(Received for publication, May 10, 1994; and in revised form, October 7, 1994)
From the
Previous studies have indicated that cell-specific expression of
the human chorionic somatomammotropin (hCS) gene may be
mediated by a placental-specific enhancer (CSEn). In the current
studies, we have analyzed the promoter elements that are required for
enhancer and promoter function in choriocarcinoma cells (BeWo).
Mutation of both hCS GHF1 sites had no effect on promoter or
enhancer activity. In contrast, mutation of the Sp1 site diminished
basal and CSEn-stimulated transcription by 75% and
56%,
respectively, indicating that Sp1 was necessary but not sufficient for
maximal basal and enhancer-mediated transcription. Deletion and
site-specific mutation of the proximal promoter region indicated that
the TATA box and an initiator site (InrE) located between nucleotides
-15/+1 of the hCS promoter were required for
maximal promoter and enhancer function. Mutations of the InrE were
associated with reduced basal and enhancer-stimulated activities and
altered transcription initiation sites. A protein of 70-kDa mass, that
was preferentially expressed in human choriocarcinoma cells (BeWo and
JEG-3), bound specifically to the InrE. The data suggest that an
initiator present in high concentrations in placental cells contributes
to the control of cell-specific hCS gene expression at the
promoter level and is required for maximal enhancer function.
Human chorionic somatomammotropin (hCS ()or placental
lactogen, hPL) belongs to the human growth hormone (hGH) gene
family which includes prolactin (hPRL) (Miller and Eberhardt, 1983).
Both hPRL and hGH are synthesized in the anterior pituitary by
lactotrophes and somatotrophes, respectively, whereas hCS is expressed
in human placental syncytiotrophoblasts. GH is an essential hormone
involved in normal growth, development, and homeostatic regulation.
Both hPRL and hCS influence mammary gland differentiation and lactation
in pregnancy (Handwerger, 1991). CS has been postulated to be a
maternal/fetal growth hormone and may increase fetal glucose
availability via its lipolytic/insulin antagonist activity (Handwerger,
1991). Lowered hCS production has been associated with abnormal
pregnancy in some but not all studies (Borody and Carlton, 1981;
Nielsen et al., 1979). Recent studies indicate that placental
lactogens from rat, mouse and human regulate their homologous
pancreatic islets directly, suggesting that these hormones are the
primary mediators of increased islet
cell function observed in
normal pregnancy and may be involved in gestational diabetes (Brelje et al., 1993). A CS receptor which binds CS, but not GH or
PRL, with high affinity has been isolated from a variety of fetal and
maternal tissues, suggesting that CS mediates direct and selective
functions, including fetal growth promotion (Freemark et al.,
1993; Freemark and Comer, 1989).
Although the exonic, intronic and
flanking regions of the hCS and hGH genes are highly
homologous (93.5% sequence identity) due to gene duplication and
gene conversion events (Hirt et al., 1987; Miller and
Eberhardt, 1983), the mechanisms controlling their cell-specific
expression are quite different. Pituitary hGH gene expression
is directed by the cell-specific transcription factor GHF1/Pit1 that
binds to the proximal hGH promoter (Bodner and Karin, 1987;
Ingraham et al., 1988). Conservation of the GHF1 bindign sites
in the hCS promoter allows efficient expression of this gene in
pituitary (GC) cells (Nachtigal et al., 1989), indicating that
complex control of these genes is required to maintain cell-specific
expression. In the case of the hCS gene, a placental-specific
enhancer, designated CSEn, located
3 kb down-stream of the hCS-2 gene appears to be responsible for its expression in
placenta (Fitzpatrick et al., 1990; Jacquemin et al.,
1994; Jiang and Eberhardt, 1994; Walker et al., 1990). CSEn is
a classical enhancer that functions in a distance- and
orientation-independent manner (Jiang and Eberhardt, 1994). Its
activity is limited to cells of placental origin (BeWo and JEG-3 cells)
with the exception of a minor activity in pituitary 18-54,SF
cells (Rogers et al., 1986). The enhancer is inactive in HeLa,
pituitary GC and HepG2 cells (Fitzpatrick et al., 1990; Jiang
and Eberhardt, 1994; Walker et al., 1990). We and others have
shown that CSEn is composed of multiple DNA elements that interact
cooperatively and are homologous to the SV40 GT-IIC and SphI/SphII enhansons (Jacquemin et al.,
1994; Jiang and Eberhardt, 1994). The latter data suggest that CSEn
function may be controlled by TEF-1 (Xiao et al., 1991),
raising the question of how cell specificity is achieved. CSEn appears
to function in a promoter-independent manner and functions equally well
with the hGH, and herpes simplex thymidine kinase promoters
and somewhat less efficiently with the Rous sarcoma virus
(RSV
) minimal promoter (Jiang and Eberhardt, 1994),
suggesting that CSEn can cooperate with different sets of promoter
elements or interacts with common basal transcription factors.
