(Received for publication, June 5, 1995; and in revised form, September 22, 1995)
From the
In previous studies of the glutamine synthetase gene, the promoter and two enhancer elements, one in the upstream region and one within the first intron, were identified. To analyze the role of the far-upstream enhancer element in the regulation of the expression of the glutamine synthetase gene, two classes of transgenic mice were generated. In GSK mice, the basal promoter directs the expression of the chloramphenicol acetyltransferase reporter gene. In GSL mice reporter gene expression is driven, in addition, by the upstream regulatory region, including the far-upstream enhancer. Whereas chloramphenicol acetyltransferase expression was barely detectable in GSK mice, high levels were detected in GSL mice. By comparing chloramphenicol acetyltransferase expression with that of endogenous glutamine synthetase in GSL mice, three groups of organs were distinguished in which the effects of the upstream regulatory region on the expression of glutamine synthetase were quantitatively different. The chloramphenicol acetyltransferase mRNA in the GSL mice was shown to be localized in the pericentral hepatocytes of the liver. The developmental changes in chloramphenicol acetyltransferase enzyme activity in the liver were similar to those in endogenous glutamine synthetase. These results show that the upstream region is a major determinant for three characteristics of glutamine synthetase expression: its organ specificity, its pericentral expression pattern in the liver, and its developmental appearance in the liver.
In the mammalian liver, many enzymes are heterogeneously
distributed according to a pattern that is related to the vascular
architecture (reviewed in (1) and (2) ). Two key
enzymes involved in ammonia metabolism, glutamine synthetase (GS) ()and carbamoylphosphate synthetase, provide a good example
of this zonal heterogeneity in gene expression. In the liver of adult
rats and mice, GS is expressed in a thin rim of cells around the
central veins(3, 4, 5) , whereas
carbamoylphosphate synthetase is expressed in a wide zone surrounding
the terminal portal veins(6) . Therefore, these two enzymes
provide an excellent model to study the topographical aspects of the
regulation of gene expression. The establishment and maintenance of the
complementary distribution patterns of GS and carbamoylphosphate
synthetase in the liver have been studied for many years. The signals
that have been considered to be involved in the determination of the
pericentral expression pattern of GS include the vascular architecture
of the liver (7) , the position of the hepatocyte on the
porto-central axis and blood-borne factors(8, 9) , the
innervation pattern(10) , the extracellular matrix and
cell-cell interactions(11, 12) , and different cell
lineages(13) . However, so far none of these potential
determinants could be unambiguously related to the regulation of the
highly specific and very stable pattern of GS expression.
Even less is known about the intracellular signal-transduction pathway that determines the expression of GS. By in situ hybridization it was shown that the localization of the GS mRNA within the liver lobule is identical to that of the GS protein, indicating that the regulation of the position-specific GS gene expression is exerted at the pretranslational, and probably at the transcriptional, level(14) . Since knowledge of the gene structure is a prerequisite for the understanding of the molecular mechanisms that underlie the topographical aspects of GS gene expression, we previously cloned and characterized the rat GS gene(15) . More recently, we characterized the regulatory regions of the GS gene by transient transfection analysis(16) . It was found that the GS gene contained two enhancer elements: one far-upstream enhancer between -2520 and -2148 bp and another within the first intron between +259 and +950 bp from the transcription start site. To investigate the role of the far-upstream enhancer in the establishment and maintenance of the very stable expression pattern of GS, two classes of transgenic mice were generated: ``GSK'' mice in which the basal GS promoter directed the expression of a reporter gene and ``GSL'' mice in which the expression of the reporter gene was driven by the basal promoter and by the upstream regulatory region, including the far-upstream enhancer. Three characteristics of GS gene expression were used to determine the in vivo function of the upstream regulatory region of the GS gene: the organ specificity of gene expression, the topography of gene expression within the liver, and the developmental changes in enzyme activity in the liver. By these criteria, the upstream regulatory region was found to play a major role in the regulation of the GS gene expression in the liver.
Figure 1: Schematic drawing of the constructs that were used for the generation of transgenic mice. In the GSK construct, the basal promoter, including sequences up to -495 bp and extending to position +59 bp of the first exon (black), is responsible for the transcription of the CAT reporter gene (white). In the GSL construct the entire upstream regulatory region of the GS gene, from -3150 to +59 bp (black), was cloned in front of the CAT gene. To both constructs, the SV40 small T-antigen intron and polyadenylation sequences (gray) were added for optimal mRNA processing. The position of the far-upstream enhancer element is indicated by an asterisk.
