(Received for publication, August 16, 1995; and in revised form, October 16, 1995)
From the
The M-CAT motif is a cis-regulatory DNA sequence that is
essential for muscle-specific transcription of several genes.
Previously, we had shown that both muscle-specific (A1) and ubiquitous
(A2) factors bind to an essential M-CAT motif in the myosin heavy chain
gene and that the ubiquitous factor is transcriptional enhancer
factor (TEF)-1. Here we report the isolation of mouse cDNAs encoding
two forms (a and b) of a TEF-1-related protein, TEFR1. The TEFR1a cDNA
encodes a 427-amino acid protein. The coding region of TEFR1b is
identical to 1a in both nucleotide and predicted amino acid sequence
except for the absence of 43 amino acids downstream of the TEA
DNA-binding domain. Three TEFR1 transcripts (
7,
3.5, and
2 kilobase pairs) are enriched in differentiated skeletal muscle
(myotubes) relative to undifferentiated skeletal muscle (myoblasts) and
non-muscle cells in culture. In situ hybridization analysis
indicated that TEFR1 transcripts are enriched in the skeletal muscle
lineage during mouse embryogenesis. Transient expression of fusion
proteins of TEFR1 and the yeast GAL4 DNA-binding domain in cell lines
activated the expression of chloramphenicol acetyltransferase (CAT)
reporter constructs containing GAL4 binding sites, indicating that
TEFR1 contains an activation domain. An anti-TEFR1 polyclonal antibody
supershifted the muscle-specific M-CAT
A1 factor complex in gel
mobility shift assays, suggesting that TEFR1 is a major component of
this complex. Our results suggest that TEFR1 might play a role in the
embryonic development of skeletal muscle in the mouse.
Commitment and subsequent differentiation of skeletal muscle involves a cascade of transcription factors that act through several different cis-regulatory elements. The best characterized of these elements are the E-box and A/T-rich motifs, which interact with the MyoD/bHLH (1) and MEF-2/MADS (2) families of transcription factors, respectively. Members of the MyoD family have been shown to be critical to the development of skeletal muscle(1, 3) . Members of the MEF-2 family are involved in both cardiac and skeletal muscle development (2, 4, 5, 6) .
In addition to
elements that are involved in muscle-lineage determination, other
cis-regulatory elements that have been shown to be involved in the
expression of muscle-specific genes include the serum response element,
SP-1 element, and M-CAT motif (6, 7, 8, 9, 10) . The
M-CAT motif was initially identified as a cis-regulatory element in the
cardiac troponin T promoter(6) . The M-CAT motif has
subsequently been shown to be required for muscle-specific expression
from the myosin heavy chain (9, 10, 11) and
-skeletal actin (7) promoters in vitro, as well as the myosin heavy
chain
(12) and
(13) promoters in
vivo.
Two mammalian M-CAT-binding proteins have been
identified, transcriptional enhancer factor-1 (TEF-1) ()(17, 20, 22) and embryonic TEA domain-containing factor
(ETF)(23) . ETF transcripts are strictly limited in their
distribution during embryogenesis, appearing predominately in the
hindbrain at embryonic day 10 by in situ hybridization
analysis. Little is known about ETF function except that it shares
binding-site specificity with TEF-1. TEF-1 is a cellular
transcriptional activator that was originally identified as a viral
cis-regulatory element-binding protein that interacts with the GT-IIC
and Sph motifs(14, 15, 16, 17) . The
GT-IIC motif is almost identical to the M-CAT motif, which TEF-1 has
been shown to bind as
well(7, 13, 18, 19, 20, 21) .
TEF-1 transcripts are present in most adult tissues, and are
particularly abundant in the kidney, skeletal muscle, heart, and lung.
During embryogenesis, TEF-1 appears to be expressed
ubiquitously(24) . TEF-1 contains a TEA DNA-binding domain (25) and an activation function that involves the cooperation
of at least three regions of the protein (acidic and proline-rich and
C-terminal regions)(26) . TEF-1 also interacts with
cell-specific co-factors, some of which are required for
activation(27) , others of which block activation(28) .