In the current studies we have examined the promoter elements present on the hCS 5`-flanking region (5`-FR) that interact with the enhancer. Previous studies indicated that Sp1 played a major role in hCS promoter basal and enhancer-stimulated activities (Fitzpatrick et al., 1990). Our studies confirmed the importance of Sp1 in mediating basal promoter activity and indicate that Sp1 is a necessary but not sufficient factor for maintaining maximal enhancer activity. Mutagenesis studies indicate that proximal elements, including the TATA box and an initiator element (InrE) located between nt -15/+1 of the hCS promoter, act cooperatively with Sp1 to achieve full basal and enhancer activity. Gel shift and UV cross-linking experiments indicate that a 70-kDa nuclear factor that is preferentially expressed in choriocarcinoma cells (BeWo and JEG-3), but is either absent or expressed at low levels in a variety of other cells, binds specifically to the InrE. Mutations on the InrE sequences not only result in major reductions of basal- and enhancer-stimulated activity, but also alter transcription start site selection. The data suggest that cell-specific expression of the hCS gene is mediated in part by an initiator that is preferentially expressed in placental cells.
The truncated promoters were generated by
digestion of EnAhCSp.LUC with SalI/PstI and SalI/MscI followed by
T4 Polymerase treatment and religation to yield EnA
282CSp.LUC and EnA
36CSp.LUC, respectively. For the EnA
19CSp.LUC construct, EnA
hCSp.LUC was digested with ApaI, treated with T4 polymerase in the presence of dNTPs, and
finally digested with BglI and BstXI. The ApaI (blunted)/BstXI fragment containing the proximal
19 bp of hCS 5`-FR and the entire LUC gene was
subcloned into SmaI/BstXI digested pA
LUC, generating 19CSp.LUC. The
CSEn fragment was inserted at the BglII site to generate EnA
19CSp.LUC. For the construction
of 493CS
APAp.LUC, 493CSp.LUC was digested by ApaI and the ends were
flushed with T4 polymerase treatment. SalI linkers were added
and the hCS 5`-FR fragment was isolated and ligated into the SalI site of EnA
pA
.LUC.
SalI/MscI digestion and religation of 493CS
APAp.LUC gave the plasmid
containing a TATA box with substitution of the sequences 3` to the TATA
box, designated 36CS
APAp.LUC.
In 493CS
TATAp.LUC, a 19-bp
region including the TATA box was removed from the hCS 5`-FR
by substituting the MscI/BstXI region of EnA
hCSp.LUC with the ApaI
(blunt)/BstXI fragment of the same plasmid.
Site-specific
mutagenesis of the sequences downstream from the TATA box were created
by inverted PCR mutagenesis utilizing the primers listed in Table 1. To improve the efficiency of PCR, a small plasmid, pEB.LUC, which contains 493 bp of the hCS 5`-FR and a
fragment of the 5`-region of the luciferase gene, was used as template.