Figure 2: Organ specificity of the expression of the CAT reporter gene in vivo in homozygous GSL and GSK mice. A, GS and CAT activity in the epididymis (EP), brain (BR), liver (LI), interscapular BAT (IB), testis (TE), kidney (KI), spleen (SP), lungs (LU), abdominal muscle (MU), and jejunum (JE) of the GSL (line 1, n = 3; line 7, n = 3) and GSK mice (n = 3). GS activity, black bars; CAT activity in GSL line 1, hatched bars; in GSL line 7, white bars; in GSK line 12, cross-hatched bars. An asterisk indicates that no CAT activity could be detected. B, ratio of CAT activity to GS activity in the same tissues of the transgenic mice (mean of GSL line 1 and GSL line 7). The CAT/GS activity ratio in the liver was arbitrarily set at 1. All animals were 12 weeks of age. CAT and GS activities were measured as described under ``Materials and Methods.''
Figure 3:
Analysis of the transcription-initiation
site of the transgenic mRNA. RNase-protection analysis of the CAT mRNAs
transcribed from the GSK and GSL constructs in the transgenic mice was
used to establish the transcription-initiation site of the transcripts.
A [P]UTP-labeled cRNA probe of 446 nucleotides (a) was used to protect a part of the CAT mRNAs transcribed
from the transgenic constructs. Lanes 1-3 show the
protected fragments in 10 µg of total liver RNA isolated from a
homozygous GSL line 1 mouse, a heterozygous GSL line 7 mouse, and a
homozygous GSL line 7 mouse, respectively. The protected fragments of
295 and 298 nucleotides (c) show that in these two GSL lines,
transcription of the CAT mRNA was correctly initiated by the GS
promoter present in the construct. In lane 4, the protected
fragments in 20 µg of the total liver RNA of a homozygous GSK mouse
were shown to be 373 nucleotides (b) and 295-298
nucleotides (c) in length, showing that both the GS promotor
of the construct as well as an upstream cryptic promoter are involved
in the transcription of the CAT reporter gene. M, the
molecular weight marker.
Figure 4:
Localization of the CAT mRNA in the liver
of GSL mice. Photomicrographs of serial sections of the liver of the
GSL transgenic mice, hybridized with either S-labeled
GS-specific cRNA (A, C, E) or with
CAT-specific cRNA (B, D, F). A and B, homozygous GSL line 1 mouse liver; C and D, homozygous GSL line 7 mouse liver; E and F, heterozygous GSL line 9 mouse liver. PV, portal
vein, identified by the presence of carbamoylphosphate synthetase mRNA
in the surrounding hepatocytes (not shown); CV, central vein,
identified by the presence of GS mRNA in the surrounding hepatocytes; bar = 100 µm.
Figure 5:
Localization of the CAT mRNA in the liver
of a GSK mouse. Serial sections of a GSK mouse liver were hybridized
with S-labeled cRNA probes as described in Fig. 4. A, GS mRNA expression; B, CAT mRNA expression; and C, carbamoylphosphate synthetase mRNA expression to delineate
periportal regions. Exposure and development of the photographic
emulsion were for 11 days and 4 min, respectively. PV, portal
vein; CV, central vein; bar, 100
µm.
Figure 6:
Comparison of the developmental changes in
CAT and GS enzyme activities in the liver of GSL mice. Developmental
profiles of the GS activity (open circles, continuous line)
and the CAT activity in the GSL transgenic lines 1 (closed
triangles, dashed line) and 7 (closed circles, dotted line) expressed as % of the adult enzyme activity.
Perinatally and from 12 neonatal days onward, the GS and the CAT
activity increase coordinately. GS and CAT activity are expressed as %
of the adult enzyme levels in the liver (adult GS activity =
465.3 ± 12.8 nmol -glutamylhydroxamate/min
mg protein;
adult CAT activity = 286.7 ± 7.6 milliunits/mg protein
for GSL line 1; adult CAT activity = 305.1 ± 13.9
milliunits/mg protein for GSL line 7). All mice were homozygous. Three
to nine samples were measured per time
point.
To investigate the role of the upstream regulatory region in
the spatio-temporal regulation of the highly characteristic expression
pattern of the GS gene, we studied two different transgenes, viz. one in which the expression of the CAT reporter gene was
directed by the basal promoter (GSK) and one in which the CAT
expression was directed by the basal promoter and 3 kilobases of the
upstream regulatory region (GSL). In contrast to findings obtained from
transient transfections(16) , the expression levels of the
reporter gene when driven by the basal promoter were hardly detectable in vivo. Similar results were found in transgenic animals in
which the basal promoter of the carbamoylphosphate synthetase gene was
used to drive the expression of a reporter gene. ()Probably,
the prerequisites to initiate transcription from the basal promoter
element are met more efficiently in transformed cells than in the cells
of an intact organ. The presence of the upstream enhancer element
increased the CAT activity in the liver up to 175-fold compared to the
transgenic construct carrying the basal promoter.
