(Received for publication, July 5, 1995)
From the
The role of the proximal promoter and the far-upstream enhancer in the hepatocyte-specific and hormonal regulation of the carbamoyl-phosphate synthetase I (CPS) gene was investigated in transient transfection assays using primary rat hepatocytes, hepatoma cells, and fibroblasts. These experiments revealed that the activity of the promoter is comparable in all cells tested and is, therefore, not responsible for tissue-specific expression. The 5`-untranslated region of the mRNA is a major, non-tissue specific stimulator of expression in FTO-2B hepatoma cells, acting at the post-transcriptional level. A 469-base pair DNA fragment, 6 kilobase pairs upstream of the transcription start-site in the CPS gene, confers strong hormone-dependent tissue specific expression, both in combination with the CPS promoter and a minimized viral thymidine kinase promoter. Sequences similar to a cyclic AMP-responsive element and a glucocorticosteroid-responsive element were found in the isolated enhancer. Substitutional mutations in these sites strongly affected hormone-induced expression. Analysis of the interaction between the enhancer and parts of the CPS promoter revealed that, in addition to the TATA box, the GAG box, a motif similar to the GC box near the TATA motif, is instrumental in conferring the enhancer activity.
Carbamoyl-phosphate synthetase (CPS) ()is the first
enzyme of the ornithine cycle. CPS expression can be detected from the
15th embryonic day onward in the liver of the rat. Expression is
initially found in a few hepatocytes only, but toward the end of the
fetal period all hepatocytes have been recruited to express
CPS(1, 2, 3) . After birth, the expression
gradually becomes confined to the hepatocytes surrounding the portal
veins(3, 4) . The only other cells producing
appreciable levels of CPS mRNA and protein are the enterocytes of the
small intestine(3, 5) . After birth, CPS enzyme and
mRNA levels change in parallel under all experimental
conditions(5, 8, 10, 11) ,
suggesting that hormonal regulation, tissue specificity, and zonal
restriction of expression of CPS is regulated at the level of
transcription. Accordingly, it was shown that glucocorticosteroids and
cyclic AMP enhance transcription of the CPS gene in adult rat
hepatocytes(6, 7, 8, 9, 12) .
The CPS gene is a single-copy, 110 gene that contains 38 exons and
is surrounded by matrix attachment
regions(13, 14, 15, 16) . The mRNA
is 5546 nucleotides in length (excluding the poly(A) tract) and
consists of a 140-nt 5`-untranslated region, an open reading frame of
4500 nt, and a 3`-UTR of 906 nt. Functional analysis of the 5`-part of
the gene showed that the minimal, fully active promoter is located
within the 161 nt upstream of the transcription initiation
site(15) . DNase I footprint analysis of this region revealed
three protected sites (sites I-III (17, 18) ), but the
actual identity of the factors occupying these sites is not
known(19) . In between the TATA motif at position -21 and
protected site I, a so called ``GAG'' element was identified (19) that resembles the element recognized by the TFIIIA-like
Cys/His
zinc finger class of transcription
factors, including Sp1, but is not a target of the Sp1 protein itself.
Two MspI restriction sites upstream of the transcription start site were found to be differentially methylated(15) . The site at -6.3 kbp becomes demethylated shortly after birth in liver and intestine. The other site, at -4.0 kbp relative to the transcription start site is fully methylated in liver and partly demethylated in postnatal small intestine. A 4-kbp fragment containing the -6.3-kbp MspI site responds to cyclic AMP and dexamethasone in transient expression assays, giving a 10-fold rise in reporter gene expression in FTO-2B hepatoma cells(15) .
The regulatory regions of the CPS gene were analyzed with respect to their role in tissue-specific expression and hormone sensitivity. A 469-bp far-upstream enhancer fragment was found to be responsible for hormone-dependent tissue-specific expression of the CPS gene. It was also investigated whether the proximal promoter and the 5`-UTR of the mRNA contributed to tissue-specific expression. Exploration of the interaction of the enhancer fragment with parts of the CPS promoter revealed an important role for the GAG element in the transduction of the hormonally induced activation signal from the enhancer.
