Regulation of Expression within a Gene Family
THE CASE OF THE RAT gamma B- AND gamma D-CRYSTALLIN PROMOTERS*

Erik Jan Klok, Siebe T. van Genesen, Azem Civil, John G. G. Schoenmakersdagger , and Nicolette H. LubsenDagger

From the Department of Molecular Biology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The six closely related and clustered rat gamma -crystallin genes, the gamma A- to gamma F-crystallin genes, are simultaneously activated in the embryonic lens but differentially shut down during postnatal development with the gamma B-crystallin gene, the last one to be active. We show here that developmental silencing of the gamma D-crystallin promoter correlates with delayed demethylation during lens fiber cell differentiation. Methylation silencing of the gamma D-crystallin promoter is a general effect and does not require the methylation of a specific CpG, nor does methylation interfere with factor binding to the proximal activator. In later development, the gamma D-crystallin promoter is also shut down earlier by a repressor that footprints to the -91/-78 region. A factor with identical properties is present in brain. Hence, a ubiquitous factor has been recruited as a developmental regulator by the lens. All gamma -crystallin promoters tested contain upstream silencers, but at least the gamma B-crystallin silencer is distinct from the gamma D-crystallin silencer. The gamma -crystallin promoters were found to share a proximal activator (the gamma -box; around -50), which behaves as a MARE. The gamma B-box is recognized with much lower avidity than the gamma D-box. By swapping elements between the gamma B- and the gamma D-crystallin promoter, we show that activation by the gamma B-box requires a directly adjacent -46/-38 AP-1 consensus site. These experiments also uncovered another positive element in the gamma D-crystallin promoter, around -10. In the context of the gamma D-crystallin promoter, this element is redundant; in the context of the gamma B-crystallin promoter, it can replace the -46/-38 element.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The mammalian genome contains a large number of gene families, which encode related proteins with similar structure and function, yet are optimized for a particular developmental and differentiation stage. The pattern of expression of gene family members varies between families. For example, in the beta -globin gene family, the paradigm of a clustered gene family, expression switches between members such that only one or two genes are active at the same time (for review, see Ref. 1). In contrast, the six clustered and closely related members of the gamma -crystallin gene family (the gamma A- to gamma F-crystallin genes), which encode abundant structural proteins of the vertebrate lens, are all simultaneously active in the embryonic lens but switched off individually during postnatal development (2-5). In the rat, at 3 months of age the gamma B-crystallin mRNA is still present at 90% of the level at birth, whereas the transcript level from the gamma D-crystallin gene has dropped to 60%, and those of the gamma E- and gamma F-crystallin genes have dropped to 5% of the level at birth (4). As lens cells do not die and as the younger lens cells overlay the older cells, the consequence of this pattern of gene expression is the creation of a gamma -crystallin gradient across the eye lens, which correlates inversely with the water gradient. This gradient in turn sets the gradient of refraction across the lens and thereby prevents optical aberration.

The mechanism of the developmental regulation of the gamma -crystallin gene expression is not known. There is a strong negative correlation between the methylation state of a gamma -crystallin gene promoter region and gene activity, suggesting that DNA methylation, or rather lack of DNA demethylation, is involved in silencing the genes (6). Differential expression or availability of transactivating factors is also likely to be causally involved in developmental regulation of expression. It is generally assumed that the closely related gamma A-F-crystallin genes share a common regulatory element that specifies the lens specificity of these genes. The prime candidate for such an element is the palindromic sequence (here denoted the gamma -box) located upstream of the TATA box (Fig. 1). Mutations of this sequence abolish promoter activity in transfection studies (7, 11, 12). Furthermore, Goring et al. (13) have shown that a pentamer of the mouse gamma F-crystallin gamma -box sequence directs lens-specific expression in transgenic mice. The expression of this construct, however, was restricted to the embryonic lens nucleus, and it was suggested that the wider range of developmental expression of the mouse gamma F-crystallin promoter is determined by upstream enhancers (13, 14).

The study of the regulatory mechanisms of crystallin gene expression is complicated by the peculiar mode of growth of the lens; the lens epithelial cells differentiate to lens fiber cells at the equator of the lens. The fiber cells of a late developmental stage but at an early differentiation state thus overlay fiber cells of an earlier developmental stage but at a later differentiation state. The lens is thus a mixture of cells at different developmental and differentiation stages. To obtain a fiber cell at a specific developmental and differentiation stage, we have made use of an in vitro differentiation system. In this system, the monolayer of epithelial lens cells, still attached to the lens capsule, is cultured in the presence of bFGF,1 which induces the differentiation of lens epithelial cells to lens fiber cells (Ref. 15; for review, see Ref. 16). The lens fiber cells follow the course of differentiation also seen in vivo, including the typical changes in morphology and the accumulation of the various crystallins. The lens epithelial cells are aware of their developmental age and differentiate to fiber cells corresponding to that developmental age (17, 18). When explants are taken from newborn rats, copious accumulation of gamma -crystallin is seen after about 10 days of in vitro culture (19-21). Lens explants isolated from older rats differentiate more slowly in vitro than those from younger rats and accumulate less gamma -crystallin mRNA and protein (18, 21); in differentiating explants from 10-day-old rats, the gamma -crystallin mRNA levels are only 1% of that seen in newborn explants,2 and below the level of detection in explants from 14-day-old rats (21).

