aldolase b enhancer in vivo 1 in vivo functional characterization of

Aldolase B enhancer in vivo

1

In vivo functional characterization of the aldolase B gene enhancer

Claudine Gregori(1), Arlette Porteu(1), Claudia Mitchell(1), Axel Kahn(1),and Anne-Lise Pichard(1, 2)

1. Département de Génétique, Développement et Pathologie Moléculaire,Institut Cochin, INSERM, CNRS et Université René Descartes – 75014 –

PARIS – France

Running title : Aldolase B enhancer in vivo

Address reprint requests to: Institut Cochin, 24 rue du Faubourg St-Jacques, 75014 PARIS, FRANCE

Fax : (33) 1. 44. 41. 24. 21 email : pichard@cochin. inserm. fr

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 28, 2002 as Manuscript M204047200 by guest on A

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Summary

A 400-bp intronic enhancer fragment in conjunction with the proximal

promoter of the aldolase B gene provided correct tissue-specific expression in

transgenic mice together with hormonal regulation in the liver. We investigated

in vivo and in cultured cells the contribution of the intronic regulatory

sequences and their interaction with the promoter elements in controlling

aldolase B gene expression. Transgene activity was completely abolished by

disruption of the two HNF1 binding sites in the enhancer, whereas mutation of

one HNF1 site had no effect in the liver but strongly decreased activity in the

kidney. Our data show that the HNF1 binding site(s) in the enhancer were key

regulators of aldolase B transgene expression both in the liver and kidney.

Deletion of the C/EBP site in the promoter completely abolished the enhancer

function in HepG2 cells. These results suggest that expression of the aldolase

B gene in the liver requires cooperative interactions between C/EBP and

HNF1. Deletion of the HNF4 binding site in the enhancer suppressed

expression in both liver and kidney in half of the transgenic lines, suggesting

that this element might play a role in chromatin opening at the insertion site.

We firmly establish that the endogenous aldolase B gene’s first response to

glucagon or cyclic AMP exposure was a transient increase in the expression

in the liver, followed by a secondary decline in the transcription, as previously

reported. This response was reproduced by all transgenes studied, indicating

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that neither HNF1 nor HNF4 binding sites in the enhancer were involved in

this biphasic cyclic AMP response.


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Introduction

The aldolase B enzyme catalyzes the reversible cleavage of fructose-1-

phosphate into dihydroxyacetone phosphate and glyceraldehyde, and is

involved in two opposite metabolic pathways, glycolysis and gluconeogenesis.

The expression of the aldolase B gene is subject to tissue-specific, hormonal

and metabolic regulation. The adult liver expresses the aldolase B gene

exclusively, while the kidney and enterocytes co-express both aldolase A and

B genes (1). In the liver, transcription of the aldolase B gene is induced by a

carbohydrate-rich diet and inhibited by fasting and glucagon, while, in the

kidney, it is almost unresponsive to dietary and hormonal regulation (2).

However, even in the liver, the expression of the aldolase B gene is never

completely abolished. In addition, it is restimulated after prolonged starvation

(our unpublished data). This behavior may reflect the dual role of aldolase B in

hepatocytes where it is required for the opposite glycolytic and gluconeogenic

pathways.

Finally, hereditary fructose intolerance is a recessive genetic disease caused

by aldolase B deficiency. Repeated ingestion of noxious sugars by

homozygotes leads to hepatic and renal injury (3), with metabolic

disturbances (including low concentrations of blood glucose) that may prove

fatal (4).

The aldolase B gene proximal promoter (-194 to +14) is sufficient to direct

cell-type-specific expression in cultured hepatoma cells, but in mice a distal

intronic enhancer fragment (+1916 to +2324) is required for transgene


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expression (5). We have previously studied both these regions extensively (6-

9), and have identified the regulatory elements and cognate binding factors

(Fig.1, Fig.3). Their respective contributions to promoter activity and enhancer

function were investigated by transient CAT assays in HepG2 hepatoma cells.

