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Endocrine Research Unit (S.-W.J., M.A.T., N.L.E.) Departments of Medicine and Biochemistry/Molecular Biology (N.L.E.) Mayo Clinic Rochester, Minnesota 55905
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) arose by relatively
recent duplication of the hGH gene (1). These homologous,
nearly identical genes are located within a short 50-kbp span of DNA on
chromosome 17q22-q24 and yet are expressed in a strict cell-specific
pattern whereby the hCS-1 and hCS-2 genes are
expressed exclusively in placental syncytiotrophoblasts (1, 2). A
chorionic somatomammotropin enhancer (CSEn) found 2 kb downstream of
the hCS-2 gene (Fig. 1A) participates in the cell-specific
control of hCS gene expression (3, 4, 5, 6). A minimal enhancer
(Fig. 2B) is contained within a 240-bp element that
stimulates hCS promoter activity in human placental BeWo and
JEG-3 and monkey kidney COS-1 cells, but not in HeLa or pituitary GC
cells (6, 7). The choriocarcinoma cell lines BeWo and JEG-3 express low
levels of hCS-1, hCS-2, and hGH-2 mRNAs, but not hGH-1 mRNA (8, 9) and
have served as the dominant model for studying cell-specific expression
of the hCS genes (3, 4, 5, 6, 7, 8, 9). The minimal enhancer has been shown
to contain several enhansons (individual DNA response elements
comprising a modular enhancer) that are homologous to the GT-IIC and
SphI/SphII enhansons in the SV40 enhancer, which are binding sites for
transcription enhancer factor-1 (TEF-1) (3, 5, 6, 7, 10). We and others
have demonstrated that the 53-kDa TEF-1 and a 30-kDa factor, chorionic
somatomammotropin enhancer factor 1 (CSEF-1) that is present in
placental and COS-1 cells, binds to the major GT-IIC-like enhansons in
CSEn (5, 7, 10). Positive enhancer activity is correlated with the
binding of CSEF-1 (7), whereas the binding of TEF-1 appears to be
associated with inhibition of enhancer activity as well as basal
promoter activity (11). The inhibition that occurs through TEF-1 is
correlated with its ability to interact with the TATA-binding protein,
TBP, and the resultant inability of the TEF-1-TBP complex to bind to
the TATA element (11).
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) that is protected from deoxyribonuclease I (DNaseI) digestion
in the presence of BeWo cell nuclear proteins (3, 5, 6). However,
several lines of evidence indicate that additional sequences are also
involved in the regulation of CSEn activity. First, footprinting
analyses with nuclear extracts from placental cells detected four
additional DNaseI-protected regions, FP-1, FP-2, FP-4, and FP-5 (Fig. 1B), within the 240-bp fragment (5, 6). Second, mutations of sequences
within these footprinted regions resulted in significant reduction of
CSEn activity (EM1-EM8, Fig. 1B), indicating that these sites are
functional (5, 6). Finally, although a single copy of a CSEn enhancer
harboring a mutation in the central Enh4 enhanson (EM5, Fig. 1B) is
virtually devoid of activity, a construct containing two copies of the
enhancer carrying the Enh4 mutation restored enhancer activity in
placental cells (S.-W. Jiang and N. L. Eberhardt, unpublished results).
These findings support the concept that functional DNA elements other
than Enh4 are required for CSEn activity. In the current studies we have performed further analysis of the additional GT-IIC- and SphI/II-like enhansons (Enh1-Enh5) within CSEn. Utilizing gel shift assays, gel supershift experiments with TEF-1 antibodies, and UV cross-linking techniques, we demonstrate that TEF-1 and CSEF-1, generated in vitro or from nuclear cell extracts, specifically bind to each of these enhansons. Interestingly, competition experiments revealed differences of as much as 85-fold in the relative binding affinities of the different enhansons. Nevertheless, each of the individual concatenated enhansons was capable of stimulating hCS promoter activity in an orientation-independent manner in transfected placental choriocarcinoma cells. These results suggest that full CSEn function depends on the cooperative binding of TEF-1 and CSEF-1 to multiple low-affinity binding sites.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) (6), are protected by nuclear extracts from GC, HeLa,
and placental BeWo cells. Mutations of individual GT-IIC- and
SphI/SphII-like sequences in an otherwise intact enhancer within FP-2
(EM3 and EM4, Fig. 1B) and FP-3 (EM5, Fig. 1B) reduced enhancer
activity dramatically (70–100%), whereas mutations within FP-1 (EM1),
FP-4 (EM6), and FP-5 (EM7 and EM8) resulted in less dramatic, but
significant (ca. 30–50%) reductions in CSEn function (6).