In a previous study (7), we analyzed the course of activation of the gamma D-crystallin promoter during the in vitro differentiation of rat lens explants isolated from newborn rats. Demethylation of this promoter occurs within the first 2 days of in vitro differentiation, long before activation of the endogenous gene. The pulse of activity of the endogenous gene, between days 10 and 12 (21), was suggested to be regulated by the balance of activity of a transactivating factor binding the gamma -box, first detected around day 6, and of a silencing factor, which appears around day 10 (7). To investigate developmental changes in these regulatory interactions, we have now followed the activation of the gamma D-crystallin promoter during in vitro differentiation of lens epithelial explants isolated from 10-day-old rats. We show here that in explants from these older rats, promoter demethylation is delayed, whereas the silencing factor appears earlier. We have further compared the gamma D-crystallin promoter with the gamma B-crystallin promoter, the promoter with the most extended developmental expression. We show that the gamma -boxes of the gamma D- and gamma B-crystallin promoters, which resemble a Maf recognition element (MARE; Refs. 22 and 23), are recognized by the same factor, possibly a Maf protein, but that the affinity of the gamma B-box for this factor is much lower than that of the gamma D-box. Activity of the gamma B-crystallin promoter requires interaction with an AP-1 binding site directly downstream of the gamma B-box. Like the gamma D-crystallin promoter, the expression of the gamma B-crystallin promoter is subject to silencing, but the silencing factor differs from the one that represses the gamma D-crystallin gene.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Culture-- Lens epithelial explants from newborn or 10-day-old (as indicated) Wistar rats were obtained essentially as described (24). Rat lenses were isolated in Medium 199 (Life Technologies, Inc.). The lens capsule together with the anterior monolayer of epithelial cells were peeled off the fiber cell mass and pinned down on a 3.5-cm Petri dish. Explants (three per dish) were cultured as described previously (7). Basic FGF (a kind gift from Scios, Inc., Mountain View, CA) was added to a final concentration of 25 ng/ml, and the cells were cultured for the indicated period prior to transfection.

Isolation of Chromosomal DNA and Ligation-mediated Polymerase Chain Reaction (PCR)-- Isolation of chromosomal DNA from lens explants and ligation-mediated PCR was performed as described previously by Dirks et al. (7).

In Vitro Methylation of DNA-- CpG-methylation of DNA using SssI methylase was essentially according to the manufacturer's protocol (New England Biolabs). Total reaction time was 4 h, whereby addition of enzyme and S-adenosylmethionine to the reaction mix was repeated after 2 h. Completeness of methylation was tested by a pilot digestion using ThaI and electrophoresis through an agarose gel.

Mutagenesis and Reporter Gene Constructs-- The template for mutagenesis was single-stranded DNA from a pBluescript II (SK-) (Stratagene) construct containing the gamma D-crystallin -1087/+48 fragment. Oligonucleotides for mutations were DB1 (5'-GCT GTT CCT GTC AAC GCA GC-3' at position -64/-45), DB2 (5'-GTT CCT GTC AAG GCA GCA GAC-3' at position -61/-41), and DB3 (5'-CTG TTC CTG TGG AGG CAG CAG-3' at position -63/-43) to obtain the mutants gamma DB1, gamma DB2, and gamma DB3, respectively. Oligonucleotide DB4 (5'-GCA GCA GTC ATG ACA GCT ATA TAT ATA GAT C-3' at position -46/-15) was used to mutate both the wild type gamma D-crystallin promoter and the mutant gamma DB3 promoter to obtain gamma D4 and gamma DB4, respectively, and oligonucleotide BD1 (5'-TGG AGG CAG CAG ACC TCC TGC TAT ATA TAC CAG-3' at position -55/-21) to obtain gamma BD1. Mutations were introduced using the Sculptor in vitro mutagenesis system version 3 (Amersham, UK). The BglII (-375)/FokI (+10) promoter fragment of each mutant was cloned into pEUCAT (25). Subsequently, replacement of the gamma D -10 region by its gamma F equivalent was performed by using the oligonucleotide DF (5'-TGT AGG GCT GGG AGC AGG GTC TAT A-3'), representing the sequence at position -23 to +1 of the gamma F promoter, and the oligonucleotide UMS (5'-TGC ATT AAA TTC CAG GAA CTT GCT TTC TGT G-3'), priming upstream from the pEUCAT multiple cloning site, in a PCR reaction using wild-type and mutant gamma D promoter constructs as templates. PCR products were cloned into pEUCAT. All mutants were checked by dideoxy sequence analysis (26). To delete the upstream region, promoter constructs were truncated at the -73 ApaI site. Other promoter constructs used have been described previously (12).