Main findings were: the promoter activity was up-regulated by hepatocyte

nuclear factor 1 (HNF1) and CAAT/enhancer binding protein (C/EBP), but

repressed by hepatocyte nuclear factor 3 (HNF3) (7). The enhancer function

was abolished by mutation or deletion of either of the two HNF1 binding sites

and reduced by deletion of the HNF4 binding site (9).

Dietary regulation of a transgene directed by 1600 bp of the 5’ flanking region

of the aldolase B gene and the 1st intron appeared paradoxical: it was down-

regulated by a high carbohydrate diet and stimulated by prolonged fasting (5).

In order to explain these results we hypothesized that the endogenous gene

possessed distinct glucose-dependent and fasting-dependent responsive

elements, only the latter being present in the transgenic construct.

The objective of the current study was to assess, in a chromosomal in vivo

context, the contribution of the different DNA elements of the intronic

enhancer to the tissue-specific expression and metabolic regulation of the

transgenes.

Our results demonstrate a major contribution of the HNF1 enhancer binding

sites to the tissue-specific expression of the aldolase B gene. Deletion of the

two HNF1 binding sites suppressed transgene expression in both liver and

kidney. In contrast, mutation in one HNF1 site did not significantly affect


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transgene expression in the liver but resulted in a 100-fold loss of activity in

the kidney. Findings suggested that the differential role of the HNF1 sites in

the different organs could be related to the abundance of C/EBP in the liver

compared to the kidney. Indeed, we show that a C/EBP binding site previously

identified in the proximal promoter is crucial for maximal expression in

hepatocytes.

Deletion of the HNF4 site seemed to render the transgene activity more

dependent on the insertion site, silencing it completely in about half of the

transgenic lines. Finally, we demonstrate that transgene activation by fasting

could be mimicked by glucagon and dibutyryl cAMP, but we have so far failed

to delineate a cyclic AMP response element in the enhancer.


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Experimental Procedures

Plasmid constructions.

Aldolase B-CAT constructs used have been previously described (9). Fig. 1A

presents a diagram of the aldolase B-CAT gene, indicating the main cis-acting

elements and cognate DNA binding proteins. The enhancer fragment (+1916

to 2329) was maintained in an intronic position, the splice sites are indicated

by the bold brackets. Fig. 1B shows the microinjected constructs indicating the

locations of the block mutations or deletions performed in the aldolase B

enhancer. Fig. 3 schematizes various constructs with deletions or mutations in

the promoter. Here, the enhancer fragment was subcloned in the Cla I site

located downstream of the CAT gene (8).

Generation and analysis of transgenic mice.

The DNA constructs were digested with restriction enzymes Cla I (cutting in 3’

in the vector) and Hind III (cutting in 5’ in the plasmid linker). The fragments of

interest were isolated by electrophoresis, electroeluted and purified by using

elutip-d columns (Schleicher and Schuell), then microinjected into fertilized

mouse eggs according to Gordon and Ruddle, (10). The progeny was

analyzed for the presence of the transgene by Southern blot.

Cell culture and transient transfection.

HepG2 cells were grown in Dubellco’s modified Eagle medium (DMEM) in the

presence of 10% (v/v) fetal calf serum, 1 µM L-triiodothyronine, 1 µM


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dexamethasone, and 10 nM insulin, at 37° in 5% (v/v) CO2. Transfections

were carried out by the calcium phosphate method (11), under experimental

conditions previously described (9). In each experiment 7.5 µg of the CAT

plasmids and 2 µg of the luciferase plasmid were cotransfected. The pRSV

luciferase standardization plasmid was used to monitor variations in

transfection efficacy. The chloramphenicol acetyltransferase (CAT) assay (12)

and luciferase assay (13) were performed as described (8).