Figure 5:
Delineation of the far-upstream enhancer.
Transient-transfection assays of constructs containing sequences
derived from an exonuclease III library of the 4-kbp enhancer fragment,
cloned upstream of the CPS promoter-containing fragment (construct
1 in Fig. 1). The open box at position A indicates
the original 4-kbp enhancer fragment(15) . The
``RI'' indicates a landmark EcoRI site in
the fragment, which is positioned 6 kbp upstream (-6 kbp) of the
transcription start site in the CPS gene. On the left, size and
position of the test sequences are represented relative to the 4-kbp
fragment (fragment A). The middle and right panels represent the activities of the fragments shown on the left after
transfection into FTO-2B hepatoma cells and into Rat-1 fibroblasts,
respectively. The bars indicate the normalized specific
luciferase activity (``Experimental Procedures'') relative to
the activity obtained from the construct containing only the CPS
promoter (-, construct 1 in Fig. 1), which is arbitrarily
set at 1. The black bars indicate activities of cells grown in
the absence of added hormones, gray bars in the presence of
dexamethasone, and hatched bars, activities in the presence of
dexamethasone, BtcAMP, and IBMX (see ``Experimental
Procedures''). Error bars indicate the S.E. of at least
three independent transfections. The hatched bars showing the
activity of enhancer fragments A, B, C, D, and F in FTO-2B cells
represent the mean of two independent transfections, with the error
bar showing the variance. ND indicates values not
determined.
Figure 1:
Functional analysis of
the CPS promoter. Transient transfection assays of CPS promoter
deletion constructs in FTO-2B hepatoma cells and Rat-1 fibroblasts.
Regions of the CPS promoter that were cloned upstream of the luciferase
reporter gene are shown to the left. The TATA box (black
box), GAG box, and sites III, II, and I of the CPS promoter (19) are indicated. The bottom-most structure, shown in gray, indicates the minimized TK promoter. The TATA box (rectangle) and the GC box of the TK promoter are given. The bars indicate the normalized specific luciferase activity
(luciferase activity/CAT activity) relative to the activity obtained
from the construct harboring the 161-bp CPS promoter, which was
arbitrarily set at 1. The black bars indicate activities of
cells grown in the absence of added hormones, while the hatched
bars show activities in the presence of dexamethasone,
BtcAMP, and IBMX (``Experimental Procedures''). Error bars indicate the S.E. of at least three independent
transfections.
All CPS promoter constructs are derived from the 299-bp PstIHindIII promoter-containing fragment (position -161 to +138, Fig. 1, HindIII site derived from polylinker of pBluescript SK+) described in (15) . To delete sites III and II from the CPS promoter, a 238-bp AflIII-HindIII fragment (position -100 to +138, Fig. 1) was cloned into pLT1. For deletion of sites III, II, and I, primer CAGCCCCTCCTCCCTCTAGAATGTCCAGAGATG (complementary to sense-strand) was used to create a XbaI site (underlined) at position -74 (Fig. 1). The resulting 212-bp XbaI-HindIII fragment was cloned into pLT1. To delete sites III, II, I and the GAG box, a 176-bp AccI-HindIII fragment (position -38 to +138, Fig. 1) was cloned into pLT1.
Deletions in the 5`-UTR of the mRNA were made by creating HindIII sites in the 5`-UTR at 50, 89, and 115 nt downstream of the transcription start site of construct 2 (see Fig. 1, construct 2) with oligonucleotides GGGAAGGAAAGCTTTGTGGAGAC, CATGAAAGCTTGTTGTCCAATTTGC and GTGACTAAGCTTAAATCACAAATATCTC, respectively (all complementary to the sense strand; HindIII sites are underlined). The T7 primer of pBluescript was used as complementary primer. All PCR products were sequenced. The putative CRE and GRE element in the 469-bp enhancer fragment (see Fig. 5, construct J) was mutated after subcloning into pBluescript SK+. Primer TTACTTTAGAATCATATTGAGGACTTATTA was used to disrupt the putative CRE and CATCAGAGAAGTTTGATCTGCTCAGCACAT to disrupt the putative GRE. Underlined nucleotides were substituted (see Fig. 6). The T3 and T7 primers were used to fill in both sides flanking the mutagenesis primer. The PCR products were sequenced in both directions.
