We searched for such elements in the 2.5-kb upstream region and in the 2.6-kb first intron of the GS gene, using FTO-2B hepatoma and C2/7 muscle cells as representatives of both cell type
Trang 1Upstream and intronic regulatory sequences interact in the
activation of the glutamine synthetase promoter
Rocio M Garcia de Veas Lovillo, Jan M Ruijter, Wil T Labruye`re, Theodorus B M Hakvoort
and Wouter H Lamers
AMC Liver Center, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
Glutamine synthetase (GS) is expressed at high levels in
subsets of cells in some tissues and at low levels in all cells of
other tissues, suggesting that the GS gene is surrounded by
multiple regulatory elements We searched for such elements
in the 2.5-kb upstream region and in the 2.6-kb first intron of
the GS gene, using FTO-2B hepatoma and C2/7 muscle cells
as representatives of both cell types and transient
transfec-tion assays as our tools In additransfec-tion to the entire upstream
region and entire intron, an upstream enhancer module at
)2.5 kb, and 5¢, middle and 3¢ modules of the first intron
were tested The main effects of the respective modules and
their combinatorial interactions were quantified using the
analysis of variance (ANOVA) technique The upstream
enhancer was strongly stimulatory, the middle intron
mod-ule strongly inhibitory, and the 3¢-intron modmod-ule weakly
stimulatory in both hepatoma and muscle cells The
5¢-intron module was strongly stimulatory in muscle cells only The major new finding was that in both cell types, the upstream enhancer and 5¢-intron module needed to be pre-sent simultaneously to fully realize their transactivational potencies This interaction was responsible for a pronounced inhibitory effect of the 5¢-intron module in the absence of the upstream enhancer in hepatoma cells, and for a strong synergistic effect of these two modules, when present sim-ultaneously in muscle cells The main difference between hepatoma and muscle cells therefore appeared to reside in tissue-specific differences in activity of the respective regu-latory elements due to interactions rather than in the exist-ence of tissue-specific regulatory elements
Keywords: enhancer; glutamine synthetase; hepatoma; muscle; transient transfection
Glutamine synthetase (GS; EC 6.3.1.2), the enzyme that
catalyses the ATP-dependent conversion of glutamate and
ammonia into glutamine, is expressed in a tissue-specific and
developmentally controlled manner GS functions to
remove ammonia or glutamate, or to produce glutamine
Cells that function primarily to remove glutamate or
ammonia, contain very high GS levels (30–160 lM),
whereas cells that synthesize glutamine contain much lower
levels (1–8 lM) [1] Another highly characteristic and
functionally important feature of GS is its topographic
distribution: in organs in which GS is present at relatively
high concentrations, it is usually expressed in a subset of
cells only, whereas in organs in which it is present at low
concentrations, it is expressed in the majority of cells
Examples of the first group of organs are the pericentral
hepatocytes in the liver, the astrocytes in nervous tissue, the
epithelial cells of the caput epididymis, and the gastric
antrum Examples of the second group are adipocytes and
muscle cells (for a review, see [1])
Because of these interorgan differences in distribution and cellular concentration of GS, and because only a single functional copy of the GS gene is present per haploid genome in rodents [2–4], it is to be anticipated that the regulation of GS expression is complex [1] Studies aimed towards delineating the transcriptional regulation of GS expression in rodents have thus far revealed upstream enhancer elements at )6.0 kb and )2.5 kb, and intron enhancer elements at +0.35 kb and +1.6 kb, by transient
or stable transfections to cultured cells [5–8] The sequence
of the far-upstream mouse GS enhancer that is active in adipocytes [9] is 80% similar to that of the far-upstream rat
GS enhancer and, like the rat far-upstream enhancer, also maps at)6.0 kb in the Celera Discovery System mouse genome database The upstream enhancer at )2.5 kb confers pericentral localization to reporter gene expression
in the liver of transgenic mice [10] The significance of the far-upstream and intron elements for the in vivo expression pattern of GS remains to be assessed
In the liver, genes are expressed in a porto-central gradient Studies with transgenic animals have shown that many of these gradients in gene expression, including that of
GS [10], are determined at the transcriptional level Porto-central gradients in gene expression have been distinguished into dynamic and stable gradients [11] The dynamic type of zonation is characterized by adaptive changes in expression
in response to changes in the metabolic or hormonal state, whereas the stable type of zonation, of which GS is an example [12,13], is characterized by the virtual absence of such adaptive changes A relatively simple model to explain
Correspondence to W H Lamers, AMC Liver Center, Academic
Medical Center, University of Amsterdam, Meibergdreef 69-71,
1105 BK, Amsterdam, the Netherlands.
Fax: + 31 20 5669190, Tel.: + 31 20 5665948,
E-mail: w.h.lamers@amc.uva.nl
Abbreviations: GS, glutamine synthetase; TK, thymidine kinase.
