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Volume 271, Number 36,
Issue of September 6, 1996
pp. 21775-21785
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Family of Developmentally Regulated Mammalian
Transcription Factors Containing the TEA/ATTS DNA Binding
Domain*
(Received for publication, April 24, 1996, and in revised form, June 14, 1996)
Patrick
Jacquemin
§¶,
Jung-Joo
Hwang
,
Joseph A.
Martial
§,
Pascal
Dollé
and
Irwin
Davidson
''
From the Institut de Génétique et de
Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP,
Collège de France, B.P. 163-67404 Illkirch Cédex,
France and the ¶ Laboratoire de Biologie Moléculaire et de
Genie Génétique, Institut de Chimie-B6, Université de
Liège, B-4000 Sart-Tilman, Belgium
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
ABSTRACT
We describe the molecular cloning of two novel
human and murine transcription factors containing the TEA/ATTS DNA
binding domain and related to transcriptional enhancer factor-1
(TEF-1). These factors bind to the consensus TEA/ATTS cognate binding
site exemplified by the GT-IIC and Sph enhansons of the SV40 enhancer
but differ in their ability to bind cooperatively to tandemly repeated
sites. The human TEFs are differentially expressed in cultured cell
lines and the mouse (m)TEFs are differentially expressed in embryonic
and extra-embryonic tissues in early post-implantation embryos.
Strikingly, at later stages of embryogenesis, mTEF-3 is specifically
expressed in skeletal muscle precursors, whereas mTEF-1 is expressed
not only in developing skeletal muscle but also in the myocardium.
Together with previous data, these results point to important,
partially redundant, roles for these TEF proteins in myogenesis and
cardiogenesis. In addition, mTEF-1 is strongly coexpressed with mTEF-4
in mitotic neuroblasts, while accentuated mTEF-4 expression is also
observed in the gut and the nephrogenic region of the kidney. These
observations suggest additional roles for the TEF proteins in central
nervous system development and organogenesis.
INTRODUCTION
Transcriptional enhancer factor-1
(TEF-1)1 belongs to a family of proteins
sharing the TEA/ATTS domain that shows a remarkable degree of
conservation between yeast and humans (Refs. 1, 2, 3, 4 and see Fig. 1). In
addition to TEF-1, this family comprises the yeast protein TEC1 that is
postulated to bind to the sterile responsive element downstream from
the Ty1 transposon long terminal repeat (5, 6), and the
Aspergillus nidulans factor AbaA, controlling a
regulatory circuit in the terminal stage of conidiophore development
essential for spore formation (7, 8 and Refs. therein). In
Drosophila, the TEF-1 homologue scalloped
(sd) is expressed in the regions of the wing disc that are
destined to become defective structures in viable sd mutants (9).
Sd is also expressed in the embryonic central nervous system
(CNS) and in peripheral sense organs. In the adult brain expression is
restricted to subsets of cells some of which are involved in taste
responses. Accordingly, viable sd mutants also display
abnormal taste behavior (10). In mouse, a TEF-1-related factor, ETF,
has been recently described (11). Like sd, ETF was reported
to be specifically expressed in the developing brain.
Fig. 1.
Evolutionary conservation of the TEA
domain. The upper panel shows the alignment of the
amino acid sequences of the human TEF-1, Drosophila
scalloped (sd), Aspergillus nidulans AbaA,
and the yeast TEC1 proteins. Highly conserved residues are
boxed. The positions of the predicted -helices are
indicated above the sequences. The arrows
indicate the positions of the degenerate oligonucleotides used for PCR
amplification of the novel TEA domains shown in the lower
panel. The lower panel shows the nucleotide and amino
acid sequences of the PCR-amplified TEA domain subregions from the
novel proteins hTEF-3, hTEF-4, mTEF-3, mTEF-4, as well as the
previously reported sequences for hTEF-1 and mTEF-1.
[View Larger Version of this Image (46K GIF file)]
The most studied member of the TEA/ATTS family, TEF-1, is a
transcriptional activator first identified in HeLa cells by its binding
to the GT-IIC and Sph(I + II) enhansons in the simian virus 40 (SV40)
enhancer (1, 12). We have shown that the TEF-1 TEA/ATTS domain is the
minimal domain required for specific binding to the GT-IIC and Sph
enhansons (13). However, in both TEF-1 and sd, DNA binding
is modulated by sequences outside of the TEA/ATTS DNA binding domain
(DBD) (13). Comparison of TEF-1 binding sites from a variety of
enhancers (see below) shows that it binds to highly degenerate
sequences (consensus 5 -(A/T)(A/G)(A/G)(A/T)ATG(C/T)N-3 ). A similar
consensus sequence has been deduced from the comparison of
AbaA binding sites (8). In contrast to the majority of
sequence-specific DNA binding proteins that bind cooperatively to
palindromic elements, TEF-1 binds cooperatively to tandem, but not
spaced or inverted, repeats of its binding sites (1). Consequently,
tandemly repeated TEF-1 binding sites have higher enhancer activity
in vivo than spaced repeats (14, 15).
TEF-1 not only activates transcription from the SV40 early promoter,
but together with large T antigen it acts to modulate SV40 late
transcription (16, 17, 18, 19, 20). Transcription of the human papilloma virus 16 E6/E7 oncogenes from the P97 promoter is also in part regulated by
TEF-1 (21). Transcriptional activation by TEF-1 requires the
cooperative action of several regions of the protein (13), a limiting
transcriptional intermediary factor(s) (2, 21), and, in
vitro, TATA-binding protein (TBP)-associated factors
(hTAFIIs) present in two chromatographically distinct TFIID
complexes (22). TEF-1 activity is also subject to negative regulation
by at least two distinct negatively acting factors (23, 24).
TEF-1 binding sites have also been characterized in several
muscle-specific promoters. Expression of the cardiac troponin C and
myosin and heavy chain genes has been shown to depend on a
GT-IIC-related enhanson (M-CAT) (25, 26, 27, 28, 29, 30, 31). This has been demonstrated
not only in gene transfer experiments but also, in the case of the
-myosin heavy chain gene, in transgenic mice (31). The M-CAT
enhanson is recognized by M-CAT binding factor, subsequently shown to
be antigenically related to TEF-1 (26). A putative chicken homologue of
TEF-1 has been isolated and shown to bind to the M-CAT enhanson
(cTEF-1, 32). Furthermore, muscle-enriched isoforms of cTEF, which have
altered transactivation properties, have been identified. TEF-1 binding
sites have also been shown to regulate the expression of vascular
smooth muscle -actin (33) and are involved in the reactivation of
the isoform of myosin heavy chain and skeletal -actin genes upon
induced cardiac hypertrophy (34, 35, 36, 37). The latter genes are normally
expressed during fetal cardiac development suggesting that TEF-1
contributes to the expression of these genes during cardiogenesis.
Indeed mice in which the murine homologue of TEF-1 has been disrupted
die during embryogenesis due to heart malformation, further supporting
the idea that TEF-1 is of critical importance for cardiac development
(38). Aside from muscle-specific gene regulation, putative TEF-1
binding sites have been noted in the placental-specific human chorionic
somatomammotropin (hCS also called placental lactogen) -B gene enhancer
(39, 40, 41, 42, 43, 44).