Hepatocytes in primary culture

Hepatocytes were isolated from the livers of adult transgenic mice carrying the

-232A100B400 wild-type transgene by perfusion with collagenase (14). Viable

hepatocytes were separated from other cells using isodensity Percoll

centrifugation (15). Hepatocytes (>95% viability) were seeded at a density of

3.5 x106 cells per dish (78 cm2). After cell attachment (4 h), the medium was

replaced by M199 medium with Earle’s salts (GIBCO/BRL) containing 20 mM

glucose, supplemented with 1 µM L-triiodothyronine, 1 µM dexamethasone,

10 nM insulin, at 37° in 5% (v/v) C02 for 3 days. The culture medium was

changed every 24 h. Cells were then cultured in the same media either with or

without 3.3 mg/L glucagon and 3.3 mg/L theophylline.

Dietary and hormonal control

Adult mice heterozygous for the transgene were used for analysis. One group

of mice was fasted 24 h prior to sacrifice; the other group was fasted 24 h


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then refed a high carbohydrate diet ad libitum for 18 h prior to sacrifice.

Organs were stored at - 80°C until use. To determine the effect of cyclic AMP

(cAMP) or glucagon, mice were either starved for 18 h or fed a high

carbohydrate diet ad libitum for 18 h, then injected intraperitoneally every hour

with either dibutyryl cyclic AMP (Bt2cAMP) (30 mg/kg of body weight), or

glucagon (0.5 mg/kg of body weight) with theophylline (30 mg/kg of body

weight), sacrificed after 2 or 4 h of treatment, then tissues were removed and

kept at - 80°C until use.

RNA isolation and Northern blot.

The mRNA extraction from liver, kidney and cultured hepatocytes was

performed using the RNAzol B reagent (Bioprobe systems) according to the

manufacturer’s instructions. Twenty µg of total RNA was electrophoresed

through a 1.5% (w/v) denaturing formaldehyde agarose gel and transferred to

Hybond N+ (Amersham). The cDNA probes were labeled by random priming

using a labeling kit, according to the manufacturer’s instructions. The CAT

cDNA probe was a Cla1-EcorR1 fragment of the PeCAT vector (5). The

murine aldolase B probe was a 379-bp fragment of cDNA, and the

phosphoenolpyruvate kinase (PEPCK) probe was a 1305-bp fragment of

cDNA (gift of Dr. B Antoine (16)).


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Results

In vivo role of the different enhancer elements.

Full and tissue-specific expression of the aldolase B transgenes results from a

cooperation between a proximal promoter and a distal intronic enhancer (5).

The enhancer activity is provided by a 400-bp sequence (+1916 to +2329)

containing binding sites for known liver-enriched nuclear factors HNF1 (+2212

to +2246 and +2275 to +2304), HNF4 (+2146 to +2184) and unknown factor

(+2195 to +2220). We analyzed the contribution of each of the cis-acting

elements to the expression of the aldolase B gene in vivo. For this purpose,

chimeric genes that contained the wild-type aldolase B promoter (-232A100)

linked to either wild-type or mutant aldolase B enhancer in its normal intronic

position, were ligated to the CAT gene (Fig. 1). Figure 2 shows the results of

the expression of the CAT transgenes directed by aldolase B gene regulatory

sequences in the liver and kidney. Expression of the aldolase B-CAT

transgenes was strictly restricted to the liver, kidney and small intestine (not

shown), no ectopic expression was detected in other tissues tested, e.g. brain

and lung (not shown). In agreement with prior data (5), the level of transgene

expression was totally independent of the copy number and strongly

dependent on the integration site. Among the six lines harboring the wild-type

aldolase B-CAT transgene, one was very weakly expressed. The HNF1-mut

transgene carries a 5 bp-block mutation of the 5’ HNF1 binding site (enhancer

element 5). In five lines with this construct, transgene expression was

conserved in the liver (although weak in two of the line) and was very low in


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the kidney, (a decrease on average of 100 fold compared to the wild-type

construct). Among 16 lines harboring a transgene with deletion of enhancer

element 7, i.e. the 3’ HNF1 binding site, only one expressed CAT activity in

the liver and, very weakly, in the kidney (Fig. 2). None of the 9 lines harboring

the transgene with deletion of both HNF1 binding sites (construct ∆ 5 to 8)

expressed CAT activity either in the liver or in the kidney (data not shown).