Figure 6: Nucleotide sequence of fragment I (Fig. 5), containing the 469-bp enhancer fragment (fragment J). The landmark EcoRI site depicted in Fig. 5and the differentially methylated MspI/HpaII site (CCGG) at -6.3 kbp (Fig. 9) are underlined. The imperfect CRE (TGACGTCA, 148-155) and the three GRE half-sites (TGTTCT, 382-401) are double underlined. On top of these sites the substitutional mutations to inactivate the putative CRE and GRE are shown. The arrow indicates the position of the most 5` nt of fragment E of Fig. 5. Sites with similarity to binding sites of liver-enriched factors HNF3 (Ca/tAAa/gTCAATA), HNF4 (GGGCCANNNa/ga/gGTCCA), and HNF5 (Ta/gTTTGc/t) are given in italics.
Figure 9:
Enhancer analysis in enterocytes.
Transient-transfection assays of constructs containing regions upstream
of the transcription start site of the CPS gene in Caco-2 cells. CPS-A and TK-A represent constructs in which a 4-kbp
fragment (fragment A in Fig. 5) was cloned upstream of
the CPS promoter (fragment 1 in Fig. 1) or the
minimized TK promoter (-81 to +52), respectively. CPS-B and TK-B represent constructs in which a 4-kbp fragment
containing the differentially methylated site at position -4.0
kbp (15) was cloned upstream of the 161-bp CPS promoter or the
minimized TK promoter. Bars indicate the normalized CAT
activity (Panel A) or luciferase activity (Panel B). Black bars show the activities of cells grown in the absence
of added hormones and hatched bars the activities of cells
grown in the presence of dexamethasone, BtcAMP, and IBMX.
The values were normalized either to the activity of construct CPS-A in
the absence of added hormones (Panel A) or to the activity of
construct TK-A in the absence of added hormones (Panel B).
Values are the mean of two independent transfections, error bars indicating the variance.
Three regions in the CPS gene appeared to be involved in the regulation of expression, viz. the 161-bp proximal promoter, the 138-bp 5`-UTR of the mRNA, and the 4-kbp far-upstream enhancer fragment(15) . These regions were systematically investigated for their contribution to tissue specificity and response to physiologically relevant hormones.
Since the activity of the CPS promoter was similar in hepatoma and fibroblast cells (Fig. 1), this sequence probably does not confer tissue specificity. To establish this conclusion more firmly, luciferase activity per transfected cell was determined. This approach (Fig. 2) clearly demonstrated that the activity of the CPS promoter was quantitatively comparable in FTO-2B and Rat-1 cells and, hence, does not confer tissue specificity. The minimized TK promoter, containing only the proximal GC box and a TATA box, was less active in the hepatoma cell lines than in the fibroblasts. The GC box, target of the Sp1 protein and the main determinant of the strength of this promoter (28, 39) may therefore be hardly functional in hepatoma cell lines, in accordance with the relatively low concentrations of Sp1 in hepatocytes(40) .
Figure 2:
Lack of tissue specificity of the CPS
promoter. Comparison of the activity of the CPS promoter (left
panel) and the minimized TK promoter (right panel) in
FTO-2B hepatoma cells and Rat-1 fibroblasts. Transfection efficiency
was determined by cotransfecting the expression vector pRSV-n-LacZ
(``Experimental Procedures''). Bars indicate
normalized luciferase activity (relative light units/mg of protein
fraction of blue cells
10
). Error bars indicate the S.E. of at least four independent transfections. Black bars represent the activities of FTO-2B cells, gray
bars the activities of Rat-1 cells. *, p (two-tail)
= 0.39, **, p (two-tail) = 0.03, using the
Student's t test for independent
samples.