Here we describe two novel human and murine factors belonging to the
TEA/ATTS family and related to TEF-1. These proteins bind to the GT-IIC
and Sph enhansons of SV40 but differ in their ability to bind
cooperatively to tandemly repeated sites. However, cooperative binding
to tandem sites is an intrinsic property of the minimal TEA/ATTS DBD
showing that differences elsewhere in the TEF factors modulate their
DNA binding properties. Reverse transcription PCR experiments show
differential expression of the human TEF factors in cultured cell
lines. In situ hybridization shows distinct expression
patterns of the mTEFs in embryonic and extra-embryonic tissues prior to
gastrulation. From mid-gestational stages onward strong mTEF-1
expression is observed in developing skeletal muscle and myocardium,
but mTEF-3 is expressed almost exclusively in skeletal muscle
precursors. These results, together with those of previous studies,
highlight the important roles played by the TEF proteins in myogenesis
and cardiogenesis. However, mTEF-1 expression is not limited to
skeletal and heart muscle as it is strongly expressed along with mTEF-4
in mitotic neuroblasts both in the brain and spinal cord. At later
stages of embryogenesis mTEF-1 and mTEF-4 show distinct expression
patterns in several developing structures such as the olfactory system,
the intestine, and the kidney.
MATERIALS AND METHODS
Polymerase Chain Reaction Amplification and Screening of cDNA
Libraries
Two degenerate oligonucleotides (5 -CCCAAGCTTGGC(A/C)G
GAA(C/T)GA(A/G)(C/T)TGAT(A/C)GC-3 and
5 -CCCAAGCTTC(A/G/C/T)A(G/A)(A/G/C/T)AC(C/T)TG(T/G/A)AT(G/A)TG-3 )
corresponding to the TEA domain amino acid sequences GRNELIA and HIQVL
were used to PCR-amplify a series of cDNA libraries including those
from HeLa cells, human fetal brain, retinoic acid-differentiated mouse
embryonic stem cells, and 10.5 dpc mouse embryos. 30 cycles (1 min at
94 °C, 1.5 min at 40 °C, and 1.5 min at 72 °C) of PCR were
performed under standard conditions in a 100-µl reaction volume with
200 pmol of each degenerate oligonucleotide primer, 2-4-µl aliquots
of each library (>109 plaque forming units/ml) and 2 units
of ampliTaq polymerase (Perkin Elmer). Amplification products of the
correct size were gel-purified and cloned into the TA cloning vector
(Invitrogen). DNA sequencing was performed on an Applied Biosystems
automated sequencer. TEF-specific probes for screening cDNA
libraries were generated by PCR using the degenerate primers and the
partial cDNAs as templates in the presence of
[ -32P]dCTP. cDNA libraries were screened by
hybridization at 42 °C in 6 × SSC, 50% formamide. Filters
were washed at 55 °C in 3 × SSC. Positive clones were picked,
purified, and excised from ZAPII (Stratagene) libraries by standard
procedures. The DNA sequences of both strands of each clone were
determined using internal primers. DNA and protein sequence analysis
were performed using the GCG (Genetics Computer Group, University of
Wisconsin) software package.
Construction of Expression Vectors
The ORFs for the novel
TEFs were amplified with appropriately positioned oligonucleotides
containing a consensus Kozak sequence replacing the translation
initiation isoleucine codons with ATG. The primers contained
EcoRI/XbaI or EcoRI/XhoI
restriction sites, and the PCR fragments were cloned between the
corresponding sites in pXJ41. The DNA sequences of the expression
vectors were verified by automated DNA sequencing.
Transfections and Preparation of Cell Extracts
Cos cells
were transfected by the calcium phosphate coprecipitation technique as
described previously (13). 48 h after transfection the cells were
harvested (from 60-mm diameter dishes) and extracts prepared by three
cycles of freeze-thaw in 200 µl of buffer A (50 mM
Tris-HCl, pH 7.9, 20% glycerol, 0.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.1% Nonidet P-40, and 1 mM dithiothreitol) containing 0.5 M KCl and 2.5 µg/ml leupeptin, pepstatin, aprotinin, antipain, and chymostatin as
described (45, 46). Generally between 2 and 8 µl of the extracts were
then used in EMSA.
Cloning and Expression of the Histidine-tagged TEF-1 TEA
Domain
The region of TEF-1 encoding amino acids 28-104 was
PCR-amplified with primers containing NdeI and
BamHI restriction sites. The PCR product was cloned between
the NdeI and BamHI sites of the pET15 vector to
generate a TEA domain tagged with 6 histidines (6His-TEA). The plasmid
was transformed into the BL21 strain, and expression was induced by the
addition of isopropyl-1-thio- -D-galactopyranoside for
1.5 h. From 400 ml of culture 20 ml of bacterial extract was
prepared in buffer A containing 0.5 M KCl and protease
inhibitors, and the fusion protein was purified by chromatography on a
2-ml nitrilotriacetic acid-Ni-agarose column (Qiagen). The column was
washed sequentially with 4-column volumes of buffer A containing 1.0 M KCl and buffer A containing 0.05 M KCl and
0.05 M imidazole. The fusion protein was then eluted with
2 × 1.5 ml of buffer A containing 0.05 M KCl and 0.25 M imidazole and loaded onto a 1-ml double-stranded DNA
cellulose column (Sigma) that was washed extensively
with buffer A containing 0.25 M KCl. The protein was then
eluted in 3 × 1 ml of buffer A containing 0.8 M KCl,
dialyzed against buffer A containing 0.1 M KCl, and frozen
in aliquots at 80 °C.
Electrophoretic Mobility Shift Assays
The oligonucleotides
containing the wild-type or mutated GT-IIC enhanson and the tandemly
repeated GT-IIC or Sph enhansons, or repeats spaced by 5 or 10 nucleotides were as described previously (1, 13). The oligonucleotides
were 32P-5 -end-labeled using polynucleotide kinase and
separated from unincorporated [ -32P]ATP by
chromatography of G-50-Sepharose. Electrophoretic mobility shift assays
(EMSA) were performed essentially as described previously (13) on 6%
polyacrylamide gels in 0.5 × standard TBE buffer.