The CSEn mutant EM1 (Jiang and Eberhardt, 1994),
which was engineered to contain a PstI site was cut with PstI and religated to remove the bulk of the enhancer, the
three polyadenylation stops in pA
LUC and
nt -493/-282 of the hCS 5`-FR. The resulting
plasmid was subjected to EcoRI/BamHI digestion,
T
Polymerase treatment and religation to yield the 3.7-kb pEB.LUC which removed the 3`-end of the luciferase gene.
Following inverted PCR mutagenesis with the oligonucleotide primers
shown in Table 1, the MscI/NarI fragments were
removed from these mutants by restriction digestion and subcloned into MscI/NarI digested EnA-hCSp.LUC generating EnA
PPM1-, EnA
PPM2-, EnA
PPM3-, and EnA
PPM4-493CSp.LUC. Plasmids
without the hCS enhancer were generated from the plasmids described
above by BglII digestion and religation. To authenticate the
transcription initiation site in primer extension assays, the plasmid hCSp.Nar/LUC, containing a 139 bp ``stuffer''
sequence in the 5` end of the luciferase gene, was constructed. The
plasmid pBluescript (Stratagene) was digested with HpaII/TaqI and the small DNA fragments (50-200
bp) were isolated and ligated to NarI digested hCSp.LUC. One construct, containing a 139-bp pBluescript
``stuffer'' was sequenced and designated hCSp.Nar/LUC.
Figure 1:
Sp1 is required for maximal CSEn
activity; however, additional promoter factors are essential for CSEn
function. A, diagram of the hCS 5`-flanking region,
highlighting the Sp1 and proximal (p) and distal (d)
GHF1 binding sites (sequences enclosed in boxes). The
mutagenesis scheme for altering the Sp1 and GHF1 sites is shown below
the sequence. Colons designate deleted nucleotides. B, the
data reflect basal- and enhancer-stimulated activity [(light
units/µg protein ± S.E.) 10
]
of wild-type hCSp.LUC and the various Sp1 and GHF1 mutants in
transiently transfected BeWo cells. Individual p values for
significance were derived as described under ``Materials and
Methods.'' ANOVA analysis for the effect of mutation was
significant at the p = 0.0001 level. C, the
data in A plotted as fold stimulation. ANOVA analysis for the
effect of mutation was significant at the p = 0.017
level.
Because the hCS Sp1 site (GGGAGG) is a variant of the canonical site (GGGCGG) and has been reported to have a lower binding affinity for Sp1 (Letovsky and Dynan, 1989), we examined whether reversion of the Sp1 site to the canonical sequence could result in a stronger promoter and increased enhancer-stimulated activity. As shown in Fig. 1, B and C, the Sp1UP mutation did increase promoter activity 1.6-fold (statistically insignificant). However, there was no corresponding effect of the Sp1UP mutation on enhancer activity (Fig. 1, B and C). These experiments suggest that the levels of Sp1 in BeWo cells may be sufficiently high to maintain a relatively high occupancy at the Sp1 site despite the anticipated lower affinity of the GGGAGG binding site (Letovsky and Dynan, 1989). These results confirm the findings of Fitzpatrick et al.(1990) which indicate that Sp1 modulates hCS basal- and enhancer-stimulated promoter activity. Nevertheless, since the enhancer activity was not eliminated upon mutation of the Sp1 site, the data suggest that other promoter factors might be involved in mediating enhancer function.
Figure 2:
The
TATA element and sequences downstream are required for maximal CSEn
function. A, diagram and sequence of the proximal hCS 5`-flanking region with mutagenesis strategy. Colons designate deleted nucleotides. B, basal- and
enhancer-stimulated activity ((light units/µg of protein ±
S.E.) 10
) of wild-type hCSp.LUC and
the various proximal promoter mutants. ANOVA analysis for the effect of
mutation was significant at the p = 0.0001 level. C, the data in B plotted as fold CSEN-stimulated
activity. ANOVA analysis for the effect of mutation was significant at
the p = 0.0001 level. D, basal- and
enhancer-stimulated activity ((light units/µg of protein ±
S.E.)
