Human Placental TEF-5 Transactivates the Human Chorionic Somatomammotropin Gene Enhancer

Shi-Wen Jiang, Kangjian Wu and Norman L. Eberhardt

Endocrine Research Unit (S-W.J., N.L.E.) Thoracic Diseases Research Unit (K.W.) Department of Internal Medicine (S-W.J., K.W., N.L.E.) Department of Biochemistry & Molecular Biology (N.L.E.) Mayo Clinic Rochester, Minnesota 55905


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human chorionic somatomammotropin (hCS) gene expression in the placenta is controlled by an enhancer (CSEn) containing SV40-related GT-IIC and SphI/SphII enhansons. These enhancers are controlled by members of the transcription enhancer factor-1 (TEF-1) family. Recently TEF-5, whose mRNA is abundant in placenta, was shown to bind cooperatively to a unique, tandemly repeated element in CSEn2, suggesting that TEF-5 regulates CSEn activity. However, expression of TEF-5 using a cDNA lacking the 5'-untranslated region and containing a modified translation initiation site was not accompanied by CSEn activation. Using nested, degenerate PCR primers corresponding to conserved TEF domains, several novel TEF-1-related cDNAs have been cloned from a human placental cDNA library. The open reading frame of one 3033-bp clone was identical to TEF-5 and contained 300- and 1423-bp 5'- and 3'-untranslated regions, respectively. The in vitrogenerated approximately 53-kDa TEF-5 polypeptide binds specifically to GT-IIC and SphI/SphII oligonucleotides. Overexpression of TEF-5 in BeWo cells using the intact 3033-bp cDNA transactivates the hCS and SV40 enhancers and artificial enhancers comprised of tandemly repeated GT-IIC enhansons, but not OCT enhansons. The data demonstrate that TEF-5 is a transactivator that is likely involved in the transactivation of CSEn enhancer function. Further, the data suggest that elements within the untranslated regions, initiation site, or both control TEF-5 expression in ways that influence its transactivation function.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previous studies have indicated that human chorionic somatomammotropin (hCS) gene expression is largely controlled by an enhancer, designated CSEn2, localized downstream of the hCS-2 gene (1, 2, 3, 4, 5, 6). Recently, we discovered that the two additional copies of this enhancer (CSEn1 and CSEn5), which are located downstream of the hCS-1 and hCS-5 genes, respectively, can function synergistically with CSEn2 to form a strong placental-specific enhancer and pituitary-specific silencer (7). We and others have shown that CSEn is composed of multiple enhansons that work cooperatively to mediate maximal enhancer activity (1, 3, 4). At least five GT-IIC and SphI enhansons were identified in CSEn2 based on 1) deletion/mutation studies of CSEn2 (3, 4); 2) sequence similarities of hCS GT-IIC and SphI/SphII enhansons with their SV40 counterparts (1, 3, 4); 3) their ability to bind to transcription enhancer factor 1 (TEF-1) (8, 9); and 4) the ability of each of the multimerized GT-IIC and SphI/SphII enhansons to stimulate hCS promoter activity in placental cells (6). In addition, we recently demonstrated that multiple copies of the various enhancers located downstream of the hCS-5, hCS-1, and hCS-2 genes act cooperatively to function as an effective silencer of hCS promoter function in pituitary GC cells (7). Thus, the composite enhancers within the hGH/hCS locus may control cell-specific repression of hCS gene expression in pituitary as well as transactivation in the placenta. Although much progress has been made concerning the fine structure of CSEn2, the identity of transcription factors that mediate its function in placental cells has remained elusive.

Our initial efforts to understand the mechanism of CSEn activation focused on TEF-1, the first cloned human GT-IIC binding factor that has been implicated in mediating SV40 enhancer function (10, 11, 12). We observed that TEF-1 is expressed in placental tissue and cultured human choriocarcinoma cell lines (BeWo and JEG-3) (8, 9). However, when TEF-1 function was tested by cotransfection with a TEF-1 expression vector and a reporter plasmid containing CSEn2, TEF-1 repressed, rather than stimulated, the reporter activity (8, 9). Also, down-regulation of endogenous TEF-1 levels with antisense oligonucleotides resulted in elevated CSEn2 as well as basal hCS promoter activity, suggesting that endogenous TEF-1 was acting as a repressor (9). We subsequently demonstrated that TEF-1 can interact in vitro with the TATA box-binding protein (TBP), inhibiting its ability to bind the TATA element (8). Thus TEF-1-TBP interactions may provide a possible mechanism by which TEF-1 exerts repressor activity on GT-IIC-containing enhancers. These data suggest that factor(s) other than TEF-1 may account for CSEn function in placental cells.