Thus, factors binding to FP-1, FP-4, and FP-5 may contribute to the
overall enhancer activity. With the exception of sequences within FP-5,
the other four regions share extensive similarities with GT-IIC and
SphI/II enhansons (Fig. 1C), suggesting that the binding of a common
factor to these regions might account for a majority of CSEn functional
activity. We therefore sought to determine whether these sequences are
recognized by TEF-1 and/or CSEF-1. To test this possibility, we
performed gel shift assays using the 5'-end labeled oligonucleotides
(Table 1) with in vitro-generated TEF-1 and
BeWo nuclear cell extracts containing both TEF-1 and CSEF-1.
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. All of the double-stranded Enh
oligonucleotides showed a pattern of migration similar to that of the
SV40 GT-IIC (GT-IICSV) oligonucleotide, whereas no retarded
band is observed in unprogrammed extracts (TNT, Fig. 2). Thus, the
shift in mobility appears to be specifically produced by TEF-1.
Although the same amount of in vitro-generated TEF-1 and
radioactivity were applied in each case, the intensity of the TEF-1-DNA
complexes was quite different. Since the specific activity of all the
probes was similar, we infer that the differences in intensity indicate
differences in the relative binding affinities of TEF-1 for each of the
enhansons, Enh1-Enh5. The relative binding affinity appears to be
GT-IICSV 
Enh4 
Enh5 Enh1
Enh3 Enh2.
We previously showed that nuclear extracts from BeWo cells contain an
additional protein that recognizes the GT-IIC- and SphI/SphII-like
motifs (7). This protein was designated CSEF-1 and was shown to have a
molecular mass of approximately 30 kDa, which produces a much more
rapidly migrating complex with the GT-IIC oligonucleotide in gel shift
analyses. As shown in Fig. 3, this more rapidly
migrating complex was observed in gel shift analyses with all of the
Enh enhansons in the presence of BeWo nuclear cell proteins. Moreover,
the general pattern of intensities with the various Enh1-Enh5
oligonucleotides was similar to that observed with the TEF-1 and CSEF-1
complexes in BeWo nuclear extracts (Fig. 3) as with in
vitro-generated TEF-1 (Fig. 2).
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). In addition, the
relative amount of supershifted TEF-1-DNA complex, as judged by the
relative intensity of the band, was in the same relative order as
observed in simple gel shift experiments (Enh4 Enh5
Enh1 Enh3 Enh2). Taken together, these results
indicate that the more highly retarded band corresponds to TEF-I.
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). A factor
with the same molecular mass was cross-linked to each of the Enh1-Enh5
oligonucleotides (Fig. 5), and the relative binding affinity, as judged
by the intensity of the band, was similar to that observed in the
previous experiments (
Figs. 2–4). These data are consistent with the
concept that CSEF-1 binds to each of the Enh1-Enh5 enhansons with an
affinity similar to that of TEF-1 binding.
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). The relative affinities of each
enhanson was estimated from the data in Fig. 6 at the point at which
50% of the binding was inhibited, and the relative binding affinities
are shown in Table 2. TEF-1 has an almost 2-fold higher
binding affinity for each of the enhansons than CSEF-1, and the order
of binding affinities is identical to that observed in the earlier
experiments (
Figs. 2–5).
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). Almost all of these constructs exhibited significant
stimulation of hCS promoter activity (1.5- to 4-fold). To
evaluate the relative activities of each of the enhansons, the
activities of constructs containing five copies of the individual
enhansons in both orientations were averaged and plotted against the
relative binding affinity as shown in Fig. 8. There was
a linear relationship between the relative activity and the relative
binding affinity for TEF-1 and CSEF-1. These results, along with our
previous mutational analyses (6), strongly support the concept that, in
addition to Enh4, Enh1, Enh3, Enh5, and possibly Enh2 contribute to
CSEn activity by acting as relatively weak binding sites for TEF-1 and
CSEF-1. The data suggest that the cooperative interaction of multiple
TEF-1 and/or CSEF-1 molecules accounts for CSEn activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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DNaseI footprinting studies of the 240-bp minimal enhancer with nuclear
extracts from a variety of cells has revealed five protected regions
(5, 6). Four of these DNaseI-protected regions contain GT-IIC- and
SphI/SphII-like sequences (Enh1-Enh5, Fig. 1A and B). Enh2 does not
reside within a previously recognized DNaseI-protected region.
