©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Dissection of the Brain-specific Rat Aldolase C Gene Promoter in Transgenic Mice
ESSENTIAL ROLE OF TWO GC-RICH BOXES AND AN HNF3 BINDING SITE (*)

(Received for publication, May 9, 1995; and in revised form, June 21, 1995)

Muriel Thomas (§) Henriette Skala Axel Kahn Françoise Phan Dinh Tuy

From the Institut Cochin de Génétique Moléculaire, Génétique et Pathologie Moléculaires, INSERM U129, Université René Descartes, 24 rue du Faubourg Saint Jacques, 75014 Paris, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The aldolase C gene product is a glycolytic isoenzyme specifically detected in brain. We have previously defined a short 115-base pair promoter fragment able to confer on a reporter chloramphenicol acetyltransferase (CAT) gene a specific expression in brain of transgenic mice. In this promoter fragment, two GC-rich regions (A/A` and B boxes) were detected by in vitro DNase1 footprinting experiments with brain, fibroblast, or liver nuclear extracts. Both A/A` and B boxes, sharing structural homology, are able to interact with Sp1, Krox20/Krox24 factors and with other proteins (Thomas, M., Makeh, I., Briand, P., Kahn, A., and Skala, H.(1993) Eur. J. Biochem. 218, 143-151). In this paper, we describe a new ubiquitous factor termed Ub able to bind the A/A` box. We also delimit a third element (box C) binding a hepatocyte-enriched protein displaced by a hepatocyte nuclear factor 3-specific oligonucleotide. The functional involvement of each binding site in brain-specific transcription of the aldolase C gene has been tested in transgenic mice carrying different mutant promoters cloned in front of the CAT gene. A promoter containing only box C was totally inactive, suggesting an essential role of the region containing A/A` and B boxes. However, mutations or deletions of either the A/A` or the B box have no significant effect on the CAT gene expression. We therefore hypothesize that the A/A` and B sites may be functionally redundant. Indeed, constructs harboring only one of these two boxes (A/A` or B) linked to the C box displayed a brain-specific CAT activity similar to that obtained with the wild-type promoter. Furthermore, a transgene with disruption of the C box, keeping intact the A/A` and B boxes, was totally inactive, suggesting a crucial role of the hepatocyte nuclear factor 3 binding site in activation of the aldolase C gene.


INTRODUCTION

Genes specifically expressed in the brain can be divided in two main groups according to the range of their specificity. A subset of these genes is active in restricted and well defined types of neurons. For instance, pcp2 gene is specifically expressed in retinal bipolar cells and cerebellar Purkinje cells(1, 2) , while the homeodomain protein Pit-1 is detected in somatotroph, lactotroph, and thyrotroph cells of the anterior pituitary(3) . In contrast, other brain-specific genes do not exhibit any sharp specificity and are expressed in many neurons, e.g. the genes for synapsin I (review (4) ), amyloid precursor protein(5) , neuron-specific enolase (6) , and Thy-1(7) . The aldolase C gene belongs to this latter group; indeed, in situ hybridization experiments allowed to detect throughout the brain different levels of aldolase C mRNA(8) . These ``nonspecific'' neuronal genes often contain housekeeping-like promoters lacking TATA and CAAT boxes and covering a GC-rich region(5, 9, 10, 11) . To understand the mechanisms underlying the widespread cerebral expression characterizing such genes, we studied the sequences involved in brain-specific transcription of the rat aldolase C gene. In previous works, we have defined a short 115-bp (^1)sequence as a minimal efficient promoter of the aldolase C gene. This promoter is indeed sufficient for driving a CAT reporter gene expression specifically in cultured PC12 cells and in brain of transgenic mice(12, 13) . Two GC-rich boxes A/A` (-197/-166) and B (-161/-138) were defined by footprinting experiments on this 115-bp sequence. Both were shown to be able to interact in vitro with Sp1 and Krox20/Krox24 factors(12) . In addition, the B box contains a direct repeat sequence located 5` to the GC-rich region and binding, in vitro, unknown X and Y proteins(12) . We report here another ubiquitous factor, termed Ub, binding the A/A` box upstream of the Sp1/Krox2O/Krox24 binding site. We also describe on the promoter, from bp -114 to -99 (Fig. 1), a third box, termed box C, detected only with liver nuclear extracts and which seems to interact with factors of the fork head/HNF3 family. In transgenic mice harboring the chloramphenicol acetyltransferase (CAT) gene controlled by various mutant versions of the aldolase C promoter, we found that boxes A and B are redundant; indeed, the presence of only one of them is sufficient to ensure a brain-specific expression. In addition, an intact box C seems to be required for the expression of the aldolase C transgenes.