gamma D-Crystallin Silencer Promoter Constructs-- Oligonucleotides DS1 s (5'-TCG AGT GCC CTG CCC CCC GCG G-3') and DS1a (5'-TCG ACC GCG GGG GGC AGG GCA C-3') were annealed (27) and cloned into the SalI site of pBLCAT2 (28) to obtain construct pBLCAT2gamma DS1. The ApaI (-73)/BamHI fragment of pBLCAT2gamma (8) was exchanged with that of pBLCAT2gamma DS1 to obtain pBLCAT2gamma DS. The gamma B- XhoI (-414)/ApaI (-73), gamma C- NheI (-183)/ApaI (-69), gamma D- PvuII (-193)/ApaI (-73), and gamma F-crystallin SpeI (-280)/ApaI (-70) blunt-ended/sticky fragments were inserted into pBLCAT2gamma by replacement of its HindIII (blunt)/ApaI fragment to obtain pBLCAT2gamma B, pBLCAT2gamma C, pBLCAT2gamma D, and pBLCAT2gamma F, respectively. Construct pBLCAT2gamma BS was obtained by deletion of the -414/-110 fragment from pBLCAT2gamma B using the SacI site at position -10.

DNA Transfection, Chloramphenicol Acetyltransferase (CAT) Assay, and beta -Galactosidase Assay-- Plasmid DNA was isolated according to the alkali lysis procedure (27) in conjunction with either the Wizard Maxiprep System (Promega) or CsCl gradient centrifugation (27). DNA was transfected to the lens cells using either Lipofectamine Reagent (Life Technologies, Inc.) or the PDS-1000/He Biolistic Particle Delivery System (Bio-Rad). When cells were transfected using Lipofectamine, per dish 2.0 µg of CAT reporter construct and 0.25 µg of CMV/beta -gal construct (29) were transfected to the cells according to the manufacturer's protocol. Using the Biolistic Particle Delivery System, 0.5 µg of CAT reporter construct and 0.125 µg of CMV/beta -gal construct was coated on 1-µm gold particles and bombarded on the cells at 450 psi helium. After culturing for 3 more days in the presence of 25 ng/ml bFGF, the cells were harvested in 100 µl of reporter lysis buffer (25 mM bicine, pH 7.8, 0.05% Tween 20, 0.05% Tween 80) per dish, and vigorously shaken for 10 min. The cell debris was pelleted in an Eppendorf centrifuge. To determine transfection efficiency, 20 µl of the supernatant was used to assay for beta -galactosidase activity (29). The remainder of the supernatant was heated for 15 min at 65 °C to inactivate cellular deacetylases. From the supernatant, 20 µl was used to assay for CAT activity as described by Gorman et al. (30) or using the Quan-T-CAT system (Amersham). Transfections were done in duplo or triplo, and two DNA isolates from each construct were tested in independent experiments.

Electrophoresis Mobility Shift Assay (EMSA)-- Nuclear extracts were prepared as described previously (12). EMSAs were performed essentially as described (31, 32). DNA restriction fragments were size-fractionated through a native 6% polyacrylamide gel and isolated by electro-elution using a Bio-Trap apparatus (Schleiger and Schuell), according to the manufacturer's protocol. Approximately 0.1-0.5 ng of end-labeled probe (10,000-20,000 cpm) was added to 5-10 µg of nuclear extract (5 µl, final concentration of 100 mM NaCl) and either 1.0 µg of poly(dGdC·dGdC) or 1.0-3.0 µg of poly(dIdC·dIdC), as indicated, in binding buffer (final concentrations 20 mM HEPES, pH 7.9, 10-50 mM KCl as indicated, 1 mM EDTA, 1 mM DTT, 4% (v/v) Ficoll) in a total volume of 20 µl. The reaction mixture was left for 10 min at room temperature, loaded on a pre-run 4% (w/v) polyacrylamide gel in 0.25 × TBE (1 × TBE = 89 mM Tris-HCl, 89 mM boric acid, 2.5 mM EDTA), which then was run for 2 h at 10 volts/cm. The gel was dried and exposed to a Fuji AX film overnight with one intensifying screen.

