(Received for publication, November 10, 1995; and in revised form, January 16, 1996)
From the
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.
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) ;
MHC (11, 12) ; cTNC(13, 14) ;
skeletal
-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 -actin (15, 16) ;
-myosin heavy
chain(11, 12, 20, 21) ;
-myosin
heavy chain(22, 23) ; vascular smooth muscle
-actin(24, 25) ;
-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)(
)(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) . ()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)(
)(36) . The
other two classes are termed DTEF-1 (for Divergent TEF-1) (55) and ETEF-1 (for Embryonic TEF-1)(37) . (
)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.
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.
Nuclear
extract from a single 100-mm plate per treatment (50 µl; 2
10
cpm for
S-labeled extracts and
4.5
10
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
, 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.
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
(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.
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.
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.
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) 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) (
)(see
``Discussion'').
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.
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.
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.
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.
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.
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.
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
-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) .