Enzymes: Glutamine synthetase (EC 6.3.1.2).
(Received 22 August 2002, revised 10 October 2002,
accepted 17 October 2002)
Trang 2such a stable expression pattern is to assume a double-lock
regulatory mechanism, meaning that GS expression depends
on the synergistic interaction of two or more factors [14]
The observation that very high levels of GS are present in
subsets of cells in some organs, and moderate-to-low levels
in all cells of other organs, in combination with the
hypothetical double-lock mechanism to account for the
stable expression of GS in pericentral hepatocytes suggested
that multiple regulatory modules would control GS
expres-sion and that at least some of these modules would be
interdependent with respect to their regulatory activity To
test this hypothesis, we examined the combinatorial effects
of modular deletions in the distal upstream and first intron
regions of the rat GS gene on reporter gene expression in
hepatoma and muscle cells We now report that interactions
of upstream and intronic regulatory elements do indeed determine the degree of activation of the GS promoter and that these interactions differ quantitatively between cells from hepatic and muscular origin
Materials and methods
Sequence of the first intron of GS The nucleotide sequences of the upstream region and of the first 1938 nucleotides of the first intron of the rat GS gene were reported [5,6] This intron sequence ends at the EcoRI restriction site (Fig 1) The remaining 877 nucleotides of the
Fig 1 Schematic representation of GS sequences used in constructs A–Q and their reporter gene activities At the top, a restriction map of the genomic GS region analysed in these experiments, is shown The exons are shown as boxes, with the arrows indicating the start sites of transcription (left) and translation (right), and pA both polyadenylation sites of the gene The upstream boundary ( )2520), the transcription start site (0) and the downstream boundary (+2774) of the genomic DNA segment that was analysed, are indicated The sequences present in the respective constructs are shown in the left portion of the figure as solid black lines The upper line shows the linkage of the 5.3-kb genomic GS segment with the luciferase reporter gene at the NcoI site (translation start site) in the second exon and with the bovine growth hormone transcription termination and polyadenylation signal (bGH) The first and second exon up to the translation start site are represented as black boxes The right portion of the figure shows luciferase activity of the respective constructs in FTO-2B hepatoma cells (light grey) and C2/7 muscle cells (dark grey) Panels I and II show activities of the respective upstream and intron elements, respectively, when present in conjunction with the minimal promoter and minimized first intron Panels III and IV show activities of combinations of upstream enhancer and the entire upstream region, respectively, in conjunction with the minimal promoter, the minimized first intron and the respective intron elements Luciferase activity (± SEM) is expressed relative to construct A containing only the GS promoter and minimized first intron, which was set at 100 Restriction sites: H, HindIII; E, EcoRV; P, PstI; Bg, BglII; S, SmaI; B, BamHI; EI, EcoRI; N, NcoI.