Recently, Jacquemin et al. (13) reported the cloning of an additional member of the hTEF-1 family, designated hTEF-5, that was shown to be related to the chicken DTEF-1 gene. They were able to show that hTEF-5 mRNA is most strongly expressed in placenta and choriocarcinoma cells (JEG-3), followed by skeletal and cardiac muscle. Jacquemin et al. (13) also demonstrated that hTEF-5 binds to several CSEn enhansons and defined a novel functional element comprised of repeated enhansons that bind TEF-5 in a cooperative manner, suggesting that hTEF-5 regulates CSEn activity. Nevertheless, attempts to express this gene product in a variety of cells, including JEG-3 cells, failed to demonstrate any CSEn activation (13). In the current study, we report the molecular cloning of an additional hTEF-5 clone from a human placental library that contains the identical open reading frame reported by Jacquemin et al. (13) but with an additional 300 bp of 5'-untranslated sequence. TEF-5 mRNA is highly expressed in placental, skeletal, and cardiac muscle tissues. The in vitro-generated TEF-5 is a polypeptide of approximately 53-kDa that specifically binds to GT-IIC and SphI/SphII enhansons. Overexpression of the complete, unmodified TEF-5 cDNA clone in the choriocarcinoma cell line BeWo resulted in activation of a variety of GT-IIC-containing enhancers. These data provide direct functional evidence that TEF-5 may play an important role in the placenta-specific expression of the hCS gene. In addition, its presence in muscle tissue indicates that it may be involved in the regulation of muscle gene expression and/or development.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of Human Placental TEA/ATTS Domain-Containing Factors
Mutagenesis studies on CSEn demonstrate that CSEn function is dependent on the GT-IIC and SphI/SphII enhansons (3). Since these enhansons are recognized specifically by the TEA/ATTS DNA-binding domain and the TEA/ATTS factor TEF-1 does not transactivate this enhancer (8, 9), we reasoned that additional members of the TEA/ATTS family might be responsible for CSEn function. We used an approach similar to that of Jacquemin et al. (18) to isolate mouse and human TEF-1 homologs from several mouse and human libraries. Accordingly, TEF sequences were amplified from a human placental cDNA library using nested, degenerate PCR primers deduced from the highly conserved TEA/ATTS and C-terminal domains (Table 1Go and Fig. 1BGo). For the convenience of the subcloning, BglII sites were fixed on each primer. After two rounds of PCR, DNA fragments between 800 and 1200 bp were purified from agarose gels. In subsequent BglII restriction digestions, some of the DNA fragments were converted to 700- and 400-bp bands, indicating the presence of an internal BglII site within certain fragments. Two classes of TEA/ATTS domain genes were identified from 18 positive clones in subsequent sequence analyses and database comparisons. These included clones identical to TEF-1 (12) and clones containing the BglII site that were related to the chicken DTEF-1 gene (19) and corresponded to the hTEF-5 cDNA clone (13), which were analyzed further.


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Table 1. Oligonucleotides

 


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Figure 1. TEF-5 Is a DTEF-1 Homolog

A, Nucleotide sequence of TEF-5 cDNA (GenBank Accession No. AF142482). The polypyrimidine stretches in the 5'-untranslated region are underlined, and their lengths are indicated by the numbers underneath. Such tracts have been implicated in the inhibition of translation initiation (21 22 ). B, Amino acid sequence comparison of TEF-5 with the other known human TEF homologs and chicken DTEF-1. The TEA/ATTS DNA binding, proline (P)-rich, serine, threonine, and tyrosine (STY)-rich, and Zn-finger-like domains are indicated by the labeled blocks that correspond to the regions below the symbols. The GeneBank accession numbers for the TEF homologs are given in parentheses: TEF-1 (M63896), TEF-3 (X94438), chicken cDTEF-1 (cDTEF-1A, U46127); the TEF-4 sequence was taken from Jacquemin et al. (16 ); its sequence is available in GenBank (accesion no. X94440). The TEF-5 sequence was derived from the clone described in this paper, and its open reading frame is identical to the TEF-5 sequence reported by Jacquemin et al. (16 ) (GenBank accession no. X94439). The multiple sequence analysis was performed by the programs PILEUP and PRETTY (GCG, Madison, WI). The boxed regions depict the amino acid sequences used to generate degenerate oligonucleotides for the PCR cloning (see also Table 1Go).

 
Using the two BglII-generated DNA fragments as probe, a human placenta {lambda}DR2 phage cDNA library (CLONTECH Laboratories, Inc., Palo Alto, CA) was screened. Approximately 1.5 million plaques were screened and 22 positive clones were identified. Sequence analysis indicate that all of these clones contained partial sequences that were missing the extreme N-terminal region. Using a probe from the 5'-end of the longest cDNA clone, we screened an additional 1 million plaques and, from 14 positive clones, isolated a 3.0-kb TEF-5 clone including more than 300 bp preceding the TEA/ATTS domain (Fig. 1AGo). Like other members of the TEA/ATTS family (12, 18, 19, 20), no initiator methionine with perfect Kozak consensus sequences was found in the 5'-region (Fig. 1AGo). Like TEF-1, TEF-5 contains an isoleucine codon (ATA) located 29 amino acids upstream of the TEA domain (starting with the sequence DAEG) with a 9 of 13 match with the Kozak consensus (GCCGCCRCCAUGG), including the three essential positions at -3, +4, and +5. Xiao et al. (12) demonstrated that the corresponding region in TEF-1 served as one of the two translation initiation sites of TEF-1. Amino acid sequence analysis indicates that TEF-5 contains a highly conserved TEA/ATTS domain and typical proline-rich and STY-rich domains (Fig. 1AGo). Alignment of TEF-5 with the other cloned TEF-related factors revealed that TEF-5 is most closely related to the chicken DTEF-1A transcription factor (Fig. 1BGo)(19).