Figure 3:
Functional analysis of the 5`-UTR. A, deletion analysis of the stimulation of reporter gene
expression by the 138-nt 5`-UTR. Constructs harboring parts of the
5`-UTR were transiently transfected in FTO-2B hepatoma cells. The bars indicate the normalized specific luciferase activity
(``Experimental Procedures'') relative to the activity
obtained from the construct containing the full-length 5`-UTR and 100
bp of the CPS promoter (UTR 138 = construct 2, Fig. 1), which is set to 1. Other constructs harbor the same
upstream promoter sequences, but lack 5`-UTR regions +115 to
+138 (U 115), +89 to +138 (U 89), +50 to +138
(U 50), and +7 to +138 (U 7) relative to the transcription
start site. The small ORF (position 97-108) is present in
constructs U 115 and U 138, and absent in the other constructs. Black bars indicate the activity of FTO-2B cells incubated in
the absence of added hormones, hatched bars in the presence of
dexamethasone, BtcAMP, and IBMX (``Experimental
Procedures''). Error bars indicate the S.E. of at least
three independent transfections. B, activity of the 5`-UTR of
the CPS mRNA in the context of the minimized TK promoter (-81 to
+52). The 5`-UTR (+3 to +138, black box) was
cloned into the BglII site (position +52) of the TK
promoter (gray) in both directions (arrows). The
activity of the TK promoter was set to 1. Black bars indicate
the activity in the absence of added hormones, hatched bars in
the presence of dexamethasone, Bt
cAMP, and IBMX. Error
bars indicate the S.E. of at least three independent
transfections.
Figure 4: Luciferase mRNA levels produced by full-length and truncated 5`-UTR constructs in FTO-2B cells. Constructs U 138, U 115, and U 50 (Fig. 3) were transiently expressed in FTO-2B hepatoma cells and assayed by determination of luciferase-SV40 hybrid mRNA concentration (``Experimental Procedures''). Lanes 1, 4, 7, 13, and 19, U 138; lanes 2, 5, 8, 14, and 20, U 115; lanes 3, 6, 9, 15, and 21, U 50; lanes 11, 12, 17, and 18, rat liver total RNA; lanes 10 and 16, 10 fg of mimic DNA. Lanes 1, 2, and 3, 200 fg of mimic DNA; lanes 4, 5, and 6: 50 fg of mimic DNA; lanes 7, 8, and 9, 10 fg of mimic DNA. Lanes 16-21, without reverse transcriptase (RT).
Figure 7:
Tissue specificity of the far-upstream
enhancer. Transient-transfection assays of the construct containing the
469-bp enhancer fragment coupled to the minimized TK promoter in FTO-2B
hepatoma cells and Rat-1 fibroblasts. Black bars represent the
activity of cells incubated in the absence of added hormones, gray
bars the activity in the presence of dexamethasone, and hatched bars the activity in the presence of dexamethasone,
BtcAMP, and IBMX. The activities indicate the normalized
specific luciferase activity relative to the activity obtained from the
minimized TK promoter and are corrected for the effect of hormonal
stimulation on the TK promoter without enhancer. Bars indicate
the S.E. of four independent transfections.
Figure 8:
Relation between expression of the
endogenous CPS gene and the far-upstream enhancer activity. Panel
A, transient transfection of the 469-bp far-upstream element (fragment J in Fig. 5) in combination with the CPS
promoter (fragment 1 in Fig. 1) in Chinese hamster
ovary cells, Rat-1 fibroblasts, and FTO-2B- and MH1C1-hepatoma cells.
The bars indicate the normalized luciferase activity relative
to the activity obtained from the basal CPS promoter without enhancer.