Trang 3first intron of GS were sequenced in both orientations The
sequence data has been deposited with the EMBL
nucleo-tide sequence data bank and is available under accession
number AF170107
Construction of plasmids
Rat GS genomic DNA sequences were cloned into the
vector pSPluc+ (Promega) The starting construct (Fig 1,
construct Q) was made by inserting the genomic GS
segment from)2520 bp (relative to the transcription start
site) to +2774 bp (corresponding to position +132 in the
GS cDNA, that is, the translation start site in the second
exon) between the HindIII and NcoI sites in the polylinker
upstream of the luciferase cDNA The 305 bp bovine
growth-hormone polyadenylation sequence (the XbaI–
PvuII fragment from pcDNA3; Invitrogen) was inserted
between the XbaI and EcoRV sites in the polylinker
downstream of the luciferase cDNA The other constructs
were generated by modular deletions of construct Q The
upstream modules were: the entire region downstream of
HindIII ()2520), the region downstream of EcoRV
()2148), the region downstream of PstI ()965), or the
upstream enhancer element ()2520 to )2148 [5]) The first
intron was subdivided into three modules: a 5¢ SmaI–
BamHI fragment (+153 to +856), a middle BamHI–
SmaI fragment (+856 to +1791) and a 3¢ SmaI–BamHI
fragment (+1791 to +2712) The extended GS promoter
(BglII ()368) to the transcription-start site [5], the first
exon, a minimized first intron, containing its 5¢-most
portion (+119 to +153) and 3¢-most portion (+2712 to
+2760), and the second exon up to the translation start
site (+132) were present in all constructs The construct
carrying only these elements (construct A) was used as
reference construct
For the transfection assays, the respective constructs were
purified in CsCl gradients or on Nucleobond columns
(Machery-Nagel, Du¨ren, Germany)
Cell culture
FTO-2B rat hepatoma cells [15] were cultured in DMEM/
F-12 medium (Gibco), supplemented with 10% (w/v)
foetal bovine serum (Gibco) C2/7 cells (generously
provided by M Buckingham, Institut Pasteur, Paris,
France) are a subclone of the C2 cell line that was
originally derived from Soleus muscle of adult C3H mice
[16] These cells were cultured in DMEM (Gibco),
supplemented with 10% (w/v) foetal bovine serum All
cells were cultured at 37C in humidified air containing
5% CO2 Cell lines were tested monthly for contamination
with mycoplasms
DNA transfection
Exponentially growing FTO-2B cells were transfected by
electroporation [17] and C2/7 cells were transfected at the
myoblast stage by the calcium-phosphate method [18] In
both cases 20 lg supercoiled plasmid was used
Cotrans-fection with 5 lg of the vector pRSVcat [19] enabled
correction for differences in transfection efficiency After
electroporation, the cell suspension was divided into two
equal parts, one being grown in culture medium and the other in culture medium supplemented with 100 nM dexamethasone Sixteen h after transfection, the cells were washed with NaCl/Piand given fresh medium In the case
of the C2/7 cells, the concentration of foetal bovine serum was reduced to 1% to induce the formation of myotubes Harvest of the cells was carried out 48 h after transfection
in the case of FTO-2B cells, and 72 h in the case of C2/7 cells, that is, when all cells were fused into myotubes Cells were lysed in 100 mM KH2PO4/K2HPO4 pH 7.6, 0.1% (v/v) Triton X-100 buffer and tested for chloram-phenicol acetyltransferase activity [20], luciferase activity [21] and protein concentration (bicinchoninic acid reagent; Pierce)
Splicing of modified first intron Constructs carrying the different modules of the GS first intron were tested for proper splicing of the mRNA RT-PCR was carried out with primers in the first exon (+1
to +18) and in the luciferase-coding region (+78 to +60 in the luciferase cDNA) All constructs generated correctly spliced mRNAs (data not shown)
Correction for experimental variation and statistics The transactivation potential of the tested DNA con-structs was expressed as the ratio between their luciferase activity (light unitsÆmg protein)1) and the chloramphenicol acetyltransferase activity (unitsÆmg protein)1) of the cotransfected pRSVcat construct The data were collected from 33 experiments with FTO-2B cells and 13 experi-ments with C2/7 cells In each experiment, different combinations of constructs were tested The number of transfections per construct was 8–20 Interexperimental variation in reporter gene activity was removed using log-transformed values and the GENERAL LINEAR MODEL/ ANOVA without interaction (SPSS version 10.0.7; SPSS Inc.)