Figure 1: Structure of the aldolase C gene promoter. The footprinted A/A` (-197/-166), B(-161/-138), and C(-114/-99) boxes detected in the 0.115-bp aldolase C promoter are indicated. For each box, recognition sites for factors are underlined, and nucleotides modified in each mutation are indicated by arrows.




MATERIALS AND METHODS

DNA Constructs

0.115/CAT, DRmut/CAT, Sp1Bmut/CAT, and A/A`mut/CAT constructs were described in Thomas et al.(12) . In 0.115/CAT, the wild-type aldolase C promoter encompassing 115 bp (nucleotides -199 to -84 with respect to the ATG translation initiator) was cloned in front of the CAT gene. To obtain the A/A`mut/CAT, DRmut/CAT, Sp1Bmut/CAT, and HNFmut/CAT constructs, mutations were introduced in the Sp1 binding site of the A/A` box, the DR subregion of the B box, the GC-rich subregion of the B box, and the C box, respectively (Fig. 1). Del.Ub (-187/-84 bp) and B/C (-167/-84 bp) fragments were obtained by polymerase chain reaction amplification and then cloned in the SmaI site of the UMS/CAT plasmid (derived from pEMBL 19), resulting in the Del.Ub/CAT and B/C/CAT constructs, respectively. Del.A/A`/B/CAT was derived from O.115/CAT by deletion of a -199/-140 fragment by HinfI digestion. In construct referred to as A/A`/C/CAT, the A/A` oligonucleotide was cloned in the BamHI recognition site of the Del.A/A`/B/CAT construct.

Nuclear Extracts

Nuclear extracts were prepared as described in (14) for the brain and in (15) for the liver.

Footprint Experiments

Oligonucleotides used as competitor were HNF3, -111/-90-bp site of the mouse transthyretin gene promoter (16) (5`-GTTGACTAAGTCAATAATCAGA-3`); HNF1, -106/-60-bp site of the rat L-type pyruvate kinase gene promoter (17) (5`-AAGAGAGATGCTAGCTGGTTATACTTTAACCAGGACTCATCTCATCT-3`); and C/EBP, -109/-86-bp site of the rat albumin gene promoter(18, 19) (5`-GGTATGATTTTGTAATGGGGTAGG-3`).

The coding strand of the 0.115/CAT plasmid was end-labeled at the XbaI site by filling in with [alpha-P]dNTP using Klenow fragment of DNA polymerase I. Then, the 0.115 probe was excised by SacI digestion and purified. After incubation with 50 µg of liver extract proteins and 250 ng of poly(dI-dC), 0.1 ng of probe was partially digested by DNase1 (Sigma). 50 ng of specific competitor were or were not added. In control experiments, probe was incubated with 2.5 µg of sonicated salmon sperm DNA prior to the DNase1 treatment. Samples were ethanol-precipitated and loaded in 8% (w/v) polyacrylamide, 8 M urea gel.

Gel Shift and Methylation Interference Assays

Oligonucleotides used as probes or competitors were A/A`, 5`-TAGACCAGTCCTGGGGAGAGGGCGGGACCAG-3` (-199/-169 bp) of the rat aldolase C gene; A/A`mut, 5`-TAGACCAGTCCTGGGGAGAGGGCGAGACCAG-3` (nucleotide at position -175 of the A/A` sequence was mutated, replacing A for G); delUb, 5`-GGGAGAGGGCGGGACCAG-3` (13 nucleotides of the A/A` oligonucleotide were deleted, from -199 to -186 bp); Sp1cons, 5`-GCATAACTCCGCCCAGTTAG-3` (sequence derived from the SV40 enhancer)(20) .

Probes were end-labeled with [-P]ATP using T4 polynucleotide kinase. Gel shift reaction mixtures included 300 ng of poly(dI-dC) (Pharmacia Biotech Inc.) as a nonspecific competitor, 0.05-0.1 ng of radiolabeled probe, and 6 µg of nuclear extract proteins in a classical binding buffer(21) . Various amounts of specific competitor were or were not added. After a 5-min incubation at 0 °C, samples were loaded on a non-denaturing 6% (w/v) polyacrylamide gel.