A transgenic mouse strain containing a null mutation of the TEF-1 gene
shows homozygous embryonic lethality around embryonic day 11 due to a
heart malformation characterized by an abnormally thin ventricle
wall(24) . However, the skeletal muscle lineage appears to be
unaffected in these mice, which suggests that TEF-1 is not essential
for the early stages of skeletal muscle development.
We have previously shown that two M-CAT (A-element) binding factors, A1 and A2, could be distinguished by gel mobility shift assays using nuclear extracts from differentiated skeletal muscle cells in culture (myotubes)(29) . The A2 factor appears to be ubiquitous, while the A1 factor is seen only in myotubes. We cloned the mouse homolog of TEF-1 (mTEF-1) and showed that it is one component of the A2 factor(20) . However, the identity of the A1 factor remained unknown. Because the A1 and A2 factors had similar DNA-binding properties(29) , we concluded that these factors might be closely related. By using TEF-1 cDNA as a probe, we isolated cDNAs for TEFR1, an M-CAT-binding transcription factor that bears a close resemblance to TEF-1. We present evidence here suggesting that TEFR1 is a candidate for the muscle-specific A1 factor. We also show that TEFR1 transcripts are enriched in the skeletal myogenic lineage during mouse embryogenesis.
Figure 3:
RT-PCR analysis of TEFR1 transcripts. A, a portion of the TEFR1 cDNAs from Fig. 1B.
Primers used for PCR (sense nt 103-124 of TEFR1a and antisense nt
703-722) are indicated by arrows. B, total RNAs from
mouse skeletal muscle (Sol8 and C2C12) and fibroblast (Swiss 3T3) cell
lines were subjected to RT-PCR using specific amplimers for either
TEFR1 (24 cycles) or -microglobulin (16 cycles). PCR
products were separated by agarose gel electrophoresis and Southern
blotted. The 620- and 491-bp bands correspond to TEFR1a- and
1b-specific PCR products, respectively. The negative control is in the
absence of RNA. Mb, myoblasts; Mt, myotubes. C, total RNAs from adult mouse tissues were subjected to
RT-PCR using TEFR1-specific (35 cycles) and
-actin-specific (22
cycles) amplimers. PCR products were separated by agarose gel
electrophoresis and Southern blotted. Ethidium bromide staining of
-actin products is shown at the bottom. The negative control is in
the absence of RNA.
Figure 1: cDNA and predicted amino acid sequence analysis of TEFR1. A, cDNA sequence of TEFR1. The sense strand is shown. The sequences of TEFR1a (upper) and 1b (lower) are numbered separately from the 5` termini of respective cDNA clones. Dashes within the TEFR1b sequence indicate identity to TEFR1a. Nucleotides which do not appear in TEFR1b are indicated by asterisks. An open box indicates the position of the probable initiation codon (ATT). A shaded box indicates the position of the termination codon (TGA). Nucleotides that correspond to the TEA DNA-binding domain are overlined. B, structures of TEFR1a and 1b cDNAs. Open and hatched boxes indicate the noncoding and coding regions, respectively. C, predicted amino acid sequence of TEFR1a (upper) and comparison with the predicted mTEF-1 sequence (lower)(20) . Between the two sequences, vertical bars indicate identity, and colons and single dots indicate strong and weak similarity, respectively. Dots within each sequence represent gaps which were introduced to maximize homology. The TEA DNA-binding domain is boxed. The 43-aa region absent from TEFR1b is underlined. D, comparison of the amino acid sequences of TEA DNA-binding domains of mouse TEFR1, human TEF-1(17) , Drosophila scalloped (Sd)(55) , Saccharomyces cerevisiae TEC-1(56) , and Aspergillus nidulans abaA(57) . The TEA domains of human, mouse(20) , and chicken (21) TEF-1 as well as ETF (23) are identical in amino acid sequence. Conserved amino acids are shaded. Dashes indicate gaps which were introduced to maximize homology. The numbers to the left of each line indicate the position of the first amino acid shown in each protein. The locations of predicted helices are indicated above the TEFR1 sequence. The locations of amino acids mutated in this study are indicated by black dots.