Mutagenesis of sequences within each of these DNaseI-protected regions
diminished CSEn activity (6), indicating that CSEn activity is governed
by the interactions of multiple elements. However, the identity of
factors binding to these elements has not been established, nor has the
function of isolated GT-IIC- or SphI/SphII-related enhansons been
tested.
In the present study, TEF-1 and CSEF-1 binding and functional
activities were examined for each of the GT-IIC- and SphI/SphII-like
regions. Each of the enhansons were shown to bind to TEF-1 and CSEF-1
with comparable relative binding affinities that varied considerably
(2 orders of magnitude) (
Figs. 2–6 and Table 2). When the
individual enhansons were concatenated (3- to 9-mers), virtually all of
the enhansons stimulated hCS promoter activity in an
orientation-independent manner when cloned upstream of the
hCSp.LUC gene and transfected into BeWo cells (Fig. 7),
indicating that each of these structures can contribute to CSEn
function. Moreover, there was a linear relationship between the
relative binding affinity of each enhanson for TEF-1 and CSEF-1 and the
relative enhancer activity (Fig. 8). This is consistent with the
concept that CSEn function is governed by the binding of multiple
copies of TEF-1 and CSEF-1 to the enhansons, Enh1-Enh5. Although Enh2
displays the weakest binding and functional activity and exists within
a CSEn domain that is not protected by DNaseI footprinting (6), it may
contribute, nevertheless, toward mediating enhancer function. The fact
that it resides within a region not protected by DNaseI may reflect a
lower bound of affinity for which DNaseI footprints may be observed.
Interestingly, mutation of either Enh3 or Enh4 results in 70–100%
loss of enhancer function in the context of the intact enhancer (6),
although there is a 20-fold difference in the TEF-1 or CSEF-1
binding affinity for the individual enhansons (Fig. 6), and Enh4 is a
more potent enhancer when multimerized (Fig. 7). This result may
reflect the importance of the spatial organization of the modular
enhancer and illustrates that a relatively weaker enhanson may possess
inordinate functional significance in the context of the intact
enhancer, which is likely the result of cooperative interactions. In
this regard it is of interest that the Enh3 and Enh4 enhansons are
centrally located, suggesting that they may comprise part of a core
enhancer structure. Taken together these data support the concept that
the cooperative interaction of TEF-1 and CSEF-1 to these multiple,
low-affinity binding sites plays an essential role in mediating CSEn
functional activity; however, they do not exclude the possibility that
other factors may be involved in enhancer function. For example,
recent evidence indicates that M-CAT (GT-IIC-like) elements
involved in both muscle-specific and non-muscle-specific
transcription may be modulated by additional factors that bind to
flanking sequences adjacent to the M-CAT motifs (12).
Although many enhancers function through the interactions of multiple,
unique proteins, several enhancers and regulatory elements appear to
operate by the binding of single transcription factors to multiple,
low-affinity binding sites. For example, ovalbumin gene regulation by
estrogen occurs by estrogen receptor binding to several
half-palindromic TGACC motifs instead of the typical palindromic
estrogen response element (ERE) (13). Cooperative binding of the
estrogen receptor to these weak, relatively widely spaced half-sites
provides for synergistic activation of the ovalbumin gene by estrogen.
In a very similar manner, estrogen regulation of the progesterone
receptor gene is governed by weak, nearly palindromic EREs that are
coupled to estrogen receptor half-sites (14). Regulation of the
immunoglobulin heavy chain µ-enhancer is modulated by the binding of
the enhancer-binding regulatory protein, NF-µNR, to four adjacent
binding sites that flank the enhancer core. Like the GT-IIC- and
SphI/II-like elements of CSEn, the individual NF-µNR binding sites
display a range of binding affinities spanning 2 orders of magnitude
(15). Nevertheless, when low- and high-affinity NF-µNR binding sites
are present on the same molecule, both sites are occupied at
concentrations of NF-µNR that would only be expected to occupy the
high-affinity site by itself. Accordingly, the juxtaposition of low-
and high-affinity binding sites within the µ-enhancer results in
cooperative binding of NF-µNR to the enhancer (15). Finally, the SV40
enhancer functions through the binding of TEF-1 to multiple GT-IIC and
SphI/SphII enhansons (16, 17, 18) that differ by 4- to 10-fold in their
ability to bind TEF-1 (GT-IIC SphI SphII) (16). These
differences in binding affinity closely mirror those observed with the
GT-IIC- and SphI/SphII-like CSEn enhansons (
Figs. 2–6 and Table 2).
Because mutation of the low affinity TEF-1/CSEF-1 binding sites results
in an enhancer with lower functional activity (6), these sites are
important for enhancer function. Consequently, it is likely that, as is
the case with the SV40 enhancer, cooperative binding of TEF-1/CSEF-1 to
the multiple low-affinity sites is important for full CSEn functional
activity.
