The Human Chorionic Somatomammotropin Enhancers Form a Composite Silencer in Pituitary Cells in Vitro

Shi-Wen Jiang and Norman L. Eberhardt

Endocrine Research Unit Departments of Medicine and Biochemistry/Molecular Biology Mayo Clinic, Rochester, Minnesota 55905


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human GH (GH) gene family includes the pituitary-specific hGH-1, placental-specific chorionic somatomammotropin (hCS-5, hCS-2, and hCS-1), and hGH-2 genes. These duplicated, nearly identical genes are localized on approximately 50 kb of DNA on chromosome 17q23-q24. An enhancer (CSEn2), located downstream of the hCS-2 gene, participates in mediating placental-specific hCS gene expression. In the preceding paper we demonstrated that CSEn2 activity derives from the cooperative binding of transcription factor-1, TEF-1, and a placental-specific factor CSEF-1 to multiple enhansons, Enh1-Enh5, that are related to the SV40 GT-IIC and SphI/SphII enhansons. Here we demonstrate that two copies of CSEn2 or a single copy of CSEn2 linked to either of the other two enhancers in the hGH/hCS locus, CSEn1 and CSEn5, act cooperatively to enhance hCS promoter activity in choriocarcinoma (BeWo) cells, but silence the promoter in pituitary GC cells. Mutation of Enh4, an essential GT-IIC-like enhanson in the context of the intact enhancer, abolishes silencer activity, and multimerized GT-IIC enhansons mimic the intact CSEn enhancer/silencer activities in BeWo and GC cells, respectively. By antibody-mediated supershift, Western, and far Western analyses, we identified TEF-1 as the GT-IIC-binding factor in pituitary cells. The data suggest that TEF-1 may be involved in pituitary-specific repression of placental GH/CS gene transcription through long-range interactions between the multiple CS enhancers present on the GH/CS gene locus.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human chorionic somatomammotropin (hCS) is a member of the GH gene family (1, 2). The gene locus contains five genes, including the pituitary hGH-1 and the placental hGH variant, hGH-2, and three hCS genes, hCS-5, hCS-1, and hCS-2 (see Fig. 1AGo). Although the multiple hCS and hGH genes share approximately 93–96% sequence identity, their differential cell-specific expression indicates that these genes are regulated by unique mechanisms. The mechanism for regulating pituitary-specific expression of the hGH gene by the POU-homeodomain containing transcription factor GHF1/Pit1 has been well established (3). A central enigma associated with this expression pattern is the fact that the GHF1/Pit1-binding sites in the hCS promoters are conserved. Morever, these promoters are as active as the hGH promoter in pituitary GC cells and function via a GHF1/Pit1-dependent mechanism (4), indicating that unique repressor mechanisms must operate to suppress hCS gene expression in the pituitary. Evidence for such repressor mechanisms has been provided by Nachtigal et al. (5), who showed that unique elements associated with hCS genes can repress their expression. Recent studies of the control of hCS placental-specific expression have focused on the characterization of an enhancer, CSEn2 (6, 7, 8, 9, 10), which is localized downstream of the hCS-2 gene at the 3'-end of the hGH/hCS gene locus (11). In the preceding paper, we demonstrated that CSEn2 is composed of multiple enhansons, Enh1-Enh5, which are homologous to the GT-IIC, and SphI enhansons that comprise the Simian Virus 40 enhancer (SVEn)(12). The studies also demonstrated that CSEn activity is dependent on the cooperative binding of transcription factor-1 (TEF-1) and a placental-specific factor, CSEF-1, to Enh1-Enh5 (12). In the current paper we demonstrate that CSEn2 in conjunction with either of two other homologous enhancers present on the hGH/hCS locus, CSEn1 or CSEn5, form a composite silencer in pituitary cells (see Fig. 1Go).



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Figure 1. Schematic Diagram of the hGH/hCS Chromosomal Locus (A) and Nucleotide Sequence Comparison of the Three CS Enhancers, CSEn1, CSEn2, and CSEn5 (B)

The localization of the individual hGH and hCS genes and the three copies of the CS enhancer are depicted. The enhancer sequence comparison (GCG PILEUP, Genetics Computer Group, Madison, WI) also shows the locations of the DNaseI-resistant footprints (FP1-FP5, open boxes), the location and orientation (arrows) of the GT-IIC-related enhansons (Enh1 and Enh4), SphI/SphII-related enhansons (Enh2, Enh3 and Enh5), and direct repeat structures (DR) as defined in previous studies (6 12 ), and the structures and location of the block mutants (EM1-EM8) that were each constructed in the context of an otherwise intact enhancer (6 ).

 
CSEn2 function was originally thought to be mediated by transcription enhancing factor-1 (TEF-1), a transactivator involved in SVEn function. Transactivation by TEF-1 was proposed to require a limiting coactivator because its overexpression led to squelching in a number of systems (7, 13, 14, 15). Nevertheless, in BeWo cells strong evidence that TEF-1 was not responsible for CSEn transactivation was obtained (7). First, transactivation was correlated with the presence of a 30-kDa protein, designated CSEF-1, that binds to the GT-IIC enhanson and competes for TEF-1 binding to that DNA element. Second, CSEn was found to be very active in COS-1 cells, which express high levels of CSEF-1 and very low levels of TEF-1. Third, down-regulation of TEF-1 levels by antisense oligonucleotides led to an increase of CSEn activity in BeWo cells. Finally, TEF-1 inhibited hCS promoter activity in BeWo cells independently of the GT-IIC binding site, suggesting that it interacted with components of the basal transcription apparatus. Subsequently, we demonstrated that TEF-1 specifically interacts with the TATA box binding protein, TBP, and that it inhibits TBP binding to the TATA box (8). Overexpression of TBP in BeWo cells alleviated the squelching effect of TEF-1, suggesting that in BeWo cells TEF-1 function is limited to transrepression (8).