TEF-5 Is Expressed Preferentially in Placenta and Muscle Tissues
TEF-5 expression in different tissues was evaluated by Northern analysis. A blot containing a panel of mRNAs from multiple human tissues was hybridized with the TEF-5 cDNA probe. A 3.0-kb band is readily detected from placenta, with somewhat lesser levels of mRNA detectable in skeletal and cardiac muscle tissues (Fig. 2AGo). After longer exposure, a faint band is also visible from liver tissue. In addition, a minor band at 6.5 kb observed from placenta during longer exposure may indicate the presence of partially spliced TEF-5 precursors. No hybridization signals were observed in brain, lung, kidney, or pancreas. Also, TEF-5 expression was characterized in several cell lines. Strong signals were found in BeWo cells, a human choriocarcinoma cell line. In addition, TEF-5 is also expressed in SV40 transformed human fibroblasts (GM0637E) and osteosarcoma (MG63) cells (Fig. 2CGo). Low levels of TEF-5 mRNA are present in cervical carcinoma (HeLa), liver tumor (HepG2), and human breast cancer (MCF-7) cells (Fig. 2CGo). GC and monkey kidney (COS-1) cells contain very low or undetectable levels of TEF-5 mRNA (Fig. 2CGo). These data indicate a restricted pattern of TEF-5 expression. Its high-level expression in placental tissue and BeWo cells indicate TEF-5 could be involved in hCS gene expression. However, the absence of a strong signal in COS-1 cells indicates that TEF-5 is unlikely to represent the source of the previously identified factor, PPf/CSEF-1, in these cells (9, 13), which appears to be a proteolytic degradation product of multiple TEF factors (13).



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Figure 2. TEF-5 mRNA Is Expressed in Human Placenta and Muscle Tissues as Well as Several Cell Lines, Including Human Choriocarcinoma Cells (BeWo)

A, Northern analysis of TEF-5 poly A mRNA in human heart, brain, placenta, lung, liver, skeletal muscle (SK), kidney, and pancreas. B, Human tissue blot in A probed with ß-actin as loading control. C, Northern analysis of TEF-5 mRNA expression in several cell lines. Total RNA was isolated from the various cell lines and analyzed as described in Materials and Methods. D, Ethidium bromide staining profile of 28S and 18S RNA of the blot in C to control for RNA loading. Under the nonstringent washing conditions used with the ß-actin probe, both the 2.1-kb ß-actin mRNA and the muscle-specific {alpha}-actin mRNAs are revealed (31 32 ).

 
TEF-5 Binds Specifically to GT-IIC Enhanson Sequences
TEF-5 cDNA was subcloned into pBluescript KS vector for in vitro transcription-translation. In SDS-PAGE, the [35S]methionine-labeled peptide showed a migration rate very close to the approximately 53-kDa bands of TEF-1 that served as a positive control in this experiment (Fig. 3AGo). Xiao et al. (12) provided evidence that in TEF-1, translation initiates from both the isoleucine (AUA codon) and methionine (AUG codon), located 29 and 14 amino acids, respectively, upstream of the TEA/ATTS domain, resulting in 53- and 51-kDa peptides. Examination of cDNA sequences reveals that DNA sequences around the AUA codon were highly conserved (12 of 13 matches) between hTEF-1 and TEF-5. However, there is no corresponding second initiation site in TEF-5, since the methionine (ATG) is replaced by arginine (CGG). The approximately 53-kDa single band of TEF-5 in vitro translation products suggests that the isoleucine as indicated in Fig. 1AGo, like the cDTEF-1, hTEF-3, and hTEF-1 homologs, is the most likely translation initiation codon. Because TEF-1 and TEF-5 contain comparable numbers of methionine (12 for TEF-1 and 10 for TEF-5), the difference in the density of in vitro translation products observed in Fig. 3AGo may be due to different translation efficiency, possibly caused by the single substitution in their translation initiation site.



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Figure 3. TEF-5 Is an Approximately 53-kDa Protein That Binds Specifically to the GT-IIC and SphI/SphII Enhansons

A, SDS-polyacrylamide gel analysis of in vitro-generated, [35S]methionine-labeled TEF-5 and TEF-1 (arrow). B, Gel shift analysis of in vitro-generated TEF-5 and TEF-1 to GT-IICSV and GT-IICCS oligonucleotides. TNT represents the coupled transcription/translation, reticulocyte lysate system (Promega Corp.). C, Competition analysis of TEF-5 binding to the GT-IICCS oligonucleotide in the presence of increasing concentrations (50-, 100-, and 300-fold excess) of the wild-type and mutant GT-IICSV and SphISV oligonucleotides (8 9 ). D, Temperature stability of in vitro- generated TEF-1 and TEF-5 as reflected in the ability to bind to the GT-IICCS oligonucleotide. The specific TEF-1- and TEF-5-DNA complexes in panels B, C, and D are indicated by the arrows.