Figure 7: Relationship between TEFR1 and M-CAT-binding factors. A, structure of GST-fusion proteins. Numbers indicate positions of amino acids in either TEFR1 or mTEF-1. Regions of identity between TEFR1 and mTEF-1 are indicated by solid bars. Hatched and shaded bars indicate specific regions of TEFR1 and mTEF-1, respectively. B, immunoprecipitation (IP) of TEFR1 and mTEF1 by anti-TEFR1 antibody. The in vitro transcription/translation products from TEFR1b and mTEF-1 cDNA clones (Pre-IP) were immunoprecipitated by anti-TEFR1 antibody in the absence(-) or presence of an equimolar amount of GST-fusion protein competitors shown in A. Proteins were separated on an 11% SDS-PAGE gel. Arrows indicate bands corresponding to full-length of TEFR1b and mTEF-1. C and D, effect of anti-TEFR1 antibody (Ab) on the binding of nuclear factors to oligo A (M-CAT motif). Three micrograms of nuclear extract (NE) from either HeLa cells or Sol8 myotubes (Mt) were preincubated with preimmune (P) or anti-TEFR1 (TR) IgY in the absence (all of C; - in D) or presence of GST-fusion protein competitors shown in A. Either 1 or 2 µl of 6 mg/ml IgY were used. Reaction mixtures were further incubated with end-labeled oligo A and separated on an 8% native PAGE gel. Arrows indicate the position of specific complexes (A1 and A2), nonspecific complex (*), supershifted complexes (S), and free probe (F).
Figure 9:
Transactivation of the p3xGAL4-BGCAT
reporter by GAL4/TEFR1-chimeric proteins. The p3xGAL4-BGCAT reporter (2
µg) containing three copies of the GAL4 DNA-binding site was
transfected with each expression vector for a GAL4/TEFR1 chimera (0.1
µg) and pCMV-lacZ (1 µg) into either Sol8 myocytes or HeLa
cells. CAT activity was normalized using the -galactosidase
activity for each transfection. Values for fold induction are given
relative to activity of the GAL4(1-147) construct. Results
represent the average of two to four separate transfection experiments.
Variation of normalized values was less than 10% between experiments.
The structure of TEFR1a is shown at the top. The region absent from
TEFR1b (aa 112-154) and the TEA domain (aa 31-98) are shown
above the TEFR1a diagram. The acidic, basic, proline-rich (Pro), and serine-threonine-tyrosine-rich (STY)
regions are indicated by different fill patterns. The positions of
mutated aa 48 (Leu
Pro) and aa 50 (Ile
Phe) in GAL4-TEFR
1M and 2M are indicated by asterisks.
The open reading frame of the TEFR1a cDNA encodes a 427 aa protein
containing a TEA DNA-binding domain (aa 31-98; Fig. 1, A-C). The initiation codon used by TEFR1 appears to be
AUU (Ile) at nt 110 (see ``Discussion''). The open reading
frame of the TEFR1b cDNA encodes a 384-aa protein, which is identical
to TEFR1a except that 43 aa downstream of the TEA domain are absent
from TEFR1b (Fig. 1, B and C). The full coding
nucleotide sequence of TEFR1b is identical to that of TEFR1a, except
that a 129-nt region (nt 443-571 in TEFR1a) is absent from TEFR1b (Fig. 1, A and B). The noncoding regions of
the two cDNAs are divergent, except for sequences proximal to the start
and stop codons, which are identical (Fig. 1, A and B). Comparison of the TEFR1 and mTEF-1 (20) predicted
amino acid sequences revealed 76% overall identity and 93% identity
within the TEA domain (Fig. 1C). The TEA DNA-binding
domain consists of three putative -helices (25) and is
highly conserved throughout evolution. Except for TEFR1, all other
vertebrate TEA proteins share 100% amino acid identity within the TEA
domain (Fig. 1D)(23) .
