(Received for publication, April 4, 1995; and in revised form, June 2, 1995)
From the
We identified a novel cDNA related to that of transcriptional
enhancer factor-1 (TEF-1) during the course of isolation and
characterization of cDNAs, whose mRNAs are preferentially expressed in
the mouse neural precursor cells. The putative polypeptide, termed
embryonic TEA domain-containing factor (ETF), deduced from the
nucleotide sequence contains 445 amino acids and shares 66% amino acid
identity with mouse and human TEF-1 proteins. The primary structure of
the TEA domain, a probable DNA-binding domain, and the specific DNA
binding activity to the GT-IIC motif of ETF are indistinguishable from
those of the known vertebrate TEF-1 proteins. However, the expression
of the ETF gene is strictly regulated in developing embryos and is
limited to certain tissues, such as the hindbrain of a 10-day-old mouse
embryo, in contrast to the ubiquitous expression pattern of the TEF-1
gene. These results suggest that ETF is a novel mammalian member of the
TEA domain-containing transcription factor family and may be involved
in the gene regulation of the neural development. We have discussed the
possible existence of multiple subtypes of the mammalian TEF-1 family
proteins, which may play different roles in cellular and developmental
gene regulation.
In mammals, relatively simple neuroepithelial cells develop into
various types of neural and glial cells through multiple steps of cell
division and differentiation to form a highly organized complex
structure of the central nervous system. As an initial approach to
elucidate the molecular mechanisms underlying the early stage of
neuronal development, we isolated and characterized cDNAs whose mRNAs
are preferentially expressed in the neural precursor cells (NPC) ( In this study, we
report the molecular cloning and analysis of a novel mouse TEA
domain-containing protein, ETF. ETF specifically binds to the GT-IIC
motif as observed for TEF-1. However, it is distinct from TEF-1 in the
primary structure, the timing of gene expression, and the tissue
distribution. Thus, ETF is a novel mammalian member of a TEA
domain-containing protein family. Furthermore, ETF mRNA is
developmentally regulated and is predominantly expressed in the
hindbrain region of a 10-day-old mouse embryo, suggesting that ETF may
be involved in the gene regulation of neural development.
Figure 1:
Primary
structure of mouse ETF cDNA. A, schematic representation of
the ETF cDNA. The sequence corresponding to the coding region is
indicated by the outlinedbox; the solidbox indicates the coding region for the TEA domain. The
cDNA inserts of clones pmETF-1 and 8.41 are shown by solidlinesbelow the schematic representation. The numbersabove the box and the solidlines indicate the positions of amino acids and
nucleotide sequences, respectively. B, nucleotide and
predicted amino acid sequences of mouse ETF cDNA. The nucleotide
sequence of the mouse ETF cDNA was determined from the cDNA inserts of
clones pmETF-1 and 8.41. The amino acid sequence deduced for mouse ETF
is shown below the nucleotide sequence. The amino acid numbers
start at the putative initiation methionine assigned to the first ATG
codon. Nucleotide residues are numbered in the 5` to 3` direction,
beginning with the first residue of the initiation codon. The
nucleotides on the 5`-side of residue 1 are indicated by negativenumbers. The termination codon TGA is indicated by asterisk. The amino acid sequence corresponding to the TEA
domain is underlined. 5`-End points of the two clones are
indicated by triangles. The cDNA sequence for the clone 8.41
and the nucleotide sequence complementary to the primers used for the
5`-rapid amplification of cDNA ends procedure are indicated by broken and doubleunderlines, respectively.
The positions of the potential polyadenylation and mRNA destabilization
signals in the 3`-untranslated region are indicated by the wavy and solidlines,
respectively.
Figure 2:
Amino acid sequence alignment of ETF and
other TEA domain-containing proteins. The positions of identical amino
acids in all five proteins are indicated by asterisks. The TEA
domains are boxed. Gaps indicated by dashes are introduced to optimize the alignment according to the
published method(27) . Sources for sequences are as follows:
mouse (m) TEF-1(2) , human (h)
TEF-1(1) , chicken (c) TEF-1A(4) , and Drosophila sd (D.sd)(8) .