Twelve lines harboring a ∆2 transgene with a deletion of the HNF4 binding

site (enhancer element 2) were generated. In 5 lines, transgene expression

was undetectable; in one it was weak and in the other 6 it was strong. Finally,

expression of a transgene with deletion of enhancer element 4, which binds a

liver-enriched but non-characterized factor (9), was much higher than that of

the wild-type transgene and had a high CAT activity in both liver and kidney in

4 lines and a lower activity in 1 line (Fig. 2).

Hypothetical role of C/EBP in the differential effect of HNF1 site mutations in

the liver and kidney.

The differential effect of the 5’ HNF1 binding site deletion from the enhancer

on transcriptional activity in different cells and tissues could reflect tissue-

specific differences in transcription factor content. We focused on transcription

factors previously demonstrated to be active on aldolase B gene expression.

The C/EBP factor was a good candidate since, its concentration is high in the

liver, low in HepG2 cells and almost zero in the kidney (17). In addition,

C/EBP like HNF1 are potent transactivators of the aldolase B promoter (7). If


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our hypothesis is correct, over-expression of C/EBP in HepG2 cells should

restore transcriptional activity of constructs with mutation or deletion of one of

the enhancer HNF1 binding sites. Indeed, when HepG2 cells were transiently

cotransfected with the wild-type aldolase B-CAT plasmid and a C/EBP

expression vector, CAT activity was stimulated only 5 fold. In contrast, under

the same conditions, the HNF1-mut and ∆7 constructs were stimulated about

200 fold (Table 1). Next, we wanted to ascertain whether the C/EBP binding

site (-170 to -140) in the aldolase B promoter mediated the observed C/EBP

effect. In fact, a construct devoid of the enhancer sequence and with deletion

of the C/EBP binding site in the promoter (-140A100) responded 10-fold less

to C/EBP than the –232A100 promoter (Table 1). We think that the residual

transactivation was due to promiscuous binding properties of C/EBP, able, at

high concentration, to bind to degenerated, low-affinity sites. Finally, in the

absence of the C/EBP element in the promoter (140A100B400wild) the

enhancer activity was abolished.

These results point to a specific role of C/EBP in the cooperation between the

promoter and the intronic enhancer of the aldolase B gene. In order to

determine whether C/EBP bound to promoter must specifically interact with

other contiguous factors to cooperate with the enhancer, we studied the CAT

activity of a series of new constructs transfected into HepG2 cells (Fig. 3).

These constructs included from 5’ to 3’ the promoter fragment, the CAT gene

and the enhancer. Fig. 3 shows that the enhancer stimulated the activity of the

190 bp wild-type promoter 30 fold.


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Deletion of the HNF4 binding site from the promoter did not change this

stimulation. Deletion or mutation of the PAB element suppressing binding of

either HNF1 or HNF3, or both HNF1 and HNF3 seemed rather to increase the

stimulatory effect of the enhancer. Finally, as expected, all mutants devoid of

the promoter C/EBP binding site were weakly sensitive to the enhancer

action. When the PAB element was replaced by a high affinity HNF1 the

enhancer lost practically all stimulatory activity, most likely because the

C/EBP/HNF1 interaction between promoter and enhancer is replaced by the

same type of interaction inside the promoter.

Dietary regulation of the transgenes.

Northern blot analysis of transgene expression as a function of diet was

performed on at least two different mouse lines for each transgene.

Feeding mice a high carbohydrate diet stimulates aldolase B gene

transcription in liver, while starvation decreases its expression. ((2) and Fig.4).

Prior transgenic analysis indicated that a transgene with 1600 bp of the 5’

flanking sequence and the whole 1st intron responds to diet in an opposite

manner (5). Northern blots in Fig.4 demonstrate that exactly the same

expression pattern was reproduced using the “wild-type” construct with only

232 bp of the promoter and the 400 bp intronic enhancer. HNF1 mut, ∆2 and

∆4 transgenes were also induced in mice fasted for 24 h and inhibited in mice

refed a high carbohydrate diet. Therefore, the investigated enhancer


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elements do not seem to be implicated in the long-term dietary regulation of

the aldolase B gene in the liver.