 
The ability of TEF-5 binding to the GT-IIC enhanson was evaluated in gel shift experiments. TEF-1- and TEF-5-programmed in vitro translation products (Fig. 3AGo) interacted with both GT-IICSV and GT-IICCS oligonucleotides, which share the core sequence (GGAATGTG) but have different flanking sequences (Fig. 3BGo). Mutation of the core sequences of GT-IICSV effectively eliminated its ability to bind TEF-1 and TEF-5, indicating that TEF-5 can interact with the GT-IIC core sequences. The binding specificity was further confirmed by a competition experiment. The TEF-5·GT-IICCS interaction was competed with increasing amounts of unlabeled GT-IICSV, SphI, and SphII, but not the corresponding mutant oligonucleotides (Fig. 3CGo). These probes were used because the binding affinity of TEF-1 to each of these sequences has been well characterized (10, 6) and the binding affinities occur in order: GT-IIC > SphI ~ SphII. In the presence of a 300-fold excess of competitor, the TEF-5 complex was completely competed by the GT-IIC oligonucleotide and greatly reduced by the SphI oligonucleotide (Fig. 3CGo). Therefore, TEF-5 has the same DNA binding specificity as the other TEA/ATTS domain factors in interacting with both GT-IIC and SphI enhansons.

In previous studies, we and others have observed that GT-IIC probes detected a two-band pattern from BeWo or JEG-3 cell extracts in gel shift experiments (4, 9, 13, 23). Based on gel supershift experiments with anti-TEF-1 antibodies, the slow migrating band contains TEF-1, and the unknown fast migrating band was designated as PPf/CSEF-1 and proposed to be a potential TEA/ATTS domain factor (9). Since TEF-1- and TEF-5·GT-IIC complexes migrate identically in these experiments (Fig. 3BGo), it is unlikely that TEF-5 and PPf/CSEF-1 are the same factors. Moreover, Jacquemin et al. (13) have shown that PPf/CSEF-1 appears to be a proteolytic breakdown product of TEF family members that is immunologically related to the TEA/ATTS domain. However, the exact precursor(s) for PPf/CSEF-1 are not known. Since PPf/CSEF-1 is much more thermostable than TEF-1 (9), we performed thermolability analyses of TEF-1 and TEF-5 to determine whether their relative thermolabilities might provide some evidence about the origin of PPf/CSEF-1. We reasoned that, if the proteolytic product were heat stable, this was likely the result of a specific structural feature that would be present in the precursor and might confer relative heat stability to the precursor protein as well. Consistent with our previous results, TEF-1 can withstand 60 C for 2 min but lost its DNA-binding activity at 65 C (Fig. 3DGo). TEF-5 GT-IIC binding activity is largely reduced at 50 C, indicating it is more sensitive to heat exposure than TEF-1 (Fig. 3DGo). This suggests that it might be a less likely precursor of the more thermostable PPf/CSEF-1.

TEF-5 Transactivates hCS and SV40 Enhancers
We next examined whether TEF-5 can transactivate the hCS and SV40 enhancers when overexpressed in BeWo cells. A cotransfection was performed with the pDR2 TEF-5 expression vector and a variety of hCSp.LUC reporter plasmids (Fig. 4AGo). While TEF-5 overexpression did not affect hCS promoter activity, it stimulated the hCSp.LUC gene carrying one copy of the SV40 enhancer 2.9-fold (Fig. 4AGo). TEF-5 overexpression increased luciferase expression 3.5-fold with constructs containing one or two copies of CSEn2 (Fig. 4AGo). In addition, TEF-5 stimulated luciferase activity 2.5-fold with a construct containing 12 copies of the GT-IIC enhanson (Fig. 4AGo), providing direct evidence that TEF-5 transactivation is mediated by interaction with GT-IIC sequences. Essentially identical results (~3-fold stimulation) were obtained with constructs containing one copy of CSEn2 with either one copy of CSEn1 (enhancer copy downstream from the hCS-1 gene) or CSEn5 (enhancer downstream of the hCS-5 gene). Since the CS enhancer contains multiple enhansons and mutation of each of the enhansons only partially removes enhancer activity (6), it is not practical to generate a mutated CS enhancer. Instead, to directly test the transactivation ability of TEF-5, we made two constructs, (GT-IIC)5-CSp.LUC and (OCT)5-CSp.LUC, containing either tandemly repeated GT-IIC elements or tandemly repeated OCT elements to serve as a control. The OCT elements are derived from a region of CSEn within the footprint 2 region (3) and neither bind any of the TEF factors nor exhibit any independent enhancer activity, but may contribute to enhancer activity, since their mutation in the context of an intact enhancer is associated with somewhat reduced enhancer activity (3). Cotransfection of TEF-5 with this pair of reporter plasmids shows that TEF-5 overexpression significantly stimulated the GT-IIC artificial enhancer but not the control (OCT)5-CSp.LUC or CSp.LUC constructs (Fig. 4BGo). These experiments, together with transfection studies using native hCS or SV40 enhancers, strongly support the concept that TEF-5 is an effective transactivator and may account for the constitutive CS and SV40 enhancer activities in placental cells.