Figure 3:
Northern blot analysis of ETF mRNA. RNAs
were denatured and electrophoresed on a 0.66 M formaldehyde,
1.2% agarose gel and transferred to a nylon membrane. A 812-bp BamHI-BglII fragment of mouse ETF cDNA was used as a
hybridization probe. The positions of both 28 and 18 S mouse ribosomal
RNAs are indicated. The control hybridization of the same filter using
the glyceraldehyde 3-phosphate dehydrogenase cDNA (28) as a
probe and the ethidium bromide staining of 28 S ribosomal RNA of the
same gel are shown at the bottom. A, developmental
changes in the levels of ETF mRNAs. 10 µg of total RNAs from
various cell lines (Neuro2a, P19, and F9 cells), whole mouse embryos
(at various embryonic days), isolated mouse NPC, the brains of neonate (P0), 7-day-old (P7), and adult mice, and 5 µg of
poly(A)
The localization of ETF
mRNA in 10-day-old mouse embryos was examined further by whole mount in situ hybridization analysis. A predominant expression of
the ETF transcript was mainly observed in the hindbrain (Fig. 4), from which the cerebellum is later derived. Thus, the
result coincides with the cerebellar expression of ETF mRNA in
7-day-old postnatal mice demonstrated by Northern blot analysis.
Additional but much weaker signals were also noted in the distal
portions of the forelimb and hindlimb buds as well as in the tail bud (Fig. 4).
Figure 4:
Expression of ETF mRNA as determined by in situ hybridization to whole mouse embryos at day 10. Whole
mount in situ hybridization analysis was carried out with the
digoxigenin-labeled antisense (A and B) and sense (C and D) cRNAs of mouse ETF. PanelsA and C illustrate lateral aspects and panelsB and D show dorsal aspects of the embryos under
dark field illumination. The largearrows indicate
distinct hybridization signals observed at the hindbrain, where they
appear in black and are concentrated along the rhombencephalic
roof portions encircling the floor of the fourth ventricle. Also note
similar but much weaker signals localized to the distal portions of the
forelimb and hindlimb buds as well as to the tail bud, as indicated by
the smallarrows. Control sense riboprobe failed to
yield specific signals, as shown in C and D. Scalebars, 1.4 mm.
Figure 5:
Specific binding of T7 tag-ETF fusion
protein to the GT-IIC oligonucleotide in a gel mobility shift assay.
The T7 tag-ETF fusion protein synthesized in vitro was
incubated with the
It has been shown that overexpression of
human and mouse TEF-1 represses the expression of a GT-IIC-containing
reporter gene in transfected HeLa cells(1, 3) . The
observed transcriptional repression is thought to be mediated by
squelching a limited amount of coactivator present in HeLa
cells(1, 22) . We thus examined whether ETF could
affect transcription of a GT-IIC-containing reporter plasmid. The ETF
expression vector containing the above T7 tag-ETF fusion DNA was
cotransfected into HeLa cells with a luciferase reporter plasmid,
comprising tetramerized GT-IIC oligonucleotides ligated upstream of the
luciferase gene under the thymidine kinase promoter. The results (not
shown) demonstrated that the cotransfection of increasing amounts of
the ETF expression vector did not significantly affect the expression
of the reporter gene activity. In the present study, we described the molecular
characterization of the novel TEA domain-containing factor, ETF. ETF is
structurally related to but distinct from the vertebrate TEF-1 proteins
and their Drosophila homologue, scalloped.
Furthermore, the spatiotemporal expression patterns and the sizes of
the mRNAs of the ETF gene are different from those of the TEF-1 gene.