In Vitro Footprint Analysis-- The appropriate DNA fragment was 32P-labeled at one end. An aliquot (600,000 cpm) was methylated using dimethylsulfate (DMS) essentially as described (33, 34). The methylated probe was incubated with 150-200 µl of nuclear extract (150-400 µg of protein) for a preparative gel retardation assay (analytical assay 30-40-fold scaled up). The complexed and the free probe were visualized by autoradiography overnight. The DNA was cut out of the gel, isolated by electro-elution as described above, cleaved by piperidine (final concentration 10% (v/v)), and size-fractionated in a 15% sequencing gel. The gel was dried, and exposed to a Fuji AX film for 18-72 h.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Demethylation of the gamma D-Crystallin Promoter Region-- In explants from newborn rats, the gamma D promoter region is fully demethylated between day 1 and 2 of in vitro differentiation (Ref. 7; see also Fig. 2A). To test whether promoter demethylation still occurs in 10-day-old rat explants, in which the level of expression of the gamma D promoter is about 50-fold lower, the state of methylation of the genomic ThaI site at position -13 of the gamma D promoter was followed during in vitro differentiation. In a parallel experiment, the methylation state of this ThaI site in explants from newborn rats was tested. In newborn rat explants, virtually complete demethylation of the ThaI site was found after 2 days of culture, in agreement with the results of Dirks et al. (7). In contrast, demethylation of this site was significantly slower during differentiation of explants from 10-day-old rats (Fig. 2A). Even after 5 days of differentiation, demethylation was only 65% complete.

To determine the effect of DNA methylation on gamma D promoter activity, a gamma D(-73/+10)CAT fusion gene was methylated using CpG methylase and transfected into explanted lens cells. We found a strongly reduced activity of the methylated construct; activity was only 1% of that of the unmethylated promoter and within background levels (Fig. 2B). Although the CAT coding region is also methylated in these experiments, other studies have shown that DNA methylation does not impede elongation (35-37) and that methylation of the CAT coding sequence does not affect transient expression (6, 38). Hence, the effect of CpG methylation is likely to block gamma D promoter activity, although from our own experiments, we cannot rule out an aspecific effect.

The gamma D promoter contains a CpG site in its proximal activator, the gamma -box, located around -50 (see also Fig. 1). To test whether methylation of the gamma -box element is sufficient to block binding of the cognate activating factor in vitro, the binding of rat lens nuclear extract factors to a methylated promoter fragment was compared with that to a nonmethylated fragment in an EMSA. Complex formation with the methylated fragment was reduced when compared with that of the unmethylated fragment, but not abolished (complex D1; Fig. 2C). This was confirmed by the fact that the methylated fragment competed for the activator complex as efficiently as the unmethylated fragment itself. In the EMSAs using the methylated gamma D promoter fragment, an additional band is seen (complex D2; note that this complex migrates slower than the faint aspecific complex seen in some of the other lanes). This band could represent binding to the methylated DNA by general MCpG binding proteins.


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Fig. 1.   Sequence alignment of the 5'-flanking regions of the six rat gamma -crystallin genes. The sequence of the gamma -box region of the mouse gamma F-crystallin promoter is shown for comparison. Only the gamma D sequence is shown in full; for the other sequences, only differences are specified. Dashes indicate gaps introduced to optimize alignment. The TATA-box is shown in bold. Transcription start sites are indicated by arrows. Asterisks mark residues involved in factor binding in lens cells, as demonstrated by in vivo footprint analysis (7). The activator element at position -57 to -46 of gamma D-crystallin promoter, the gamma -box, is indicated as suggested by Peek et al. (8) as is the silencer region. Note that the nucleotide sequence of the mouse gamma F-crystallin gamma -box is identical to that of the rat gamma D-crystallin gamma -box except for the A, which is present in the rat gamma A- and gamma C-crystallin gamma -box sequences as well. Also note that the gamma B-crystallin equivalent of the gamma -box, and the region directly downstream, contains the most nucleotide changes relative to that of the gamma D-crystallin, and that the gamma E- and gamma F-crystallin promoters lack the G/C-rich -10 region present in the gamma D-crystallin promoter. Sequences and their alignment are according to Den Dunnen et al. (9); the mouse gamma F-crystallin promoter sequence is from Lok et al. (10).