Figure 2:
Northern blot analysis of TEFR1
transcripts. A and B, total RNAs were isolated from
either cell lines (A) or adult mouse tissues (B).
Thirty micrograms of total RNAs were separated on a 1% agarose gel and
transferred to a nylon filter. The full-length coding region of the
TEFR1a cDNA clone was used as a probe. The positions of 28 S and 18 S
ribosomal RNAs are indicated. The positions of mRNAs (7,
3.5,
and
2 kb) hybridized with the TEFR1a probe are indicated by open arrowheads. Ethidium bromide staining of 28 S ribosomal
RNA prior to transfer of the gel is shown at the bottom. Mb,
myoblasts; Mt, myotubes. C, poly(A)
RNAs were prepared from Sol8 myotubes (Mt) and 3T3
fibroblasts. Three micrograms of poly(A)
RNAs were
separated on a 0.8% agarose gel and transferred to a nylon filter. The
filter was hybridized with a probe containing the full-length coding
region of the TEFR1a cDNA (left). The same filter was
sequentially rehybridized with a labeled EcoRI fragment of
mTEF1 cDNA (right) and a labeled GAPDH cDNA (bottom)(20) . The positions of mRNAs hybridized with
the TEFR1a probe and the mTEF1 probe (
12 kb) are indicated by open and solid arrowheads,
respectively.
RT-PCR was used to distinguish between TEFR1a and 1b transcripts (Fig. 3). Both TEFR1a (620 bp) and 1b (491 bp) PCR products were seen in Sol8 and C2C12 myotubes (Fig. 3B). Both TEFR1a and 1b were also observed in adult lung, skeletal muscle, heart, and kidney (Fig. 3C). A very small amount of TEFR1 was seen in Sol8 and C2C12 myoblasts and non-muscle cells (3T3) (Fig. 3B). TEFR1b has consistently appeared to be more abundant than 1a in all tissues and cell lines tested under the RT-PCR conditions used here, except in 3T3 cells. In 3T3 cells, TEFR1a has consistently appeared to be more abundant than 1b.
We also examined
the expression of TEFR1 transcripts during mouse development by in
situ hybridization using antisense riboprobes made by in vitro transcription of the full coding region of the TEFR1a cDNA. TEFR1
transcripts are enriched in the myotome at embryonic day 9 (Fig. 4, A-E), co-localized with -cardiac
actin (Fig. 4, C and F). At embryonic day
14.5, TEFR1 transcripts are enriched in embryonic skeletal muscle (Fig. 5B), co-localized with
-cardiac actin (Fig. 5A).
-Cardiac actin has been shown to be a
marker for embryonic striated muscle(34) . In situ hybridization using sense riboprobes showed background levels of
hybridization over tissue sections (Fig. 5, C and D).
Figure 4:
In situ hybridization to serial
sections of an embryonic day 9 mouse embryo, showing somites. Antisense
riboprobes complimentary to either TEFR1 (A, B, D, and E) or -cardiac actin (C and F) transcripts were used. B, C, E,
and F are views under dark-field optics. A and D are bright-field views of B and E, respectively. D, E, and F are higher magnification views
of A, B, and C, respectively; the box in A indicates the magnified area. Solid arrowheads in A indicate somites. Arrowheads in B indicate
artifactual debris. DT, dermatome; MT, myotome; NT, neural tube. Bar, 50
µm.
Figure 5:
In situ hybridization to
parasaggital sections of an embryonic day 14.5 mouse embryo, showing
tongue. All views are under dark-field optics. The following riboprobes
were used: -cardiac actin antisense (A) or sense (C); TEFR1 antisense (B) or sense (D). All
dark-field photographs were taken at the same exposure level. Bar, 100 µm.