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Figure 4. TEF-5 Transactivates Enhancers Containing GT-IIC Enhansons

A, BeWo cells were transfected with 5 µg of each of the reporter plasmids in the presence (+) or absence (-) of 500 ng of the pDR2 TEF-5 expression plasmid. Csp.LUC is the control plasmid that contains 500 bp of the hCS proximal promoter and 5'-flanking DNA, but no enhancer; SVEn CSp.LUC and CMV CSp.LUC contain SV40 and CMV enhancers, respectively; CSEn2 CSp.LUC and (CSEn2)2 CSp.LUC contain one and two copies of hCS2 gene enhancers, respectively; (GT-IIC)12 CSp.LUC contains 12 tandemly repeated GT-IIC enhansons; CSEn1 CSEn2 CSp.LUC and CSEn5 CSEn2 CSp.LUC are plasmids containing consecutive homologous enhancers associated with the hCS1, hCS2, and hCS5 genes, either CSEn1 and CSEn2 or CSEn5 and CSEn2, respectively. B, BeWo cells were transfected with 5 µg of each of the reporter plasmids in the presence (+) or absence (-) of 500 ng of the pDR2 TEF-5 expression plasmid. (GT-IIC)5_CSp.LUC and (OCT)5_CSp.LUC contain five copies of either tandemly repeated GT-IIC or OCT (Materials and Methods) sequences. C, BeWo cells were transfected with 5 µg of each of the reporter plasmids in the presence (+) or absence (-) of 5 µg of the pDR2 TEF-5 expression plasmid. The data in panels A, B, and C represent the results from four independent transfections, expressed as light units/µg protein ± SE. The data were analyzed by Student t tests to determine statistical significance as indicated (p); N.S., nonsignificant.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The hCS enhancer, CSEn2, is a multipartite enhancer comprised of enhansons related to the SV40 GT-IIC and SphI/SphII enhansons (1, 3, 4, 6). Originally, this suggested that the GT-IIC-binding factor, TEF-1, might be involved in CSEn2 function. However, overexpression of TEF-1 in BeWo cells resulted in significant repression of both the basal and CSEn2-stimulated hCS promoter activity (8, 9). Also, such repression was observed with the thymidine kinase promoter, suggesting that the repression works through basal transcriptional factor(s) (8). Using an antisense oligonucleotide approach, we showed that endogenous TEF-1 represses hCS basal and enhancer-stimulated activity in placental cells (8, 9), further implicating TEF-1’s primary role as a repressor. Subsequently, we found that TEF-1 interacts with the TATA-binding protein and inhibits its ability to bind to the TATA element (8). While enhancer activity has correlated with the binding of another factor, designated PPf/CSEF-1, to the GT-IIC and SphI/SphII enhansons within CSEn2 (4, 9, 13, 23), Jacquemin et al. (13) have presented evidence that PPf/CSEF-1 is likely to represent a proteolytic byproduct of TEF family member digestion by an enzyme that is activated during cell extract preparation and is therefore less likely to be involved in activation of the enhancer. More recently we have found that multiple copies of CSEn1, CSEn2, and CSEn5 act as silencers of hCS promoter activity in pituitary GC cells (7). The silencer activity in GC cells is correlated with the binding of TEF-1, providing additional evidence that TEF-1 can act as a repressor and that other factors, possibly related to TEF-1 are responsible for the enhancer activity of CSEn.

To isolate additional TEA/ATTS family members we used a degenerate oligonucleotide approach based on conserved domains within TEF-1 and its family members (18). We isolated a clone that contained an open reading frame identical to the TEF-5 homolog that was recently reported by Jacquemin et al. (13). TEF-5 is related to the chicken DTEF-1 family (19) (Fig. 1Go). In agreement with Jacquemin et al. (13), high levels of TEF-5 mRNA were found in human placental tissue and BeWo cells (Fig. 2Go), and its gene product was found to bind specifically to GT-IIC sequences from both the hCS and SV40 enhancers (Fig. 3Go). In contrast to Jacquemin et al. (13), who were unable to demonstrate functional activity after overexpressing TEF-5 in HeLa or JEG-3 cells, we demonstrate that it is a transactivator of CSEn function in BeWo cells (Fig. 4Go). TEF-5 stimulates reporter constructs containing single copies of CSEn2 or multiple enhancers, including CSEn5 and CSEn1, that occur downstream of the hCS-5 and hCS-1 genes, respectively (Fig. 4Go). Also, TEF-5 stimulated reporter constructs containing the SV40 enhancer or an artificial enhancer comprised of GT-IIC multimers. Taken together, these studies demonstrate that TEF-5 is a transactivator in BeWo cells that operates through binding GT-IIC and SphI/SphII enhansons and provide strong support to the notion that TEF-5 plays a major role in mediating hCS gene expression through the placental enhancer.

