©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Spatio-temporal Control of the Expression of Glutamine Synthetase in the Liver Is Mediated by Its 5`-Enhancer (*)

(Received for publication, June 5, 1995; and in revised form, September 22, 1995)

Heleen Lie-Venema (1) Wil T. Labruyère (1) Marian A. van Roon (2) Piet A. J. de Boer (1) Antoon F. M. Moorman (1) Anton J. M. Berns (2) Wouter H. Lamers (1)(§)

From the  (1)Department of Anatomy and Embryology, University of Amsterdam, Academic Medical Centre, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands, and the (2)Department of Molecular Genetics, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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.


MATERIALS AND METHODS

Manipulation of DNA and RNA

Standard techniques for isolation and the handling of DNA and RNA (17, 18) were used throughout this study.

Animal Care

Animals were housed with a 12-h light and 12-h dark cycle and permitted ad libitum access to water and a standard pellet-type diet. This study was performed in accordance with the Dutch guidelines for the use of experimental animals.

Generation and Screening of Transgenic Mice

Two constructs were used for the generation of transgenic mice (Fig. 1). Both constructs are based on the pCT1 vector, a pBluescript-based vector containing the promoterless CAT reporter gene in conjunction with the SV40 small T-antigen intron and polyadenylation signal optimized for expression(19, 20) . This reporter gene cassette is flanked by NotI restriction sites to allow isolation of the insert. The GSK construct contains 495 bp of the 5`-flanking region of the rat GS gene, including the basal promoter (from the BclI site at -495 bp to the Ksp632I site at +59 bp) cloned into the multiple cloning site of pCT1. The GSL construct contains the 5`-flanking region of the rat GS gene up to -3150 bp (from the PstI site to the Ksp632I site at +59 bp), including the 5`-enhancer and the basal promoter cloned into the multiple cloning site of pCT1. The construct DNA was injected into pronuclei of zygotes of FVB/N mice(21) , and the injected embryos were implanted into pseudopregnant foster mothers(22) . Initially, transgenic offspring was identified by Southern blot analysis of tail-tip DNA(23) . Later, a direct polymerase chain reaction on tail-tip lysates with primers specific for the SV40 sequence (SV40i, CAGGCATAGAGTGTCTGC and SV40pA, CTGGGGATCCAGACATGA) was used to analyze the offspring of the founders for the presence of the transgene. The polymerase chain reaction was performed in duplicate in a total volume of 50 µl containing 100 mM Tris-HCl (pH 9.0), 15 mM MgCl(2), 50 mM KCl, 0.1% gelatin, 1% Triton X-100, 0.1 unit of Taq polymerase (SuperTaq, HT Biotechnology Ltd., Cambridge, UK), 100 µM dNTPs, 10 pmol of each primer, 10% Me(2)SO, 0.05% bovine serum albumin, and 1 µl of the tail-tip lysate. The reaction mixtures were covered with mineral oil (Sigma, M-5940). The polymerase chain reaction was executed for 30 cycles, each consisting of the following steps: 92 °C for 1 min, 1 min at 57 °C, and 2 min at 72 °C and followed by a final extension for 5 min at 72 °C. The reactions were performed in 96-well roundbottom microtiter plates covered with an adhesive plastic in a PTC-100 thermocycler (MJ Research Inc., Watertown, MA). The copy numbers of the heterozygous transgenic animals were determined by Southern blot analysis of 5, 10, and 20 µg of tail-tip DNA digested with PstI. Unlike the tail-tip DNA used for the screening of the transgenic mice, the DNA used for the determination of the copy number was purified by extraction with phenol/chloroform/isoamylalcohol (25:24:1). Calibration standards consisted of 1-50 pg of the 1662-bp BamHI restriction fragment of pCT1 hidden in herring testis DNA.


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.