In the current study we provide additional evidence that TEF-1 plays a major role in transrepression and that it may function as part of a molecular enhancer/silencer switch. We discovered that multiple copies of CSEn2 acted cooperatively in BeWo cells to synergistically activate the hCS promoter, but repressed the activity of the same expression construct in pituitary GC cells. The DNA element mediating GC cell-specific repression was localized to the GT-IIC motif by mutation analysis and confirmed by demonstrating enhancer/silencer function of constructs containing multiple GT-IIC motifs. A single protein-GT-IIC complex was detected in GC cells, and TEF-1 was identified as the GT-IIC binding factor by gel supershift and Western blot analyses using TEF-1 antibodies. The CSEn-mediated transrepression did not depend on specific hCS promoter elements and may be mediated by direct interaction with basal transcription factors, consistent with the TEF-1-TBP interaction identified in BeWo cells (8). Interestingly, combinations of CSEn2 and the additional copies of the CSEn1 or CSEn5, which are associated with the placental GH/CS gene locus, acted cooperatively to repress hCS promoter activity in GC cells, suggesting that silencing might be an in vivo function of the multiple enhancers on the GH/CS locus. Accordingly, CSEn pituitary-specific silencing functions may participate with the pituitary-specific repressor elements (PSF) associated with the placental hGH/hCS members (5) to ensure absence of hCS promoter activity in the pituitary.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Multiple CSEn and SVEn Repress hCS Promoter Activity in Pituitary Cells
In previous studies we and others established that a single copy of CSEn was able to stimulate the hCS promoter in BeWo cells, but lacked any activity in pituitary GC cells or HeLa cells (6, 9). Curiously, in footprinting studies, the identical protection patterns were observed in both BeWo, HeLa, and GC cells (see Fig. 1BGo for footprint locations and associated enhansons). Mutation of these protected regions resulted in loss of enhancer activities in BeWo cells (6), and we demonstrated that these regions contain multiple enhansons that cooperatively bind TEF-1 and CSEF-1 (12). The presence of identical footprinting patterns over functional enhancer regions raised the question of why CSEn lacked activity in HeLa and GC cells. We had previously shown that multiple copies of CSEn2 acted synergistically to stimulate hCS promoter activity in BeWo cells (6) and COS-1 cells (7). We therefore decided to reassess the activities of promoter constructs containing one and two copies of CSEn2 in COS-1, HeLa, and GC cells (Fig. 2Go).



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Figure 2. Multiple Copies of the CSEn2 and SV40 Enhancers Exhibit Selective Repressor Activity in Rat Pituitary GC Cells

GC (A), BeWo (B), COS-1 (C), and HeLa (D) cells were transfected with 15 µg of the designated plasmids, CSp.LUC, CSEn2 CSp.LUC, (CSEn2)2_CSp.LUC, SVEn CSp.LUC, (SVEn)2 CSp.LUC, CMVEn CSp.LUC, and (CMVEn)2 CSp.LUC as described in Materials and Methods. Luciferase reporter activity was normalized to protein concentration and expressed as light units/µg protein ± SE. ANOVA analyses for each of the experiments A-D were significant (P < 0.0001). Individual comparisons were made by posthoc Dunnett’s t tests (P < 0.05) for values less than the mean (§) or greater than the mean (¶) of the control, CSp.LUC.

 
Confirming previous results (6, 7), CSEn2 produced strong synergistic stimulation in BeWo and COS-1 cells. Remarkably, when the same set of plasmids were introduced into pituitary GC cells, the constructs containing two copies of CSEn2 were repressed significantly (Fig. 2AGo). To exclude any possibility that this represents a nonspecific phenomenon resulting from insertional alteration of the plasmid structure, we subcloned two copies of the cytomegalovirus (CMV) enhancer and SV40 enhancer at the same positions as the CSEn-containing constructs. Unlike CSEn, one or two copies of CMVEn produced strong and cooperative stimulation of hCS promoter in GC cells (Fig. 2AGo), indicating that some sequences specific to CSEn were involved in repression. Interestingly, like CSEn, two copies of the SV40 enhancer significantly repressed hCS promoter activity in GC cells (Fig. 2AGo). These data indicate that CSEn and SVEn are functionally related and suggest that some common feature accounts for this pituitary cell-specific negative regulation.

CSEn Repressor Action Is Mediated through GT-IIC Enhansons
As the first step to understand the mechanism of the negative regulation, we determined which CSEn DNA motifs were required for this silencing function. Site-specific CSEn mutants of the individual enhansons in the context of the intact enhancer (EM1-EM8, see Fig. 1BGo) were subcloned at BglII and PstI sites of hCSp.LUC to generate plasmids containing two copies of the individual mutants. These constructs were then compared with wild type CSEn in transfected BeWo and GC cells (Fig. 3Go). Consistent with our previous results obtained with a single copy of CSEn mutants (6), mutations EM3, EM4, EM5, EM6, and EM7 cause significant reduction in enhancer activity in BeWo cells. However, the previously observed loss of enhancer activity with single copies of EM1 and EM8 are largely restored after duplication, a phenomenon observed previously with SV40 enhancers (16, 17). In GC cells, while duplicate copies of the wild type CSEn mediate 3- to 4-fold repression, significant derepression is observed only with the duplicate EM5 mutant (Fig. 3AGo). In contrast, mutations of several of the enhansons significantly affect enhancer function in placental cells (Fig. 3BGo). These results indicate that Enh4, the GT-IIC-related enhanson (Fig. 1BGo), is the only essential element for silencer function in pituitary cells. The data suggest that GT-IIC binding factors present in GC cells are crucial for silencer function.



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Figure 3. The GT-IIC Enhanson of CSEn2 Is Essential for Pituitary Silencer and Placental Enhancer Activity

GC (A) and BeWo (B) cells were transfected with 15 µg of the designated plasmids, CSp.LUC, (EM1)2 CSp.LUC through (EM8)2 CSp.LUC, which contain block mutations in individual CSEn enhansons (6 ) as described in Materials and Methods. The differences in luciferase reporter activities (light units/µg protein ± SE) were significant by ANOVA analyses (P < 0.0001 for data in A and B). Individual comparisons were made by posthoc Dunnett’s t tests (P < 0.05) for values less than the mean (§) of the control, CSp.LUC (A), or (CSEn2)2_CSp.LUC (B).

 
Since EM5 specifically disrupts Enh4, the highest affinity binding site for TEF-1 (12), we tested whether multiple copies of this GT-IIC-like enhanson exhibited independent repressor activity. The artificial enhancers containing GT-IIC 12-mers and 16-mers stimulate hCS promoter activity in BeWo cells (Fig. 4BGo). The GT-IIC 8-mer stimulated hCS promoter activity 2.7-fold in BeWo cells, a statistically insignificant effect. In GC cells, the GT-IIC 8-mer repressed hCS promoter activity as much as the 12-mers and 16-mers in GC cells (Fig. 4AGo). Thus the multimerized GT-IIC enhansons are efficient at mimicking silencer activity in GC cells, indicating that this enhanson is an essential silencer element. These experiments indicate that the isolated GT-IIC enhansons can independently mediate both enhancer and silencer activity, suggesting that GT-IIC binding factors may be responsible for the switch mechanism between placental and pituitary cells.