Reverse Transcription PCR
Total cytoplasmic RNA was
isolated from human cell lines by lysis with buffer B (50 mM Tris-HCl, pH 7.9, 0.1 M KCl, 0.5 mM EDTA, and 0.2% Nonidet P-40) and subsequent
phenol/chloroform extractions and ethanol precipitations. To test the
integrity of the RNA preparations RT-PCR was performed using primers in
the hRBP17 subunit of the RNA polymerases generating a 630-nucleotide
fragment (Ref. 47 and data not shown). Reverse transcription was
performed with 2.5 µg of RNA for 30 min at 40 °C with 5 units of
Moloney murine leukemia virus reverse transcriptase using the following
TEF-specific antisense primers; hTEF-1, 5 -CTTTAGCTTGGAATGAAAA-3 ;
hTEF-3, 5 -AGCTTGGCCTGGATCTCG-3 ; hTEF-4, 5 -CTTGGACTGGATTTCCCT-3. The
products of reverse transcription were then amplified using the same
antisense primers and the following sense primers: hTEF-1,
5 -GACTCTGCAGATAAGCCA-3 ; hTEF-3, 5 -AGTGGAGCTCTCCCACCT-3 ; hTEF-4,
5 -GGGGGTGACGGGGGCCCG-3 . The 5 and 3 primers were chosen in separate
exons to distinguish the cDNA product from contaminating genomic
DNA. RT-PCR generated a 270-nucleotide fragment for hTEF-1, 316 nucleotides for hTEF-3, and 255 nucleotides for hTEF-4. Control PCR
reactions were performed using 10 pg of the appropriate expression
vectors. 30 cycles of PCR were performed for 1 min at 94 °C, 1.5 min
at 53 °C, and 1.5 min at 72 °C in a 60-µl volume. 15 µl of
the reaction was then electrophoresed, transferred to nitrocellulose,
and hybridized to the homologous 32P-5 -end-labeled TEA
domain probes generated by PCR using the degenerate oligonucleotide
primers shown in Fig. 1.
In Situ Hybridization
The mTEF antisense riboprobes were
generated by in vitro transcription of the Bluescript SK
plasmids recovered by in vivo excision of the ZAP II
libraries using T7 or T3 RNA polymerase as appropriate. Control sense
riboprobes were synthesized from the same templates again using T3 or
T7 RNA polymerase as appropriate. The probes contained the entire
coding region as well as the 5 and 3 noncoding regions. To verify
that there was no cross-hybridization between probes due to the
conserved TEA domain, in situ hybridization was performed
using probes lacking the TEA domain and 5 regions, and results similar
to those shown in Figs. 7, 8, 9 were obtained (data not shown). Probe
length was reduced by a 1-h alkaline lysis, and in situ
hybridization was performed as described (48).
Fig. 7.
Expression of mTEFs at 6.5-8.5 dpc.
A-D, three serial sections through a 6.5 dpc (egg cylinder
stage) mouse embryo sectioned in utero. As in the following
figures, the left-hand side panel is a bright-field view of
the section showing the histology, and the right-hand side
panels are dark-field views of sections hybridized, respectively,
to mTEF-1 (B), mTEF-3 (C) and mTEF-4
(D) riboprobes. Dark-field views reveal the hybridization
signal grain in white. Scale bar, 450 µm. The
insets show high power magnifications of the conceptus.
Scale bar, 160 µm. E-H, serial sections
through a 7.5 dpc (primitive streak stage) embryo sectioned in
utero. The arrow in (F) points to the strong
labeling of mTEF-1 in cells of the ectoplacental cone. Scale
bar, 600 µm. I-L, serial sections of a 8.5 dpc
embryo (early somite stage, prior to embryo turning) sectioned in
utero. These sections cross the developing heart and trunk region,
as well as more lateral and caudal regions of the embryo toward the
upper left side of the cavity. Scale bar, 750 µm. Abbreviations: a, amniotic membrane; AMP,
anti-mesometrial pole; d, decidual tissue; em,
embryonic germ layers; epc, ectoplacental cone;
ex, extra-embryonic layers; h, heart;
MP, mesometrial pole; my, myometrium;
nt, neural tube.
[View Larger Version of this Image (155K GIF file)]
Fig. 8.
Expression of m-TEFs at 10.5-15.5 dpc.
A-D, sagittal sections of a 10.5 dpc embryo, hybridized as
indicated to mTEF-1 (B), mTEF-3 (C), and mTEF-4
(D) riboprobes. A-B and D
are two adjacent sections that cross the body axis slightly laterally
to the midline. In these sections, the spinal cord is sectioned only in
the trunk region due to skewing of the embryo axis. Section
C is a more lateral plane that shows the mTEF-3 segmented
labeling pattern in presumptive myotomes. Scale bar, 600 µm. E-H, serial sections through the lateral regions of
the head and trunk of a 12.5 dpc embryo. Scale bar, 750 µm. I-L, serial sections through the lateral regions of
the head and trunk of a 15.5 dpc embryo. Scale bar, 1.5 mm.
Abbreviations: ao, aorta; br, (first) branchial
arch; ce, cerebellum; e, eye; fb,
forebrain; fm, facial mesenchyme; g, herniated
gut; h, heart; hb, hindbrain; hl,
hindlimb; li, liver; lu, lung; mb,
midbrain, ml,: mantle layer; s, stomach;
sc, spinal cord, vl, ventricular layer;
w, whisker follicles.
[View Larger Version of this Image (157K GIF file)]
Fig. 9.
Differential expression of mTEFs during late
gestation. A-C, sagittal section through the shoulder
region of a 15.5 dpc fetus. These views illustrate mTEF-1 and mTEF-3
labeling patterns in the shoulder, cervical, and upper intercostal
muscles (not all of which are indicated). Scale bar, 600 µm. D-F, sagittal sections crossing the olfactory
epithelium of a 16.5 dpc fetus, hybridized to mTEF-1 and mTEF-4 probes,
respectively. Scale bar, 500 µm. G-I, sagittal
sections through the kidney and adrenal gland of a 16.5 dpc fetus.
Scale bar, 500 µm. J-L, coronal sections
through the lower abdominal cavity and urinary bladder of a 16.5 dpc
fetus. The asterisks point to mTEF-4 labeling in the
endothelium of hepatic blood vessels. Scale bar, 500 µm.
Abbreviations: ad, adrenal gland; bl, urinary
bladder; di, diencephalon; hu, humerus;
in, intestine; ki, kidney; m, muscles
(non-exhaustive); nc, nasal cavity; nz,
nephrogenic zone; oc, oral cavity; olf, olfactory
region; li, liver; ri, ribs; to,
tongue.
[View Larger Version of this Image (181K GIF file)]
RESULTS
Cloning and Characterization of Novel Human and Murine
Transcription Factors with TEA/ATTS Domains
The amino acid sequences of the known TEA/ATTS (hereafter TEA)
domains show highest conservation in putative -helices 2 and 3 (Fig.
1). Two degenerate oligonucleotides (see ``Materials
and Methods'' and Fig. 1) deduced from the sequence encoding GRNELIA
and the complement of the sequence encoding HIQVL were used in
polymerase chain reaction (PCR) amplification experiments with cDNA
libraries from either human or mouse cells. Amplification products of
the expected size were cloned and their DNA sequence determined. In
addition to partial cDNAs for the human and murine TEF-1 (hTEF-1
and mTEF-1 in Fig. 1), four other partial cDNAs were obtained
(hTEF-3, hTEF-4, mTEF-3, and mTEF-4 in Fig. 1, hTEF-2 has already been
ascribed to an unrelated protein recognizing the SV40 GT-IC enhanson.
Refs 1, 49). These cDNAs encode the same amino acid sequence as
TEF-1 but have a distinct nucleotide sequence, with the exception of
mTEF-3 that also contains a Tyr to His substitution (in bold in
Fig. 1). hTEF-1 was amplified from HeLa and human fetal brain cDNA
libraries, hTEF-3 from the HeLa library, and hTEF-4 only from the human
fetal brain library. The mTEFs were all amplified from a retinoic
acid-differentiated embryonic stem cell library and a 10.5-day mouse
embryo library.