Chloramphenicol Acetyltransferase Activity Assay

CAT activity in the organs of the transgenic animals was determined in tissue homogenates, essentially as described by Seed and Sheen(24) . Organs (liver, total brain, epididymis, testis, brown adipose tissue (BAT) of the interscapular fat pad, kidney, spleen, lungs, abdominal muscle, and jejunum) were isolated and immediately frozen in liquid nitrogen. Homogenates were prepared at 0 °C by use of an Elvejhem type tissue homogenizer in 200 mM Tris-HCl (pH 8.0), 150 mM KCl, and 0.1% Triton X-100. After centrifugation at 4 °C in a microfuge for 10 min, total protein contents were measured in the supernatants using the BCA protein assay reagent of Pierce as indicated by the manufacturer. CAT activity-interfering enzymes were heat-inactivated for 30 min at 70 °C. This heat inactivation was important for the accurate measurement of the CAT activity, especially in liver homogenates. Afterward, the homogenates could be flash-frozen in liquid nitrogen and stored at -80 °C until use. CAT activity of 100 µg total protein was determined at 37 °C in a buffer containing 250 µM butyryl-CoA (Sigma B1508), 100 mM Tris-HCl (pH 8.0), 100 mM chloramphenicol, and 30-60 pM [^3H]chloramphenicol (DuPont NEN). At a temperature of 37 °C, the reaction time was 2 h for the liver, brain, and epididymis of the homozygous GSL transgenic mice and 5 h for all other tissues. Linearity of enzyme activity with protein content and reaction time for this period was demonstrated in preliminary tests. The reaction was stopped by mixing with 4 volumes of xylene. The organic phase was extracted twice with 100 µl of TE, and the radioactivity of the organic phase was counted in a liquid scintillation counter for 1 min. The detection limit for CAT activity was defined as three times background radioactivity, which was 50 cpm (equivalent to approximately 0.05 milliunits). Calibration curves for the amount of enzyme present in the samples consisted of 0-50 milliunits of CAT (Sigma) dissolved in 100 mM Tris-HCl (pH 8.0) and supplemented with 100 µg of heat-inactivated total liver protein from a negative Swiss mouse. Calibration curves prepared with 100 µg of heat-inactivated total protein of tissues other than liver showed the same percentages of chloramphenicol conversion as those prepared with liver protein. CAT activity is expressed in pmol product/minbulletmg protein at 37 °C

Glutamine Synthetase Activity Assay

The activity of the endogenous GS was determined by measuring the -glutamyltransferase activity, basically as described by Levintow (25) . The homogenates were prepared as described for the CAT activity assay, with omission of the heat-inactivation step. GS activity of 100 µg total protein was assessed in a reaction mixture containing 50 mML-glutamine, 160 µM ADP, 25 mM hydroxylamine, 25 mM sodium arsenate, 50 mM imidazole, and 1.5 mM MnCl(2)bullet4H(2)O at 37 °C. The reaction was stopped after 15-120 min, depending on the GS activity of the sample measured in a preliminary test, with 2.5 volumes of 90 mM FeCl(3)bullet6H(2)O, 2.2% HCl, and 7.2% perchloric acid. A calibration series was prepared with 0.25-10 µmol of -glutamylhydroxamate dissolved in the homogenization buffer. After centrifugation, the absorbance at 520 nm was determined spectrophotometrically. GS activity is expressed in nmol of -glutamyl hydroxamate/minbulletmg protein at 37 °C.

In Situ Hybridization

Fixation of freshly isolated liver tissue was done overnight at 4 °C in 4% (w/v) paraformaldehyde in phosphate-buffered saline (10 mM sodium phosphate (pH 7.4), 150 mM NaCl). The tissue was dehydrated in a series of ethanol followed by a final dehydratration step in butanol for 18 h. Subsequently, the tissue was embedded in paraffin, and serial sections of 7 µm in thickness were prepared. Serial liver sections were probed for the presence of CAT and GS mRNAs by in situ hybridization with the respective S-labeled cRNAs according to laboratory protocols(26) , with a few modifications (27) . The [S]UTP-labeled GS cRNA was transcribed from the 700-bp 5`-EcoRI fragment of the mouse cDNA in pGEM1(28) , linearized with PvuII. To prepare CAT cRNA, a 1065-bp fragment (Ksp632I-HindIII) of pCT1, encoding the CAT gene, was subcloned into pBluescript. Double-labeled antisense cRNA ([S]UTP and [S]CTP) was transcribed from this fragment after linearization with Asp718. The in situ hybridization was followed by exposure to an autoradiographic emulsion (Ilford Nuclear Research Emulsion G-5) for 6 days and development for 8 min, unless stated otherwise.