TEF-1 is ubiquitously expressed by both embryos and adults except for
cells of hematopoietic lineage(1, 2, 3) . By
contrast, the expression of the ETF gene is mostly embryonic and is
strictly controlled in a developmental manner. The distribution of ETF
mRNA is also limited to certain tissues, such as the hindbrain of
10-day-old embryos, cerebellum and testis of postnatal 7-day-old mice,
as well as neural precursor cells but is barely detectable in any adult
tissue. The expression pattern of ETF mRNA appears to overlap partly
but not coincide with those of a large number of transcription factors
and cell signaling molecules known to be expressed in the developing
mouse hindbrain(24) , although further studies on the spatial,
temporal, and cellular expression of ETF are required for more detailed
comparison. Thus, these characteristic features indicate that ETF
belongs to a novel subtype of the mammalian TEA domain-containing
transcription factors, which may play a role in neural development,
although its transcriptional activity and physiological function remain
to be determined. The primary structure of the TEA domain and the
specific DNA binding activity of ETF are indistinguishable from those
of other vertebrate TEF-1 proteins. However, the cotransfection
experiments with ETF have so far failed to detect any obvious effects
on the expression of the GT-IIC-containing reporter gene. Lack of
transcriptional activity may reflect lack of expression of T7 tag-ETF
protein in HeLa cells. However, we believe this is not likely because
our preliminary Western blotting experiments with T7 Until recently,
TEF-1 was the only known TEA domain-containing protein in mammals.
However, the present results clearly demonstrate that at least two
distinct subtypes of TEF-1 proteins exist in mammals. Although the
chicken TEF-1 is thought to be a homologue of mammalian TEF-1, several
lines of evidence support the notion that the chicken TEF-1 may belong
to a distinct subtype different from those of mammalian TEF-1 and ETF.
First, the similarity between chicken and mammalian TEF-1 (76%
identity) is much lower than that observed between mouse and human
TEF-1 (99% identity). Second, the tissue distribution of chicken TEF-1
mRNA showed enrichment in cardiac and skeletal muscles but
substantially reduced or barely detectable levels in liver and brain,
respectively(4) , in contrast to the ubiquitous expression
pattern of TEF-1 mRNA(1, 2, 3) . Third, the
172-bp insert of EST 683 human cDNA clone 32B5 (GenBank accession No.
T25108), which was obtained by systemic sequencing of a colorectal
cancer cDNA library (26) , showed the highest similarity (79%
identity) to the sequence of chicken TEF-1 cDNA in the GenBank data
base (data not shown). Moreover, 52 out of the 56 amino acids,
potentially encoded by the largest open reading frame of the same DNA
sequence, coincided with the corresponding sequence from Arg Finally, identification and characterization of
cellular target gene(s) of ETF and analysis of ETF function in vivo by gene disruption in embryonic stem cells should help define the
role of this molecule in mammalian development.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
D50563[GenBank].
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)of 10-day-old mouse embryos by using the method of
subtraction and differential hybridization. During the course of these
studies, we identified a novel cDNA, which exhibits a significant
sequence similarity to transcriptional enhancer factor-1 (TEF-1)
cDNAs(1, 2, 3, 4) . Transcription
factors play a crucial role in determining spatial and temporal
patterns of gene expressions during embryonic development. TEF-1 is a
transcriptional activator initially isolated from human HeLa cells and
binds specifically to the GT-IIC and Sph motifs in the SV40 enhancer
sequence(1) . TEF-1 belongs to a family of transcription
factors that share a common DNA-binding motif, the TEA domain, in the
amino-terminal regions of these proteins(5) . Among these, TEC1 of yeast Saccharomyces cerevisiae is involved in
transcriptional activation of the transposon Ty1 element (6) , AbaA of the filamentous fungus Aspergillus nidulans regulates development of the asexual spores(7) , scalloped (sd) of Drosophila controls
differentiation of the sensory neuronal organs(8) , while the
mouse and chicken TEF-1 homologues have been implicated in
cardiogenesis and skeletal muscle-specific gene
expression(3, 4, 9) . Therefore, the TEA
domain-containing proteins may play important roles in developmental
gene regulation among a wide variety of species.
Construction, Subtraction, and Differential
Screening of Mouse NPC cDNA Library
About 1.15
10
NPC were prepared from 420 10-day-old mouse embryos
according to the method described by Kitani et
al.(10) . A unidirectional cDNA library was constructed
from NPC poly(A)
RNA in
Uni-ZAP
XR
vector by using the ZAP-cDNA synthesis kit and the Gigapack II Gold
packaging extracts according to the instructions by the
manufacturer (Stratagene). After a portion of the library, representing
1.2
10
independent clones, was amplified and
converted to single-stranded circular DNA, 2 µg of the DNA was
subjected to two cycles of hybridization with 20 µg of adult brain
poly(A)
RNA(11) , which had been biotinylated
with Photoprobe (long arm) biotin (Vector laboratories)(12) .