TEF-1 is a member of a highly conserved family of regulatory proteins that includes the yeast factor TEC1 (19), the Aspergillus nidulans factor AbaA (20), and the Drosophila scalloped (sd) gene product (21). In addition to TEF-1, a number of distinct TEF-1 homologs from humans (22), mice (22), and chicken (23, 24) have been cloned. TEFs are involved in the regulation of a diverse set of processes, including skeletal and cardiac muscle gene expression (12, 23, 24, 25, 26), SV40 (16, 17, 18), CSEn (3, 4, 5, 6), and human papillomavirus type 16 E6 and E7 (27) enhancer control. The mouse TEF-1, TEF-3, and TEF-4 homologs display a complex pattern of expression during development that implicate these homologs in myogenesis and cardiogenesis, as well as central nervous system development and organogenesis (22). Despite the diversity of these different products, all the members of this family contain a very highly conserved TEA/ATTS DNA-binding domain that recognizes the DNA sequences related to the prototypic GT-IIC and SphI/SphII enhansons (28).
Although the exact structure of the TEA/ATTS domain is not yet known,
it has been proposed that this 80-amino acid-containing domain contains
either three 
-helices or one -helix and two ß-sheet structures
(20, 21, 28). Mutational analysis of the putative -helical and/or
-helical/ß-sheet structures demonstrates that the first
-helical and third -helical/ß-sheet are critical for DNA
binding; however, the carboxyl terminus of TEF-1 can also modulate
DNA-binding affinity (18). Nevertheless, expression of a synthetic
DNA-binding domain containing all three of the -helical and/or
-helical/ß-sheet motifs established that these structures are
sufficient to determine the binding specificity to the unrelated GT-IIC
and SphI/SphII enhansons (18). Thus DNA-binding specificity of these
family members resides within the TEA/ATTS domain. Given the striking
similarity in the relative binding affinities of CSEF-1 and TEF-1 for
the unrelated GT-IIC and SphI/SphII enhansons (
Figs. 2–6 and Table 2),
it seems likely that CSEF-1 is an as yet unidentified member of this
family. It is noteworthy that the TEA/ATTS domain is sufficient for
cooperative binding to tandemly repeated GT-IIC and Sph enhansons and
that the cooperativity is required for binding to low-affinity
enhansons (22). This lends further support to the concept that the
multiple, low-affinity GT-IIC- and SphI/SphII-like sites within CSEn
may act via cooperative binding of TEF-1 and/or CSEF-1.
It is possible that multiple low-affinity binding sites with or without interspersed high-affinity sites provide a mechanism for generating a transcriptional rheostat that senses different levels of physiological signals and generates fine-tuned control of gene expression. Such a mechanism may help to explain hCS gene expression during pregnancy, which is gradually up-regulated from nearly silent expression during early pregnancy to maximal expression in the third trimester. It should be emphasized that the mechanism by which TEF-1 and/or CSEF-1 mediate enhancer function is unknown. Overexpression of intact TEF-1 in several cell lines (11, 17) results in a dominant negative inhibition of reporter gene activity, suggesting that limiting cofactors are required for its transactivating functions (17). Using GAL4-TEF-1 chimeras, Hwang et al. (18) have been able to demonstrate that three distinct, but interdependent, domains were required for both transactivation and squelching functions, providing additional support for the limiting cofactor model. In contrast, we previously demonstrated that TEF-1-mediated transrepression may be accounted for by interactions with TBP that inhibit TBP from binding the TATA element. Interestingly, the same three TEF-1 activation/squelching domains identified earlier (18) were required for TBP binding (11). These latter results suggest an alternate model in which TEF-1 is a repressor, whose binding to the enhancer may allow it to interact with TBP and negatively regulate transcription initiation. Further evidence for such a repressor model is presented in the accompanying article in which it is shown that TEF-1 binding to multiple enhancers (CSEn2 and CSEn1 or CSEn2 and CSEn5) is associated with a composite silencer activity in pituitary GC cells (30). Accordingly, a cofactor might be required as part of a switch mechanism in cell types in which TEF-1 acts as a transactivator, but not presumably in BeWo cells in which CSEF-1 appears to mediate transactivation. Further studies will be required to elucidate these mechanisms.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Plasmid Construction
All reporter plasmids were cloned into
pA3LUC (29). Construction of
CSp.LUC, which contains the hCS 496-bp promoter,
was described previously (6). Complementary oligonucleotides harboring
CSEn sequences protected in footprinting experiments (6) were
synthesized at the Molecular Biology Core Facility, Mayo Clinic (Table 1). We devised a simple and efficient PCR-based method to subclone all
the synthetic oligonucleotides in tandem repeats with defined spacing,
orientation, and repeat number (31). Briefly, phosphorylated individual
enhanson monomers that contained GG and CC overhangs at the 3'-ends
(Table 1) were mixed with a synthetic linker containing a
BglII site and similarly affixed 3'-GG and 3'-CC overhangs.