The cDNA libraries were rescreened with the novel partial
cDNAs, and full-length clones encoding hTEF-3, mTEF-3, mTEF-4, and
a truncated clone encoding hTEF-4 were isolated. Alignment of the amino
acid sequences of hTEF-1, hTEF-3, and hTEF-4 (Fig. 2)
showed that overall hTEF-3 is 76% identical to hTEF-1, while hTEF-4 is
67% identical to hTEF-1. Nevertheless, conservation is not homogeneous
throughout the protein. The TEA domains of hTEF-3 and hTEF-4 are
identical to that of hTEF-1, and the carboxyl 200 amino acids are well
conserved in all the hTEFs. In contrast, the N-terminal regions and the
regions immediately following the TEA domain are much more
divergent.
Fig. 2.
Alignment of the amino acid sequences of the
hTEF proteins. The deduced amino acid sequences of the three hTEF
proteins are aligned. Amino acids conserved in all three proteins are
boxed. In the 3 region where no hTEF-4 sequence is
available amino acids conserved in hTEF-1 and hTEF-3 are
boxed. The position of the TEA domain is indicated. The
numbers to the left indicate the amino acid
coordinates. For hTEF-4 we have assumed an extended homology with
mTEF-4.
[View Larger Version of this Image (73K GIF file)]
Alignment of the amino acid sequences of the mTEFs (Fig.
3A) showed a similar pattern with respect to
the overall homology and the regions of conservation and divergence.
One exception to this, however, is the TEA domain of mTEF-3 that
contains five amino acid substitutions (underlined in Fig.
3A). Three of these changes, D39E, S62T, and D63E, are
conservative and are at positions that are less well conserved in the
TEA domains of AbaA and TEC1. On the other hand, the Q42R
and the Y77H changes are less conservative. Moreover, Tyr-77 is
invariant in all other known TEA domains (see Fig. 1).
Fig. 3.
A, alignment of the amino acid sequences
of the mTEF proteins. Amino acids conserved in all three proteins are
boxed. The position of the TEA domain is indicated. The
amino acid substitutions in the TEA domain of mTEF-3 are
underlined. B, comparison of the hTEF-3 and
mTEF-3 and hTEF-4 and mTEF-4 amino acid sequences. Only the comparison
of the divergent region following the TEA domain is shown.
[View Larger Version of this Image (41K GIF file)]
The interspecies comparison showed that, as described previously (28,
50), mTEF-1 is 99% identical to hTEF-1. hTEF-3 is most closely related
to mTEF-3. This is clearly seen by comparing the divergent regions of
the proteins (see above) that are greater than 90% identical between
hTEF-3 and mTEF-3 (Fig. 3B), whereas they are only 10-40%
identical between mTEF-3 and the other h- or mTEFs (Figs. 2 and
3A). By the same criteria, mTEF-4 is the counterpart of
hTEF-4 (see Fig. 3B).
Another striking feature of the amino acid sequences is that with the
exception of mTEF-4, which has an extended open reading frame (ORF)
upstream of the TEA domain beginning with an AUG codon, all the ORFs
initiate with an AUU codon encoding isoleucine (Ref. 2, and data not
shown). Analysis of the nucleotide sequences of the 5 -untranslated
regions showed that in each case the ORFs are preceded by upstream stop
codons and that there are no in frame AUG codons downstream of these
stops (data not shown). In addition, the 5 -untranslated regions are
highly divergent, and sequence homologies between family members begin
only immediately upstream of the ORFs.
The TEFs Differ in Their Ability to Bind Cooperatively to Tandemly
Repeated Sites
To assess the ability of the members of the TEF-1 family to bind
to the GT-IIC and Sph(I + II) enhansons of the SV40 enhancer, their
ORFs were cloned into the eukaryotic expression vector pXJ41 and
transfected into Cos cells. The transfected cell extracts were then
tested in EMSA. In extracts from cells transfected with vectors
expressing hTEF-1 and hTEF-3, a specific complex (complex B) was formed
with an oligonucleotide containing a single wild-type GT-IIC enhanson,
whereas no complex was observed with extracts from mock-transfected
cells or with a mutated GT-IIC enhanson (Fig.
4A, lanes 1-6). hTEF-1 bound
cooperatively to tandem repeats of the GT-IIC enhanson as judged by the
preferential formation of complex A in which both binding sites are
occupied (Fig. 4A, lane 8; Fig. 4B,
lane 5; Refs. 1, 2 and see below). Note that in some lanes
minor complexes A and B , formed by the binding of a
truncated proteolytic degradation product of the TEFs, and complex
C, a dimer formed between the full-length and truncated
TEFs, can be observed. hTEF-1 also bound cooperatively to the tandem
Sph(I + II) enhansons (Fig. 4A, lane 11, and
Fig. 4B, lane 9). In contrast, formation of
complex A on the tandemly repeated GT-IIC enhanson was much less
efficient with hTEF-3 (compare complexes A and B formed with hTEF-1 and
hTEF-3 in Fig. 4A and with lower amounts of extract in
panel B) indicating that hTEF-3 binds noncooperatively. As
cooperativity is required for efficient binding to the low affinity Sph
enhansons (Ref. 1, and see below), binding of hTEF-3 to the Sph
enhansons was much weaker than hTEF-1 (compare Fig. 4A,
lanes 11-12, and Fig. 4B, lanes 9 and
10). Thus, although hTEF-3 recognizes the GT-IIC enhanson,
cooperative binding to tandem repeats is impaired resulting in reduced
binding to the low affinity Sph enhansons.
Fig. 4.
A, autoradiography of EMSA performed
with extracts from Cos cells transfected with vectors expressing hTEF
proteins. The hTEF overexpressed in each cell extract is indicated
above each lane. Reactions in lanes 1, 4, 7, and
10 were performed with extract from mock-transfected cells.
The oligonucleotides used in each reaction are indicated
below each lane. GT-IIC wt contains the wild-type
(wt) enhanson, Mut the mutated enhanson,
GT-IICX2 the tandemly repeated enhanson, and
SphI+II the tandemly repeated Sph enhansons from
the SV40 enhancer as described previously (13). F indicates
the position of the free oligonucleotides; B indicates the
position of the complex formed by binding of the full-length TEF
proteins to one binding site; and A indicates the complex
formed by the simultaneous binding of protein to two binding sites.
A and B are equivalent to A- and
B-formed proteolytic degradation products of the TEFs, and
C is the dimer formed between full-length and truncated TEFs
on tandemly repeated sites. B, comparison of the binding of
the hTEFs and mTEF-4 to the GT-IIC and Sph enhansons. The overexpressed
protein is indicated above each lane. A, B, C,
and F are as in panel A. Oct indicates
the complex formed by the binding of cellular Oct-1 protein to the Oct
enhanson overlapping the Sph enhansons, and D indicates the
position of the higher order complex formed by the binding of mTEF-4.