The activity of a specific construct (X,Y) containing upstream module (X) and intron module (Y) can be modelled to consist of the sum of a basal activity (produced
by the promoter and minimized intron), the effects of the respective upstream (X) and intron (Y) modules, and the interaction (X,Y) between these modules:
activityconstructðX;YÞ¼ activityconstruct A
þ effectupstream module ðXÞ
þ effectintron module ðYÞ
þ effectinteraction ðX;YÞ
In this model, the value of the main effects of the upstream and intron modules, and that of their interactions can be calculated with an approach based on the analysis of variance (ANOVA) technique To normalize the data, the activity of reference construct A was set to 100 arbitrary units (AU) in these calculations The activity of the respective modules and their interactions, including 95% confidence intervals, was expressed relative to construct A Whenever a difference is mentioned in the text, it is significant at the 5% level
Trang 4We based the design of our analysis of the regulatory
properties of sequences in the upstream region and within
the first intron of the rat GS gene on the assumption that
two or more interdependent regulatory elements were
responsible for transactivation of the GS promoter [14]
For this reason, the study was designed to reveal which
DNA sequences do interact with respect to transactivation
of this promoter We also wished to avoid that changes in
the position of the regulatory sequences might affect the
regulatory behaviour of the DNA modules For that reason,
the experimental constructs were made by modular
ortho-topic additions to construct A (Fig 1)
To delineate regulatory elements upstream of the GS
structural gene, the upstream sequence present in construct
A was extended to)965 nucleotides (construct B), to )2148
nucleotides (construct C), or to)2520 nucleotides (construct
M) (Fig 1, panel I) None of these upstream modules
significantly enhanced the activity of the GS promoter in
either FTO-2B hepatoma cells or C2/7 myotubes Previous
experiments had shown that the upstream region was able
to transactivate the heterologous thymidine kinase (TK)
promoter, and that this activity was localized between
)2520 and )2148 bp [5] When placed directly in front of
the GS promoter, this distal upstream enhancer element
(construct H, Fig 1, panel III) caused a small but significant
increase in reporter gene expression (1.4-fold in FTO-2B
and 1.8-fold in C2/7 cells)
The transactivational capacity of the first intron of the GS
gene was tested as such (construct G), as a 703-bp 5¢ intron
module (construct D), a 935-bp middle intron module
(construct E), a 921-bp 3¢ intron module (construct F), or
after deleting all intron sequences except 35 nucleotides at the
5¢ end and 48 nucleotides at the 3¢ end (construct A, the
reference construct) (Fig 1, panel II) In FTO-2B cells, the
entire intron (construct G) decreased reporter gene activity
significantly to 40% of that of construct A The inhibitory
activity was found to reside in the 5¢ and middle intron
fragments (constructs D and E) In muscle cells, the entire
intron depressed reporter gene activity to 50% of that of
reference construct A When the intron fragments were
tested individually, the 5¢ intron fragment (construct D) was
without effect on the promoter, whereas the middle fragment
slightly decreased reporter gene activity (to 70%) and the 3¢
fragment (construct F) stimulated promoter activity 1.9-fold
Interactions between upstream and intron regulatory
modules
When different combinations of upstream and intron
sequences were tested for transactivation of the GS
promo-ter, the highest activities were observed for constructs
containing the upstream enhancer (constructs H-L), whereas
the lowest activities were consistently associated with the
presence of the middle intron module (constructs E, J and O)
(Fig 1, panels II–IV) The effect of partner choice appeared
to matter most for constructs containing the 5¢-intron
module (constructs D, I and N) These findings
demonstra-ted that the degree of transactivation of the GS promoter
depended to a substantial degree on interactions between the
upstream and intron regulatory sequences We therefore
used an approach that is based upon theANOVAtechnique to quantify the main (that is, intrinsic) effects of upstream and intron modules, and to segregate these effects from those due
to interaction between the respective elements In this approach, the activity of construct A was set at 100 AU FTO-2B hepatoma cells
The computation of the main effects revealed that the presence of the upstream enhancer increased promoter activity with 87 AU in hepatoma cells, whereas this number was slightly lower (67 AU) for the entire upstream region (Fig 2, upper panel) Both effects were statistically signifi-cant The 5¢-and 3¢-intron modules both increased promo-ter activity with 23 AU The middle intron module decreased promoter activity with 84 AU Although the effect of the entire intron on promoter activity was not significant (14 AU), it neutralized the negative effect of its middle fragment The actual activity of the respective constructs often resulted from less than additive effects between the upstream and intron modules Such negative interactions were observed for constructs containing the upstream enhancer, but lacking the 5¢-intron fragment (constructs H, J and K), and vice versa (constructs D, G) Furthermore, the effects of the upstream region and the entire intron were not additive (construct Q) The other combinations did not show significant interactions, meaning that the main activities of their components accounted for the observed effect These findings demonstrate that the simultaneous presence of the upstream enhancer and the 5¢-intron module is necessary for full transactivation of the GS promoter in hepatoma cells