The DNA-RNA duplex was then subtracted by binding biotinylated RNA to
Vectrex Avidin matrix (Vector Laboratories)(13) . The
unhybridized single-stranded DNA was recovered and introduced into Escherichiacoli. About 14,400 clones of the
subtracted library were transferred to Hybond
-N
nylon membranes (Amersham Corp.) in duplicates and screened by
differential hybridization with
P-labeled cDNA probes of
NPC or adult brain. The cDNAs, made from NPC and adult brain
poly(A)
RNAs by using reverse transcriptase and an
oligo(dT) primer, were labeled with
[
-
P]dCTP (Amersham) to a specific activity
of 1.2
10
cpm/µg by the random primer
method(14) . The nucleotide sequence of the 5`-end of the cDNA
insert was determined for the selected clones by using an Applied
Biosystems model 373A DNA sequencer.
RNA and DNA Analyses
Total RNA was
extracted from cultured cells, mouse tissues, and embryos by the method
previously described(15) . Poly(A) RNA was
prepared from total RNA by using Oligotex-dT30<super> (Nippon
Roche, Japan). Northern blot analysis was performed according to the
procedure described(16) . The nucleotide sequence was
determined for both strands of the cDNA presented in Fig. 1according to standard procedure(17) . The 5`-rapid
amplification of cDNA ends system (Life Technologies, Inc.) with
poly(A)
RNA from 10-day-old mouse embryos was employed
to isolate 5`-extended ETF cDNA clones, together with the two
oligonucleotide primers, whose sequences are complementary to
nucleotide residues 31-50 and 797-816 of ETF cDNA shown in Fig. 1B(18) .
Whole Mount in Situ
Hybridization
Preparation and fixation of 10-day-old mouse
embryos followed by whole mount in situ hybridization to
digoxigenin-labeled RNA probes were performed essentially as
described(19) . The 1,063-bp StuI DNA fragment of ETF
cDNA subcloned into the EcoRV site of pBluescript II
SK(+) (Stratagene) was transcribed with T7 or T3 RNA polymerase in vitro to synthesize sense or antisense riboprobe in the
presence of digoxigenin-11-UTP. The bound probes were visualized by
alkaline phosphatase-conjugated anti-digoxigenin antibody (Boehringer
Mannheim), which reacted with 5-bromo-4-chloro-3-indolyl phosphate and
nitro blue tetrazolium to form a blue precipitate. The embryos were
then photographed under dark field illumination.Gel Mobility Shift Assay
The 77-bp XbaI-BamHI fragment of pET-3b (Novagen) coding for
the first 12 NH-terminal amino acids of the T7 gene 10
protein and the 1439-bp BamHI-StuI fragment of ETF
cDNA covering the entire coding region except for the first two
NH
-terminal amino acids were ligated in frame into the
pSP64(poly(A)) vector (Promega) to produce the T7 tag-ETF fusion
protein. In vitro transcription with SP6 RNA polymerase and
translation with rabbit reticulocyte lysate were done according to the
manufacturers Stratagene and Promega, respectively. The binding
reaction using 1 fmol of
P-labeled wild type GT-IIC probe
and 50 nl of the in vitro synthesized fusion protein was
carried out essentially as described(20) , except that the
incubation temperature was 25 °C. DNA-protein complexes were
electrophoresed on a 6% Long Ranger gel(AT Biochem) in 44.5 mM
Tris, 44.5 mM boric acid and 1 mM EDTA. The sequences
of the oligonucleotides used were adopted from Xiao et al. (1) as follows: wild type GT-IIC is
5`-TCGGGCAGTGGAATGTGTGGAATGTGTCTC-3` annealed to
5`-CCGAGAGACACATTCCACACATTCCACTGC-3` (identical to OGT2- 56 in (1) ); mutant GT-IIC is 5`-TCGGGCAGTTTAATGTGTTTAATGTGTCTC-3`
annealed to 5`-CCGAGAGACACATTAAACACATTAAACTGC-3` (identical to
OGT2-58 in (1) ). The double-stranded wild type GT-IIC
oligonucleotide was labeled with [
-
P]dCTP
by filling in the 5`-overhangs using DNA polymerase. T7
Tag
antibody (Novagen), the mouse monoclonal antibody directed against the
12-amino acid gene 10 leader peptide, was used to detect the T7 tag-ETF
fusion protein in the complex. Control mouse monoclonal antibody
against type II collagen was kindly provided by Dr. K. Kakimoto
(University of Occupational and Environmental Health, Japan).