The activities are corrected for the effect of added hormones on the
basal CPS promoter without enhancer. Black bars indicate the
activity of cells cultured without added hormones, hatched bars the activity of cells to which dexamethasone, BtcAMP,
and IBMX were added. Error bars indicate the S.E. of at least
three independent transfections. Panel B, Western blot
analysis of CPS protein content in the respective cell lines in the
absence(-) and the presence of dexamethasone, Bt
cAMP,
and IBMX (+). In each lane 50 µg of total protein was
applied.
To verify the validity of the use
of hepatoma cells as a model for assessing tissue-specific expression,
the enhancer was also tested in freshly isolated hepatocytes. In these
cells the CPS promoter was stimulated 4.6-5.0-fold (n = 2) by dexamethasone, BtcAMP, and IBMX. When
combined with the 469-bp enhancer, the expression was reduced to 27
± 3% (n = 5) of that of the promoter alone in
the absence of added hormones. In the presence of hormones, however,
the expression was stimulated 63-79-fold (n = 2)
compared to the promoter, the hormonal induction being more than
250-fold.
Figure 10: Delineation of promoter elements involved in relaying the activation signal of the enhancer. Hormonal inducibility of reporter gene expression by the far-upstream enhancer in combination with CPS promoter deletion constructs in FTO-2B hepatoma cells. The promoter fragments, shown in the left panel were coupled to the 1003-bp enhancer fragment (fragment H in Fig. 5) or the 469-bp enhancer fragment (fragment J in Fig. 5). The TATA box (black rectangle), GAG box, and sites I-III of the CPS promoter (19) are indicated. In gray the minimized TK promoter is indicated, with the TATA box (rectangle) and the GC box. Hormonal inducibility is expressed as the ratio of the activity in the presence of added hormones over the activity in absence of added hormones after correction for the effect of hormones on the promoter-deletion construct.
When the distance between the enhancer and the minimized TK promoter was decreased, luciferase expression levels increased strongly (Fig. 11). Such distance effects were virtually absent in combination with the CPS promoter ( Fig. 5and Fig. 11). Whether or not sites upstream of the TATA box of the CPS promoter are functioning as coupling elements between the enhancer and the promoter was tested. A promoter fragment in which all sites upstream of the TATA box were deleted, leaving only the TATA box and 5`-UTR (Fig. 1, construct 4) was coupled to either the 1003-bp enhancer fragment (H in Fig. 5) or the 469-bp fragment (J in Fig. 5) and transfected to FTO-2B cells (Fig. 11). The truncated promoter was more sensitive to increasing distance than the CPS promoter, but not as sensitive as the TK promoter. These results underline the role of the GAG box.
Figure 11:
Effects of distance between promoter and
enhancer on the effectiveness of the enhancer. The distance (bp)
between the TATA box and the center of the 469-bp enhancer fragment is
depicted on the horizontal axis and reporter gene activity on
the vertical axis. FTO-2B hepatoma cells were incubated in the
presence of the hormones dexamethasone, BtcAMP, and IBMX.
All activities were normalized to the activity of the CPS promoter
fragment which was set to 1. Circles (
) represent the
activity of the CPS promoter containing elements I-III, the GAG box,
and the TATA box (construct 1, Fig. 1). Triangles (
) represent the activity of the CPS promoter containing
only the TATA box (construct 4 of Fig. 1) and squares (
) represent the activities of the minimized TK
promoter (construct TK in Fig. 1). Promoters were combined with
the 469-, 547-, 1003-bp, and the 2-kbp enhancer fragments (Fig. 5, fragments J, I, H, and D,
respectively). The solid lines are curves proportional to
(bp)
. Theoretically, the exponent q is a
measure for the probability of a DNA site to be in the vicinity of the
second. Mathematically, for random distribution in three dimensions, q = -1.5 (the Gaussian limit(55) ). q (CPS) = -0.2, q (truncated CPS) =
-1, q (TK) =
-3.5.