The dietary response of the aldolase B transgenes resembles that of

neoglucogenic genes such as the PEPCK gene. Since such genes are known

to be responsive to glucagon and cAMP, we tested the effects of the hormone

and of cAMP analogues on the expression of the –232A100B400wild-CAT

transgene. Figure 5A shows that Bt2 cAMP induced parallel accumulation of

PEPCK and CAT transgenic mRNA 2 and 4 h after injection into

carbohydrate-fed mice. In fasted mice, these messengers were already

abundant before Bt2cAMP treatment, consequently the Bt2cAMP effect was

weak or nil (Fig. 5B).

As expected, the effect of Bt2cAMP in stimulating the transgene was

mimicked by glucagon (Fig. 5C). The response to glucagon was conserved for

the ∆2 and HNF1-mut transgenes, characterized by a deleted HNF4 binding

site and a mutated HNF1 binding site, respectively (not shown). Although the

endogenous aldolase B gene has been described rather as a typical

“glycolytic gene”, up-regulated by carbohydrates and down-regulated by

fasting and glucagon (2), the results observed with the aldolase B-CAT

transgenes prompted us to reevaluate more precisely the response of the

endogenous gene to glucagon. Fig. 5C shows that, in fact, endogenous

aldolase B mRNA accumulated 2 and 4 h after glucagon administration to

carbohydrate-fed animals, in parallel with accumulation of the PEPCK mRNA.


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In primary hepatocytes isolated from the liver of mice bearing the

-232A100B400wild-CAT transgene and cultured for 3 days in the presence of

25 mM glucose and 20 nM insulin, addition of glucagon led first to aldolase B

mRNA accumulation, with a peak at 3 h and thereafter a decline. Aldolase B,

and PEPCK mRNA inductions were parallel. In contrast, the response of the

aldolase B-CAT transgene was delayed, with a peak at 24 h (Fig. 6).


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Discussion

Tissue-specific transcription of the aldolase B gene is governed by a short

200-bp promoter stimulated by a 400-bp intronic enhancer (9), (5). In previous

studies using transient transfection assays, both the promoter and the

enhancer were shown to be cell-specific (18), (9). The nuclear factors mainly

involved in liver-specific gene transcription include HNF1 (19), HNF3 (20),

HNF4 (21), HNF6 (22), C/EBP (23) and DBP (24). Both the aldolase B gene

promoter and the enhancer fragments contain HNF1 and HNF4 binding sites;

in addition, a binding site for C/EBP is present in the promoter. Therefore the

aldolase B gene was an attractive model for identifying, in vivo, the promoter

and enhancer elements necessary for tissue-specific, dietary-regulated gene

expression.

In the present study we have shown that mutation of the 5’ HNF1 binding site

in the enhancer, preventing binding of HNF1, reduced expression of the

transgene (-232A100B400HNF1mut) in the kidney but did not alter its

transcription in the liver. A similar role for HNF1 in the differential control of

PEPCK gene transcription in the liver and kidney has been reported (25). In

contrast, the aldolase B gene was normally transcribed in the liver (26) and in

the kidney of HNF1 knock-out mice (Pontoglio & Yaniv, personal

communication). However, these results are not inconsistent with our own

data since the two isoforms HNF1α and HNF1β, binding to the same element,

are coexpressed in the liver and the kidney, HNF1β being increased by a

compensatory mechanism in HNF1α -/- (26).


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Deletion of the 2nd HNF1 site in the enhancer (element 7) led to an inactive

transgene in 11 out of the 12 lines tested. In the only line expressing the –232

A100B400∆7 transgene, expression was weak, but easily detectable in the

liver and almost nil in the kidney, which confirms that here again abrogation of

the HNF1 binding site has a more severe effect in the kidney than in the liver.