Figure 6: In vitro transcription/translation of TEFR1 and mTEF-1. A, SDS-PAGE of in vitro translated mTEF-1 and TEFR1. The mTEF-1 and TEFR1a and 1b RNAs were synthesized in vitro, using either T7 or T3 RNA polymerase, and then translated using rabbit reticulocyte lysate. Three microliters of the in vitro translation products were separated on an 11% SDS-PAGE gel. B, gel mobility shift assay of in vitro translated products using end-labeled oligo A. Three microliters of either unprogrammed rabbit reticulocyte lysate (Lysate), mTEF-1, or TEFR1 translation products, or 2 µg of nuclear extracts from HeLa cells or Sol8 myotubes (Mt) were incubated with end-labeled oligo A and separated on an 8% native PAGE gel. Arrows indicate the positions of specific complexes (A1 and A2), nonspecific complexes (*), and free probe (F). C, competition for complex formation between in vitro translated products and oligo A. TEFR1 translation products were incubated with end-labeled oligo A in the absence(-) or presence of unlabeled competitor oligo A or MutA (M) at a 50-fold molar excess over the labeled probe.
We then conducted gel mobility shift assays using nuclear extracts from either HeLa cells or Sol8 myotubes in the presence of either preimmune or anti-TEFR1 antibody (Fig. 7C). Using Sol8 myotube nuclear extract, addition of anti-TEFR1 antibody resulted in the disappearance of the A1 complex band, reduction of the intensity of the A2 complex band, and formation of a supershifted complex (lanes 6 and 8). However, using HeLa cell nuclear extract, addition of anti-TEFR1 antibody reduced the formation of the A2 complex and did not result in the formation of a supershifted complex (lanes 2 and 4). Supershifted complexes are generally thought to contain probe, DNA-binding protein, and specific antibody. To confirm whether the muscle-specific A1 complex contains TEFR1, we examined the ability of GST-fusion protein competitors (Fig. 7A) to block the activity of the anti-TEFR1 antibody (Fig. 7D). A GST-fusion protein containing only TEFR1-specific residues (GST-TEFR(1-24)) completely blocked the activity of the anti-TEFR1 antibody (lane 4), preventing the formation of the supershifted complex and returning the A1 and A2 complex band intensities to those observed in the presence of preimmune antibody (lane 1). The GST-TEFR1 immunogen (GST-TEFR(1-38)) also completely blocked the activity of the anti-TEFR1 antibody (lane 3). In the presence of either GST alone (lane 6) or a GST-mTEF-1 competitor (GST-mTEF(1-29); lane 5), no competition was observed with the anti-TEFR1 antibody. These results suggest that the A1 complex contains TEFR1.
Figure 8:
Effect of overexpression of TEFR1 and
mTEF-1 on reporter plasmids containing multiple binding sites. Each
reporter (2 µg) was co-transfected with expression vector (0.2 or
1.0 µg) and pCMV-lacZ (1 µg) into either Sol8 myocytes (A) or HeLa cells (B). The 5GTIIC-TKCAT and 3A-TKCAT
plasmids contain five copies of a GTIIC tandem repeat and three copies
of the A element (21 bp), respectively, upstream of the herpes simplex
virus thymidine kinase promoter in pTKCAT(20) . The CAT
activity was normalized using the -galactosidase activity for each
transfection. All CAT activities are given relative to the value
obtained for the p5GTIIC-TKCAT, which is set at 100% for each cell
type. Results represent the average of two to four separate
transfection experiments. Variation of normalized values was less than
10% between experiments.