C, binding of mTEF-3 to the tandemly repeated GT-IIC or Sph
motifs. Increasing quantities (0.5, 1.5, and 3 µl) of extract from
cells transfected with the expression vectors for the TEF proteins
shown above each lane were used in EMSA. The positions of
the A, B, and C, complexes are indicated.
[View Larger Version of this Image (37K GIF file)]
Similar experiments performed with increasing quantities of mTEF-3
transfected cell extracts resulted in the formation of complex B on the
GT-IIC enhanson (Fig. 4C, lanes 4-6). However,
when compared with hTEF-1, the formation of complex A on the tandemly
repeated enhansons was much less efficient (compare lanes 3 and 5 where there is equal formation of complex B with
lanes 15 and 17 and 21 and
23). Comparison of the ratio between complexes A and B shows
that even at the highest concentration of mTEF-3 more of complex B than
A is formed, whereas with hTEF-1 formation complex A is favored
(compare lanes 18 and 24 with 15 and
21). These results show that, despite the five amino acid
substitutions within its TEA domain, mTEF-3 binds to the GT-IIC and Sph
enhansons but, as with hTEF-3, cooperative binding to tandemly repeated
sites is impaired.
In EMSA, mTEF-4 bound to both the GT-IIC and Sph enhansons (lanes
3, 7, and 11 in Fig. 4B). Strikingly, in
addition to complexes A and B, complex D, possibly a trimer or a
tetramer, was formed on tandemly repeated sites. Complex D has
fortuitously the same electrophoretic mobility as the complex formed by
the cellular Oct-1 factor binding to the Oct enhanson overlapping the
Sph enhansons (complex Oct in lanes 9-11; and
see Refs. 1, 2, 51). Complex D was the most abundant species formed
with mTEF-4 indicating that its formation was highly cooperative. In
addition, multimeric complexes were formed that did not enter the gel
(see top of lanes 7 and 11). Thus,
unlike hTEF-3 and mTEF-3 that showed a reduced ability to bind
cooperatively, mTEF-4 cooperatively formed multimers on tandemly
repeated binding sites.
Cooperative Binding to Tandemly Repeated Sites Is an Intrinsic
Property of the TEA Domain
We next asked if the TEA domain alone would bind cooperatively to
tandemly repeated sites. A 6 histidine-tagged TEA domain fusion protein
(6His-TEA) was purified from Escherichia coli (see
``Materials and Methods'' and Fig. 5A) and
used in EMSA. The 6His-TEA protein bound to both the tandemly repeated
GT-IIC and Sph enhansons to generate complex B in which only one of the
two sites is occupied and complex A in which both are occupied (Fig.
5B, lanes 1 and 19). Complex A was
efficiently formed at the lowest concentration of fusion protein
despite the presence of an excess of free oligonucleotides, indicating
that binding was highly cooperative. However, complex B was formed more
readily on the high affinity GT-IIC enhanson than on the low affinity
Sph enhansons. These observations indicate that the TEA domain binds
cooperatively to tandemly repeated GT-IIC or Sph enhansons.
Fig. 5.
A, purification of the histidine-tagged
hTEF-1 TEA domain fusion protein. Lane 1 shows the starting
bacterial extract, lane 2 the flow-through (FT)
fraction from the nickel-agarose chromatography, lane 3 the
fraction eluted from the nickel-agarose column with 0.25 M
immidazole, lane 4 the flow-through fraction from the
double-stranded DNA-cellulose column, and lane 5 the 0.8 M KCl eluate from the DNA-cellulose. The gel was stained
with Coomassie Blue. Lanes 6-8 show an aliquot of the same
material as in lanes 3-5 stained with silver nitrate. The
positions of prestained molecular size markers are indicated to the
left of the panel. The arrow to the
right indicates the purified 6 His-TEA protein.
B, binding of the purified 6 His-TEA protein to tandemly
repeated and spaced binding sites. A, B, and F
are as described in Fig. 4. The tandem repeats are schematized by the
arrows, and the presence of a 5- or 10-nucleotide spacer is
indicated below the arrows. Each series of
reactions shows increasing concentrations of protein from 5-50
ng.
[View Larger Version of this Image (43K GIF file)]
EMSA was also performed with oligonucleotides containing directly
repeated elements spaced by 5 or 10 nucleotides. Strikingly, compared
with the efficient binding of 6His-TEA to tandemly repeated Sph
enhansons, almost no binding to the spaced repeats was observed at low
protein concentrations (Fig. 5B, lanes 7-9 and
13-15), confirming that cooperativity is required for
binding to these low affinity enhansons. Only at high concentration of
the fusion protein was formation of complex B observed, but almost no
complex A was formed (lanes 11-12 and 17-18).
Insertion of 5 or 10 nucleotides between the GT-IIC enhansons had no
effect on the formation of complex B, but at low concentrations of
fusion protein the formation of complex A was dramatically reduced
(lanes 24-26 and 29-31). Complex A was observed
only at high concentrations of fusion protein when the pool of free
oligonucleotides was limiting (lanes 27-28 and
32-33). Thus, while the TEA domain binds cooperatively to
tandemly repeated enhansons, binding to the spaced repeats is
noncooperative. These results clearly demonstrate that the ability to
bind cooperatively to tandem, but not spaced repeats, is an intrinsic
property of the TEA domain.
Differential Expression of the hTEFs in Cultured Cell Lines
We next investigated the expression of the hTEF proteins in human
cell lines of various origins by RT-PCR with exon- and hTEF-specific
primers (see ``Materials and Methods''). hTEF-3 was expressed in all
the cell lines tested (Fig. 6A), and hTEF-1
was absent only in HepG2 hepatoma cells (Fig. 6A, lane
4). hTEF-4 had a more restricted pattern of expression, being
strongly expressed only in the ovarian carcinoma cell line Ovcar-3
(Fig. 6B, lane 11), while weaker expression was
detected in Intestine 407 cells, in placental JEG-3 choriocarcinoma
cells, and in embryonic kidney 293 cells (Fig. 6B,
lanes 5, 10, and 6, respectively). In contrast to
hTEF-1 and hTEF-3, no hTEF-4 expression was detected in HeLa, HepG2,
Molt 4, IMR-32, and CaCo2 cells (Fig. 6B, lanes 2-4,
9, and 12). These results show that the hTEF proteins
have distinct expression patterns in cultured cells.
Fig. 6.
A, expression of hTEF-1 and hTEF-3 in
human cell lines. RT-PCR was used to amplify fragments of the hTEF-1
and hTEF-3 mRNAs. After electrophoresis on a 6% acrylamide gel, an
aliquot of the PCR reactions was transferred to nitrocellulose and
hybridized with the homologous TEA domain probe. Lanes 1 and
2 show the PCR fragments generated using 10 pg of the
corresponding expression vectors as template and lane 8 the
PCR performed in the absence of added template. The source of the RNA
used in the other reactions is indicated above each lane.
HeLa cells are derived from a cervical carcinoma, HepG2 from
a hepatocarcinoma, Molt4 from a T-cell leukemia,
IMR32 from a neuroblastoma, OVCAR-3
from a ovary adenocarcinoma, JEG-3 from a
choriocarcinoma, CaCo-2 from a colon
adenocarcinoma. Intestine (In) 407 cells are from embryonic
intestine and 293 cells are transformed embryonal kidney
cells. B, expression of hTEF-4 in human cell lines.