C2/7 muscle cells The upstream enhancer significantly increased promoter activity in muscle cells with 121 AU, whereas this number was not significant (13 AU) for the entire upstream region (Fig 2, lower panel) The intron modules all had significant effects on the promoter: the 5¢-and 3¢-intron modules increased promoter activity with 127 AU and 47 AU, respectively, whereas the middle intron module decreased promoter activity with 58 AU The entire intron did not affect promoter activity significantly The inhibitory effect of the middle intron module therefore outweighed the strongly stimulatory effects of the 5¢-and 3¢-intron modules The interaction between the upstream enhancer and the 5¢-intron fragment produced a more than additive transactivational effect on the promoter Similar to liver cells, the stimulatory effect of either element was largely lost if the other element was absent Furthermore, and again similar to liver cells, the effects of the upstream region and the entire intron were not additive These findings show that the upstream enhancer and the 5¢-intron modules are mutually dependent for full activity of the GS promoter in both liver and muscle cells Comparison of FTO-2B with C2/7 cells
The comparison of both cell lines revealed that the main activities of the upstream enhancer and 5¢-intron modules, as well as their interaction, were higher in C2/7 cells than in FTO-2B cells Both cell types resembled each other in that
Trang 5the upstream enhancer and 5¢-intron module had to be
simultaneously present for the highest level of reporter gene
expression, whereas significant negative interactions were
observed if either element was absent Apparently as a result
of the latter effect the upstream enhancer increased the
inhibitory effect of the middle intron module, but did not
support the stimulatory effect of the 3¢-intron module in both
cell types In C2/7, but not in FTO-2B cells, the upstream
enhancer and the 5¢-intron modules lost their stimulatory
activity in the context of the upstream region and the entire
intron, respectively As the presence of both the entire
upstream region and the entire intron negatively affected
promoter activity in both cell types, the 5.3-kb region
encompassing the entire upstream region and first intron,
was threefold less active in muscle than in hepatoma cells The differences between hepatoma and muscle cells therefore can be explained by tissue-specific differences in activity
of the respective regulatory elements due to interactions rather than in the use of distinct, tissue-specific regulatory elements
Glucocorticoid sensitivity of the regulatory sequences All modules were tested for sensitivity to glucocorticoid treatment Only constructs containing the middle intron module showed a threefold induction of reporter gene activity when tested in C2/7 cells (data not shown) In hepatoma cells, no effects of the hormone were observed
Fig 2 Main activity and interactions of GS upstream and intron modules in FTO-2B hepatoma and C2/7 muscle cells Main activities and interactions (±95% confidence interval) were calculated from the activities of constructs A and D-Q, using the ANOVA technique Main activities were expressed
as increase in activity upon addition to reference construct A, which was set at 100 arbitrary units Interactions were expressed as the difference between the observed activity of a construct and the main activities of its components Main activities and interactions marked in bold differ significantly (P < 0.05) from construct A and from 0 The activity of a construct (e.g I: FTO-2B: 220; C2/7: 430) can be calculated from the figure
as the sum of the basal activity (¼ 100), the main activity of the upstream element (UE; FTO-2B: 87; C2/7: 121), the intron element (5¢; FTO- 2B: 23; C2/7: 127) and their interaction (FTO-2B: 10; C2/7: 82).
Trang 6We have studied the capacity of the upstream region and
first intron of the GS gene to transactivate its promoter,
as well as the effect of interactions between these regions
on GS promoter activity Furthermore, we aimed to
determine if any of the regulatory elements in these
regions behaved differently in cells which can express high
levels of GS (hepatocytes) and in cells which do express
low levels of the gene (muscle) The application of the
ANOVA technique allowed us to segregate the main
(intrinsic) activities of the respective elements from the
effects of their interactions Using this approach, the
upstream enhancer was identified as a strongly stimulatory
element in both hepatoma and muscle cells, whereas the
5¢-intron module was strongly stimulatory in muscle cells
only In both cell types, however, the upstream enhancer
and 5¢-intron module depended on each other for effective
transactivation of the GS promoter Other intriguing
findings were that the upstream enhancer lost most of
its activity when present in the context of the entire
upstream region in muscle cells, but not in hepatoma cells
Furthermore, the inhibitory effect of the middle intron
module appeared to be constitutive in hepatoma cells, but
dependent on glucocorticoids in muscle cells Apparently,
both positive and negative elements, and extensive