Figure 12:
Functional identification of
hormone-responsive elements in the enhancer. Panel A,
functional identification of putative CRE by transient transfection of
the enhancer into FTO-2B hepatoma cells. Cells were cultured in the
absence of added hormones. Black bars indicate the activities
of the CPS promoter (construct 1, Fig. 1) combined with
the 469-bp enhancer (wt) or the 469-bp enhancer containing a mutation
in the putative CRE (CREmut). Hatched bars show activities in
the presence of 4 µg each of the co-transfected expression vectors
encoding C of protein kinase A, CREB, and CBP. The activity of the
CPS promoter was set to 1. Error bars represent the S.E. of at
least three independent transfections. Panel B, functional
identification of the putative GRE by transient transfections into
FTO-2B hepatoma cells. Bars indicate the relative luciferase
activity of the CPS promoter (construct 1, Fig. 1) in
conjunction with the 469-bp enhancer (wt), carrying a mutation in the
putative GRE site (GREmut) or the putative CRE site (CREmut), in the presence of 4 µg of a cotransfected
expression vector encoding the glucocorticosteroid receptor. The black bars indicate the activity in the absence, and hatched bars in the presence of dexamethasone. The activity of
the CPS promoter was set to 1. The activities of hormonally stimulated
cells were corrected for the effect of added hormones on the CPS
promoter without enhancer. The ratio of the activity in the presence of
dexamethasone (hatched bars) over the activity in the absence
of dexamethasone (black bars) is depicted above the bars.
Error bars represent the S.E. of at least three independent
transfections.
The aim of this study was the functional characterization of the regulatory regions of the CPS gene with respect to their role in tissue-specific expression and hormone sensitivity. Three regions were functionally analyzed. The CPS promoter, comprising a TATA box, a GAG box, and elements I-III, associated with the binding of presently unknown proteins(19) , the 138-bp 5`-untranslated region of the mRNA, and the far-upstream enhancer at -6.3 kbp.
Our data show that, in vitro, the CPS promoter has comparable strength in hepatoma cells, which do express CPS, and in fibroblasts, which do not express CPS, demonstrating that it is not involved in tissue-specific regulation of transcription. Furthermore, our findings clearly show that sites I, II, and III (-150 to -79 nt) are not essential for basal promoter activity or transduction of the hormonally induced activation signal from the enhancer. The GAG box, however, is quantitatively important because its absence reduces both the promoter activity and the effect of the enhancer on the promoter 2-3-fold. These data suggest that the protein(s) binding to the GAG box are involved in transducing the activation signal from the enhancer to the promoter as well as in the functioning of the promoter itself.
A promoter fragment lacking the 5`-UTR of CPS mRNA (+7 to +138) is hardly active in FTO-2B cells ( (15) and Fig. 3). Such a finding can be due to an incorrect mapping of the transcription start site, or to the involvement of downstream sequences in the basal transcription complex(15) . These possibilities can now be virtually ruled out because addition of up to 115 nt of the 5`-UTR downstream of the transcription start site does not restore expression levels to those obtained with the full-length 5`-UTR (Fig. 3A). When combined with the minimized TK promoter (Fig. 3B), the full-length 5`-leader of CPS mRNA inhibits, rather than stimulates expression. This result makes the presence of cis-elements in the 5`-UTR DNA, which, in combination with a promoter, are able to stimulate transcription, rather unlikely. Quantification of RNA levels (Fig. 4) shows that the 5`-UTR acts at the translational level. The 5`-UTR contains a small upstream open reading frame (uORF) of four codons (Met-Arg-Tyr-Leu) starting with the initiation codon at position 97 (position relative to the transcription start site) in a suboptimal context compared to the more downstream initiation codon of the CPS reading frame (50 and 70% similarity to the consensus sequence(43) , respectively), followed by three stop codons. uORFs are thought to be able to suppress translation of downstream cistrons(44, 45, 46) . Strikingly, other eukaryotic CPS genes of which the 5`-UTR sequences are known, the human CPS I gene(47) , the shark CPS III gene(48) , and the yeast CPA1 gene(46) , also contain one or more uORFs. The uORF of the CPA1 gene is known to suppress translation in a regulated manner. Constructs U 115 and U 138 (Fig. 3) both contain the uORF, but differ markedly in luciferase activity. Deletion of the 23 nt between the uORF and the luciferase start codon affects inter-cistronic length, the sequence context at the uORF stop codon, and secondary structure, parameters which were found to be important for translational control (49) .