There are two
functional DNA-binding domains in both GAL4-TEFR 1 and GAL4-TEFR 2. It
has been reported that GAL4/AP2, SP1/(CTF/NF1), and GAL4/TEF-1
chimeras, which contain more than one functional DNA-binding domain,
are weak transactivators, although they contain strong activation
domains(26, 41, 42) . To determine whether or
not strong activation functions in GAL4-TEFR 1 and 2 were being masked
by the presence of two functional DNA-binding domains, we mutated two
conserved amino acids within the first putative -helix of the
TEFR1 TEA DNA-binding domain, aa 48 (Leu
Pro) and 50 (Ile
Phe) (Fig. 1D). Mutations in this region result in loss
of DNA-binding activity (26) (data not shown). Transfection of
GAL4-TEFR 1M or GAL4-TEFR 2M which contain mutated TEFR1a and 1b,
respectively, strongly activated the expression of the p3xGAL4-BGCAT
reporter in both Sol8 myotubes (
100-fold) and HeLa cells
(
150-fold) (Fig. 9). Deletion of aa 1-111 of TEFR1a
(GAL4-TEFR 3), including acidic and basic regions and the entire TEA
domain, dramatically increased the expression of the reporter plasmid
(
400 fold in Sol8;
800-fold in HeLa). Strong activation was
still observed when aa 1-206, including the proline-rich region,
were deleted (GAL4-TEFR 5). However, deletion of aa 1-302,
including one of two STY-rich regions, resulted in total loss of
activation function (GAL4-TEFR 6). In addition, there was no activation
when the C-terminal 28 aa (aa 400-427) were removed from
GAL4-TEFR 4 (GAL4-TEFR 7). Therefore, TEFR1 apparently contains one or
more activation domains, at least one of which is located in the
C-terminal half of the protein (aa 207-427).
TEF-1 is considered to be the transcription factor that is
primarily responsible for the transcriptional activation through the
M-CAT cis-regulatory element found in certain muscle-specific genes.
This seems reasonable for both non-muscle and cardiomyocyte cells, as
in both cases there is a single M-CAT-binding activity seen in gel
mobility shift assays, which has been attributed to TEF-1 by antigenic
criteria(7, 13, 17, 19) . However,
we had shown previously that in differentiated mouse skeletal muscle in
culture two M-CAT-binding activities could be detected, which we
designated A1 and A2(29) . The M-CATA2 factor-complex is
ubiquitous and contains mTEF-1 as a major component(20) . Here
we report the isolation of a TEF-1-related transcription factor, TEFR1,
which appears to be a major component of the muscle-specific
M-CAT
A1 factor-complex.
We have isolated two TEFR1 cDNAs,
TEFR1a and 1b, which have identical nucleotide sequences within their
coding and proximal noncoding regions, but for the absence of 129 nt
(43 aa) in TEFR1b. Southern blotting of CD1-mouse genomic DNA with the
full-length TEFR1b cDNA clone showed hybridization to a single,
10-kb EcoRI fragment, which suggests that TEFR1a and 1b
are splice forms of a single gene (data not shown). In all cell types
and tissues examined by RT-PCR, TEFR1b transcripts have been more
abundant than 1a, except in the case of 3T3 cells, where the ratio is
inverted (Fig. 3). It has been reported that the function of a
transcription factor can be altered by structural changes occurring in vivo, such as alternative splicing (43, 44) and phosphorylation(45) . Further
work will be required to determine whether TEFR1a and 1b are
functionally distinct.
TEFR1 appears to use AUU (Ile) at nt 110 as an initiation codon, similar to TEF-1(17) . Mutation of this codon to UGG (Trp) resulted in no translational initiation in vitro from nt 110, while mutation to AUG (Met) increased translational efficiency (data not shown). There are several AUG codons in the 5` portion of the TEFR1 cDNAs. However, comparison of the flanking sequences surrounding these various codons indicated that the AUU at nt 110 lies in the best Kozak sequence context(46) . Furthermore, none of the AUG codons is appropriately positioned to account for the fact that TEFR1 contains a TEA DNA-binding domain, as indicated by its ability to bind to the M-CAT motif (Fig. 6). Also, only initiation at the AUU codon at nt 110 can account for both the molecular weights of the in vitro translated TEFR1 products (Fig. 6) and the existence of the amino-terminal residues which were detected by the anti-TEFR1 antibody (Fig. 7).