Preparation of RNA

Total RNA was isolated from the liver of the transgenic mice by use of the single-step RNA isolation procedure of Chomczynski and Sacchi(29) , modified by additional removal of residual DNA by precipitation in 2 M LiCl for 18 h at 4 °C(18) . Following centrifugation at low speed (2000-4000 rpm in a microfuge), the pellet was washed twice with 70% ethanol, air-dried, and dissolved in water containing 0.1% SDS.

Ribonuclease Protection Analysis

The RPAII ribonuclease protection assay kit of Ambion Inc. (Austin, TX) was used as instructed by the supplier. The [P]UTP-labeled antisense RNA probe was transcribed in vitro with T7 polymerase in a total volume of 10 µl from a pBluescript-based subclone of the GSK construct, which contained the restriction fragment from the StyI site at -70 bp of the GS promoter to the EcoRI site at +250 bp of the CAT sequence. The CAT-specific RNA probe was 446 nucleotides in length.


RESULTS

Transgenic Lines, Copy Numbers, and Expression of the Reporter Gene in the Liver

In vitro, the 5`-enhancer of the GS gene was previously demonstrated to be more active in HepG2 hepatoma cells than in mouse embryonic fibroblasts (16) . To investigate whether this 5`-enhancer also showed tissue-specific activity in vivo and, in particular, whether it could direct the expression of a reporter gene to the pericentral hepatocytes in the liver, two classes of transgenic animals were generated. In GSK mice, carrying the GSK construct, the basal GS promoter up to -495 bp directs the expression of the CAT reporter gene. In mice carrying the GSL construct, the complete 3-kilobase upstream regulatory region drives the expression of the reporter gene. Both constructs are depicted in Fig. 1. Initially, nine GSL transgenic founder mice and five GSK founders were available. As can be seen in Table 1, the carriers of the GSK construct showed hardly any expression of the CAT reporter gene. In contrast, the addition of the upstream regulatory region, including the 5`-enhancer, resulted in a 125-175-fold increase in reporter gene expression in the three GSL lines that showed the highest liver CAT expression. Only the lines that expressed CAT at a relatively high level were further analyzed. The copy number of their inserts is indicated in Table 1. No clear relation between the copy number of the inserts and the level of CAT expression in the liver was found. GSK line 12, being the only GSK line available with detectable CAT activity in the liver, and GSL lines 1 and 7 were bred to homozygosity. When compared to heterozygous mice, homozygous transgenic mice showed twice as much CAT activity in the liver (Table 1).



Tissue Distribution of the Expression of the CAT Reporter Gene in GSL and GSK Transgenic Mice

To investigate whether the upstream regulatory sequences of the GS gene, including the 5`-enhancer, give rise to an organ-specific expression of the reporter gene, CAT activity was determined in several organs of the two GSL transgenic lines and in the single GSK transgenic line that were bred to homozygosity. Organ-specific expression was determined by comparing the CAT activity with the endogenous GS activity in the same organ. No differences with respect to the expression levels of GS in the various organs were found between the two GSL transgenic mouse lines, the single GSK line and non-transgenic Swiss mice, showing that the presence of the transgenic constructs did not interfere with the endogenous GS expression (results not shown). Fig. 2A shows that the highest GS expression was found in the epididymis, followed by the brain and the liver. The CAT activity in tissues of the GSK mice did not exceed a low basal level of about 0.5-1 milliunit per mg total protein, except for a slightly higher expression of CAT in the brain (6 milliunits of CAT/mg of protein). In the epididymis, interscapular BAT, and lungs of GSK mice, CAT activity was not detectable. In contrast, high levels of CAT activity were found in the epididymis and in the liver of the transgenic mice carrying the GSL construct. In the brain of GSL mice, the CAT activity was 3-fold lower than in the liver and epididymis. A perhaps more relevant way to assess the organ specificity of the expression of the transgene is to compare its activity with that of the endogenous GS in the same organ. The ratio between CAT and GS activity in the liver was arbitrarily set at 1. Using this ratio, three different groups of organs could be identified in the GSL mice (Fig. 2B). The first group included organs with ratios varying between 0.3 and 1.0. In the second group of organs the ratios between the CAT and the GS activity was less than 0.1, while in the third group this ratio was 4.3.