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Fig. 2.   Demethylation of the gamma D-crystallin promoter during development. A, analysis of the methylation state of the genomic ThaI site at position -13. Lens explants from both newborn and 10-day-old rats were cultured in the presence of bFGF and harvested at several stages during differentiation. Chromosomal DNA was isolated as described previously (7) and digested with both ThaI (cuts at -13) and Sau3A (cuts at -23). The DNA was amplified by ligation-mediated PCR using three primers in succession (from +85/+67, +42/+21, and +42/+19; for details see Ref. 7) and visualized by electrophoresis and autoradiography. PCR products from methylated and unmethylated DNA are indicated. Both the autoradiograph and the quantitated data are shown. B, activity of methylated (mutant) gamma D- or gamma C-crystallin promoters. Wild-type and mutant -73/+10 gamma D-crystallin promoter constructs or the -70/+28 gamma C promoter construct were in vitro methylated using CpG methylase (SssI methylase). Methylated and mock-methylated constructs were transfected to explants pre-cultured for 10 days in the presence of bFGF. The explants were cultured for three more days before harvesting (see "Experimental Procedures" for details). Activities of the non-methylated gamma D and gamma C promoter constructs were set at 100%; the activity of gamma DB2 or gamma DF is given relative to that of gamma D. The bars indicate the standard deviation. C, factor binding to the CpG-methylated gamma -box. Methylated and unmethylated gamma D (-73/+45) fragments were used as probes, as indicated. Completeness of DNA methylation was tested by digestion (ThaI), and factor binding was examined in the absence (-) and presence (+) of nuclear extract from newborn rat lenses. Binding was in the presence of 50 mM KCl and 50 ng/µl poly(dGdC·dGdC). Complex D1 represents the gamma D-crystallin activator complex (gamma -box complex), as confirmed by methylation interference footprint analysis using the -73/+45 fragment (results not shown). Complex D2 might represent binding of MCpG-binding protein, as it is found only with the methylated probe. Specific competitor DNA was added in a 100-fold molar excess (right two lanes).

These data suggest that methylation of the gamma -box is not sufficient to suppress promoter activity. We therefore tested the effect of methylation on the activity of mutant constructs, lacking either the CpG site at -50 or the CpG sites between -20 and -10. The activity of these mutant gamma D promoters, when methylated, was also in the background range (Fig. 2B). Similarly, the activity of the gamma C promoter was also very low when methylated. Together, our results indicate that the reduction in gamma -crystallin promoter activity by methylation is a general effect and not due to methylation of a specific site.

Appearance of Trans-acting Factors during in Vitro Lens Cell Differentiation-- We have previously proposed that the differentiation stage-specific expression of the gamma D-crystallin gene during fiber cell differentiation was regulated by the phased appearance of first an activating and then a silencing factor (7). The reduced activity of the gamma D promoter in explants of 10-day-old rats could be due to a changed expression profile of these factors. Therefore, the activities of the gamma D(-73/+45)CAT fusion gene and of a silencer-tkCAT construct were followed during the course of differentiation of 10-day-old explants. The gamma D(-73/+45)CAT construct was active at all stages of differentiation, with a maximum around day 12 (Fig. 3A). The timing of up-regulation of the gamma D activating factor in these explants is very similar to that in explants from newborn rats (see Ref. 7). However, the activity of the gamma D(-73/+45)CAT construct in 10-day-old explants was around 50% of that in explants from newborn animals (data not shown), indicating that the level of the activating factor is decreased in the older explants.


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Fig. 3.   Activities of the proximal gamma D-crystallin promoter and its silencer element during differentiation of explants from 10-day-old rats. Transfections were done as described under "Experimental Procedures." The dotted lines represent the activities obtained using newborn rat lens explants as reported previously (7). A, activity of gamma D(-73/+45)CAT transfected into explants from 10-day-old rats at several stages of bFGF-directed differentiation. Activities are shown relative to that of the maximum level (100%). The bars indicate the standard deviation. B, activity of pBLCAT2gamma (8), containing four copies of the -85/-67 silencer region in front of the tk promoter, transfected to explanted lens cells from 10-day-old rats at several stages of differentiation. Activities are shown relative to that of the parental construct pBLCAT2, which was set at 100% (not shown). The bars indicate the standard deviation.

Rather different results were obtained when the presence of the silencing factor was assayed for; the construct containing four copies of the silencing element in front of the HSV tk promoter was inactive even in early differentiated cells from 10-day-old rats (Fig. 3B), indicating that the silencing factor is present throughout differentiation of these cells. In contrast, in explants from newborn rats, silencing activity was maximal only after 10 days of in vitro differentiation (Ref. 7; see also Fig. 5B). The level of silencing in fiber cells from 10-day-old rats was not significantly different from that during late differentiation of cells from newborn animals. The earlier appearance of the silencing factor might well explain the reduced activity of the gamma D gene in the 10-day-old explants.