For methylation interference assays(22, 23) , partially methylated 5`-labeled or 3`-labeled A/A`mut probe was used. Bound and free DNA fractions were separated by gel shift electrophoresis and then eluted from the gel, cleaved at modified guanine residues with 10% (v/v) piperidine, and loaded on a 15% (w/v) polyacrylamide, 8 M urea gel.

Production and Analysis of Transgenic Mice

Each transgene 0.115/CAT, DRmut/CAT, Sp1Bmut/CAT, A/A`mut/CAT, Del.Ub/CAT, Del.A/A`/B/CAT, A/A`/C/CAT, B/C/CAT, and HNFmut/CAT was excised from its corresponding plasmid by XbaI and ClaI digestions. After electrophoresis on 0.8% (w/v) GTG-seakem-agarose gel (Life Technologies, Inc.), fragments were electro-eluted and purified by using elutip-d columns (Shleicher & Schuell). 3-10 ng of fragment diluted in a 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA buffer were microinjected into fertilized B6D2 mouse eggs according to (24) . Transgenic founders and offsprings were identified (25) by Southern blot, and transgene copy numbers were estimated by quantitative analysis on a phosphorimager system (Molecular Dynamics). CAT assays were performed with 200 µg of F1 animal tissue extract proteins (liver, heart, lung, and brain) according to (26) .


RESULTS

Binding Activity of an Evolutionarily Conserved Sequence in the 5`-Region of A/A` Box

In the A/A` box (nucleotides -197/-166), we have previously defined upstream of the Sp1 binding site a 15-bp sequence that is 100% conserved between rat and human promoter (nucleotides -196/-181)(12) . To investigate the existence of putative additional binding sites in this conserved sequence, the oligonucleotide A/A`mut, consisting in the A/A` sequence mutated at position -175 (A instead of G) (Fig. 1), was used as a probe in gel shift experiments to suppress any binding of Sp1 (Fig. 2A) and of Krox20/Krox24 proteins (not shown). A complex, termed Ub, was detected in brain, liver, and fibroblast extracts. This complex specifically vanished when the homologous unlabeled A/A`mut oligonucleotide was added in excess; in contrast, it was not modified by a large amount of the Sp1cons oligonucleotide, a high affinity recognition site for Sp1(20) . These data indicate that the A/A`mut sequence is a recognition site for an ubiquitous protein (Ub) different from Sp1. We then assessed the Ub binding activity on the wild-type A/A` oligonucleotide by competition experiments with increasing amounts of A/A` or A/A`mut competitor (Fig. 2B). According to this experiment, the A/A` sequence displayed the same affinity for Ub than the A/A`mut box. As expected, an excess of A/A` competitor (but not of A/A`mut) competed for the binding of Sp1 on the Sp1cons probe (Fig. 2C). Therefore, the A/A` sequence is the recognition site for at least three types of cellular factors: Sp1, Krox20/Krox24, and Ub.


Figure 2: Detection of a new binding activity upon A/A`mut oligonucleotide. A, radiolabeled A/A`mut oligonucleotide was incubated with 6 µg of nuclear proteins prepared from 3T6 fibroblasts, or from whole organs (liver or brain). Homologous or heterologous competitors (20 ng) were or were not added. The complex named Ub is indicated by the arrow. Free, probe incubated without nuclear extracts. B and C, A/A` mut or Sp1cons oligonucleotide was used as radiolabeled probe and incubated with 6 µg of brain nuclear proteins. As indicated, 20 ng or increasing amounts (5, 10, and 20 ng) of homologous or heterologous competitors were or were not added to the reaction mixture. Free, probe incubated without nuclear extracts. Ub and Sp1 complexes are indicated by arrows. The fast migrating N.S complex obtained with A/A`mut probe appeared nonspecific since it was unaffected by addition of unlabeled homologous competitor.



We further investigated which nucleotides were contacted by the Ub factor by in vitro methylation interference assays using radiolabeled A/A`mut oligonucleotide and nuclear proteins extracted from adult brain, in which the gene is active, and from adult liver, in which the gene is inactive. In both extracts, methylation at guanines -195 and -194 of the non-coding strand interfered with Ub complex formation strongly and weakly, respectively (Fig. 3). On the coding strand, three residues were mainly contacted by the Ub factor: nucleotides -192, -187, and -182, while three minor interferences were detected at positions -186, -185, and -184 (Fig. 3). These results indicate that the 5`-region of the A/A` box, which contains a 15-bp sequence conserved between rat and human, is involved in the binding of the Ub complex, both in the brain and the liver.