Isolation of Novel cDNA Related to
TEF-1
To obtain cDNAs representing mRNAs preferentially
expressed in mouse embryonic brain, we screened about 14,400 clones of
the subtracted NPC cDNA library by differential hybridization to P-labeled cDNAs from NPC and adult brain (for details, see
``Experimental Procedures''). A total of 104 clones were
selected for positive hybridization to cDNA probes of the NPC but
negative or reduced hybridization to those of the adult brain, and the
cDNA inserts from these clones were further characterized by
restriction mapping analysis and partial sequence determination of
their 5`-ends. One of the clones, designated pmETF-1, contained a novel
sequence highly related to TEF-1
cDNAs(1, 2, 3, 4) . The complete
nucleotide sequence of pmETF-1 cDNA consisted of 2,073 bp excluding the
poly(A) tail (Fig. 1). Further screening of the original NPC
cDNA library by hybridization with the 676-bp BamHI-PstI fragment of pmETF-1 cDNA as a probe gave
rise to eight positive clones isolated from about 2.5
10
independent plaques. However, none of them contained an insert
larger than that of pmETF-1 (data not shown). Since the sequence
contained a large open reading frame, which was open at its 5`-end, we
attempted to isolate additional 5`extended cDNA clones by the 5`-rapid
amplification of cDNA ends procedure. Clone 8.41 contained the largest
insert among the clones thus obtained (Fig. 1A),
extending 42 bp upstream from the 5`-end of the pmETF-1 cDNA, but the
sequence was still open at its 5`-end. The sequence with a total of
2,115 bp derived from the two cDNAs are shown in Fig. 1B. The size of this sequence is similar to that
of the major transcript (2.3 kilobases) detected in Northern blot
analysis (see below), assuming a length for the poly(A) tail of
approximately 200 nucleotides. The ATG at position 1-3 is the
first ATG triplet within the cDNA sequence determined, and the open
reading frame starting with this triplet can encode the largest one
without multiple termination codons. Therefore, we tentatively assigned
the translational initiation site to the methionine codon ATG at
position 1-3, although the nucleotide sequence surrounding this
ATG codon does not exactly match the optimal context for translational
initiation proposed by Kozak(21) , and no in-frame stop codon
is found in the 5`-sequence upstream from this ATG triplet. The reading
frame shown in Fig. 1B consists of 1,335 nucleotide
residues encoding a polypeptide of 445 amino acids, with a calculated
molecular weight of 49,039. A possible polyadenylation signal, AATAAA,
found 18 nucleotides upstream from the poly(A) tail, and the presence
of two potential mRNA destabilization signals, ATTTA, were noted in the
3`-untranslated region (wavy and solidunderlining, respectively, in Fig. 1B).