10
) of wild-type hCSp.LUC and the mutants of the InrE region. ANOVA analysis for
the effect of mutation was significant at the p =
0.0001 level. E, the data in D plotted as fold
CSEn-stimulated activity. ANOVA analysis for the effect of mutation was
significant at the p = 0.0005
level.
Removal of sequences downstream of the
TATA box (nt -19/+1, 493CSAPA
p.LUC and EnA
493CS
APA
p.LUC)
had a significant effect on the basal activity (35% reduction) but
decreased enhancer-stimulated activity by 60% (Fig. 2, B and C) in the context of a normal 493CS 5`-FR
containing the Sp1 site, suggesting that sequences around the
transcription initiation site might be required for promoter activity.
Deletion of the Sp1 site in this latter construct (36CS
APA
and EnA
36CS
APA
)
resulted in a marked reduction of basal activity (4.5% wild-type, Fig. 2B) and enhancer-stimulated activity (15.4%
wild-type, Fig. 2C). Thus in the presence of the Sp1
binding site, the sequences downstream of the TATA box appear to be
more important for mediating promoter function than the TATA box
itself. As in the case with the Sp1 mutant, mutation of the proximal
promoter elements did not result in a complete loss of enhancer
activity, suggesting that no single promoter element was essential for
enhancer function.
Figure 3:
A
nuclear protein that is preferentially expressed in choriocarcinoma
cells binds to the hCS InrE. A, gel shift analysis of
protein-DNA complexes (H1-H6) formed with a P-labeled DNA fragment (PCB/MCB) containing
nucleotides -20/+1 of the hCS 5`-flanking DNA and
nuclear extracts from BeWo, GC, HeLa, GM0637E (SV40 transformed human
fibroblasts), MG63 (osteosarcoma), COS, and JEG-3 cells. B,
effect of extended incubation (INC, 37 °C for 20 min),
freeze-thaw (FT, four cycles), and mixing 4 µg each of
BeWo and GC or HeLa cell nuclear extracts on the profiles of the
complexes H1, H2, and H4-H6.
The specificity of the protein-DNA complexes formed with BeWo cell nuclear proteins was characterized further by oligonucleotide binding and competition experiments. For these experiments three additional oligonucleotides containing mutations within a 4-5 bp region from nt -12/+1 were examined (Fig. 4A). Both PCB/MCB-MUT1 and PCB/MCB-WT oligonucleotides formed complex H1 equally well, whereas PCB/MCB-MUT2 and PCB/MCB-MUT3 failed to form complex H1 (Fig. 4A). Similarly, PCB/MCB-MUT1 and PCB/MCB-WT, but not PCB/MCB-MUT2 and PCB/MCB-MUT3, competed for H1 complex formation to PCB/MCB (Fig. 4B). Similar results were obtained with competition experiments with DNA fragments corresponding to the proximal promoter mutants (PPM1-PPM4) that were employed in the functional studies (data not shown). These experiments indicate that the binding to the proximal promoter is sequence-specific and that the recognition site is localized between nt -12/+1. Thus the sequences that are important for mediating basal and enhancer-stimulated activity co-localize with the sequences required for formation of the BeWo nuclear protein-DNA complex. Taken together, these data suggest that a factor binding at the InrE just downstream from the TATA box is required for hCS promoter function.
Figure 4:
The InrE-binding protein present in
choriocarcinoma cells recognizes specific sequences located between
nucleotides -12/-1 of the hCS promoter. A, binding of BeWo cell nuclear extract to P-labeled PCB/MCB and
P-labeled mutant
oligonucleotides, PCB/MCB-MUT1, PCB/MCB-MUT2, and PCB/MCB-MUT3. The
sequences of the oligonucleotides are shown to the right of the gel
shift experiment. B, competition analysis of the BeWo nuclear
protein-DNA complex H1 formed with the
P-labeled PCB/MCB
probe and with the wild-type and mutant oligonucleotides PCB/MCB-MUT1 (MUT1), PCB/MCB-MUT2 (MUT2), and PCB/MCB-MUT3 (MUT3). The fold excess (weight) of the competing
oligonucleotides is given (FOLD).