It was possible to establish the role of the upstream regulatory region with respect to the organ specificity, position specificity within the liver, and developmental changes in GS gene expression by comparing the expression of the transgene with that of the endogenous GS.
On the basis of the ratio of CAT and GS activity in GSL mice, three groups of organs could be distinguished. The first group, which is represented by the liver, epididymis, brain, and lungs, is characterized by a relatively high expression of both CAT and GS. In the second group of organs (interscapular BAT, testis, and kidney), the CAT to GS ratio is 3-10-fold lower than in the first group, whereas in the third group (spleen, muscle, and jejunum), the CAT to GS ratio is about 4-fold higher than in the first group. Apparently, the expression of the GS gene is differentially regulated in these three groups of organs. In the first group of organs, relatively high expression levels of both the endogenous GS and the transgenic CAT gene indicate that the upstream regulatory region is mainly responsible for the expression of the GS and CAT gene. In the second group of organs, the low CAT to GS ratios suggest that the GSL construct lacks a positive regulatory element that is responsible for the relatively high levels of GS in these organs. This positive regulatory element might well be the enhancer element found in the first intron of the GS gene(16) . Conversely, the GSL construct induces relatively high levels of CAT expression in the third group of organs. This finding suggests that the construct misses a negative regulatory element of the GS gene that acts in these tissues. Because the level of CAT activity shows that the far-upstream enhancer functions fairly well in these organs, the activity of this negative element does not directly affect the functioning of either the far-upstream regulatory region or the basal promoter. Possibly, this third element is involved in the regulation of GS mRNA or protein stability in these organs.
The expression of the CAT reporter gene in GSL mice was restricted to the pericentral cells of the liver lobule as was shown by in situ hybridization experiments. In contrast, very low levels of expression of the CAT reporter gene were found in a large periportal zone in GSK mice. The latter result shows that, in the absence of the upstream regulatory region, the CAT reporter gene expression is not directed to the pericentral zone of the liver lobule. These findings, therefore, unambiguously demonstrate that sequences within the upstream regulatory region of the GS gene direct its expression to the hepatocytes surrounding the central vein. In previous transient transfection analysis of the GS promoter(16) , we demonstrated that the -2500 to -2100 enhancer fragment confers hepatocyte specificity on the basal promoter. Because the sequences that separate the enhancer from the basal promoter have no enhancer activity and do not seem to contain well defined recognition sites for transcription factors(16) , the pericentral localization can probably be ascribed to the activity of the far-upstream enhancer. It should be noted that the pericentral region in which the GSL transgene is expressed seems to be somewhat wider and the gradient somewhat less steep than that of the endogenous GS. In this respect, it should also be mentioned that the levels of expression of the reporter gene are considerably lower than that of the endogenous GS, both at the mRNA (see Fig. 4) and at the protein level (e.g. in the liver, approximately 90 fmol of CAT and 60 pmol of GS is present per mg total protein). Possibly, additional elements in the GS gene contribute to these phenomena.
The shapes of the developmental profiles of the endogenous GS activity and of the transgenic CAT activity in the liver of the GSL transgenic mice that were found in the present study show great similarity. The upsurges in CAT activity coincided with those in GS activity and were in accordance with the upsurges in GS expression described for the developing rat liver(30) . The difference in the levels of GS and CAT activity between neonatal days 2 and 12 can probably be ascribed to the lower stability of the CAT protein and/or mRNA(31, 32, 33) . From these results we may conclude that the developmental changes in GS expression are governed largely by the upstream regulatory region of the GS gene, including the 5`-enhancer. Unfortunately, it was not possible to determine the developmental changes in the expression of the CAT activity in the GSK mice because of the extremely low CAT activity and CAT mRNA levels in these animals.
So far, little is known about the transcription
factor(s) that are responsible for the specific activity of the
regulatory elements in the GS gene and for the establishment and
maintenance of the strictly pericentral expression of GS. No consensus
sequences for the binding of known transcriptional activators could be
identified within the 5`-enhancer element, except for a GC box at
-2343 bp and a sequence at -2390 bp that shows a weak
homology to the CCAAT/enhancer binding protein alpha (C/EBP)
consensus site. The finding that the C/EBP
mRNA can be induced in
the pericentral hepatocytes by treatment with
dexamethasone(34) , a treatment that also increases the GS mRNA
levels(35) , may provide a clue to further elucidate the role
of the 5`-enhancer in the three features of GS expression described
above.
In summary, the upstream regulatory region of the GS gene has been shown to have a functional role in the organ-specific expression pattern of GS, in the strictly pericentral expression pattern of GS within the liver, and in the regulation of the developmental appearance of GS in the liver. To ascertain that the regulation of these three characteristics of GS expression can be ascribed to the far-upstream enhancer, transgenic mice carrying a reporter gene driven only by the basal promoter and the upstream enhancer, without the intervening sequences, are currently being generated.