Since the mutation in the 1st HNF1 site changed 5 bp while deletion of the 2nd

site excised 47 bp from the transgene, we cannot deduce from our results that

the two HNF1 binding sites in the enhancer play different roles. Indeed, it

could be that the 47-bp sequence binds factors other than HNF1 or destroys a

binding site 5’ to the deletion, although we did not detect such a site either by

in vivo footprinting (9), or by gel shift analysis (not shown).

A transgene deleted from the HNF4 site was silent in both livers and kidneys

in half of the mouse lines. It is possible that the HNF4 mutation affects the

recruitment of chromatin-remodeling factors (27), (28). Accordingly, the –

232A100B400∆2 transgene could remain silent when inserted into a closed

chromatin region, and would be active when integrated into a permissive,

open chromatin region. Although both HNF1 (29), (30) and HNF4 binding sites

(31), (32) have been reported to play a role in the response of some genes to

cAMP, the corresponding elements present in the enhancer do not seem to be

involved in the dietary and hormonal regulation of the aldolase B gene.

Indeed, the response of our mutated transgenes to fasting and cAMP was

conserved.


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To explain the differential effect of enhancer HNF1 binding site mutations

according to the cells and tissues, we hypothesized that HNF1 is involved in a

cooperative interaction of the enhancer with another DNA element able to

bind a factor differentially abundant in the liver, hepatoma cells and kidneys.

C/EBPα, present and abundant in adult hepatocytes, at low concentration in

hepatoma cells, and essentially absent from kidney (33) is a good candidate.

Mouse models also suggest a major role for C/EBPα in liver-specific

expression of aldolase B. The liver of albino lethal mice shows reduce

aldolase B and C/EBP levels (34), whereas other transcription factors are

normally expressed. In contrast, aldolase B is normally expressed in the

kidney of these animals. Aldolase B gene expression is also depressed in the

liver of C/EBP α-/- mice (35). A C/EBP binding site (-170 to –140) has been

characterized in the aldolase B promoter (6), and both C/EBP and HNF1 have

been shown to be potent transactivators of the promoter (7). To confirm that

the different levels of C/EBP in different tissues could account for the tissue-

specific cis-effects of the enhancer HNF1 binding sites, we studied by

transient transfection in HepG2 cells the inducibility by C/EBPα of aldolase

B–CAT constructs carrying either the wild-type enhancer or enhancers devoid

of one of the two HNF1 binding sites. The constructs with mutations of the

HNF1 binding site(s) in the enhancer were highly responsive to

overexpression of C/EBP (100-fold induction) compared to the 5-fold induction

of the construct with an intact enhancer. Furthermore, deletion of the C/EBP

site in the promoter impaired the enhancer function, whereas mutations or


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deletions of the other known sites (binding HNF1, HNF3 or HNF4), alone or in

combination, did not affect the enhancer function. These findings suggest that

liver-specific expression of the aldolase B gene requires cooperation between

the C/EBP site in the promoter and at least one HNF1 site in the enhancer. In

contrast, expression in the kidney (lacking C/EBP) requires the presence of

the two enhancer HNF1 binding sites. Cooperation between HNF1 and C/EBP

in the control of gene expression in the liver has already been reported for the

PEPCK gene (25), the phenylalanine hydroxylase gene (36), the

apolipoprotein B gene (37) and the albumin gene (38).

Our data indicate that neither HNF1 nor HNF4 binding sites mediate the

aldolase B gene regulation by fasting and cAMP. However C/EBP was a

possible alternative candidate since its involvement in the response to cAMP

has been established for several genes, in particular the PEPCK gene (39),

(40). Unfortunately, although the wild-type aldolase B-CAT construct was

perfectly responsive to glucagon in hepatocytes isolated from transgenic mice,

the same construct transiently transfected into HepG2 cells was insensitive to

cAMP. This insensitivity could not be reversed by cotransfection with

expression vectors C/EBPα, and (or) the PKA catalytic subunit. Consequently,

further experiments will be needed to determine whether C/EBP and C/EBP-

binding sites are involved in the regulation of the aldolase B gene by fasting,

glucagon and cAMP.