Lanes 1 and 8 show the amplification product
generated using 10 pg of the corresponding expression vectors as
templates and lane 7 with no added template. The cell lines
used for the other reactions are indicated above each
lane.
[View Larger Version of this Image (28K GIF file)]
Differential Expression of the mTEFs during Mouse
Embryogenesis
As indicated by their presence in cDNA libraries from 10.5-day
embryos, all the mTEFs should be expressed during embryogenesis. The
expression patterns of mTEF-3 and mTEF-4 were therefore investigated
and compared with that of mTEF-1 by in situ hybridization of
mouse embryos and fetuses from 6.5 to 18.5 days post-coitum (dpc).
Early Gestational Stages (6.5 to 8.5 dpc)
All three TEFs were
differentially expressed during early development in embryonic as well
as extra-embryonic and maternal cell lineages. In the maternal decidua,
mTEF-3 and mTEF-4 expression was somewhat complementary at 6.5 and 7.5 dpc. mTEF-3 transcripts were abundant in the embryonic
(antimesometrial) pole and decreased gradually toward the ectoplacental
(mesometrial) pole (Fig. 7, C and
G). In contrast, weaker expression of mTEF-4, restricted to
the ectoplacental pole of the outer decidua, was observed. mTEF-1 was
also expressed in decidual cells (Fig. 7, B, F,
and H), but at a lower level than mTEF-3. At 8.5 dpc, mTEF-3
was strongly expressed throughout the decidua, although dispersed cells
or cell clones were unlabeled (Fig. 7K).
Distinct expression patterns of the mTEFs were also observed in the
early conceptus. At 6.5 dpc (pregastrula, egg-cylinder stage), mTEF-1
and mTEF-3 were expressed in distinct extra-embryonic regions, mTEF-1
in the ectoplacental cone and mTEF-3 in the extra-embryonic layers
(Fig. 7, B and C). Nevertheless, at 7.5 dpc
(early gastrula stage), mTEF-1 expression was observed in the embryo as
well as in the extra-embryonic tissues, where particularly intense
signals were observed in some ectoplacental cells (Fig. 7F).
At 8.5 dpc mTEF-1 was expressed in the entire embryo (Fig.
7J). At 7.5-8.5 dpc mTEF-3 was expressed at low levels in
the entire conceptus (Fig. 7, G and K). In
contrast, mTEF-4 was strongly expressed in the entire conceptus at 6.5 dpc (Fig. 7D) and remained strongly expressed in the embryo
until 8.5 dpc, whereas its expression in the extra-embryonic region
progressively disappeared (Fig. 7, D, H,
L).
Mid-gestational Stages (9.5-12.5 dpc)
Both mTEF-1 and mTEF-4
were ubiquitously expressed in 9.5 dpc embryos, but from 10.5 dpc these
two genes showed preferential expression in specific tissues, some
being common to both genes. Both mTEF-1 and mTEF-4 were strongly
expressed in the ventricular layer of the neuroepithelium that contains
the mitotic neuroblasts. This was observed both in the developing brain
and spinal cord (compare Fig. 8, B,
D, and F, H). Strikingly, no labeling
was observed in the surrounding mantle layer that contains post-mitotic
neural precursors. In contrast, the distribution of mTEF-1 and mTEF-4
transcripts differed in various mesenchymes, for example the facial and
gut mesenchymes where accentuated mTEF-4 expression was observed (Fig.
8, F and H). On the other hand, mTEF-1, but not
mTEF-4, was preferentially expressed in the developing myocardium as
early as 10.5 dpc (Fig. 8B). Note also that the heart
chambers, the cavities of the outflow tract, and the descending aorta
were unlabeled by all mTEF probes suggesting that these genes are not
expressed in embryonic blood. At later stages strong mTEF-1 expression
was also seen in various muscle anlagen, both in facial and at other
axial levels (Fig. 8J).
Strikingly, mTEF-3 signals were only detected in the developing
myotomes (the myogenic compartment of the somitic mesoderm) and
appeared in the cranio-caudal progression from cervical levels (at 9.5 dpc, data not shown) to trunk and caudal levels (10.5 dpc, Fig.
8D). Indeed, skeletal muscle precursors were specifically
labeled by the mTEF-3 probe at 11.5-12.5 dpc including head, axial,
body wall, and limb muscle anlagen (Fig. 8G, and data not
shown). The developing myocardium was never labeled at any stage by the
mTEF-3 probe (Fig. 8D, and data not shown).
Late Developmental Stages (13.5-18.5 dpc)
In skeletal
muscle, mTEF-3 expression persisted during late gestational stages
(Fig. 8K shows the head, neck, shoulder, intercostal,
abdominal wall, and hindlimb muscles). Interestingly, the mTEF-3 probe
yielded a ``spotted'' labeling in 15.5 dpc and older developing
muscles, some of the cells showing much more intense labeling
(illustrated in Fig. 9C for the shoulder
muscles). Nevertheless, mTEF-3 transcripts were detected in several
discrete regions outside the developing muscle from 15.5 to 18.5 dpc,
namely the liver, the lung, the salivary gland, and nasal gland
epithelia, and the small intestine presumably in the duodenal region
(Fig. 8K and data not shown). mTEF-1 expression also
persisted in late developing muscles where the labeling was more
homogeneous than that of mTEF-3 (compare Fig. 8,
J-K, and Fig. 9, B and C).
As at earlier stages the mTEF-1 signal remained high in the
differentiating myocardium (data not shown).
At later stages, strong expression of mTEF-1 and mTEF-4 persisted in
the ventricular zone of the CNS (see the 15.5-dpc forebrain ventricles
in Fig. 8, J and K) that tends to become thinner
as development proceeds. Both genes also showed accentuated expression
in the developing lungs at 15.5 dpc (Fig. 8, J and
L) and at later stages (not shown). However, distinct mTEF-1
and mTEF-4 expression patterns were observed in a number of developing
organs or tissues, some examples of which are illustrated. mTEF-1 was
strongly expressed in the entire nasal epithelium (both in the
olfactory and respiratory regions) as well as in the surrounding
mesenchyme (Fig. 9E; note that mTEF-1 transcripts appear
more abundant toward the apical layers of the olfactory epithelium). In
contrast, mTEF-4 transcripts were clearly more abundant in the basal
cell layer of the olfactory epithelium (Fig. 9F). mTEF-1 was
rather uniformly expressed in the developing kidney (metanephros),
whereas accentuated mTEF-4 expression was restricted to the cortical
region corresponding to the nephrogenic zone where new nephrons are
being generated (Fig. 9, G-I; note also the
higher expression of mTEF-1 in the adrenal gland).