Figure 3: Protein/DNA contact points of brain- and liver-Ub complexes. Binding reactions were performed with brain or liver nuclear extracts and with partially methylated A/A`mut oligonucleotide end-labeled on the coding or non-coding strand. G, Maxam-Gilbert sequence reaction. Residues at which methylation interfered with protein binding are indicated by circles and summarized in the bottom of the figure. Solidcircles represent a greater degree of interference than opencircles. Nucleotides conserved between rat and human sequences are indicated.



Box C, a Proximal Element of the Aldolase C Promoter, Interacts with Liver-enriched Factors

Footprinting experiments showed that the A and B boxes observed with brain extracts (12) were also protected with liver nuclear extracts (Fig. 4). A third region termed box C (nucleotides -114/-99), undetectable with brain extracts, was here well protected and characterized by the presence of a strong hypersensitive site in the middle of the footprint. This C sequence was reminiscent of the binding site for HNF3 described in promoters of various liver-specific genes (e.g. transthyretin and aldolase B genes)(27, 28, 29) , and the inner hypersensitive site is, indeed, a feature of HNF3-dependent footprints(30, 31) . Accordingly, footprint C was specifically displaced by an excess of an HNF3-specific oligonucleotide (i.e. the transthyretin gene HNF3 binding site) and not by oligonucleotides specific to HNF1 (17) or C/EBP (18, 19) (Fig. 4).


Figure 4: Detection of a liver-specific footprint on the 115-bp promoter. The 115-bp fragment used as probe was excised from the 0.115/CAT plasmid by XbaI and SacI digestion. Prior to the attack with DnaseI, this fragment was incubated with 50 µg of liver nuclear extract proteins and, as indicated, with 30 ng of unlabeled oligonucleotides representing the binding sites for HNF1 (box L1 of the pyruvate kinase gene promoter(17) , HNF3 (sequence derived from the transthyretin gene promoter)(16) , and C/EBP (site D of the albumin gene promoter)(18, 19) . G+A, Maxam-Gilbert sequence reaction; Free, digestion of the probe without extracts. Borders of the footprinted sequences are indicated.



Functional Dissection of the 115-bp Promoter in Transgenic Mice

Since the A/A` box is the recognition site for Ub, Sp1, and Krox20/Krox24 factors, we have generated two types of mutated transgenes: 1) the A/A`mut/CAT transgene with the point mutation at -175 suppressing Sp1 (Fig. 2) and Krox20/Krox24 binding (data not shown) and 2) the Del.Ub/CAT transgene whose region implicated in Ub fixation was deleted (Fig. 1). In transgenic mice obtained with either mutated transgene, CAT activity was specifically detected in brain, similar to that obtained with the wild-type transgene 0.115/CAT (Fig. 5).


Figure 5: Distribution of CAT activity in transgenic mice carrying wild-type or mutated transgene. A, scheme of the different transgenes injected. B, CAT activities, expressed in cpmbulletminbullet(µg protein), were measured on 200 µg of proteins from brain and other tissues (liver, heart, and lung), previously heated 10 min at 65 °C to eliminate endogenous deacetylase activity. For each line, the transgene copy number was estimated by Southern blot hybridization of transgenic mouse DNAs and of standard amounts of the injected transgene. CAT activities obtained with the 0.115/CAT fragment were more extensively described in (12) and (13) ). F indicates that founders were sacrificed before mating. For line 39, carrying the A/A`/C/CAT transgene, an ectopic CAT expression was also observed with heart extracts.



To test the in vivo involvement of the B region in brain-specific expression of the aldolase C gene, the DRmut/CAT and Sp1Bmut/CAT constructs previously described (12) were used to generate several independent transgenic lines. The DRmut mutation prevents the binding of the X and Y proteins(12) , while the Sp1Bmut mutation suppresses the binding of Sp1 (12) and Krox20/Krox24 (not shown) (Fig. 1). Fig. 5shows that a comparable brain-specific expression was observed with either wild-type or mutant transgenes.

To test the role of A/A` and B boxes in the promoter activity, both regions were deleted, yielding the construct Del.A/A`/B/CAT (Fig. 5). In six independent lines carrying the Del.A/A`/B/CAT transgene, no significant CAT activity was detected (Fig. 5), suggesting an essential involvement of the region containing the A/A` and B boxes in the expression of the transgene. As independent mutations of binding sites in either A or B box had no effect, we then supposed that these two sequences could have a redundant role in transcriptional activity. To further test this hypothesis, each box (A/A` or B) was independently cloned in front of the C box, resulting in A/A`/C/CAT and B/C/CAT plasmids. CAT activities obtained in transgenic mice were comparable to that obtained with wild-type construct (Fig. 5). The presence of only one box (A/A` or B) in front of the C region was therefore able to restore a specific activity of the aldolase C promoter.