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Figure 4. Multiple Copies of the GT-IIC Enhansons Are Sufficient to Generate Pituitary Silencer and Placental Enhancer Activity

GC (A) and BeWo (B) cells were transfected with 15 µg of the designated plasmids, CSp.LUC, (GT-IIC)8 CSp.LUC, (GT-IIC)12 CSp.LUC, and (GT-IIC)16 CSp.LUC as described in Materials and Methods. Basal expression of the CSp.LUC gene was 5.9 ± 0.3 (SE) x 104 and 18.1 ± 3.1 (SE) x 104 light units/µg protein in GC and BeWo cells, respectively. Differences in luciferase reporter activities (light units/µg protein ± SE) were significant by ANOVA analyses (P < 0.0001 for data in A and B). Individual comparisons were made by posthoc Dunnett’s t tests (P < 0.05) for values less than the mean (§) or greater than the mean (¶) of the control, CSp.LUC.

 
Specific hCS Promoter Elements Are Not Required for Enhancer-Silencer Switching
Functional elements mediating hCS promoter activity in GC and BeWo cells have been well established in previous studies. In transfected GC cells the GHF1, Sp1, and TATA box elements are important for optimal hCS promoter activity (4, 18). Despite the conservation of the pituitary-specific GHF1 sites, the hCS genes are not expressed in the pituitary, indicating that negative regulatory mechanisms suppress their expression. Accordingly, it is possible that CSEn silencer activity in pituitary cells functions through specific CS promoter elements. Consequently, mutated hCS promoters were coupled to luciferase expression vectors containing two copies of CSEn, and their functional activities were examined in transfected BeWo and GC cells. Like the results with a single copy of CSEn in BeWo cells (18), the proximal, distal, or both GHF1 mutations had no impact on either basal (data not shown) or CSEn-stimulated hCS promoter activity (Fig. 5BGo). In contrast, mutation of the TATA and Sp1 elements significantly reduced basal (data not shown) and CSEn-stimulated activity (Fig. 5BGo). In GC cells, mutations of the proximal, distal, or both GHF1 sites caused a 2- to 3-fold reduction in promoter basal activity (data not shown), but had no effect on CSEn-mediated silencer activity (Fig. 5AGo). Mutation of the TATA and Sp1 elements also decreased the basal promoter activity 3- and 8-fold, respectively (data not shown). While CSEn repressor activity was not affected by the TATA box mutation, it was significantly increased with the Sp1 site mutation (Fig. 5AGo). These data indicate that Sp1, while not an essential element, exerts a reciprocal modulatory effect on enhancer/silencer function, serving to increase enhancer activity in placental cells and diminish silencer activity in pituitary cells.



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Figure 5. Sp1 Elements Present on the CS Promoter Contribute to CSEn2 Cooperative Interactions that Result in Synergistic Enhancer Action, but Antagonize the Silencer Activity

GC (A) and BeWo (B) cells were transfected with 15 µg of the designated plasmids, CSp.LUC, pGHF1p.LUC, dGHF1p.LUC, pdGHF1p.LUC, SP1p.LUC, and TATAp.LUC and their cognate constructs that contain two copies of CSEn2 as described in Materials and Methods. The measured light units for the least active (EN2)2_Sp1p.LUC gene in GC cells was 1929 ± 267 (SE) light units with a signal-noise ratio of 11.0 ± 1.5 (SE), a value within the linear range of the instrument as determined by limiting dilutions of partially purified luciferase (250–5 x 106 light units). Basal expression of the CSp.LUC gene was 7.6 ± 0.7 (SE) x 104 and 11.5 ± 1.5 (SE) x 104 light units/µg protein in GC and BeWo cells, respectively. The fold differences in luciferase reporter activities (light units/µg protein ± SE) for each cognate pair were calculated and the differences were significant by ANOVA analyses (P < 0.0001 and < 0.0006 for GC (A) and BeWo (B) cell data, respectively). Individual comparisons were made by posthoc Dunnett’s t tests (P < 0.05) for values less than themean (§) or greater than the mean (¶) of the control, (En2)2 CSp.LUC.

 
CSEn1 and CSEn5 Cooperate with CSEn2 to Generate Both Enhancer and Silencer Function
The human hGH/hCS locus contains two other copies of CSEn, designated CSEn1 and CSEn5, that are located downstream of the hCS-1 and hCS-5 genes, respectively (Fig. 1AGo). The CSEn1 and CSEn5 enhancers contain substitutions within or near individual enhansons that have been reported to render these enhancers nonfunctional (see Fig. 1AGo)(9). Nevertheless, since these enhancers are composed of multiple elements, it is possible that these duplicated sequences could interact with each other. To study the potential cooperativity among these native enhancers, single copies of CSEn1 and CSEn5 were inserted into expression vectors containing either one or no copy of CSEn2. In BeWo cells, CSEn2, CSEn1, and CSEn5 alone stimulate hCS promoter activity by 8-, 5-, and 3-fold, respectively. All of these effects were statistically significant compared with the activity of the hCSp.LUC gene (multivariate ANOVA P < 0.0001; posthoc Dunnett’s t test P < 0.05), suggesting they are competent enhancers, albeit less effective than CSEn2 (Fig. 6BGo, compare with CSEn2 CSp.LUC in Fig. 1Go) This is contrary to the results of Jacquemin et al. (9) that indicated that CSEn1 and CSEn5 lack enhancer activity. This discrepancy may be due to the different sensitivity of the chloramphenical acetyl transferase and luciferase reporter systems used in the two studies. Importantly, constructs containing the multiple enhancer combinations, CSEn1-CSEn2 and CSEn5-CSEn2, produced strong cooperative stimulatory activity (~ 15-fold) in BeWo cells. In fact, constructs containing CSEn1-CSEn2 or CSEn5-CSEn2 were nearly as strong as constructs containing two copies of CSEn2 (Fig. 6BGo). These results point to the possibility that the multiple CSEn enhancers present on the hGH/hCS locus might be able to interact in vivo to synergistically stimulate placental hCS gene expression.



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Figure 6. The Additional Enhancers, CSEn5 and CSEn1, Present on the hGH/hCS Genomic Locus Can Participate in Cooperative Enhancer and Silencer Function with CSEn2

GC (A) and BeWo (B) cells were transfected with 15 µg of the designated plasmids, (CSEn2)2_CSp.LUC, CSEn1 CSp.LUC, CSEn5 CSp.LUC, CSEn1-CSEn2 CSp.LUC, and CSEn5-CSEn2 CSp.LUC as described in Materials and Methods. Basal expression of the CSp.LUC gene was 4.5 ± 0.8 (SE) x 104 and 14.9 ± 2.0 (SE) x 104 light units/µg protein in GC and BeWo cells, respectively. Differences in luciferase reporter activities (light units/µg protein ± SE) were significant by ANOVA analyses (P < 0.0001 for data in A and B). Individual comparisons were made by posthoc Dunnett’s t tests (P < 0.05) for values less than the mean (§) of the control, (En2)2 CSp.LUC.