The gamma D-Crystallin Silencing Factor-- The gamma D silencer was originally found by chance, when testing the effect of the -84/-71 G/C-rich region conserved among the gamma -crystallin promoters. This region suppresses gene activity in non-lens cells such as retina, skin, and brain (12). In vivo footprinting in lens cells, however, showed that nuclear factor contacts extended further upstream to -88 (7). In view of its importance in the developmental shut down of the gamma D gene, we have reexamined this region. Methylation interference footprinting showed a contacted region between -91 and -79 in the upper strand and between -90 and -78 in the lower strand in both a lens and a brain nuclear complex, suggesting the presence of the same factor in lens and brain cells (Fig. 4, A and B). This was further confirmed by measurement of the molecular weight of the factors in an EMSA-based method (39), in which the mobility of the complexes in gels of different polyacrylamide concentrations were compared. The molecular masses of both the lens and brain complexes, including the oligonucleotide probe, were estimated at 105 kDa (Fig. 4C). These results strongly suggest that the lens and brain factors are the same and bind the gamma D silencer element at position -91 to -78 in both tissues. Apparently, this ubiquitous, or at least not lens-restricted, factor has been recruited by the lens to function in the differentiation and developmental control of the gamma D-crystallin promoter.


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Fig. 4.   Nuclear factor from lens or brain binds the gamma D-crystallin silencer at the same site. A, methylation interference footprint analysis of the gamma D-crystallin -80 region complexed by either lens or brain nuclear factors. gamma D-crystallin -106/+45 promoter fragment (containing mutation -46Gright-arrowT, thus abolishing factor binding to the gamma -box; see Ref. 7) was 32P labeled at either end and used to bind nuclear factors from either newborn rat lens or brain. Results using both free (F) and bound (B) DNA are shown. Sites of protein contacts are indicated by brackets. B, summary of footprint analyses shown in A. Residues involved in factor binding are specified by asterisks. Nucleotide sequence is as reported by Den Dunnen et al. (9). C, estimate of the molecular weights of gel-retarded DNA/protein complexes as described by Orchard and May (39). A cloned 32P-labeled oligonucleotide containing the -91/-78 gamma D-crystallin silencer element (gamma DS1; see Fig. 5) was bound to either lens or brain nuclear extracts. EMSAs were run together with native standard proteins in a series of gels with increasing polyacrylamide concentration. The relative migration of the DNA/protein complexes (B, complex brain; L, complex lens) and standard proteins (BSA, bovine serum albumin; CA, carbonic anhydrase) were plotted against the polyacrylamide concentration (left). Rf is the migration distance relative to the migration distance of bromphenol blue. The negative slopes of the curves from the standard proteins were then plotted against their molecular weights in a Ferguson plot (39), from which the molecular weight of the lens and brain nuclear complexes was determined (right).

A synthetic copy of the -91/-78 element silenced the heterologous tk promoter by about 65% (Fig. 5A; gamma DS1), which corresponded to the silencing activity of a larger promoter fragment (gamma D), whereas the conserved G/C-rich region (-84/-68; gamma ) silenced by about 25%. Thus, the -91/-78 element is the silencer element within the gamma D promoter. The in vivo activity of the original tetramer silencer construct (Ref. 7; see Fig. 3B), although containing only part of the silencer element is probably due to the fact that the multimerization of the -84/-68 sequence by chance partially provided the missing part of the element.


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Fig. 5.   The gamma D-crystallin -91/-78 promoter element is a functional silencer in lens cells. Constructs containing the gamma D-crystallin promoter sequences fused to the tk promoter as indicated in the figure were transfected to explants from newborn rats precultured in the presence of bFGF for 11 days (A) or as indicated (B). After 3 days, the cells were harvested, and the CAT activities were determined as described under "Experimental Procedures." CAT activities are shown relative to that of pBLCAT2 (100%). The bars indicate the standard deviation. The silencer sequence is underlined. Sequences are according to Den Dunnen et al. (9).

The Common Proximal Activator of the gamma -Crystallin Promoters-- The alignment of the proximal promoters of the gamma -crystallin genes shows that the gamma -box is a well conserved element (Fig. 1) and predicts that all gamma -crystallin promoters, with the possible exception of the gamma B promoter, bind the same factor. Indeed, nuclear factor binding to the gamma D promoter is efficiently competed for by the gamma C and the gamma F promoter (data not shown). The gamma B promoter fragment, however, competed poorly for binding (Fig. 6A). As the gamma B promoter is also the one with the most extended developmental expression, we analyzed the gamma B promoter element in more detail.