Primary Structure of ETF and Its Comparison to Other
TEF-1-related Proteins
The amino acid sequence deduced from
the cDNA exhibited a significant sequence similarity to those of human,
mouse, and chicken TEF-1 and of Drosophilasd as
shown in Fig. 2. It is noteworthy that the 72 consecutive amino
acids (Asp-Lys
of the deduced protein
sequence) located toward the NH
-terminal portion of the
proteins, covering the TEA domain(5) , are completely conserved
among all five proteins, except that the alanine at position 58 of the
deduced amino acid sequence is replaced by serine in Drosophila
sd. The TEA domain is demonstrated to be a DNA binding domain (1, 22) and is originally marked by a block of
66-68 amino acids highly conserved between TEF-1, TEC1,
and abaA(5) . Therefore, we termed the putative
protein encoded by the novel cDNA the embryonic TEA domain-containing
factor (ETF). The sequence conservation is also observed in the
COOH-terminal region between ETF and the other proteins (Fig. 2). Although the NH
-terminal segment and the
middle portion (between the TEA domain and the COOH terminus) of ETF
diverge in their sizes and amino acid sequences among these proteins,
they retain some common structural features often found in the
activation domains of different transcription factors; the NH
terminus contains many acidic residues followed by the basic TEA
domain, and the middle part possesses a proline-rich character. In
addition, the NH
terminus of ETF from Met
to
Pro
contains 14 glycine residues out of 39 amino acids
(36%). The degree of overall sequence identity is 66, 66, 65, and 58%
for ETF/human TEF-1, ETF/mouse TEF-1, ETF/chicken TEF-1A, and
ETF/Drosophila sd comparisons, respectively. On the other
hand, human or mouse TEF-1 bears 76% identity to chicken TEF-1A (4) and 68% to Drosophila sd(8) , while 99%
identity exists between human and mouse TEF-1 (2) . Thus, ETF
is the most divergent of the vertebrate TEF-1 family proteins. These
structural features indicate that ETF is a new mammalian member of the
TEA domain-containing protein family and suggest that ETF may also
function as a transcription factor.
Expression of ETF mRNA in Developing Mouse Embryos
and Various Tissues
Northern blot analysis of total or
poly(A) RNA from different mouse tissues, embryos, and
cell lines was performed to elucidate the spatiotemporal expression
pattern of ETF mRNA. The analysis revealed the presence of a major ETF
transcript of 2.3 kilobases and a minor transcript of 4.0 kilobases (Fig. 3). Since the level of 28 S rRNA stained with ethidium
bromide seemed to vary in each lane, indicating possible differences in
RNA loading, we could not accurately quantitate the amount of mRNA.
However, the relative amount of ETF mRNA showed an obvious change
during the development of the mouse embryo, reached a plateau at day
10, and subsequently decreased to a much lower level on embryonic day
18 (Fig. 3A). A low level of ETF mRNA was still
detected in the brain at 0 and 7 days after birth but not in the adult
brain, although it was detectable at a low level in the
poly(A)
RNA from the adult brain (Fig. 3A). In the brain at postnatal day 7, the
cerebellum seemed to be the major site of expression for ETF mRNA; the
other brain regions failed to produce a detectable amount of the
transcript (Fig. 3B). Interestingly, a similar level of
the transcript was observed in the testis of mice of the same age (Fig. 3B). However, ETF mRNA was undetectable in total
RNAs from any of the adult tissues examined (Fig. 3B),
in marked contrast to the rather ubiquitous expression of TEF-1
mRNA(1, 2, 3) . Furthermore, a relatively
high level of expression of the ETF transcript was observed in RNAs
from the mouse embryonal carcinoma cell lines, P19 and F9, as well as
in that of NPC, but not in an RNA of the mouse neuroblastoma cell line,
neuro2A (Fig. 3A), suggesting that ETF may also
function in early stage development as reported for
TEF-1(1, 9, 23) .
RNA from adult mouse brain were analyzed. B, tissue distribution of ETF mRNAs. 10 µg of total RNAs
extracted from P19 cells or various tissues of 7-day-old postnatal and
adult mice were analyzed. Tissue names are indicated above each lane.
Binding of ETF Protein to GT-IIC
Motif
Because the TEA domain, a possible DNA-binding
region, of ETF is completely identical to those of the known mammalian
TEF-1 proteins, we used gel mobility shift analysis to examine whether
ETF binds to the GT-IIC motif. To detect the ETF protein in the
complex, we generated an epitope-tagged derivative, T7 tag-ETF, which
contains a 12-amino acid fragment derived from the leader peptide of
the T7 gene 10 protein that was fused to the NH terminus
(Asp
) of ETF and can be detected by the monoclonal antibody
(T7
Tag antibody). The in vitro transcription/translation
product of T7 tag-ETF formed specific complexes with a
GT-IIC-containing oligonucleotide (Fig. 5, bandsA and B); these complexes were competed by the same wild
type DNA but not by DNA containing a mutant GT-IIC motif and were
supershifted with T7
Tag antibody (Fig. 5, bandC). The results thus indicate that ETF can specifically
bind to the GT-IIC motif.