As first observed at 12.5 dpc, mTEF-4 was strongly expressed in the
mesenchyme of the intestinal loops, whereas lower mTEF-1 expression was
observed in both the mesenchymal and epithelial components (Fig. 9,
J-L). Pronounced mTEF-1 expression was detected
in the most internal layer of the urinary bladder epithelium as well as
in the external layer of the mesenchyme, whereas mTEF-4 transcripts
were more evenly distributed in the entire bladder mesenchyme and
epithelium (Fig. 9, J-L). Interestingly,
specific expression of mTEF-4 along the lining of some hepatic blood
vessels was observed (Fig. 9L). As no such endothelial
labeling was detected in other blood vessels outside the liver, it
appears specific to the portal system.
DISCUSSION
A Novel Family of Transcription Factors Sharing a Common DNA
Binding Domain, but with Differential DNA Binding Properties
We
report here the molecular cloning of four novel mammalian members of
the TEA domain family of transcription factors with extensive homology
to TEF-1, notably in the TEA domain and in the carboxyl 200 amino
acids. Several regions of TEF-1 have distinctive amino acid
compositions often shared in other transcription factors, a
proline-rich region between amino acids 143 and 204, a region rich in
serine, threonine, and tyrosine between amino acids 306 and 328, and a
region with the potential to form a zinc finger at the extreme C
terminus (2). We have subsequently shown that these three regions are
involved in transcriptional activation, although the potential of the
carboxyl region to form a zinc finger was not required for
transactivation (13). Comparison of the amino acid sequences of the TEF
proteins shows that, although each contains a proline-rich region, its
primary amino acid sequence is among the least well conserved. The
serine, threonine, and tyrosine-rich region is well conserved; however,
despite the overall high homology in the carboxyl regions, the cysteine
residues in the putative zinc finger are not all conserved. We have
attempted to compare the transcriptional properties of the TEFs by
transfection in HeLa cells. However, in agreement with the high
conservation in the regions involved in transactivation, each TEF had a
dominant negative phenotype2 due to a
transcriptional interference/squelching effect as observed with TEF-1
(2, 13).
We determined the relationship between TEF-3/4 and other previously
described TEA domain proteins. For AbaA and TEC1 the
sequence similarities outside the TEA domain were too low to allow any
relationships to be determined. However, sd was more related
to TEF-1 than to the other TEFs.2 Strikingly, cTEF-1/RTEF-1
(32, 52) is 90% identical to hTEF-3 but only 77% identical to hTEF-1.
Thus, the cTEF isolated by Stewart et al. (32) should be
considered as a counterpart of TEF-3 rather than of TEF-1, whose avian
counterpart has been designated NTEF-1 (52). mTEF-4 is clearly
identical to ETF (11) that was reported to be specifically expressed in
the developing brain. Although our results also show strong expression
of mTEF-4/ETF in mitotic neuroblasts, it is clearly expressed in other
tissues (see below). While this manuscript was in preparation, an
additional chicken TEF gene, DTEF-1, was described (52). We have also
isolated a human cDNA (hTEF-5) homologous to DTEF-1 and are
presently characterizing its properties.2
As expected from the fact that the TEF proteins share a common DNA
binding domain, each binds to the GT-IIC and Sph enhansons from the
SV40 enhancer. This is true even of mTEF-3 whose TEA domain contains
five amino acid substitutions one of which changes a highly conserved
Tyr residue. However, our results do not exclude the possibility that
the amino acid changes in the mTEF-3 TEA domain may confer the ability
to recognize additional unrelated sequences.
Although all the TEF proteins bind to the previously defined consensus
binding site exemplified by the SV40 GT-IIC and Sph enhansons, they
exhibit differences in their ability to bind cooperatively to tandemly
repeated sites. mTEF-4 not only binds cooperatively to tandemly
repeated sites, but it cooperatively generates higher order complexes,
probably trimers or tetramers. In contrast, h- and mTEF-3 bind
essentially noncooperatively. Consequently, binding of these proteins
to the low affinity Sph sites is inefficient when compared with hTEF-1
or mTEF-4. Although the changes within the TEA domain of mTEF-3 may
contribute to the diminished cooperativity, this cannot be the case for
hTEF-3 whose TEA domain is identical to that of TEF-1. Moreover, as the
TEA domain alone binds cooperatively to tandemly repeated sites, the
above variations must result from the differential abilities of other
regions of the TEFs to either decrease or enhance cooperativity. Thus,
although the TEA domain is the minimal domain required for specific and
cooperative DNA binding, its activity is modulated by other regions of
the TEF proteins. Such a result is not without precedent as a mechanism
for intramolecular modulation of DBD activity has been described in the
Ets-1 factor (53, 54).
The mTEFs Are Differentially Expressed during Early Embryonic
Development
The mTEFs are differentially expressed in the early
conceptus at 6.5 dpc. mTEF-1 and mTEF-3 were specifically expressed in
the extra-embryonic tissues, notably a strong expression of mTEF-1 in
the ectoplacental cone, whereas mTEF-4 was expressed in both the
embryonic and extraembryonic tissues. The extraembryonic tissues are
derived from the primitive endoderm and trophectoderm lineages that are
the first to form in mammalian embryos (reviewed in Ref. 55).
Consequently, our present observations suggest that, although no
relevant target genes have been described, members of the TEF family
are expressed early in embryogenesis (see also Refs. 1, 2, 12, 14, 38,
56, 57, reviewed in 58) and may play a role in specification of the
extra-embryonic lineages.
While analyzing the expression of the TEF factors in early
post-implantation embryos, we also noted differential expression of the
TEFs in the maternal decidua where a strong mTEF-3 expression was
observed. As mTEF-3 was not expressed in the surrounding myometrium, it
is possible that mTEF-3 expression is directly related to the
decidualization reaction. Further identification of the TEF-3-regulated
genes will be required to understand its role in the decidua.
At Mid-gestational Stages mTEF-3 Expression Is Largely Restricted
to Developing Skeletal Muscle
As described in the Introduction
TEF-1 and/or related factors have been implicated in muscle-specific
gene expression and cardiogenesis. The importance of the TEF factors in
these processes is further supported by the results presented here.
From 10.5 dpc, mTEF-1 was expressed in the developing myocardium and in
skeletal muscle precursors. Even more strikingly, mTEF-3 was
specifically expressed in skeletal muscle precursors as early as 9.5 dpc, but not in the myocardium. mTEF-3 and mTEF-1 were expressed in the
developing skeletal muscles derived from epaxial and hypaxial lineages
(59) as well as the head muscles derived from the nonovertly segmented
paraxial mesoderm. The expression pattern of mTEF-3 is therefore
similar, but not identical, to that of other myogenic factors, such as
MyoD in both its specificity and onset of expression (reviewed in Refs.
60 and 61). The expression of mTEF-1 and mTEF-3 in muscle is maintained
at late stages of embryogenesis where a heterogeneity of mTEF-3, but
not mTEF-1, expression is seen, beginning around 15.5 dpc.
Interestingly, this corresponds to the time at which muscle innervation
and fiber type differentiation begin suggesting that mTEF-3 may be
differentially expressed in different fiber types.