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.''



Site of Transcription Initiation of the GSL and GSK Constructs in Homozygous Transgenic Lines

Upon injection into the male pronucleus, the heterologous DNA integrates at random in the genome of the pronucleus. Therefore, a promoter, which might influence the expression of the transgene, could be located upstream of the integration site of the construct. To determine the transcription start site of the CAT reporter genes, total liver RNA from the GSK line 12 and the GSL line 1 and line 7 mice were analyzed by ribonuclease protection. In Fig. 3, it can be seen that in both GSL lines, the liver CAT mRNA is transcribed from position +1 of the rat GS sequence in the construct. In the liver RNA of GSK mice, two protected fragments were found, one corresponding to transcription initiation at position +1 and one corresponding to transcription initiation upstream of the GSK construct (this site was not found in GSL mice). This finding shows that in the GSK mice both the basal GS promoter and an upstream cryptic promoter are responsible for the transcription of the reporter gene.


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.



Localization of the CAT mRNA in GSL and GSK Transgenic Mice

To gain further insight into the role of the 5`-regulatory regions of the GS gene with respect to its characteristic pericentral pattern of GS expression in the liver(3, 4, 5, 14) , the localization of the expression of the transgenes was examined by in situ hybridization. In all three GSL lines analyzed, the CAT reporter gene was expressed exclusively in a small and distinct pericentral zone. As can be seen in Fig. 4, the expression pattern of the transgenic mRNA was virtually identical to that of the endogenous GS mRNA in the GSL lines 1 and 7. Only in the livers of GSL line 9 mice, the expression of the CAT mRNA seemed to extend somewhat outside the GS-positive boundaries. In the livers of the sole GSK line that expressed the CAT gene, very low levels of CAT mRNA were detected in a large periportal zone of hepatocytes (Fig. 5).


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.



Expression of the CAT Reporter Gene during Development

To establish whether the GS upstream regulatory sequences are also responsible for the regulation of the developmental changes in GS expression in the liver(30) , GS and CAT activity were measured in GSL transgenic mice ranging in age from 2 days before birth to 2 months after birth. From Fig. 6, it can be seen that the developmental increases in CAT activity in the livers of GSL line 1 and line 7 mice coincide with those in GS activity. For both enzymes, upsurges in activity were found perinatally and from 12 neonatal days onward. Adult levels of enzyme activity were reached about 3 weeks after birth for both GS and the reporter gene product. The more pronounced decrease in CAT activity in the first neonatal week compared to that in GS activity most probably results from differences in the stability of both enzymes and of their mRNAs. Whereas the half-life of GS protein in the liver is approximately 5 days(31) , that of CAT amounts to approximately 2 days in mammalian tissues(32, 33) .


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/minbulletmg 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.




DISCUSSION

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. (^2)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/EBPalpha) consensus site. The finding that the C/EBPalpha 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 31-20-5664927; Fax: 31-20-6976177.

(^1)
The abbreviations used are: GS, glutamine synthetase (EC 6.3.1.2); CAT, chloramphenicol acetyltransferase (EC 2.3.1.28); BAT, brown adipose tissue; bp, base pair(s).

(^2)
V. Christoffels and W. H. Lamers, unpublished results.


ACKNOWLEDGEMENTS

J. B. Daalhuisen and D. V. M. Klappe-Banse took care of the transgenic mice; J. A. M. Korfage prepared the serial sections of the liver. C. E. Gravemeijer, C. J. Hersbach, and A. van Horssen-Medema made the photographs and illustrations. L. de Vijlder helped with the experiments concerning the developmental profiles. We gratefully acknowledge their contributions to this publication. We thank Prof. R. Charles for critically reading the manuscript.


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