Enhanson monomers and synthetic linker (10:1) were mixed and ligated
with T4 DNA ligase, and 200- to 300-bp DNA fragments containing
tandemly repeated monomers and linkers were selected on a 2% agarose
gel. Oligomers recovered after the gel had been subjected to several
freeze/thaw cycles were amplified by PCR using the synthetic linker
oligonucleotides as primers. Self-priming, due to the repetitive nature
of the oligomers, generated large DNA PCR products. After
BglII restriction digestion, clear bands corresponding to
DNA fragments containing different numbers of repeated monomers bounded
by the linker oligonucleotide were visible on the gel. These oligomers
were ligated to the BglII-treated
pA3LUC to generate constructs
containing Enh1-Enh5 sequences in different numbers and orientations.
Positive clones were screened by BglII digestion, and the
sequence of the insert was confirmed by dideoxy-nucleotide sequencing
(Molecular Biology Core Facility, Mayo Clinic).
Data Analysis
Data were subjected to multivariate ANOVA using post
hoc Bonferroni t tests to assess individual differences
among the multiple comparisons.
Gel Shift Assays
The pXJ40-TEF-1A plasmid (17), generously provided by
Dr. Pierre Chambon and Irwin Davidson (University of Strasbourg,
Strasbourg, France), was used to generate TEF-1 protein by in
vitro translation (TNT, Promega, Madison, WI) using pBluescript in
a mock translation reaction for negative control as described
previously (7). Large-scale nuclear extracts were isolated from
cultured BeWo cells according to the method of Dignam et al.
(32).
Gel shift probes, which contain the same sequences as the
oligonucleotides listed in Table 1, except for the absence of
protruding 5'-GG and 3'-CC ends, were 5'-end labeled with
[
-32P]ATP (Amersham Corp., Arlington Heights, IL) and
with polynucleotide kinase to a specific activity 2 x
106 cpm/pmol. The labeled probe was purified through a
Bio-Gel P-60 (Bio-Rad, Richmond, CA) column. Probe (30,000 cpm) and 2.5
µl of in vitro-generated TEF or 20 µg of BeWo nuclear
extract were used for gel shift analyses under conditions described in
detail previously (33).
The TEF-1 and CSEF-1 affinities to Enh1-Enh5 were measured by gel shift competition experiments. Increasing concentrations (0.05 to 500 nM) of unlabeled double-stranded oligonucleotides were mixed with labeled GT-IICSV probe (30,000 cpm) and BeWo cell nuclear extracts (20 µg). After autoradiography, the TEF-1-DNA and CSEF-1-DNA complexes were analyzed by densitometry (NIH Image).
Rabbit anti-chicken TEF-1 antibody was generously provided by Drs. Charles Ordahl and Iain Farrance (University of California San Francisco) and used for gel supershift experiments. Compared with normal gel shift analyses, less nuclear extract (10 µg) and more DNA probe (80,000 cpm) was used to enhance the sensitivity. After a 30-min incubation, 3 µl TEF-1 antibody were added to the binding reaction and the incubation was continued for an additional 15 min. After electrophoresis, the gel was dried and exposed to Kodak x-ray film for 2 days.
UV Cross-Linking
For cross-linking studies, 120,000 cpm of DNA probe and 80 µg
BeWo nuclear extracts were incubated and subsequently resolved by
electrophoresis. The wet nondenatured gel was placed on ice and
irradiated with UV light (Stratalinker, Stratagene, La Jolla, CA) for
1 h. Autoradiography was performed overnight at 4 C with an
intensifying screen on top of the gel. Gel slices containing the CSEF-1
complexes were excised and soaked in 200 µl 2x SDS-PAGE loading
buffer (100 mM Tris·HCl (pH 7.6), 300 mM KCl,
1 mM EDTA, 10 mM dithiothreitol) at 4 C for 30
min. The cross-linked CSEF-1-DNA complex was resolved by 10% SDS-PAGE.
The gel was dried and exposed to Kodak x-ray film with intensifying
screens at -20 C for 2 days.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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 22, 1997.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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