TEFR1 is
the third member of the TEA family to be found in mammals. The
expression patterns of ETF (23) and TEFR1 are
tissue-restricted, in contrast to the widespread expression of
mTEF-1(20) . Comparison of the predicted amino acid sequences
of mTEF-1, TEFR1, and ETF indicated that ETF is equally divergent,
65% identity, from either mTEF-1 or TEFR1, while mTEF-1 and TEFR1
share 76% identity. Comparison of TEFR1 to the 172-bp EST 683 human
cDNA clone 32B5 (GenBank
accession no. T25108) (47) showed that they share 95% identity at the amino acid
level. The sequence of EST 683, which was identified by systematic
sequencing of a colorectal cancer cDNA library, coincides with TEFR1 aa
261-316. EST 683 might correspond to the human analog of mouse
TEFR1, but this must be confirmed by cloning and sequencing of the
full-length cDNA. EST 683 also shows a high degree of homology to
chicken TEF-1 (cTEF-1)(21) . Comparison of the cTEF-1 predicted
amino acid and nucleotide sequences to those of mTEF-1 and TEFR1
revealed an unexpected result. At the amino acid level, TEFR1 has an
overall identity with cTEF-1 of 87%, but only 76% with mTEF-1, while
mTEF-1 and cTEF-1 share 77% identity. Comparison of the nucleotide
sequences of the TEA domains revealed that TEFR1 shares 83% identity
with cTEF-1, but only 76% with mTEF-1, while mTEF-1 and cTEF-1 share
78% identity. These comparisons suggest that cTEF-1 is the chicken
analog of TEFR1 rather than mTEF-1. However, the structures of the
activation domains of TEFR1 (Fig. 9) and cTEF-1 (21) are
significantly different. One possibility is that cTEF-1, mTEF-1, and
TEFR1 represent three distinct members of the TEA family. If, on the
other hand, cTEF-1 and TEFR1 are homologs, then differences between the
activation domains of cTEF-1 and TEFR1 might be due to differences in
co-factors between avians and mammals (see below). A third alternative
is that, in avians, there might be a single transcription factor
(cTEF-1) responsible for transcriptional activation through the M-CAT
motif in all striated muscle.
TEFR1 contains multiple putative
activation domains, most of which are shared structurally with TEF-1,
such as acidic(48, 49) , proline-rich (41, 42) , and STY-rich (50, 51) regions. However, the acidic and proline-rich
regions which are essential for TEF-1 activation function (26) are dispensible for TEFR1 activation function (Fig. 9). In transfection assays using TEFR1 expression vectors
and CAT reporter constructs containing either M-CAT or GT-IIC elements,
we observed a dose-dependent repression of reporter gene activity (Fig. 8). One possible explanation for this observation is that
TEFR1 is an M-CAT-binding repressor. However, two observations make
this possibility unlikely. First, accumulation of transcripts of some
contractile proteins in differentiated skeletal muscle is dependent
upon the M-CAT positive cis-regulatory element. Second, the formation
of the M-CATA1 factor (TEFR1) complex occurs in skeletal muscle in vitro as differentiation proceeds(29) . An
alternative explanation is that overexpression of TEFR1 results in
``squelching'' (52) , which implies that TEFR1
requires a co-activator(s) to function, as has been determined
biochemically for TEF-1(27, 28) . The dose-dependence
of the squelching effect and minimal structural requirements for
activation are different between TEF-1 and TEFR1. Therefore, TEF-1 and
TEFR1 might interact with overlapping but nonidentical sets of
co-factors. Further work will be required to determine whether the
structural differences between TEF-1 and TEFR1 indicate in vivo functional differences.