Figure 5:
A BeWo cellular protein of 70 kDa binds
to the hCS 5`-flanking region in the vicinity of the InrE.
BeWo and GC cell nuclear protein-DNA complexes formed with P-labeled PCB/MCB DNA were UV cross-linked as described
under ``Materials and Methods,'' and the products were
separated on SDS-polyacrylamide gels and visualized by autoradiography.
The molecular mass of the DNA fragment was
20
kDa.
Figure 6:
Sequences near the hCS gene InrE
are required for accurate transcription initiation site selection and
define a probable initiator element (InrE). A, primer
extension analysis of RNA from BeWo cells transfected with the
wild-type hCSp.LUC, pALUC (promoterless control) and hCSp.Nar/LUC genes (containing
a 139-bp insert to assess specificity of the transcription initiation
site). Cellular RNA was from mock-transfected BeWo cells. A and G represent dideoxynucleotide sequencing reactions
utilizing the same primer used for primer extension with the wild-type hCSp.LUC plasmid DNA. B, primer extension analysis of
total RNA from BeWo cells transfected with 493CSp.Nar/LUC and 36CSp.Nar/LUC. C and T represent
dideoxynucleotide sequencing reactions utilizing the same primer used
for primer extension. C, primer extension analysis of total
cellular RNA (PPM1-PPM4) from BeWo cells transfected
with the 493CS
PPM1-PPM4p.LUC genes. T and C represent dideoxynucleotide
sequencing reactions utilizing the same primer used for primer
extension. D, sequences and transcription initiation site
selection in the various hCS proximal promoter mutants derived
from the data in C.
Initiation sites from truncated and mutated promoters are shown in Fig. 6, B and C, and are summarized in Fig. 6D. Deletion of the upstream sequences, including the Sp1 site, to nt -36 (36CSp.Nar/LUC) did not alter the transcription initiation site (Fig. 6B), supporting the concept that initiation site selection is solely determined by the core promoter. The PPM1 and PPM2 mutations just upstream from the region required for InrE binding by the BeWo cell nuclear protein did not significantly affect the use of the authentic start site (Fig. 6C). However, the PPM3 and PPM4 mutations between nt -10/+5 shifted the transcriptional start sites 2 and 3 nt downstream, respectively (Fig. 6C). Thus loss of initiation site focus is correlated with the effects of these proximal promoter mutations on basal- and enhancer-stimulated promoter activity and factor binding at the InrE, providing additional evidence that the InrE-binding factor is involved in transcription initiation. These results suggest that Inr elements are required for accurate transcription initiation from the CS promoter in placental cells.
The hCS enhancer is composed of multipartite enhansons which bear striking similarities with SV40 enhansons (Jacquemin et al., 1994; Jiang and Eberhardt, 1994), particularly the GT-IIC and SphI/SphII enhansons. The unique location of CSEn at the extreme 3`-end of the hGH/hCS gene cluster, placing it in proximity to the placenta-expressed genes, and the highly restricted function of this enhancer to placental cells suggest that CSEn might play a dominant role in the control of hCS gene expression. Nevertheless, the similarity of CSEn to the SV40 enhancer, and the possibility that it might be controlled by the ubiquitous factor TEF-1 (Jacquemin et al., 1994; Jiang and Eberhardt, 1994; Walker et al., 1990) that binds the GT-IIC and SphI/SphII enhansons (Davidson et al., 1988; Xiao et al., 1991), raise questions about possible cell-specific control mechanisms. For example, if CSEn is controlled by TEF-1, why is it not functional in cell types which are known to have abundant TEF-1 activity? Previous data had also shown that the ubiquitous Sp1 played an important role in mediating enhancer function (Fitzpatrick et al., 1990), providing additional questions about how this enhancer could be controlled in a cell-specific manner. Consequently, we decided to study the interaction of the enhancer with the hCS promoter to ascertain if clues about the cell-specific mechanism could be obtained. We reasoned that the enhancer stimulatory effect results from the synergistic action of enhancer and promoter elements and that cell-specific factors might be dependent on such interactions. In the current studies, we provide evidence that in addition to Sp1, the TATA box and an initiator element (InrE) located near the transcription initiation site are required for optimal enhancer activity. The InrE binding factor may be a cell-specific factor since it is preferentially expressed in choriocarcinoma cells (BeWo and JEG-3 cells), but is absent or present in very low concentrations in a variety of other cell types (Fig. 3).