In lines 70 (Del.Ub/CAT), 1 and 80 (Sp1Bmut/CAT), and 20, 32, 29, and 73 (A/A`/C/CAT), the CAT transgene was totally inactive, probably owing to a position effect. We already reported that for the 115/CAT transgene the CAT expression was dependent on the integration site and not correlated with the transgene copy number(13) .

The involvement of the C box in transcription was tested by replacing the genuine box by an HNFmut sequence mutated at 5 nucleotides, -111, -109, -107, -104, and -103 (Fig. 1), resulting in the HNFmut/CAT transgene. In the 6 lines obtained, the transgene was inactive in all tissues tested (Fig. 5).

We can therefore assume that an integral C box associated with at least one of the two A/A` or B boxes is necessary to ensure a brain-specific activity of the aldolase C promoter.


DISCUSSION

The DNA-Protein Interactions on the 115-bp Aldolase C Gene Promoter Fragment

In this paper, we further analyzed the protein interactions occurring on the 115-bp aldolase C gene promoter. We found that the A/A` box, in addition to Sp1 and Krox20/Krox24 factors, can bind a ubiquitous protein termed Ub on a 15-bp subregion located upstream of the Sp1 binding site and 100% conserved between rat and human aldolase C gene promoters. In addition, a third box termed box C was detected by footprinting experiments between bp -114 and -99 with liver nuclear extracts; its AT-rich sequence, the presence of a strong inner hypersensitive site, and the competition with an HNF3-specific oligonucleotide suggest that box C binds proteins of the fork head/HNF3 family. In summary, the 115-bp aldolase C gene promoter fragment can be subdivided into at least three boxes: 1) the A/A` and B boxes, which share structural similarities; both encompass GC-rich sequences, able to bind Sp1/Krox20/Krox24 factors, overlapping with more 5` recognition sites for widespread expressed factors (X and Y for the B box (12) and Ub for the A/A` box); 2) the C box detected with liver nuclear extract and able to bind factors of the fork head/HNF3 family. HNF3 factors are supposed to be the rodent homologues of the Drosophila fork head factors (32, 33) and are known to be early markers of endodermal differentiation, essential to the development of primitive intestine and gut derivatives(34, 35) . In addition, HNF3-beta and HNF3-alpha are also expressed at early stages of brain differentiation, especially in the floor plate of mouse embryos where HNF-3beta is indispensable to ensure brain development(36, 37, 38) .

Functional Studies of A/A`, B, and C Boxes in Transgenic Mice

To investigate their functional involvement, the different protein binding sites determined in the A/A` and B boxes were independently altered by mutations or small deletions. Each of the corresponding mutant promoter constructs displayed a brain-specific CAT expression similar to that obtained with the wild-type 0.115/CAT transgene. However, the region containing the A/A` and B boxes plays a crucial role in brain-specific transcription; indeed, the Del.A/A`/B/CAT transgene where these two sequences were deleted was inactive. Results reported here show that the association of only one region, either A/A` or B, with the C box was sufficient to ensure an active and brain-specific promoter (Fig. 5). In line with their similar structural organization, we therefore assume a redundant involvement of the A/A` and B boxes in the brain-restricted transcription of the aldolase C gene. Neutrality of single-site mutations has already been described as a consequence of a functional redundancy between several similar sites closely interspersed within a short sequence(39, 40) .

We had previously shown that a CAT construct harboring a consensus Sp1 binding site in place of the whole A/A` box (Sp1cons/CAT) appears very weakly and ubiquitously expressed in transgenic mice(12) . The A/A` box was then assumed to be essential to achieve a brain-specific expression (12) . Present results show, in fact, that the B box is also able to ensure a brain-specific activity in the absence of the A/A` box. Considering this result, we can now assume that, in the Sp1cons/CAT construct, the presence of a high affinity binding site for the ubiquitous Sp1 protein may have induced a new array of interactions upon this promoter region, responsible for the widespread activity of the Sp1cons/CAT transgene.