 
In GC cells, like CSEn2, neither a single copy of CSEn1 or CSEn5 exhibited significant repressor activity on its own (Fig. 6AGo, compare CSEn2 CSp.LUC activity in Fig. 1AGo; CSEn1 CSp.LUC and CSEn5 CSp.LUC activities not significantly different than hCSp.LUC, ANOVA P > 0.05). However, when combined with an additional copy of CSEn2, both CSEn1 and CSEn5 exhibited strong cooperative repressor function. Indeed, the CSEn1-CSEn2 CSp.LUC and CSEn5-CSEn2 CSp.LUC genes were about as active as the (CSEn2)2 CSp.LUC gene and were not statistically different from two copies of CSEn2 (Fig. 6AGo). These results are consistent with the observation that in CSEn1 and CSEn5 the essential GT-IIC motif required for silencer function (Fig. 3AGo) is perfectly conserved (Fig. 1BGo). These data suggest that CSEn1, CSEn2, and CSEn5 comprise a composite placental-specific enhancer and pituitary-specific silencer whose functions are mediated by cooperative interactions.

GC Cell TEF-1 Binds the CSEn GT-IIC Enhanson
As demonstrated above, the GT-IIC enhanson was essential for CSEn2-mediated silencer activity in GC cells (Fig. 3AGo), suggesting that TEF-1 binding to this enhanson might mediate repressor activity. In gel shift experiments using GC cell nuclear extracts, a single complex was detected with GT-IICSV and GT-IICCS oligonucleotides (Fig. 7Go), and this complex comigrated with complexes formed by BeWo and HeLa nuclear extracts that were previously shown to contain TEF-1 (7). Since the GT-IICS and GT-IICSV probes contain divergent flanking sequences, the factor must recognize the TGGAATG core sequences present in both of these enhansons. This view is supported by the fact that mutation of the TGTGGAATGTGT sequences blocks formation of this complex (Fig. 7Go). Also, the complex is competed away by an 80-fold excess of unlabeled GT-IICSV oligonucleotide and to a lesser extent by a 320-fold excess of an SphISV oligonucleotide, but not a 320-fold excess of the mutated GT-IICSV oligonucleotide (data not shown). Since the complex was competed by SphISV oligonucleotide, albeit with relatively lower affinity, the GC factor also recognizes SphI sequences, a property that is shared by TEF-1 (19).



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Figure 7. GC cell TEF-1 Is the Major Factor Binding to the GT-IIC Enhanson

GC cell nuclear proteins (15 µg) that interacted with the GT-IIC enhanson were analyzed by gel shift analysis and compared with the same amount of protein from BeWo, COS-1, and HeLa nuclear extracts in the gel shift assay as described in Materials and Methods. Probe GT-IICSVMUT contains mutations in the core consensus, but identical flanking sequences as the wild type GT-IICSV probe (7 ). Specific TEF-1 and CSEF-1 (COS-1 and BeWo cells) complexes are marked.

 
To establish that TEF-1 was contained in the GT-IIC complex from GC cells, the complex was further characterized using a TEF-1-specific antibody. In gel shift experiments, addition of the antibody generated a new complex with slower migration and a correspondingly decreased density of the original complex (Fig. 8AGo). No supershift band was observed in either nonimmunized serum or nonextract controls. Presence of TEF-1 in GC cells was established by Western blot analysis which showed the presence of a 53 kDa protein that reacted with the antibody (Fig. 8BGo) and by Northern blot analysis with the TEF-1 cDNA probe demonstrating the characteristic 12- and 3.5-kb TEF-1 transcripts (data not shown). Also, mild heat treatment (55 C for 2 min) of GC nuclear extracts abolished specific complex formation (data not shown), another property characteristic of TEF-1 (7). To further establish that TEF-1 was present in the complex, the protein-DNA complex was isolated from a gel shift experiment (Fig. 8CGo) and transferred onto nitrocellulose membrane, and far Western analysis using the TEF-1 antibody indicated that a band at the same position as TEF-1-GT-IIC complex was visualized in the GT-IIC probe lane, but not the GT-IIC mutant lane (Fig. 8DGo). Taken together, these results confirm that TEF-1 is the major factor present in GC cells that binds to the GT-IIC enhanson. These data provide strong support for the concept that TEF-1 may be involved in mediating the CSEn repressor function in pituitary cells.



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Figure 8. TEF-1 Is Expressed in GC Cells and Specifically Binds to the GT-IIC Enhanson

A, Gel supershift experiment with wild type GT-IICSV (W) and mutant GT-IICSVMUT (M) enhanson probes and 7.0 µg GC cell nuclear extract. Different amounts of the TEF-1 antibody were included in each binding reaction along with a nonimmune serum control (NI). TEF-1- and TEF-1-antibody-GT-IICSV complexes are marked. B, Western blot analysis. GC cell nuclear extract (70 µg) was resolved by SDS-PAGE, transferred to nitrocellulose, and detected by TEF-1 antibody as indicated in Materials and Methods. C and D, Far Western blot analysis. The protein-GT-IIC complex from a large scale gel shift experiment (C) was excised, transferred to nitrocellulose, and incubated with TEF-1 antibody as described in Materials and Methods (D). A positively reacting protein was detected by the antibody in the complex containing the wild type GT-IICSV (W), but not the mutant GT-IICSVMUT (M) probe.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In previous studies we demonstrated that the CS enhancer, CSEn2, was composed of multiple enhansons that were related to those comprising the SV40 enhancer (6, 12). The finding of SVEn GT-IIC enhanson-like sequences in CSEn2 suggested that TEF-1 might be involved in its function. We and others subsequently provided evidence that a protein distinct from TEF-1, CSEF-1, or "f" bound to the GT-IIC enhanson and was responsible for CSEn2 transactivation in BeWo (6, 9) and COS-1 cells (6). Since TEF-1 has been proposed to be a principal transactivator for the SV40 enhancer and it is ubiquitously expressed, we could not understand why CSEn2 lacked significant enhancer activity in certain cell types, including pituitary GC cells and HeLa cells. Since we had demonstrated that multiple copies of CSEn2 produced a synergistic response in BeWo (6) and COS-1 cells (7), we reasoned that reexamination of these reporter constructs with their increased sensitivity might reveal additional information about TEF-1-mediated enhancer function.

