©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Characterization of cDNA Encoding a Novel Protein Related to Transcriptional Enhancer Factor-1 from Neural Precursor Cells (*)

(Received for publication, April 4, 1995; and in revised form, June 2, 1995)

Michio Yasunami (1) Kazuo Suzuki (1) Takeshi Houtani (2) Tetsuo Sugimoto (2) Hiroaki Ohkubo (1)(§)

From the  (1)Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto 862, Japan and the (2)Department of Anatomy, Kansai Medical University, Moriguchi, Osaka 570, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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.

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.


EXPERIMENTAL PROCEDURES

Construction, Subtraction, and Differential Screening of Mouse NPC cDNA Library

About 1.15 10^8 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-ZAPXR 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 10^6 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 [alpha-P]dCTP (Amersham) to a specific activity of 1.2 10^9 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) .


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.



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(2)-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(2)-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 [alpha-P]dCTP by filling in the 5`-overhangs using DNA polymerase. T7bulletTag 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).


RESULTS

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 10^5 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(2)-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(2)-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(2) terminus contains many acidic residues followed by the basic TEA domain, and the middle part possesses a proline-rich character. In addition, the NH(2) terminus of ETF from Met^1 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.


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) .



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) .


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) 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.



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.



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(2) terminus (Asp^3) of ETF and can be detected by the monoclonal antibody (T7bulletTag 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 T7bulletTag antibody (Fig. 5, bandC). The results thus indicate that ETF can specifically bind to the GT-IIC motif.


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 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.



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.


DISCUSSION

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 T7bulletTag 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(2)-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(2)-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.

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 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). (^2)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.

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.


FOOTNOTES

*
This work was supported in part by research grants from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank&#174;/EMBL Data Bank with accession number(s) D50563[GenBank].

§
To whom correspondence should be addressed: Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kuhonji 4-24-1, Kumamoto 862, Japan. Tel.: 81-96-373-5346; Fax: 81-96-373-5350.

^1
The abbreviations used are: NPC, neural precursor cells; TEF-1,transcriptional enhancer factor-1; ETF, embryonic TEA domain-containing factor; bp, base pair(s).

^2
M. Yasunami, K. Suzuki, and H. Ohkubo, unpublished observations.


ACKNOWLEDGEMENTS

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.


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