To directly address the question of whether TEFR1 is a component of the muscle-specific A1 complex, we generated a polyclonal antibody to the amino terminus of TEFR1. Gel mobility shift assays in the presence of this antibody resulted in the formation of a supershifted complex and reduction in both A1 and A2 complex band intensities (Fig. 7C). This effect could be eliminated by competition with a segment of the immunogen which was specific to TEFR1 (GST-TEFR(1-24); Fig. 7D, lane 4). These results, along with previous data indicating that components of the A1 complex are antigenically distinct from TEF-1(20) , suggest that a major component of the muscle-specific A1 complex is TEFR1. It is possible that the A1 complex does not contain TEFR1, but rather a protein that contains a region that is antigenically identical to the amino terminus of TEFR1. This is unlikely, though, because the region corresponding to TEFR(1-24) is highly divergent among the three mammalian TEA-domain proteins identified so far, TEF-1(17, 20, 22) , TEFR1, and ETF(23) . Further work will be required to determine whether the A2 complex in differentiated skeletal muscle also contains TEFR1 and to determine whether TEFR1a, 1b, or both are present in the muscle-specific A1 complex.
Skeletal muscle cell lines exhibit many of the properties of embryonic and perinatal muscle(53, 54) . The enrichment of TEFR1 transcripts in differentiated skeletal muscle cells in culture suggested that TEFR1 might also be enriched in embryonic skeletal muscle. In situ hybridization analysis of mouse embryos at embryonic day 9 (Fig. 4) and embryonic day 14.5 (Fig. 5) showed that TEFR1 transcripts are relatively abundant in the skeletal myogenic lineage. Our Northern analysis of adult tissue RNA showed that TEFR1 transcripts are expressed weakly in adult skeletal muscle. Therefore, TEFR1 might be transiently required for some early stage of myofiber maturation, such as appropriate accumulation of key components of the contractile apparatus.
Northern analysis showed that both mTEF-1 (20) and TEFR1 (Fig. 2B) are present in adult heart. Our in situ hybridization results showed no enrichment of TEFR1 transcripts in embryonic heart (data not shown). Our initial isolation of TEFR1 was from an adult mouse cardiac cDNA library. However, after multiple screenings of this library, only a single, partial TEFR1 cDNA clone was isolated, while we recovered multiple mTEF-1 cDNA clones. This suggests that TEFR1 transcripts are less abundant than those of mTEF-1 in the adult heart. Also, there appears to be a single cardiac M-CAT-binding activity(7, 13, 19) . Although these observations imply a minor role for TEFR1 in the heart, we cannot rule out the possibility that TEFR1 is involved in cardiac gene regulation. In particular, we do not know whether TEFR1 transcripts are present at the time when contractile protein gene transcription is initiated in the heart, prior to embryonic day 8(34) .
In the adult mouse, TEFR1 transcripts are present at a higher level in lung than in any other tissue we tested. Mouse TEF-1 transcripts are also present at a high level in adult lung, approximately equivalent to striated muscle and kidney(20) . Our in situ hybridization data indicated that TEFR1 is not enriched in the embryonic lung at either embryonic day 9 or 14.5 (data not shown). This suggests that TEFR1 might not be involved in early development of the lung. Though TEFR1 and possibly other members of the TEA family might be involved in lung-specific gene expression, especially postnatally, no lung-specific promoters have been identified yet that contain functional M-CAT motifs.
As more transcription factors have been characterized, it has become clear that the existence of networks of factors interacting at single cis-regulatory elements is common. The E-box and A/T-rich motifs have been shown to interact with distinct families of transcription factors, the MyoD/bHLH (1) and MEF-2/MADS (2) families, respectively. The M-CAT motif appears to be another such element, interacting with members of the TEA family of transcription factors. Our results indicate that TEFR1 and TEF-1 are closely related members of this TEA family and that TEFR1 might play a role in the embryonic development of skeletal muscle.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L26343 [GenBank](TEFR1a) and L26344 [GenBank](TEFR1b).