The observed early stimulation of both transgenes and the endogenous

aldolase B gene by glucagon in hepatocytes in primary culture prompted us to


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reevaluate our previous presentation of this gene as being negatively

regulated by glucagon and cAMP (2). In fact, our former results were taken

from mRNA measurements performed 12 to 24 h after glucagon

administration. We now find that the early positive response to glucagon,

maximal at the 3rd h, is followed by a decline that reached undetectable levels

at the 24th h. Accordingly, the transcription rate of the aldolase B gene

measured by run-on-assay showed first a transient increase in transcription

10 min after glucagon injection, followed by transcriptional inhibition (41).

Hormonal regulation of the aldolase B gene reflects its dual role in both

glycolysis and gluconeogenesis. Glucagon and cAMP are involved in

hypoglycemic and stress conditions in which a rapid increase in glucose

production is essential. This requires, in particular, a transient increase in

aldolase B gene transcription, leading to an increase in mRNA and enzyme

abundance. However, this gene must also be stimulated to allow for a proper

adaptation to a shift from a carbohydrate-poor to a carbohydrate-rich diet. As

previously discussed, the regulatory domains responsible for the response to

cAMP (and fasting) are retained in the –232A100B400CAT transgene, while

the region required for the positive response to carbohydrates is absent from

this construct.

In conclusion, we report in this paper the complex, tissue-specific functional

interplay between the promoter and intronic enhancer of the aldolase B gene,

which can be considered to be both a glycolytic and gluconeogenic gene.


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Footnotes

The abbreviations used are: bp, base pair(s); kb, kilobase pair(s); CAT,

chloramphenicol acetyltransferase; HNF, hepatic nuclear factor; C/EBP,

CCAAT/enhancer binding protein;

PEPCK, phosphoenolpyruvate carboxykinase,


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Legends

Table 1. Effect of C/EBP- overexpression on activity of aldolase B CAT

vectors containing an impaired HNF1 binding site in the enhancer. Hep G2

cells were transfected with 7.5 µg of aldolase B CAT vectors, 2.5 µg of

luciferase plasmid and, when indicated, 2.5 µg of expression vector for

C/EBP-α. CAT activity was normalized according to transfection efficiency by

measuring the luciferase activity. When more than four independent

experiments were performed, results are given as means ± SE; otherwise, the

means are given, with the number of experiments in parentheses.

Fig. 1. Diagram of the aldolase B-CAT transgenes. (A) Map of the non-

mutated transgene (wild) with the location of the regulatory elements and

transcription factors known to bind to these sites. (B) Map of the various

transgenes with precise indication of the deleted or mutated elements.

Fig. 2. Level of expression of aldolase B CAT transgenes in the liver and

kidney of transgenic mice. CAT activity was assayed as described in liver

(black) and kidney (grey) homogenates from chow-fed animals. Each value

represents the mean of independent assays on different mice (at least 3). The

CAT activity in the kidney of –232A100B400HNF1mut transgenic mice is

shown on a large scale in the inset.


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Fig. 3. Analysis of the element in the promoter required for enhancer function.

At the top is an illustration of the wild construct with the location of the

regulatory elements and transcription factors known to bind to these sites. 7.5

µg of the indicated constructs mutated in the promoter linked or not to the

enhancer fragment were transfected into HepG2 cells with 2.5 µg of the

reference plasmid RSV luciferase. CAT activity was normalized with respect

to the transfection efficiency by measuring luciferase activity. Results give the

ratio of the CAT activity of the construct with and without the enhancer

fragment.

Fig. 4. Northern blot analysis of aldolase B and CAT transcripts in livers of

transgenic mice under different nutritional conditions. 20 µg of total liver

mRNAs were electrophoresed in formaldehyde-agarose gel, transferred onto

a Hybond N+ membrane and hybridized with the indicated probes. Animals

were fasted 24 h (fasted state) then fed a 75% carbohydrate diet for 18 h (fed

state). The northern blots are representative of two different experiments.