Surprisingly, we discovered that multiple copies of CSEn2 produced a significant silencing activity in GC cells (Fig. 2Go). The observed repression is not caused by insertional alteration of the reporter gene structure because subcloning of two copies of CMVEn at the same positions resulted in high level stimulation of hCS promoter activity. In addition, multiple copies of the SV40 enhancer exhibited similar repressor activity on the hCS promoter (Fig. 2Go), suggesting that the repressor activity was caused by a structural feature common to these two related enhancers. CSEn mutation analysis indicated that the GT-IIC enhansons were essential for repressor activity, a finding confirmed by demonstrating that multiple GT-IIC enhansons also exhibited silencer activity (Figs. 3Go and 4Go). A single GT-IIC complex with mobility identical to that produced by authentic TEF-1 was detected from GC cell nuclear extracts. Gel supershift, Western, and far Western blot analyses of this complex using TEF-1 antibodies confirmed that TEF-1 from GC cells specifically bound to the GT-IIC motif of CSEn (Figs. 7Go and 8Go). These results, together with our earlier observation that TEF-1 negatively regulates CSEn activity in BeWo cells (7, 8), support the concept that TEF-1 may be a dominant transrepressor in selected cell types.

TEF-1 was identified initially as a transactivator, by virtue of its binding to the essential GT-IIC and SphI enhansons for the SV40 enhancer and its ability to activate in vitro transcription (19). Nevertheless, overexpression of TEF-1 in transfected cells negatively regulated wild type SV40 and artificial GT-IIC-containing enhancers, a phenomenon attributed to squelching of a specific cofactor (13, 14, 15). We observed that overexpression of TEF-1 in BeWo cells only led to decreased CSEn2 enhancer activity while down-regulation of TEF-1 levels resulted in increased enhancer activity, suggesting that TEF-1 was a transrepressor in BeWo cells (7). Consistent with these observations, Takahashi et al. (20) reported that overexpression of TEF-1 in human SV40 transformed keratinocytes repressed involucrin gene expression. Immunohistochemistry demonstrated high levels of TEF-1 in basal cells, which do not express involucrin, whereas in the suprabasal keratinocytes, TEF-1 expression is repressed and involucrin expression is abundant.

In follow-up studies, we demonstrated that TEF-1 interacted with the TATA box binding protein, TBP, and this interaction interfered with in vitro TBP binding to the TATA element (8). Three lines of evidence supported the concept that TBP is involved in TEF-1-mediated transrepression in vivo. First, the TEF-1 proline-rich and zinc finger domains that were shown to be involved in TEF-1 squelching effects (15) were required for TBP binding. Second, overexpression of TBP alleviated the TEF-1-mediated squelching in transfected BeWo cells. Finally, the TATA and initiator (InrE) elements (18) play an essential role in mediating TEF-1 function (8). Although Sp1 is clearly capable of augmenting enhancer (18, 21) (Fig. 4Go) and antagonizing silencer function (Fig. 4Go), its absence does not abrogate enhancer function. Several factors, including p53 (22, 23), E1A (24), Dr1 (25), even-skipped (26, 27) and Tax1 (28), have been found to exert their negative function through interaction with TBP, suggesting that TBP may be a common target for negative regulation. It is not clear how the same set of factors can also serve as transactivators in certain cells. It is possible that participation of additional factor(s), including cell-specific TAFs or other coactivators, may change their mode of interaction with TBP.

Since both CSEn and SVEn contain multiple GT-IIC and SphI motifs, it is perhaps not surprising to observe a similar pattern of function of these two enhancers in different cells, including the silencer activity in GC cells (Fig. 1Go). Several previous studies have implicated the GT-IIC enhanson in negative regulation in plants, yeast, and mammalian systems, suggesting that this might represent a primordial regulatory mechanism. Evidence for an activator-repressor switch mechanism mediated by GT-IIC-related sequences emerged from studies of the small subunit of pea ribulose-1,5-bisphosphate carboxylase gene expression in transgenic tobacco plants (29). This gene contains a light-inducible transcriptional enhancer with four SV40 core enhansons that represses transcription in the dark. The SV40 core enhancer also functions as a repressor in yeast. Zhang-Keck et al. (30) demonstrated that when positioned adjacent to a yeast upstream activator sequence), the SV40 core sequence inhibited CYC1 promoter activity. Southwestern analysis revealed a protein of 68 kDa that bound to the SV40 core enhanson; however, the identity of this factor(s) or its relationship to TEF-1, if any, has not been established. Interestingly, SVEn repressor activity has been observed previously in pituitary cells. Dana and Karin (31) used SV40 promoters as controls in early studies of cAMP regulation of the hGH gene and observed that reporter constructs containing SVEn were expressed with 20–30 fold lower activity than constructs containing only the SV40 promoter. In our current study, the factor involved in GT-IIC-mediated repression was identified, and the data provide a possible physiological role for this negative regulatory pathway.

We propose that the multiple CS enhancers act collectively as a switchable enhancer/silencer in placental syncytiotrophoblast and pituitary, respectively. The molecular control of the enhancer/silencer switch is due to factors that bind to the essential GT-IIC enhanson, since it is required for both enhancer and silencer activity (Fig. 3Go). The simplest model would be that TEF-1 and CSEF-1 bind to the GT-IIC enhanson in a mutually exclusive manner and the overall CSEn activity would be determined by competition between these factors. In placental and COS-1 cells, which contain relatively low levels of TEF-1, the GT-IIC enhansons will be dominantly occupied by CSEF-1, resulting in strong CSEn stimulation. In GC cells, which contain abundant TEF-1, but no detectable CSEF-1 binding activity, CSEn is switched to a repressor. This model assumes that TEF-1 acts dominantly as a transrepressor; however, it cannot be excluded that TEF-1 functions as a transactivator via the interaction with other coactivators in certain cell types. For example, this mechanism might explain why CSEn and SVEn act positively, albeit weakly, in HeLa cells (Fig. 2Go), which appear to lack or contain very low CSEF-1-like binding activity (Fig. 7Go).

Clearly, other factors may be involved in controlling GT-IIC-containing enhancers. Recently, additional GT-IIC binding factors that are distinct from TEF-1, but share the TEA/ATTS DNA binding domain, have been identified from different tissues (32). Unlike TEF-1, which appears to be expressed ubiquitously, these factors are expressed in restricted tissues and are involved in control of a variety of genes. GT-IIC binding factors distinct from TEF-1 participate in activation of muscle-specific genes through binding to the M-CAT motif, a GT-IIC variant found in myosin heavy chain genes (33, 34, 35). Inamdar et al. (36) found that a Drosophila factor, sd, is essential for taste development. Yasunami et al. (37) cloned several embryonic factor cDNAs from neural precursor cells. Preliminary studies suggest that the expression of these genes is restricted to certain stages of brain development. Therefore, it is probable that multiple GT-IIC binding factors exist in different tissues, although their transactivation and/or transrepression functions remain to be determined.