Previous studies had indicated that Sp1 appeared to play a dominant
role in CSEn function in Jar and JEG-3 cells (Fitzpatrick et
al., 1990). Since Sp1 serves as an enhanson in several enhancers,
including those for bovine papilloma virus (Li et al., 1991),
chicken histone H5 (Sun et al., 1992) and mouse pro-2(I) collagen genes (Pogulis and Freytag, 1993), it
was possible that Sp1 was an integral and essential component of
enhancer function. We confirmed the earlier observations that Sp1
contributes in a major way to hCS promoter activity (Fig. 1B) in human placental cells (Fitzpatrick et
al., 1990). However, our studies demonstrate that CSEn maintains
significant, albeit reduced, enhancer activity in the absence of the
Sp1 site (Fig. 1C), suggesting that CSEn interactions
with other parts of the basal transcription machinery are required. Sp1
does not participate in the selection of the transcription initiation
site, since a core hCS promoter without Sp1 binding site can
direct faithful initiation in placental cells (Fig. 6B). Similar results, demonstrating that neither
the number nor orientation of Sp1 binding sites affected initiation
site selection on other TATA box-containing promoters, have been
observed (Jones and Tjian, 1985; Segal and Berk, 1991). Thus the role
of Sp1 can be distinguished from those factors that direct initiation
site selection. Importantly, as discussed below, the evidence indicates
that hCS promoter function requires an InrE-binding factor which is
involved in transcription initiation.
Support for the concept that factors in addition to Sp1 were required for optimal enhancer-stimulated activity was obtained in subsequent mutational studies of the hCS proximal promoter (Fig. 2) which indicated that the TATA element and downstream sequences played an important role in hCS promoter and enhancer function. Direct evidence for an initiator factor was obtained from gel shift experiments ( Fig. 3and Fig. 4) and UV cross-linking experiments (Fig. 5) which demonstrate that a BeWo cell-specific protein of 70 kDa binds to the hCS promoter near the InrE. The specificity of the hCS InrE-protein interaction was established by a series of binding and competition experiments with wild-type and mutated oligonucleotides (Fig. 4). Site-specific mutation of the region between nt -15/+1 resulted in significantly reduced basal- and enhancer-stimulated activity (Fig. 2, D and E) and resulted in altered transcription initiation site selection (Fig. 6C), providing additional evidence that an InrE was required for basal- and enhancer-mediated hCS promoter activity. Since the InrE-binding ability and initiation site selection are strongly correlated, we propose that the 70-kDa protein binding to the hCS InrE might play a crucial role in the formation of the initiation complex.
The requirement of the DNA sequences around the InrE for
faithful and efficient transcription is well known (Nakatani et
al., 1990; Smale and Baltimore, 1989; Talkington and Leder, 1982).
Several families of InrEs based on consensus sequences have been
described (reviewed in Weis and Reinberg(1992)). These families include
the TdT-InrE (YYCAYYYYY
), PBGD-InrE (
CA
and
TCCTGGTTAC
), DHFR-InrE (
ATTTCGCGCCAAACTT
), the ribosomal
protein InrE (
CTTCCCTTTTCC
) and the
adeno-associated virus p5-InrE (CTCCATTTT). All of these
sequences are pyrimidine rich (>63%), but there is no obvious
relationship in the sequences between nt -15/+1 of the hCS promoter (38% pyrimidine content) with any of these InrE
families, suggesting that the hCS InrE may be a unique element.