The existence of specific transcriptional factors able to bind both A/A` and B boxes and responsible for brain-specific expression of the aldolase C gene remains to be established. Two types of hypotheses can be proposed. 1) The brain-specific activity of the aldolase C promoter could result from interaction between ubiquitous transcriptional factors (like Sp1) and brain-restricted factors not yet identified. Such an implication of Sp1 in tissue-restricted transcription has been already described(41, 42, 43) . Mutations in the Sp1 site in A/A`/C/CAT or in B/C/CAT construct should help to test this hypothesis. 2) The brain-specific activity of the aldolase C promoter should result from direct interaction of a brain-specific factor with A/A` and B boxes.

Fig. 6shows that a GGGAG sequence is shared by A/A` and B boxes. Interestingly, a similar element is located in the Pcp2 gene promoter involved in the widespread expression of a reporter gene in almost all neurons of transgenic mice(44) . The aldolase C sequence is also similar to an element of the neuron-specific enolase gene promoter (6) and with a hemipalindrome described in the heavy neurofilament gene promoter (NF-H) (45) (Fig. 6). The palindromic sequence of this latter promoter was described to be important in brain-specific enhancement of in vitro transcription. We are currently investigating whether the role of the A/A` and B boxes in the brain-specific transcription of the aldolase C gene can indeed be ascribed to this conserved G-rich sequence.


Figure 6: Similarities between A/A` and B boxes of the aldolase C gene promoter and sequences present in other brain-specific promoters. G-rich and GGGA core elements found in A/A` and B boxes, and shared by brain-specific promoters of several genes, are underlined.



It should be observed that the results obtained in vivo in transgenic mice are significantly different from those obtained ex vivo in cultured PC12 cells; in these cells, a single mutation in the Sp1/Krox20/Krox24 binding site of the A/A` box was sufficient to inactivate the gene, and mutations of the B box also decreased expression(12) . In fact, it appears that the redundance between boxes A/A` and B observed in vivo was not operative ex vivo, where integrity of both boxes was required. This could be explained by a lower level of differentiation in PC12 cells than in the brain in vivo, such that a strict cooperation between the functionally redundant boxes A/A` and B would be required ex vivo and dispensable in vivo. A similar observation was reported for the cell-specific elastase I enhancer where three distinct enhancer domains are all required in transfected cells but have a redundant function in transgenic mice (46) . Other examples point out the differences between activity of cis-acting sequences tested ex vivo, in transient transfection assays, and in vivo, in the normal developmental and chromosomal context of transgenic animals(47, 48) .

Mutations performed in the aldolase C gene promoter HNF3 binding site led to the complete inactivity of this promoter in transgenic mice (Fig. 5). To explain this unexpected result, two hypotheses could be proposed. 1) The C box is a binding site for a positive brain-specific activator related to the HNF3/fork head family. Such a protein, essential in developing brain, has been described as the brain factor-1 (BF-1) and the brain factor-2 (BF-2)(49, 50) . However, by footprint experiments (12) as well as by gel shift assays (data not shown), we failed to detect any binding activity on the C box with adult brain nuclear extracts. Further experiments (e.g.in vivo footprinting experiments) are in progress to study in more details protein interaction on the C box in fetal and adult brains. 2) The binding of an HNF3-like factor leads to the formation of an active aldolase C promoter by a chromatin structure modification at an early stage of development. In line with this hypothesis, HNF3 factors have been suggested to be essential for the establishment of the phased nucleosome arrangement in the albumin gene enhancer, necessary for gene activation(51) . Since HNF3 proteins are synthesized early during development, especially in derivatives of the neural crest, we hypothesize that such a protein can contribute to make the aldolase C promoter accessible to other factors potentially interacting with the A/A` and/or B boxes and involved in brain-specific expression. Although further investigations are required to test this model, the present paper is the first report suggesting that a fixation of an HNF3-related protein could be involved in the activity of a brain-specific gene promoter.

In conclusion, activity and brain specificity of the aldolase C gene promoter require the presence of an AT-rich element, box C, that seems to be able to bind factors of the HNF3 family and of at least one of two composite GC-rich elements, boxes A/A` and B.


FOOTNOTES

*
This work was supported by grants from the Ministère de la Recherche et de l'Espace, l'association de Recherche sur le Cancer, and l'Université Paris V-René Descartes. 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.: 33-1-44-41-24-24; Fax: 33-1-44-41-24-21.

(^1)
The abbreviations used are: bp, base pair(s); HNF, hepatocyte nuclear factor; CAT, chloramphenicol acetyltransferase; C/EBP, CCAAT enhancer binding protein.


ACKNOWLEDGEMENTS

We thank Alexandra Henrion for kindly reading the manuscript.


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