Our results have important implications for the tissue-specific control of the linked, nearly identical hGH and hCS genes, which has long been considered enigmatic. The hCS promoter retains the two essential GHF-1 elements, required for pituitary-specific hGH expression. Indeed, these sites mediate high level hCS promoter activity in transfected GC cells (4). Therefore why aren’t the closely linked hCS genes expressed in the pituitary? Nachtigal et al. (5) have found that PSF elements associated with the placental members of hGH/hCS gene family can repress hCS promoter activity in transfected pituitary cells. Two proteins, PSF-A and PSF-B, present in pituitary but not in placental nuclear extracts, were found to bind to the PSF elements. Thus it is possible that cooperative interactions between CSEn1, CSEn2, and CSEn5 operating in conjunction with the PSF-A and PSF-B repressors could ensure the absence of hCS gene expression in the pituitary.

While previous studies have shown that CSEn1 and CSEn5 exhibit minor activity in placental cells and may not play significant roles in hCS expression, our findings suggest this may not be the case. Although single copies of CSEn1 and CSEn5 are weak enhancers in choriocarcinoma cells (Fig. 6Go), in cooperation with CSEn2, they can work almost as efficiently as CSEn2. This is especially true in terms of their repressor activity in GC cells. Neither CSEn1, CSEn2, or CSEn5 exhibit silencer activity on their own, but in combination they cooperate to repress hCS promoter activity (Fig. 6Go). Clearly, for such a mechanism to operate in vivo, long distance interactions between the CSEn5 [locus position: 16,689 bp (Genebank Accession No. J03071)], CSEn1 (locus position: 31,422 bp) and CSEn2 (locus position: 54, 237 bp) would be required. Comparing DNAse I sensitivity of the hGH-1 and hCS genes, Nickel and Cattini (38) have shown that all the genes on the hGH/hCS locus are maintained in a relatively open configuration in pituitary tissue. Thus such long range interactions among the multiple CS enhancer/silencers in pituitary are not precluded. Further studies will be required to establish the physiological significance of this mechanism.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Transfection and Luciferase Assays
BeWo cells were maintained in RPMI 1640 (GIBCO, Grand Island, NY). Rat anterior pituitary tumor (GC), COS-1 (ATCC, Rockville, MD) and HeLa (S3, ATCC) cells were maintained in DMEM (GIBCO). All media were supplemented with 10% FBS (BioWhittaker, Walkersville, MD), 100 U/ml penicillin (GIBCO), 100 µg/ml streptomycin (GIBCO), and 2 mM L-glutamine (GIBCO). Cells were grown at 37 C in an atmosphere containing 5% CO2 and 100% humidity. Cell transfections were performed by electroporation at optimal capacitance and voltage using 15 µg double CsCl gradient purified plasmid DNA and 5 x 106 cells. In all cases the data reflect a minimum of three independent transfections, and luciferase activities were normalized to cell protein content. Cell plating, harvesting, and luciferase and protein assays have been described in detail (6).

Data Analysis
Data were subjected to multivariate ANOVA with posthoc Dunnett’s one-tailed t tests for values greater than or less than the mean of the control to assess individual comparisons.

Plasmid Constructions
All reporter plasmids were constructed with the firefly luciferase expression vector pA3LUC (39). The parent plasmid 493CSp.LUC contains the hCS-1 5'-flanking region (493 bp) preceding the reporter gene. En2 CSp.LUC and (En2)2_CSp.LUC contain one and two copies of CSEn2, respectively, subcloned at the BglII (nucleotide -993 relative to the transcription initiation site) and PstI sites (nucleotide -285) and were constructed as described previously (6). CSEn1 and CSEn5 sequences were PCR amplified from bacteriophage {lambda} clones {lambda}EB-11 and {lambda}EB-4 (40) using primers CSEn p31 and CSEN p32 (Table 1Go). After BglII digestion, CSEn1 and CSEn5 were ligated to BglII-digested 493CSp.LUC to generate En1 CSp.LUC and En5 CSp.LUC. Similarly, ligation of the CSEn1 and CSEn5 BglII fragments with BglII-treated (En2)2 CSp.LUC results in the replacement of the upstream En2 to yield En1-En2 CSp.LUC and En5-En2 CSp.LUC.


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Table 1. Oligonucleotides Used in Current Studies

 
The CSEn2 mutants (EM1-EM8) were created by mutagenesis and subcloned at the BglII site as described previously (6). Except for EM1 and EM7, the mutant enhancer fragments were PCR amplified by PA3_p11 and PA3_p12 primers (Table 1Go), digested with PstI, and ligated to their corresponding parental plasmid (EM1-EM8), which were digested with PstI to give (EM2)2_CSp.LUC, (EM3)2_CSp.LUC,(EM4)2 CSp.LUC, (EM5)2_ CSp.LUC, (EM6)2 CSp.LUC, and (EM8)2 CSp.LUC. Since PstI sites had been introduced into the EM1 and EM7 mutations for the convenience of screening, the PstI site of the 493CSp.LUC vector was first converted to KpnI and the EM1 and EM7 enhancer fragments were subcloned at the KpnI site. An additional copy of EM1 and EM7 were inserted at the BglII site to obtain (EM1)2 CSp.LUC and (EM7)2 CSp.LUC.

Reporter plasmids carrying mutations at the hCS GHF-1, Sp1, and TATA elements, either containing or lacking CSEn2 at the BglII site, were described previously (18). Insertion of an additional copy of CSEn2 at the PstI site of these promoter mutants generated (En2)2 pGHF1p.LUC, (En2)2_dGHF1p.LUC (En2)2 pdGHF1p.LUC, (En2)2 SP1p.LUC, and (En2)2 TATAp.LUC.

A fragment containing the SVEn 72-bp repeats was isolated by GeneClean (BIO 101, Vista, CA) from BamHI/HindIII-digested pED4Neo (41). The CMVEn was PCR amplified from the pUHD 15–1 plasmid (42) using CMV1 and CMV2 primers (Table 1Go). After T4 DNA polymerase treatment and BglII linker addition, these fragments were subcloned into the BglII site of 493CSp.LUC to generate SVEn CSp.LUC and CMVEn CSp.LUC. Like CSEn, these viral enhancers were also inserted at the PstI site through linker addition, to yield (SVEn)2 CSp.LUC and (CMVEn)2 CSp.LUC.