Scanning sequences up to 12 kbp
upstream and 4 kbp downstream of the promoter fragment, only one
fragment located at 6 kbp from the transcription start site could be
found that had the capacity to stimulate reporter gene expression in
FTO-2B cells and Rat-1 fibroblasts(15) . This enhancer element
has now been confined to a 469-bp fragment (Fig. 5). Transgenic
mice, harboring the CPS promoter and 12-kbp upstream DNA in combination
with the CAT reporter gene, give rise to hepatocyte-specific expression
of CAT mRNA, which co-localizes with the endogenous CPS mRNA in the
liver. Mice harboring only the proximal promoter show extremely weak
CAT activity, which is not tissue-specific. ()Combination of in vivo and in vitro results suggests that the
proximal promoter and the 469-bp far-upstream enhancer are both
necessary and sufficient for tissue-specific CPS expression.
Sites homologous to an imperfect CRE, with the structural characteristics of a so called ``low affinity site'' (50) and a GRE were found to be essential elements in the minimal enhancer fragment (Fig. 12); in the absense of added hormones, the enhancer was inactive, while mutations of the CRE and GRE significantly decreased hormone-dependent enhancer activity. Expression of the construct carrying the CRE mutation was still responsive to glucocorticoids (Fig. 12B), indicating that the CRE and GRE are not functionally linked. On the other hand, when sequences upstream of position 339, including the CRE (Fig. 6) are deleted (construct E of Fig. 5), the enhancer looses all activity. The deleted area contains nearly perfect consensus sequences for HNF3 and, to a lesser extent, for HNF4.
In our transfection
analysis the hormonal stimulation by the CPS enhancer was found to be
independent of the distance between the CPS promoter and enhancer,
whereas this distance was important when combining TK promoter and
enhancer (Fig. 11). This promoter-specific effect is probably
highly relevant in vivo, because the CPS promoter and the
enhancer are approximately 6-kbp apart. One way to explain the
differences is to hypothesize that transcriptional activity is related
to the probability of the enhancer to contact the promoter. The curves,
proportional to (bp), represent this probability and
exponent q the slope in a double logarithmic plot (Fig. 11). Mathematically, when the promoter and the enhancer
are randomly distributed in space, q = -1.5 (the
Gaussian limit(55) ). q
0 (CPS promoter) means
that the probability is
1, i.e. that the promoter and the
enhancer are connected independently of distance. q =
-1 (truncated CPS promoter) approaches a random distribution and q = -3.5 (TK promoter) indicates hindrance of
enhancer-promoter interaction. The main difference between the CPS and
the truncated CPS promoter is the GAG box (see also Fig. 10),
indicating that this motif is instrumental in conferring the hormonal
activation of the enhancer to the transcriptional complex. The main
difference between the minimized TK promoter and the truncated CPS
promoter is the GC box. This GC box might be the actual cause of the
distance dependence of the minimized TK promoter in conjunction with
the CPS enhancer, possibly by preventing interaction of enhancer and
promoter through steric hindrance.
In summary, our data support a model in which the tissue-specific expression of CPS is determined by the far-upstream enhancer. The activity of this enhancer is strictly dependent on the presence of glucocorticoids and cyclic AMP. The activated enhancer complex will stimulate transcription through interaction with factors bound to the GAG box and the TATA region.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X90476[GenBank].