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Fig. 6.   The gamma B-crystallin gamma -box region. A, EMSAs showing binding of the 32P-labeled gamma D-crystallin -73/+45 promoter fragment in lens nuclear extracts (+), competed with the equivalent gamma B-crystallin promoter fragment. Binding was in the presence of 50 mM KCl and 50 ng/µl poly(dGdC·dGdC). Specific competition DNA was added in a 10-200-fold molar excess, as indicated. In the first lane (-), no extract was added. Only the region of the autoradiograph with bound probe is shown. See "Experimental Procedures" for details. B, reciprocal competitive EMSAs using the 32P-labeled gamma B-crystallin -73/-16 promoter fragment as a probe. See A for further details. Note the appearance of an additional, specific complex (B2) migrating faster then B1. C, in vitro methylation interference footprints of the upper (coding) strand. 32P-labeled gamma B-crystallin -73/-16 fragment was methylated and bound to nuclear factors from newborn rat lenses. See "Experimental Procedures" for further details. Sequence ladders of both bound (B) and free (F) probes are shown. Positions of the G residues relative to the transcription start site are indicated. Sites of protein contact are marked by brackets. D, summary of the results shown in C. G residues involved in protein interaction are marked by asterisks. Binding sites in complexes B1 and B2 are indicated. AP-1 consensus binding sites are specified by arrows. Nucleotide sequence and numbering are according to Den Dunnen et al. (9).

A promoter fragment containing the gamma B-box yielded two complexes in an EMSA. The lower complex (B2) was competed for by the gamma B but not by a gamma D fragment and thus appeared to be gamma B-specific (Fig. 6B). The upper complex (B1) comigrated with the single complex formed by the equivalent gamma D promoter fragment (not shown) and was also competed for efficiently by this gamma D fragment (Fig. 6B), suggesting that this complex represents the gamma B -57/-46 activator (gamma B-box) complex. Competition by the gamma B fragment for either the gamma D-box (Fig. 6A) or the gamma B-box complex (Fig. 6B) was very poor, confirming the relatively low affinity of this gamma B promoter element for factor binding. To confirm the conclusion that complex B1 (see Fig. 6B) represents binding to the gamma B -57/-46 region, this complex was mapped by in vitro footprinting. As shown in Fig. 6C (left panel), the B1 complex is indeed the result of factor interaction between positions -55 and -46. In vitro footprinting of complex B2 showed that the binding site in this complex is the -46 to -38 region (Fig. 6C, right panel), directly adjacent to the gamma B-box. The B2 footprint corresponds to a consensus AP-1 site (Fig. 6D). A second AP-1 site is located directly downstream, but no interaction with this site was seen in vitro.

To understand the functional significance of the low affinity binding by the gamma B-box, the gamma D-box was exchanged for the corresponding gamma B element. Mutating the gamma D-box in the gamma D(-375/+10)CAT construct successively to the equivalent gamma B sequence led to a gradual decrease in promoter activity (Fig. 7A, left panel), showing that the gamma B-box is a lesser activator than the gamma D-box. The drop in activity is sharpest between mutant constructs gamma DB1 and gamma DB2, nicely corresponding to the in vitro binding affinity of these mutants; gamma DB1 still efficiently competes with the gamma D sequence, but gamma DB2 no longer does so (Fig. 7B). An even more dramatic effect of mutating the gamma D-box to the gamma B-box is seen when the upstream region is deleted from the -375/+10 constructs (Fig. 7A, right panel). Deletion to -73 in the wild-type gamma D promoter has only a slight effect on promoter activity. However, introducing two nucleotide substitutions in the gamma D-box (gamma DB2) now results in activity barely above background. Hence, elements in the upstream region contribute to promoter activity, but this effect is only seen experimentally when the gamma -box sequence is less than optimal.


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Fig. 7.   gamma B-crystallin promoter elements. A, functional comparison of the gamma B- and gamma D-boxes and mutant intermediates. The gamma -box sequence in a gamma D(-375/+10)CAT construct was successively mutated to its gamma B equivalent yielding constructs gamma DB1 to gamma DB3 (left panel). gamma B-like sequences are underlined. These constructs and a gamma B(-414/-16)CAT construct were transfected into explanted lens cells from newborn rats, following a preculture period of 10 days in the presence of bFGF. Cells were cultured for three more days before harvesting. Promoter activities were determined as described under "Experimental Procedures." Similar experiments were performed with constructs in which the upstream sequences were deleted to -73 (right panel). All activities shown are relative to that of the parental gamma D(-375/+10) promoter construct (100%). The bars indicate the standard deviation. B, EMSA showing binding of 32P-labeled wild-type gamma D(-73/+10) fragment and lens nuclear extract (+) competed with the gamma D fragment, the equivalent gamma B fragment, or mutant fragments (see A). Binding was in the presence of 50 mM KCl and 50 ng/µl poly(dGdC·dGdC). Specific competitor DNA was used in 100-fold molar excess. In the first lane, no extract was added (-). Free (F) and bound (B) probes are indicated. C, functional analysis of the gamma B -46/-38 (B2) region. By in vitro mutagenesis, the gamma B -40 region was exchanged for its gamma D counterpart yielding construct gamma BD1. The reciprocal experiment yielded construct gamma D4. Subsequently, the gamma B-box was introduced in the latter construct, yielding construct gamma DB4. gamma B-like sequences are underlined. Arrows point to mutated nucleotides. Constructs were transfected into explanted lens cells from newborn rats, which had been precultured for 10 days in the presence of bFGF. After three additional days of culture, the cells were harvested, and promoter activities were assessed as described under "Experimental Procedures." Activities are shown relative to the wild-type gamma D construct (100%). The bars indicate the standard deviation.