P-labeled wild type GT-IIC
oligonucleotide probe. DNA-protein complexes were analyzed by gel
mobility shift assay. Arrows indicate the positions of the
specific complexes (A and B), the unbound probe (F), and the antibody-dependent supershift (C). The
wild type (wt) or mutant (m) GT-IIC oligonucleotide
used as a competitor and the antibodies (Ab) added to the
binding reaction are indicated above each lane.
Tag antibody
suggest the presence of a product of
55 kDa in HeLa cells
transfected with T7 tag-ETF DNA, the size of which is similar to that
of in vitro synthesized T7 tag-ETF protein (data not shown).
However, these results need further confirmation with purified ETF
protein and its specific antisera. On the other hand, the
transcriptional activation and interference of TEF-1 requires
cooperative function of the following three interdependent regions: the
acidic NH
-terminal, proline-rich middle, and COOH-terminal
regions(22) . The amino acid sequences of the two proteins are
highly conserved in the COOH-terminal region but diverge in the
NH
-terminal and middle portions except for their acidic and
proline-rich nature, respectively. In the case of the two alternatively
spliced isoforms of chicken TEF-1, TEF-1A, and TEF-1B, the 13-amino
acid exon in the middle portion encoded only by the latter is shown to
be responsible for the transactivation function exhibited by TEF-1B but
not by TEF-1A(4) . Furthermore, Oct-1 and Oct-2, the
homeobox-containing transcription factors, differ in their activation
potentials mainly due to differences in their activation domains even
though they bind to the same DNA sequence(25) . Thus, the
differences in the transcriptional activities of TEF-1 and ETF may also
be ascribed to differences in activation domains, which may selectively
interact with distinct auxiliary proteins, such as a transcriptional
intermediary factor(1) . On the other hand, the TEA domain does
not seem to be the sole determinant of the DNA binding specificity of
TEF-1(22) . Therefore, it is also plausible that differences in
other regions that contribute to the sequence-specific DNA binding may
cause a subtle change in the binding affinity of ETF to the GT-IIC
motif, resulting in the apparent lack of effect observed for the
cotransfection assay with HeLa cells containing endogenous TEF-1. The
transcriptional activity of ETF needs to be explored further by using
different cells and chimeric constructs of ETF and different
transcription factors, such as TEF-1 and yeast GAL4.
to Pro
of chicken TEF-1 protein in Fig. 2.
The degree of similarity (93% identity) between these two proteins is
comparable to that observed for human and mouse TEF-1 comparison,
further indicating the presence of a distinct mammalian isotype of
chicken TEF-1. In addition, we have recently isolated another mouse
cDNA related to TEF-1 but distinct from all of the above mentioned
cDNAs by using a polymerase chain reaction procedure with mixed
oligonucleotides primed amplification of cDNA (MOPAC). (
)All
of these findings strongly suggest the existence of multiple subtypes
of a TEA domain-containing transcription factor family in mammals,
which may play different roles in regulating cellular and developmental
gene expression.
We thank Dr. Satoru Kuhara (Kyushu University,
Fukuoka, Japan) for help and useful discussions on multiple sequence
alignment and Dr. Masaki Takiguchi (Kumamoto University, Kumamoto,
Japan) and Dr. Ryoichiro Kageyama (Kyoto University, Kyoto, Japan) for
valuable discussions. We also acknowledge Dr. Chiaki Setoyama (Kumamoto
University, Kumamoto, Japan) for providing F9 RNA, Dr. Chiri Nagatsuka
for useful comments on the manuscript, and Dr. Yoshi Yamada (NIDR,
National Institutes of Health) for critical review of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.