Complementary oligonucleotides containing two GT-IIC motifs (OGT2–56)(13) were synthesized (Molecular Biology Core Facility, Mayo Clinic) to construct reporter plasmids containing multiple GT-IIC binding sites. After phosphorylation, annealing, and ligation of monomers, 4-mers containing eight copies of the GT-IIC motif were selected by eluting the 120-bp DNA fragment from a 2% agarose gel using GeneClean. BglII linkers were added and the DNA fragments were cloned into 493CSp.LUC to yield (GT-IIC)8 CSp.LUC. Subsequently, additional 2-mers and 4-mers containing four and eight GT-IIC motifs, respectively, were cloned into the PstI sites of these plasmids to generate (GT-IIC)12 CSp.LUC and (GT-IIC)16 CSp.LUC. Sequences of all plasmid constructions were confirmed by dideoxy DNA sequencing (Molecular Biology Core Facility, Mayo Clinic).

Gel Shift and Gel Supershift Assays
The sequences, labeling, and purification of double-stranded oligonucleotide probes, GT-IICCS, GT-IICSV, GT-IICSVMUT, were described by Jiang and Eberhardt (7). Crude nuclear extracts from BeWo, GC, HeLa, and COS-1 cells were isolated from cultured cells according to Dignam et al. (43). After DNA-protein binding using 15,000 cpm of probe and 15 µg nuclear extract, samples were resolved on 4.0% polyacrylamide gels and the DNA-protein complexes were visualized by exposure to Kodak x-ray film at -70 C for 1–2 days. To increase the sensitivity of the gel supershift experiment, 60,000 cpm probe and 7 µg GC cell nuclear extracts were used. After the initial incubation, TEF-1 antibodies were added and incubation continued for 15 min longer before electrophoresis. The rabbit anti-chicken TEF-1 antibody was generously provided by Drs. Charles Ordahl and Iain Farrance (University of California, San Francisco).

Western and Far Western Blot Analyses
For direct detection of TEF-1, 70 µg GC cell nuclear extracts were resolved by 9% SDS-PAGE and transferred onto 0.2 µm nitrocellulose membrane for antibody detection (7). To obtain sufficient DNA-protein complexes for far Western analysis, gel shift assays with GC cell extracts were scaled up 3-fold. After electrophoresis, the wet gel was exposed to Kodak x-ray film at 4 C overnight to localize the position of the DNA-protein complex. The gel band containing the complex was excised, electrotransferred onto 0.2-µm nitrocellulose membrane, and subjected to primary antibody binding (1:1500 dilution), secondary alkaline phosphatase-conjugated antibody binding, and color development as described previously (7).


    ACKNOWLEDGMENTS
 
The authors express their appreciation to Drs. Charles Ordahl and Iain K. G. Farrance for the anti-chicken TEF-1 antibodies, Dr. Randall Kaufman for the pED4neo vector, Dr. Hermann Bujard for the pUHD 15–1 plasmid, and Nicole Henry and Ruth Kiefer for secretarial and editorial help.


    FOOTNOTES
 
Address requests for reprints to: Dr. Norman L. Eberhardt, Ph.D., 4–407 Alfred, Mayo Clinic, Rochester, Minnesota 55905.