The expression of mTEF-3 is similar, but not identical, to that
reported for its avian homologue RTEF-1, although preferential
expression of cTEF-3/RTEF-1 was observed in skeletal muscle, and,
unlike mTEF-3, cTEF-3/RTEF-1 was also expressed in both fetal and adult
heart (32). Further studies will be required to determine whether
mTEF-3 is expressed in the adult heart. However, our present results
support those of Farrance and Ordahl (62) who indicated a key role for
cTEF-3/RTEF-1 in muscle-specific gene transcription in chicken. Thus,
while previous studies on myogenesis have concentrated on the MyoD and
MEF2 families of factors (63), the correlation between the expression
of mTEF-1 and mTEF-3 and that of known muscle-specific target genes
provides strong evidence that these TEF factors are also likely to be
important for myogenesis.
The Overlapping Expression Zones of the mTEFs Suggest Partially
Redundant Roles in Several Developmental Processes
The expression
of mTEF-1 is clearly not limited to muscle tissue but is not
ubiquitous. Strong expression was observed in mitotic neuroblasts as
well as in a number of developing organs. In many, but not all of these
expression zones, accentuated expression of mTEF-4 was also detected.
Despite the fact that mTEF-1 is expressed at early stages of
embryogenesis and is subsequently expressed in several tissues, the
only major defects in mTEF-1 null mice concerned cardiogenesis,
skeletal muscle and the central nervous system being apparently
unaffected (38). As all the mTEFs bind the same consensus site, the
restricted phenotype of the TEF-1 null mice may potentially be
explained by the overlapping mTEF expression zones presented here. In
the early conceptus and in the CNS, lack of expression of mTEF-1 may be
compensated by the expression of mTEF-3 and/or mTEF-4. In skeletal
muscle its expression may be compensated by expression of mTEF-3. Such
redundancy has been previously noted for other myogenic factors, such
as myf-5 and MyoD (Ref. 64 and references therein). However, as the
lack of mTEF-1 in the developing myocardium is not compensated by
expression of other mTEFs, TEF-1 null embryos die at 11.5-12.5 dpc,
and their premature death prevents investigation of mTEF-1 function(s)
at later stages.
In addition to zones of expression overlapping with mTEF-1, there are
several regions where mTEF-4 is the predominantly expressed member of
the family. mTEF-4 is strongly expressed throughout the 6.5 dpc embryo
and remains so until 9.5-10.5 dpc, after which a more specific pattern
of expression is established. Later in development mTEF-4 may play a
role in organogenesis. For example, accentuated mTEF-4 expression was
observed in the intestinal mesenchyme and the nephrogenic region of the
kidney where it may contribute to the mesenchyme-epithelial transition
during nephron development. The identification of TEF-regulated genes
in the CNS and the above organs will help to clarify the function of
the TEFs in these tissues.
mTEF-4/ETF was initially reported to be a neural-specific factor based
on the results of whole mount in situ hybridizations and
Northern blots (11). Our results, using in situ
hybridization on sectioned embryos, more precisely define the
mTEF-4/ETF expression zone to the mitotic neuroblasts. Nevertheless,
mTEF-4/ETF is not a neural-specific factor since, as described above,
it is strongly expressed in the embryo from 6.5 to 8.5 days, and at
later times it is also strongly expressed in several other non-neural
tissues.
It is interesting to compare the specific expression of the mTEFs
during embryogenesis with the expression pattern of the human TEFs seen
in cultured cell lines. Consistent with the fact that mTEF-1 is widely
expressed in embryogenesis, but is not expressed in liver, hTEF-1 is
expressed in all of the cell lines tested with the exception of the
HepG2 hepatocarcinoma cells. In contrast, it is striking that hTEF-3
expression is much wider than would have been expected from observation
of mTEF-3 in the mouse embryo. Analogous results have been seen with
myogenic MEF2 factors that are specifically expressed during
embryogenesis but are ubiquitously expressed after birth and in
cultured cell lines (Refs. 65, 66 and references therein). Consistent
with the observation that mTEF-4 was expressed in neural tissue, hTEF-4
was cloned from a human fetal brain library, but it was not expressed
in IMR32 neuroblastoma cells. Similarly, mTEF-4 was expressed in the
developing gut mesenchyme, and hTEF-4 was expressed Intestine 407 cells
derived from embryonic intestine.
Our results imply that the TEF family of transcription factors may be
involved in several developmental processes from early times of
embryogenesis. Further gene targeting experiments will help to define
more precisely the functions of these factors.
FOOTNOTES
*
This work was supported by Grants PAI P3-042 and PAI P3-044,
from the Services Féderaux des Affaires Scientifiques,
Techniques, et Culturelles (Belgium), the CNRS, the INSERM, the Centre
Hospitalier Universitaire Régional, the Ministère de la
Recherche et de la Technologie, the Association pour la Recherche
contre le Cancer, and the Collège de France. The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
``advertisement'' in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X94438[GenBank], X94441[GenBank], X94440[GenBank], X94442[GenBank].
§
Supported by short-term fellowships from the EMBO, the Human
Science Frontier Organisation, and a long-term fellowship from the
Association pour la Recherche contre le Cancer (A.R.C.).
Present address: The Norris Cancer Center, 1441 Eastlake Ave.,
Los Angeles, CA 90033-0800.
''
To whom correspondence should be addressed: Tel.: 33 88 65 34 40 (45); Fax: 33 88 65 32 01; E-mail: irwin{at}titus.u-strasbg.fr.
1
The abbreviations used are: TEF-1,
transcriptional enhancer factor-1; dpc, days post-coitum; RT, reverse
transcription; PCR, polymerase chain reaction; EMSA, electrophoretic
mobility shift assays; CNS, central nervous system; ORF, open reading
frame; DBD, DNA binding domain; h, human; m, murine.
2
P. Jacquemin, J.-J. Hwang, J. A. Martial, P. Dollé, and I. Davidson, unpublished data.
Acknowledgments
We thank M. Vigneron, J. Acker,
R. Roy, and E. Bellefroid for different materials used in this study;
P. Chambon for support and critical reading of the manuscript; B. Schuhbaur for invaluable help with in situ hybridization, L. Carré for technical assistance, J.-M. Garnier and T. Lerouge for
providing the cDNA libraries, S. Vicaire and P. Hamman for DNA
sequencing, the oligonucleotide facility, the staff of cell culture
facility for providing the cell lines; B. Boulay, J. M. Lafontaine, R. Buchert, S. Metz, and C. Werlé for illustrations.
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S.-W. Jiang, M. Dong, M. A. Trujillo, L. J. Miller, and N. L. Eberhardt
DNA Binding of TEA/ATTS Domain Factors Is Regulated by Protein Kinase C Phosphorylation in Human Choriocarcinoma Cells
J. Biol. Chem.,
June 22, 2001;
276(26):
23464 - 23470.
[Abstract]
[Full Text]
[PDF]
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H. Sugimoto, M. Bakovic, S. Yamashita, and D. E. Vance
Identification of Transcriptional Enhancer Factor-4 as a Transcriptional Modulator of CTP:Phosphocholine Cytidylyltransferase alpha
J. Biol. Chem.,
April 6, 2001;
276(15):
12338 - 12344.
[Abstract]
[Full Text]
[PDF]
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Copyright © 1996 by the American Society for Biochemistry and Molecular Biology.
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