The disparity in our TEF-5 functional analyses vs. those of Jacquemin et al. (13) are likely to be explained by the different strategies that were used to express the protein and analyze the protein. Thus, differences in the choriocarcinoma cell lines JEG-3 (13) and BeWo, which were used in the current studies, may well be a factor contributing to the difference in behavior. In addition, in constructing their TEF-5 expression vector, Jacquemin et al. (13) modified the 5'-untranslated region and translation initiation site of their TEF-5 cDNA clone, replacing the ATA isoleucine codon with ATG, replacing the imperfect Kozak sequence (9 of 13 matches) with a perfect Kozak sequence, and deleting any upstream 5'-untranslated sequences. This was the identical strategy used for TEF-1 expression (12). Our TEF-5 cDNA clone contained about 300 bp of 5'-untranslated sequence and a much larger 3'-untranslated region, which was not modified: the pDR2 TEF-5 expression vector was obtained from the original {lambda}DR2 bacteriophage clone via loxP-dependent cre excision (15). These differences in expression strategy suggest that expression efficiency could be the result of engineering a more efficient translation initiation site (ATG vs. AUA and perfect Kozak consensus vs. 9 of 13 matched consensus) in the constructs. In addition, the 5'- or 3'-untranslated regions could contain information that either controls mRNA abundance or modifies translation efficiency. In this regard it is interesting that the 5'-untranslated region of TEF-5 contains several polypyrimidine tracts (see Fig. 1AGo) that are similar to those that have been shown to negatively regulate translation intitiation in a number of genes, including the androgren receptor (20) and ribosomal protein rpL32 gene (21). Since most proteins that are derived from mRNAs with non-AUG initiation codons contain methionine as the initiating amino acid (30), the question of whether a unique NH2-terminal structure may contribute to TEF-5 structure/function is considered less likely. While the translation initiation site for TEF-5 has not been directly measured, based on the size of the expressed protein product, its size relative to TEF-1, its amino acid sequence homology with TEF-1, and the relative absence of other likely translation initiation sites, the isoleucine initiation codon (AUA, nucleotide 306, Fig. 1AGo, GenBank Accession No. AF142482) is the most probable. Consistent with this interpretation is the observation that Jacquemin et al. (13) did observe squelching with their pXJ41 TEF-5 expression construct in JEG-3 cells. These observations are consistent with the concept that, like TEF-1, TEF-5 may operate through the obligate requirement of a cofactor that is present in limited abundance. Nevertheless, when higher concentrations of TEF-5 expression plasmid were used in otherwise identical experiments, several reporter constructs containing CSEn2 were stimulated to even higher levels of activity (Fig. 4CGo), indicating that TEF-5 stimulates over widely varying concentrations. Thus, if expression efficiency explains the difference in functional behavior between these two otherwise identical clones, either large differences in the translational efficiency or negative regulatory sequences in the 5'- and/or 3'-untranslated regions or both are very likely to be involved.

DTEF-1 was cloned originally from chicken skeletal muscle by Azakie et al. (19) and was found to be expressed at high levels in cardiac muscle, but low levels in skeletal muscle. Moderate expression of this gene was also found in gizzard and lung, and lower levels were present in kidney. The transcriptional potential of this gene has not been examined. The alignment of TEA/ATTS domain genes (Fig. 1Go) indicates that DTEF-1 and TEF-5 are most closely related, sharing 87% identity and 91% similarity at the amino acid level. This suggests that TEF-5 is the human homolog of chicken DTEF-1. However, the tissue-specific distribution between the two species appears to be different. In humans, TEF-5 is expressed at comparable levels in cardiac and skeletal muscle, and no expression was observed in lung and kidney even after long exposure. The relatively high level expression of TEF-5 in muscle tissues suggest that TEF-5 may participate in muscle gene expression and development.

Currently the TEA/ATTS gene family is comprised of four members, including TEF-1, TEF-3 (RTEF-1), TEF-4, and TEF-5 (DTEF-1). These factors are thought to play important, but partially redundant roles in myogenesis, cardiogenesis, and central nervous system development. Stewart et al. (22) cloned RTEF-1 from human heart. RTEF-1 was shown to be expressed at high levels in skeletal muscle and pancreas and at lower levels in placenta. Interestingly, RTEF-1 shares 98% identity with human TEF-3, a gene whose mouse homolog was found to be expressed mainly in embryonic mouse skeletal muscles (18). From the human placental library, we isolated a clone that is identical to hTEF-3 (data not shown), confirming the expression of this homolog in placenta reported by Stewart et al. (22). Thus at least three of the TEA/ATTS homologs, TEF-1, RTEF-1/TEF-3, and TEF-5, are expressed in the placenta and could all play varying roles in mediating CSEn function and placental development.

In addition to the human TEA/ATTS domain factors described above, a mouse gene ETRF-1, closely related to RTEF-1, has been isolated (7). However, its ubiquitous expression pattern in all tested tissues, including lung, kidney, muscle, heart, liver, brain, thymus, spleen, and testis, argues against its assignment as the mouse homolog of hRTEF-1. Similarly, another mouse gene, ETRF-2, showed high level expression in lung, a situation unique to all the other TEA/ATTS domain factors (7). Two more mouse TEA/ATTS factors, FR19 (26) and TEFR-1 (27), were isolated from NIH3T3 cells and heart libraries, respectively. Both genes were shown to be expressed in lung and muscle tissues. More recently, Kaneko and associates (28, 29) have demonstrated that mouse mTEAD-2, the hTEF-4 homolog, is expressed throughout early mammalian development before the onset of zygotic gene expression, followed by expression of all the other TEF family members, suggesting a selective role for this homolog in development. With the exception of TEFR-1, which was shown to have M-CAT (chloramphenicol acetyltransferase) binding ability and function as transactivator in HeLa cells, the transcriptional potential of these gene products has not been characterized.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nested PCR Amplification of TEA/ATTS Domain-Related Factors
Two sets of primers, corresponding to the highly conserved TEA/ATTS domain amino acid sequences and a carboxyl-terminal domain, were designed for nested PCR with the lowest possible degeneracy (Table 1Go). BglII sites were incorporated into each oligonucleotide at the 3'-end to facilitate efficient subcloning. In the first round PCR, the outside pair of oligonucleotides, TIC11 and TIC12, with a total sequence complexity of 256, were used to amplify two human placental libraries subcloned in {lambda}gt11 (Stratagene, La Jolla, CA) and {lambda}DR2 (CLONTECH Laboratories, Inc., Palo Alto, CA). The reactions contained 10 µl 10x PCR extender buffer (Stratagene), 2.5 µg each primer, 10 mM deoxynucleoside triphosphates, 2.0 µl library DNA template in a total volume of 100 µl. After 5 min of initial denaturing at 94 C, 10 U of Taq DNA polymerase and 10 µl of Taq Extender (Stratagene) were added, and 25 cycles of PCR were performed at the following conditions: denaturation, 94 C for 1 min; annealing, 50 C for 1.5 min; and elongation, 72 C for 2 min. PCR products were extracted with phenol-chloroform, precipitated with ethanol, and resolved on 1.5% agarose gels. Faint DNA bands corresponding to about 800-1200 bp were excised from the gel, purified by GeneClean (BIO 101, Vista, CA), and dissolved in 10 µl 10 mM Tris·HCl (pH 7.5), 1 mM EDTA.