Fig. 5. Effects of cAMP and glucagon on the level of expression of the

-232A100B400 wild-CAT transgene and the endogenous aldolase B gene in

the liver. Mice were fed a 75% carbohydrate diet for 18 h (panel A and C) or

fasted for 24 h (panel B) prior to intraperitoneal injection of either Bt2cAMP or

glucagon as indicated under Experimental Procedures. Total liver RNA were

prepared at the indicated time after the first injection and northern blots were


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28

analyzed as described in Fig. 4 legend. PEPCK served as a positive control of

the hormonal status.

Fig. 6. Effect of glucagon on aldolase B gene expression in primary cultured

hepatocytes. Hepatocytes isolated from –232A100B400 wild-CAT transgenic

mice were cultured for 3 days without glucagon in medium defined under

Experimental Procedures, then incubated 3 to 24 h in the presence or

absence of glucagon and theophylline. Total RNAs were extracted and

analyzed for the level of transgenic CAT mRNA, endogenous aldolase B and

PEPCK mRNAs. The northern blots are representative of two culture

experiments with two different lines of mice.


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Table 1.

-232A100 -232A100B400 wild

-232A100B400

HNF1“mut”

-232A100B400 ∆7

-140A100 -140A100B400 wild

WithoutC/EBP-α

70±25 2003±312 54±9 64±11 50±19 46 (3)

WithC/EBP-α

15000 (4) 10794 (4) 10268 (4) 10355 (4) 1500 (4) 2439 (3)


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BB

AA

+1

HNF4C/EBP DBP

HNF1HNF3

NFY

promoter enhancer

-232A100

CAT gene

B400

2 4 5 7HNF4 HNF1 HNF1

AldolaseB-CAT Transgene

-232A100B400 wild

-232A100B400 2

-232A100B400 4

-232A100B400 7

-232A100B400 5-7

-232A100B400 HNF1 mut

-232A100

Fig.1



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Fig. 2

-232A100B400wild

0

500

1000

1500

2000

2500

10 4 42 7 8 30line #

-232A100B400 7

4 8 10 11 16 17 28 29 34 40 41 44 46 47 480

100

200

300

400

43

line #

line #

0

2000

4000

6000

8000

10000

14 12 26 1 25

-232A100B400 4-232A100B400

0

500

1000

1500

2000

2500

2 25 42 24 35 43 31 18 28 29 10 38line #


-232A100B400HNF1mut

line #2 3 1 13 7

0

100

200

300

400

2 3 1 13 70

10

20


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Ratio CAT activity+enhancer /- enhancer

30

33

100

146

155

6

+1

HNF4C/EBP DBP

HNF1HNF3

NFY

CAT gene

HNF4 HNF1 HNF1

promoter enhancer

+1

+1

+1

+1

+1

+1

-190 wild

-190 HNF4-

-190HNF1- /HNF3+

-190 HNF1+ /HNF3-

-190HNF1- /HNF3-

-190 C/EBP-(-142 PAB)

constructs

cat gene +or -enhancer

+1

+1

-142 HNF3C/EBP-

-142 HNF1C/EBP-

11

1.5

Fig.3



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-232 A100 B400HNF1 mutCAT mRNA

-232 A100 B400 WildCAT mRNA

-232 A100 B400 ∆2CAT mRNA

-232 A100 B400∆4CAT mRNA

PEPCK mRNA

murine aldolase B mRNA

Fig. 4



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glucagon

CAT mRNA


PEPCK mRNA

CC

2 h0 4 h

Bt2cAMP

FED FASTED

CAT mRNA

PEPCK mRNA

AA BB

2 h0 4 h 2 h0 4 h

Fig. 5



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CAT mRNA


PEPCK mRNA

hour 0 3 6 24 24

glucagon - + + + -

Fig. 6



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Claudine Gregori, Arlette Porteu, Claudia Mitchell, Axel Kahn and Anne-Lise PichardIn vivo functional characterization of the aldolase B gene enhancer

published online May 28, 2002J. Biol. Chem.

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