcriptional regulation in muscle and in non-muscle cells (see below). A tissue-specific regulatory role for complex 3 is supported by the increased amount of this complex in lung, kidney, and gizzard relative to striated muscle tissues and brain.

The human chorionic somatomammotrophin gene contains a placental specific enhancer. Within this enhancer are multiple M-CAT sites that are required for enhancer function and that bind nuclear factors present in extracts from placenta and from cell lines(48, 49, 50) . Recently, Jiang and Eberhardt (48) have shown that COS cells and BeWo cells, a choriocarcinoma cell line, contain two factors that bind M-CAT sites: TEF-1 and the previously undescribed CSEF-1. CSEF-1 was distinct from TEF-1 based on its migration on mobility shift gels, biochemical characteristics, molecular mass (30 kDa for CSEF-1 and 55 kDa for TEF-1), and nonreactivity to the TEF-1 antiserum used here. The tissue distribution of CSEF-1 was not determined, although it was absent in HeLa cells. We do not find any M-CAT DNA-binding proteins in the chicken tissues tested that are not recognized by the TEF-1 antibody (Fig. 1B and Fig. 5; see legend to Fig. 1B). However, oviduct was not tested here.

Role of Other TEF-1 Family Members in Gene Regulation

The results presented in this study support the idea that RTEF-1A is a component of a M-CAT-binding complex that plays an essential role in muscle-specific expression of M-CAT-dependent genes. The role of other RTEF-1 mRNA isoforms is not yet clear. We found, previously, that a GAL4-RTEF-1B fusion protein activated transcription of a GAL4-dependent promoter while a GAL4-RTEF-1A fusion protein did not. This lack of transactivation by RTEF-1A could result from the lack of the TEA and amino-terminal domains of RTEF-1A in these constructs. These portions of the protein may contain domains or modification sites stringently required for transactivation by RTEF-1A but less so for RTEF-1B. Indeed, the amino terminus of the 57-kDa TEF-1 polypeptide is required for its phosphorylation since the 52-kDa TEF-1 polypeptide, which lacks this portion of the protein, is not phosphorylated. While RTEF-1B mRNA is present in muscle cells at 20% of the levels of RTEF-1A mRNA^6 (data not shown), RTEF-1B protein is not found at comparable levels in muscle cells. The TEF-1 antiserum did not detect any protein that comigrated with overexpressed RTEF-1B protein on SDS gels. Overdevelopment of Western blots did result in the appearance of a band at the molecular weight expected for RTEF-1B. However, it is not known whether this represents authentic RTEF-1B protein or background reactivity.

As mentioned above, RTEF-1 is a member of a multigene family. These other family members also likely play a role in gene expression. Insertional mutagenesis of mouse NTEF-1 is is lethal by embryonic day 10.5(56) . The expression patterns of other TEF-1 family members indicate that they may regulate gene expression in muscle and non-muscle tissues. Chick DTEF-1 is expressed at highest levels in cardiac muscle, at lower levels in lung, gizzard, and kidney, and virtually absent in skeletal muscle and brain(55) . Mouse ETF-1 is expressed at highest levels in adult brain and at lower levels in heart and skeletal muscle(37) . Also, M-CAT sites are involved in gene regulation in non-muscle cells. Non-muscle promoters with functional M-CAT sites include the VSM alpha-actin promoter in AKR-2B fibroblasts (24, 25) , the human papilloma virus enhancer in keratinocytes and SiHa cells(51) , and the enhancer/promoter of SV40 in HeLa cells(29, 52) .

Interactions Required for Muscle-specific Gene Expression

The above results support the notion that complex 1 plays an essential role in muscle-specific gene expression. The enrichment of complex 1 in muscle, however, is not sufficient to account for full activity of M-CAT-dependent promoters in muscle. Muscle-specific gene expression directed by multimerized M-CAT elements is dependent upon sequences flanking the M-CAT sites as well as spacing between M-CAT motifs. (^7)Also, high levels of expression of the cTNT gene in cardiac muscle cells requires both M-CAT sites from the proximal region of the promoter and upstream sequences contained in a cardiac element(8) . Function of this element requires binding sites for the MEF-2 and the GATA families of transcription factors. (^8)Those results and the results presented here show that combinatorial interactions between the TEF-1 family of transcription factors, its DNA binding sites, and members of other transcription factor families, are required for the regulation of muscle-specific gene expression in the proper developmental stage, in the appropriate fiber type and in response to physiological cues.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL-35561 and HL-43821 (to C. P. O.) and a Postdoctoral Fellowship from the National Institutes of Health (to I. K. G. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Cardiovascular Research Institute and V.A. Medical Center, 111-C8, 4150 Clement St., San Francisco, CA 94121. Tel. 415-221-4810 (ext. 3206); Fax: 415-750-6950; iaink{at}itsa.ucsf.edu.

(^1)
The abbreviations used are: TEF, transcription enhancer factor; MES, 4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.

(^2)
P. C. Simpson, personal communcation.

(^3)
In this manuscript the term TEF-1 refers to mRNA or cDNA or proteins encoded by any member of the TEF-1 gene family.

(^4)
W. R. Thompson, personal communication.

(^5)
J. A. Winkles, personal communication.

(^6)
S. Ausoni, A. Stewart, and S. B. Larkin, unpublished results.

(^7)
S. B. Larkin, I. K. G. Farrance, and C. P. Ordahl, manuscript submitted for publication.

(^8)
S. Ausoni and C. P. Ordahl, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Nina Kostanian and Monique Benoualid for expert technical assistance on this project and Drs. Paul Simpson and Holly Ingraham for critical reading of the manuscript. The antibody against rat NTEF-1 was kindly provided by M.-H. Disatnik and P. C. Simpson.


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