For second-round PCR reactions, 1.0 µl templates from the first-round PCR reactions were reamplified using 2.5 µg primers TIC11 and TIC12 (Table 1Go) with a combined complexity of 1024. PCR was performed for 35 cycles under the same conditions as first-round PCR. After agarose gel electrophoresis, 1100-bp products were cut out and purified by GeneClean. Restriction digestion with BglII, the site used for subcloning, rendered some of the 1100-bp DNA to 700- and 400-bp fragments, indicating the presence of an internal BglII site. These DNA fragments were purified and subcloned individually into BglII-digested pA3LUC vector. Eighteen positive clones were purified and sequenced by automatic dye termination sequencing (Molecular Biology Core Facility, Mayo Clinic) using M13 and pA3 P12 primers (Table 1Go). Sequencing results were analyzed using the FASTA program (GCG, Madison, WI).

Screening of cDNA Libraries
The 700-bp and 400-bp DNA fragments of TEF-5 were purified from agarose gels and used as probes to screen a {lambda}DR2 cDNA library. The 700-bp and 400-bp DNA fragments (20 ng) were combined and labeled by [32P]dCTP (8000 Ci/mmol, Amersham, Arlington Heights, IL) using a random primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN) and purified on BioGel P-60 columns. Plaques (~50,000 per 15-cm plate) were transferred onto nitrocellulose membranes (Protron, Schleicher & Schuell, Inc., Keene, NH) as described (14). The membranes were hybridized with 1.5 x 106 cpm/ml of denatured probe dissolved in ExpressHyb solution (CLONTECH Laboratories, Inc.) and washed according to the manufacturer’s recommendations. The membranes were exposed to Kodak x-ray film (Eastman Kodak Co., Rochester, NY) for 18–36 h. Positive plaques were purified by two additional rounds of selection and subjected to lox-mediated automatic excision for releasing of pDR2 plasmid (15). DNA sequences from several clones were organized by contig analysis, and the longest sequence was identified. A more complete TEF-5 clone was obtained by using the most 5'-sequences as probes to screen the library for additional clones. This clone, which represents the most complete human TEF homolog cDNA cloned to date, was sequenced at least three times over its entire length on both strands.

Northern Blot Analysis
Cells grown to 50% confluence were washed three times with PBS (Gibco BRL, Gaithersburg, MD), scraped off, and collected by centrifugation. Total RNA was purified from cell cultures using a RNA STAT 60 kit (Tel-Test, "B", Friendswood, TX) according to the manufacturer’s instructions. RNA (20 µg) was resolved in a 1% denaturing agarose gel and stained with ethidium bromide for evaluation of RNA loading and integrity. The RNA was transferred onto a nylon membrane (Boehringer Mannheim) by capillary elution and immobilized by UV cross-linking. The TEF-5 cDNA-containing pDR2 plasmid was digested with BamHI and XbaI restriction enzymes, and TEF-5 cDNA was purified by GeneClean from agarose gel. TEF-5 cDNA probes were labeled, purified, and hybridized to the cellular RNA blot or a human multiple tissue Northern blot (MTN, CLONTECH Laboratories, Inc.) as described above. Blots were washed two times each sequentially with 2x SSC and 1x SSC at 42 C, a nonstringent condition which, in the case of the ß-actin probe, detects the 2.1-kb ß-actin mRNA and the muscle-specific 1.6- to 1.7-kb {alpha}-actin mRNA in muscle tissues only (31, 32). Autoradiography was performed at -70 C for 1–3 days with two intensifying screens. To assess RNA loading, the multiple tissue blot was also probed with a ß-actin cDNA probe under identical conditions.

Cell Culture, Transfection, and Luciferase Assays
BeWo cells purchased from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 (Gibco BRL). The medium was supplemented with 10% FBS (BioWhittaker, Walkersville, MD), 100 U/ml penicillin (Gibco BRL), 100 g/ml (Gibco BRL), and 2 mM L-glutamine (Gibco BRL). Cells were maintained at 37 C in an atmosphere containing 5% CO2 and 100% humidity. For transfection studies, BeWo cells grown to confluence in T175 flasks were rinsed with 10 ml PBS and harvested by treatment with 5 ml 1x trypsin (Gibco BRL). Cells were transfected by electroporation at 960 µFarads and 250 V using a GenePulser (Bio-Rad Laboratories, Inc.). After 18–20 h of incubation, cells were harvested for luciferase and protein assays, which have been described in detail previously (3). Transfection data expressed as relative light units/µg protein were analyzed by Student’s t test. Luciferase reporter plasmids used in transfection were constructed in pA3LUC (16) as described previously (3, 8, 9). All plasmid DNAs were grown in Escherichia coli HB101 and purified by double CsCl2 gradient ultracentrifugation.