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

Received for publication November 12, 1996. Revision received March 18, 1997. Accepted for publication May 23, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Miller WL, Eberhardt NL 1983 Structure and evolution of the growth hormone gene family. Endocr Rev 4:97–130[Medline]
  2. Chen EY, Liao YC, Smith DH, Barrera-Saldana HA, Gelinas RE, Seeburg PH 1989 The human growth hormone locus: nucleotide sequence, biology, and evolution. Genomics 4:479–497[Medline]
  3. Theill LE, Karin M 1993 Transcriptional control of GH expression and anterior pituitary development. Endocr Rev 14:670–689[Medline]
  4. Nachtigal MW, Nickel BE, Klassen ME, Zhang WG, Eberhardt NL, Cattini PA 1989 Human chorionic somatomammotropin and growth hormone gene expression in rat pituitary tumour cells is dependent on proximal promoter sequences. Nucleic Acids Res 17:4327–4337[Abstract]
  5. Nachtigal MW, Nickel BE, Cattini PA 1993 Pituitary-specific repression of placental members of the human growth hormone gene family. A possible mechanism for locus regulation. J Biol Chem 268:8473–8479[Abstract/Free Full Text]
  6. Jiang SW, Eberhardt NL 1994 The human chorionic somatomammotropin gene enhancer is composed of multiple DNA elements that are homologous to several SV40 enhansons. J Biol Chem 269:10384–10392[Abstract/Free Full Text]
  7. Jiang SW, Eberhardt NL 1995 Involvement of a protein distinct from transcription enhancer factor-1 (TEF-1) in mediating human chorionic somatomammotropin gene enhancer function through the GT-IIC enhanson in choriocarcinoma and COS cells. J Biol Chem 270:13906–13915[Abstract/Free Full Text]
  8. Jiang SW, Eberhardt NL 1996 TEF-1 transrepression in BeWo cells is mediated through interactions with the TATA-binding protein, TBP. J Biol Chem 271:9510–9518[Abstract/Free Full Text]
  9. Jacquemin P, Oury C, Peers B, Morin A, Belayew A, Martial JA 1994 Characterization of a single strong tissue-specific enhancer downstream from the three human genes encoding placental lactogen. Mol Cell Biol 14:93–103[Abstract]
  10. Lytras A, Cattini PA 1994 Human chorionic somatomammotropin gene enhancer activity is dependent on the blockade of a repressor mechanism. Mol Endocrinol 8:478–489[Abstract]
  11. Walker WH, Fitzpatrick SL, Saunders GF 1990 Human placental lactogen transcriptional enhancer. Tissue specificity and binding with specific proteins. J Biol Chem 265:12940–12948[Abstract/Free Full Text]
  12. Jiang SW, Trujillo MA, Eberhardt NL 1997 Human chorionic somatomammotropin enhancer function is mediated by cooperative binding of TEF-1 and CSEF-1 to multiple, low-affinity binding sites. Mol Endocrinol 11:1223–1232[Abstract/Free Full Text]
  13. Xiao JH, Davidson I, Matthes H, Garnier JM, Chambon P 1991 Cloning, expression, and transcriptional properties of the human enhancer factor TEF-1. Cell 65:551–568[Medline]
  14. Ishiji T, Lace MJ, Parkkinen S, Anderson RD, Haugen TH, Cripe TP, Davidson I, Chambon P, Turek LP 1992 Transcriptional enhancer factor (TEF)-1 and its cell-specific co-activator activate human papillomavirus-16 E6 and E7 oncogene transcription in keratinocytes and cervical carcinoma cells. EMBO J 11:2271–2281[Abstract]
  15. Hwang JJ, Chambon P, Davidson I 1993 Characterization of the transcription activation function and the DNA binding domain of transcriptional enhancer factor-1. EMBO J 12:2337–2348[Abstract]
  16. Herr W, Clarke J 1986 The SV40 enhancer is composed of multiple functional elements that can compensate for one another. Cell 45:461–470[Medline]
  17. Herr W, Gluzman Y 1985 Duplications of a mutated simian virus 40 enhancer restore its activity. Nature 313:711–714[Medline]
  18. Jiang SW, Shepard AR, Eberhardt NL 1995 An initiator element is required for maximal human chorionic somatomammotropin gene promoter and enhancer function. J Biol Chem 270:3683–3692[Abstract/Free Full Text]
  19. Davidson I, Xiao JH, Rosales R, Staub A, Chambon P 1988 The HeLa cell protein TEF-1 binds specifically and cooperatively to two SV40 enhancer motifs of unrelated sequence. Cell 54:931–942[Medline]
  20. Takahashi H, Kobayashi H, Matsuo S, Iizuka H 1995 Repression of involucrin gene expression by transcriptional enhancer factor 1 (TEF-1). Arch Dermatol Res 287:740–746[Medline]
  21. Fitzpatrick SL, Walker WH, Saunders GF 1990 DNA sequences involved in the transcriptional activation of a human placental lactogen gene. Mol Endocrinol 4:1815–1826[Abstract]
  22. Ragimov N, Krauskopf A, Navot N, Rotter V, Oren M, Aloni Y 1993 Wild-type but not mutant p53 can repress transcription initiation in vitro by interfering with the binding of basal transcription factors to the TATA motif. Oncogene 8:1183–1193[Medline]
  23. Liu X, Miller CW, Koeffler PH, Berk AJ 1993 The p53 activation domain binds the TATA box-binding polypeptide in Holo-TFIID, and a neighboring p53 domain inhibits transcription. Mol Cell Biol 13:3291–3300[Abstract]
  24. Song CZ, Loewenstein PM, Toth K, Green M 1995 Transcription factor TFIID is a direct functional target of the adenovirus E1A transcription-repression domain. Proc Natl Acad Sci USA 92:10330–10333[Abstract]
  25. Inostroza JA, Mermelstein FH, Ha I, Lane WS, Reinberg D 1992 Dr1, a TATA-binding protein-associated phosphoprotein and inhibitor of class II gene transcription. Cell 70:477–489[Medline]
  26. Austin RJ, Biggin MD 1995 A domain of the even-skipped protein represses transcription by preventing TFIID binding to a promoter: repression by cooperative blocking. Mol Cell Biol 15:4683–4693[Abstract]
  27. Um M, Li C, Manley JL 1995 The transcriptional repressor even-skipped interacts directly with TATA-binding protein. Mol Cell Biol 15:5007–5016[Abstract]
  28. Caron C, Rousset R, Beraud C, Moncollin V, Egly JM, Jalinot P 1993 Functional and biochemical interaction of the HTLV-I Tax1 transactivator with TBP. EMBO J 12:4269–4278[Abstract]
  29. Kuhlemeier C, Fluhr R, Green PJ, Chua NH 1987 Sequences in the pea rbcS-3A gene have homology to constitutive mammalian enhancers but function as negative regulatory elements. Genes Dev 1:247–255[Abstract]
  30. Zhang-Keck ZY, Kibbe WA, Moye-Rowley WS, Parker CS 1991 The SV40 core sequence functions as a repressor element in yeast. J Biol Chem 266:21362–21367[Abstract/Free Full Text]
  31. Dana S, Karin M 1989 Induction of human growth hormone promoter activity by the adenosine 3',5'-monophosphate pathway involves a novel responsive element. Mol Endocrinol 3:815–821[Abstract]
  32. Jacquemin P, Hwang JJ, Martial JA, Dolle P, Davidson I 1996 A novel family of developmentally regulated mammalian transcription factors containing the TEA/ATTS DNA binding domain. J Biol Chem 271:21775–21785[Abstract/Free Full Text]
  33. Shimizu N, Smith G, Izumo S 1993 Both a ubiquitous factor mTEF-1 and a distinct muscle-specific factor bind to the M-CAT motif of the myosin heavy chain beta gene. Nucleic Acids Res 21:4103–4110[Abstract]
  34. Gupta MP, Gupta M, Zak R 1994 An E-box/M-CAT hybrid motif and cognate binding protein(s) regulate the basal muscle-specific and cAMP-inducible expression of the rat cardiac alpha-myosin heavy chain gene. J Biol Chem 269:29677–29687[Abstract/Free Full Text]
  35. Stewart AF, Larkin SB, Farrance IK, Mar JH, Hall DE, Ordahl CP 1994 Muscle-enriched TEF-1 isoforms bind M-CAT elements from muscle-specific promoters and differentially activate transcription. J Biol Chem 269:3147–3150[Abstract/Free Full Text]
  36. Inamdar M, Vijayraghavan K, Rodrigues V 1993 The Drosophila homolog of the human transcription factor TEF-1, scalloped, is essential for normal taste behavior. J Neurogenet 9:123–139[Medline]
  37. Yasunami M, Suzuki K, Houtani T, Sugimoto T, Ohkubo H 1995 Molecular characterization of cDNA encoding a novel protein related to transcriptional enhancer factor-1 from neural precursor cells. J Biol Chem 270:18649–18654[Abstract/Free Full Text]
  38. Nickel BE, Cattini PA 1996 Nuclease sensitivity of the human growth hormone-chorionic somatomammotropin locus in pituitary and placenta suggest different mechanisms for tissue-specific regulation. Mol Cell Endocrinol 118:155–162[CrossRef][Medline]
  39. Maxwell IH, Harrison GS, Wood WM, Maxwell F 1989 A DNA cassette containing a trimerized SV40 polyadenylation signal which efficiently blocks spurious plasmid-initiated transcription. Biotechniques 7:276–280[Medline]
  40. Hirt H, Kimelman J, Birnbaum MJ, Chen EY, Seeburg PH, Eberhardt NL 1987 The human growth hormone gene locus: structure, evolution, and allelic variations. DNA 6:59–70[Medline]
  41. Kaufman RJ, Davies MV, Pathak VK, Hershey JW 1989 The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs. Mol Cell Biol 9:946–958[Medline]
  42. Gossen M, Bujard H 1992 Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:5547–5551[Abstract]
  43. Dignam JD, Lebovitz RM, Roeder RG 1983 Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475–1489[Abstract]