inter-actions between them, regulate GS promoter activity
In addition to transcriptional control, translatability of
the GS mRNA and stability of the GS protein appear to
be important post-transcriptional levels of control [1,22]
In fact, we did show that it is necessary to consider these
post-transcriptional control levels when analysing the
expression of GS in transgenic animals [23,24] However,
as both the reporter gene and the transcription
termin-ation and polyadenyltermin-ation sequences that were used are
not normally expressed in either liver or muscle,
post-transcriptional control is an unlikely level of control to
explain the observed differences between hepatoma and
muscle cells
The similarity of the activity of constructs A, B and C
argues against the presence of an inhibitory sequence within
the upstream region Nevertheless, the activity of the
upstream enhancer in muscle cells and, to a lesser extent, in
hepatoma cells, is clearly mitigated in the context of the entire
upstream region, i.e by the interposition of 1780 bp We
interpret the increase in activity of the upstream enhancer
when positioned in close proximity to the promoter as the
consequence of a distance effect (see [25,26]): apparently, the
upstream enhancer has difficulty contacting the promoter
when the 1780 bp intervene We have previously observed
such a distance effect for the carbamoylphosphate
syn-thetase enhancer in conjunction with the TK promoter, but
not with the carbamoylphosphate synthetase promoter
alone [26]
The 5¢ and middle intron modules of the first intron of GS
correspond tp two DNaseI-hypersensitive sites [5] Three
studies [6–8] have analysed the enhancer activity of these
modules in conjunction with the heterologous TK promoter
[27] The 5¢ intron module behaves as a conditional enhancer
element when positioned downstream of the promoter (this
study), but as a constitutive stimulatory element when tested
upstream of the TK promoter [6] In this configuration, the
activity of the 5¢ intron element resided between positions +153 and +627 [6] In contrast, the strong and consistently inhibitory effect of the middle intron module on GS promoter activity does not appear to be context sensitive,
as it was also observed when tested upstream of the TK promoter [8] This inhibitory activity resided in a 325-bp fragment (position +1466 to +1791) and was, similar to our finding, relieved by glucocorticoids [8] A putative GRE (glucocorticoid-responsive element) was identified at posi-tion +1656 to +1670 The middle intron regulatory element
in the mouse GS gene [7] was tested in stable transfection assays in a differentiating adipocyte cell line Its core activity was found to be limited to a 310-bp fragment, the sequence of which corresponds with that of position +1450 to +1752 in the rat GS intron This sequence was found to contain C/ EBP and HNF3 consensus-binding sites at position +1580
to +1592 The middle intron regulatory module may therefore qualify as a glucocorticoid-responsive unit (see [28]) The presence of an inhibitory GRU (glucocorticoid-responsive unit) in the GS gene and a strongly stimulatory one in the carbamoylphosphate synthetase gene [28] may explain the frequently reciprocal behaviour of both genes with respect to expression [13]
Co-operative interactions in the binding of transcription factors to arrays of response elements within an enhancer module appear to be the rule rather than the exception The explanation for these co-operative effects is that the binding
of a factor to an element within such an array entails an increase in the affinity of adjacent elements for their corresponding transcription factors Due to the presence
of protein–protein interactions within an enhancer–promo-ter complex, the transcription factor-binding sites do not have to be adjacent [29,30] However, co-operative interac-tions between distant enhancer modules as now reported for
GS are described infrequently Reported examples include the synergistic interaction between a far-upstream and an upstream enhancer [31], between an upstream and an intron enhancer [32,33], and between an intron and a downstream enhancer [34] Interestingly, the GS gene itself may present yet another example of an interaction between distant regulatory modules, as both the )6.0 kb far-upstream enhancer and the middle intron element enhance reporter gene activity in stably transfected adipocytes [7,9] Notwith-standing this association, it remains to be shown that these two elements do indeed interact Whether the cooperative interactions between distant enhancers obey the same rules as observed for elements within a single enhancer and for enhancer–promoter interactions, remains to be established
In transgenic animals, the spatio-temporal expression pattern of a reporter gene that is driven by the GS upstream region, revealed several discrepancies between the expression
of the endogenous GS gene and the reporter gene [23,24] This finding suggested that one or more regulatory elements that were not present in this transgene, were responsible for the expression pattern of endogenous GS Furthermore, our modelling of gene expression patterns in the liver had predicted that an interaction between at least two regulatory elements was necessary to generate the remarkably stable expression gradient of GS [14] The present study has identified the upstream enhancer and the 5¢-intron module as two such interacting regulatory elements
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