Javahery et al.(1994) have provided evidence that many
reported Inrs fit the consensus YYA(+1)N(T/A)YY at the
transcription initiation site; however, this does not fit the hCS initiation site (CTA
GGA), providing additional
evidence that the hCS InrE may be unique. It is interesting that the
region containing the hCS InrE contains an imperfect
palindrome (
AGAGACCGGCTCT
) and
that the InrE binding protein fails to bind to oligonucleotides
containing mutations in the region -8/-5 and
-5/-1; however, the significance of this palindromic
structure is unknown.
By footprinting and gel shifting assays, several InrEs have been shown to interact with specific nuclear proteins (Du et al., 1993; Means and Farnman, 1990; Nakatani et al., 1990; Purnell and Gilmour, 1993). Two different InrE-binding factors from HeLa cells, YY1 (adeno-associated virus p5-InrE-binding factor) (Shi et al., 1991) and TFII-I (TdT-InrE-binding factor) (Roy et al., 1991) have been extensively characterized, demonstrating that InrE function is dependent on unique transcription factors. Although InrEs are essential for the function of TATA-less promoters, they also have been shown to function on TATA-containing promoters. Cooperative interactions between TATA and Inr elements may be important for the functioning of several promoters (Conaway et al., 1990; Nakatani et al., 1990; Purnell and Gilmour, 1993; Smale and Baltimore, 1989; Tokunaga et al., 1984), but not the major histocompatibility complex E1 promoter (Mantovani et al., 1993). Such cooperative interactions appear to be important for the hCS promoter (Fig. 2C).
Although the exact mechanism(s) involved in InrE-mediated transcription are not known, evidence has been obtained that TBP is required for TATA- and InrE-mediated transcription. Smale et al.(1990) demonstrated that a TFIID-containing fraction restored TdT-InrE-mediated transcription in a heat-inactivated nuclear extract. Subsequently, Carcamo et al.(1991) showed that transcription from the Ad-ML promoter lacking the TATA element was TBP-dependent and Pugh and Tjian(1991) demonstrated that polyclonal anti-TBP antibodies inhibited transcription from both TATA-containing and TATA-less promoters. It has been proposed that InrE-binding proteins may tether TBP to the transcription initiation complex through protein-protein interactions (Mantovani et al., 1993; Pugh and Tjian, 1991). On promoters like hCS containing both TATA and Inr elements, TBP may be more stably associated with the initiation complex by binding to the TATA element and by its interaction with the InrE-binding protein. This could explain why mutation or deletion of the TATA or Inr elements result in reduction of hCS promoter function and enhancer-stimulated activity. Accordingly, our data are consistent with a model in which the TATA and Inr elements form a basic initiation complex which directs a low level, faithful transcription whose initiation frequency is increased by Sp1 and the other enhancer elements.
Our results indicate that no single, specific promoter element is responsible for enhancer activity. All of the promoter elements that were found to be necessary for hCS enhancer stimulation also made significant contributions to the basal promoter activity. Thus the enhancer may interact with ubiquitous transcription factors that form part of the basal transcriptional machinery. This idea is supported by the data showing that the enhancer functions with a core promoter containing the TATA and Inr elements (Fig. 2C) and has an absolute requirement for a promoter, since the enhancer cannot initiate transcription by itself (Fig. 6A). These results are consistent with our earlier observation that CSEn can stimulate cell-specific transcription from the heterologous thymidine kinase and RSV promoters (Jiang and Eberhardt, 1994), which lack a structure similar to the hCS InrE. Thus the InrE-binding factor may contribute to hCS promoter efficiency which provides for optimal enhancer modulation of promoter activity. Although there is no evidence for a direct interaction between the InrE-binding protein and the enhancer complex, it cannot be excluded that the InrE-binding protein may be utilized in promoters which lack the InrE binding site by basal factor- or enhancer-mediated tethering mechanisms (Zenzie-Gregory et al., 1992). Although these data do not completely explain cell-specific hCS gene expression, the data provide the first evidence for an InrE in any member of the human growth hormone gene family, provide evidence that the InrE accounts in part for cell-specific expression of the hCS promoter and demonstrate that this element is essential for maximal basal- and enhancer-mediated hCS transcription in placental cells.