Csp.LUC is the control plasmid and contains 500 bp of the hCS proximal promoter and 5'-flanking DNA, but no enhancer (7); SVEn CSp.LUC and CMV CSp.LUC contain SV40 and CMV enhancers, respectively (7); CSEn2 CSp.LUC and (CSEn2)2_CSp.LUC contain one and two copies of hCS2 gene enhancers, respectively (7); (GT-IIC)12 CSp.LUC contains 12 tandemly repeated GT-IIC enhansons (7); CSEn1 CSEn2 CSp.LUC and CSEn5 CSEn2 CSp.LUC are plasmids containing consecutive homologous enhancers associated with the hCS1, hCS2, and hCS5 genes, either CSEn1 and CSEn2 or CSEn5 and CSEn2, respectively (7). The (GT-IIC)5 CSp.LUC and (OCT)5 CSp.LUC genes were constructed using a ligation-coupled PCR protocol with the oligonucleotides GT5 and OCT (Table 1Go) that places each of the tandemly arrayed sequences in the same orientation as described (17). The amplified inserts were subsequently cloned into the BglII site of the hCSp.LUC, upstream of the polyadenylation repeats that prevent upstream transcription initiation (16).

In Vitro Translation
TEF-1 expression plasmid pXJ-40-TEF1A was kindly provided by Drs. Pierre Chambon and Irwine Davidson (Institut de Chemie Biologique, Strasbourg CEDEX, France). The 3.3-kb TEF-5 BamHI/XbaI fragment was resolved by agarose gel electrophoresis and purified by GeneClean treatment. The fragment was ligated into BamHI/SpeI- digested pBluescript KS (Stratagene) to yield pKS TEF-5. The construct was confirmed by DNA sequencing and used for in vitro expression of TEF-5.

Standard in vitro transcription-translation reactions were performed with TNT-coupled rabbit reticulocyte lysate (Promega Corp., Madison, WI). Reactions were carried out at 30 C for 2 h with 25 µl TNT, 2 µl reaction buffer, 1 µl T7 polymerase (for TEF-1) or T3 (for TEF-5) RNA polymerase, 1.5 µl ribonuclease (RNase) inhibitor (RNasin, 40 U/µl, Boehringer Mannheim), 1 µl amino acid mixture lacking methionine, 1 µg of pXJ-40-TEF1A, or pKS TEF-5 template, and 4.0 µl [35S]methionine (1000 Ci/mmol, Amersham) in a total volume of 50 µl. To characterize the reaction products, 5 µl from each reaction were mixed with the same volume of 2x SDS loading buffer, heated to 95 C for 2 min, and resolved on a 10% SDS polyacrylamide gel. Prestained protein standards (10 µl, Bio-Rad Laboratories, Inc.) were loaded alongside to estimate molecular size. After electrophoresis, the gels were dried and autoradiography performed at -80 C for 18 h. For gel shift experiments, in vitro-generated TEF-1 and TEF-5 were prepared as described above except that labeled methionine was omitted and the amino acid mixture was supplemented with unlabeled methionine.

Gel Shift Assays
Double-stranded oligonucleotides containing wild-type or mutated GT-IIC sequences were labeled as described previously (8, 9). In vitro-generated TEF-1 and TEF-5 (5 µl) were mixed with 28 µl binding buffer containing 15 µl 20 mM HEPES (pH 7.9), 100 mM KCl, 20% glycerol, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM dithiothreitol, 10 mM MgCl2, and 100 µg/ml poly dI·dC. Binding reactions were initiated by addition of 30,000 cpm DNA probes in 2 µl 10 mM Tris·HCl (pH 7.5), 1 mM EDTA. After incubation on ice for 30 min, the samples were loaded immediately onto a 4.0% polyacrylamide gel that had been prerun for 1 h. Electrophoresis was performed in 0.85x TBE buffer at room temperature for 2 h at 220 V (constant voltage), with cold water circulation. After electrophoresis, gels were dried in vacuo and exposed to Kodak x-ray film (Eastman Kodak Co.) at -80 C with intensifying screens for 16 h. To characterize their heat stability, in vitro-generated TEF-1 and TEF-5 were heated in a PCR cycler at various temperatures for 2 min. Treated samples were cleared by centrifugation at 13,000 rpm x 5 min, and supernatants were used in gel shift experiments.


    ACKNOWLEDGMENTS
 
The authors wish to express their appreciation to Ruth Kiefer for secretarial and editorial assistance.


    FOOTNOTES
 
Address requests for reprints to: Norman L. Eberhardt, Ph.D., 4–407 Alfred, Mayo Clinic, Rochester, Minnesota 55905. E-mail: eberhardt{at}mayo.edu

This work was supported by NIH Grants DK-41206 and DK-51492 (N.L.E.).

Received for publication June 11, 1998. Revision received February 9, 1999. Accepted for publication March 16, 1999.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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