The functional significance of the AP-1 consensus site at -46/-38 in the gamma B promoter was tested by mutating this sequence to the corresponding gamma D sequence (gamma BD1; Fig. 7C). This mutation severely decreased promoter activity of the gamma B promoter, showing that the AP-1 site acts as an activator (Fig. 7C). This was confirmed by the reciprocal construct, in which the -46/-38 gamma B element was introduced at the corresponding site in the gamma D promoter (gamma D4); again, this element functioned as an activator, as a 2-fold increase in promoter activity was the result. Finally, we tested the effect of a combination of both the gamma B -57/-46 and -46/-38 elements in the gamma D promoter. As expected, this construct (gamma DB4) had the same low activity as the gamma B-promoter itself.

The gamma D-Box Is a MARE-- Kataoka et al. (22) first suggested that the gamma -box might be a Maf recognition element (MARE). This suggestion is supported by the experiments reported by Ogino et al. (40). We have therefore tested whether the factor binding to the gamma D-box also binds the MARE consensus sequence. As shown in Fig. 8, in an EMSA using lens nuclear extract, a gamma D promoter fragment competes efficiently with binding to a consensus MARE. In addition, the mobility of the gamma D-box complex is the same as that of a MARE complex (data not shown), suggesting that the gamma D-box does indeed bind a Maf, at least in vitro.


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Fig. 8.   The gamma D-box resembles a MARE. EMSA showing binding of a double-stranded MARE consensus oligonucleotide (TGCTGACTCAG) and lens nuclear extract from newborn rats (+) competed for with a gamma D(-73/+10) fragment. As control, competition with the MARE sequence is also shown. Binding was in the presence of 50 mM KCl and 50 ng/µl poly(dGdC·dGdC). Specific competitor DNA was added in 10- and 100-fold molar excess. In the first lane, no extract was added (-). Free (F) and bound (B) probes are indicated.

Two Maf sequences have been reported thus far to be present in the rat lens, Maf-1 and Maf-2 (41, 42). Maf-2 is expressed in lens fibers but not in epithelium, whereas Maf-1 is also present in epithelial cells. Cotransfection of expression constructs for Maf-1 or Maf-2 and gamma D(-73/+10)CAT into differentiating lens explants showed that the gamma D promoter activity was stimulated about 2-fold by Maf-2 but not Maf-1 (data not shown). Hence, the factor binding the gamma D-box might well belong to the Maf family.

The gamma D -10 Element-- The data presented above show that the gamma B-box can function as an activator only in conjunction with the downstream -46/-38 element. Yet, the gamma DB3 mutant, which lacks this activator, still retains activity, albeit low (see Fig. 7A). We therefore wondered whether an additional activating element is present in the -73/+10 gamma D promoter, which, in cooperation with the low affinity gamma DB3-box, drives promoter activity in this mutant. Genomic footprinting of the rat gamma D promoter had revealed a protected site downstream of the TATA box: the GC-rich -10 region (Ref. 7; see Fig. 1). The nucleotide sequence of this region is unique to the gamma D promoter and absent from the otherwise nearly identical gamma E and gamma F promoters. To test the effect of the gamma D -10 element, we constructed gamma D/gamma F promoter chimeras by replacing the gamma D TATA box and downstream region with the gamma F equivalent, causing the mutations -21(Tright-arrowC), -18(Cright-arrowT), and -15 to -12(CGCGright-arrowT---). The latter series of four mutations is situated within the in vivo footprint sequence mentioned above (see Fig. 1). In addition, for practical reasons, the 5' noncoding sequence was truncated from +10 to +1.

The activity of the -375/+1 gamma DF construct was not significantly lower than that of the wild-type gamma D construct when transfected into explanted lens cells (Fig. 9). However, shortening to -73 caused a drop in activity to about 20% of the corresponding gamma D construct. Introduction of the gamma B-box in the -73/+1 gamma DF promoter inactivated it, whereas mutation of the G/C-region in construct gamma D4 (see also Fig. 7C) strongly decreased promoter activity. Hence, the gamma D -10 region acts as an activator. These data further show that the gamma D-box is a relatively poor activator and needs additional elements for full activity. In the gamma D promoter, such elements are located between -375 and -73 and around -10. In the context of our experiments, these elements are redundant. Finally, our results confirm the observation that the gamma B-box is inactive in the absence of other positive elements. However, the positive element does not need to be positioned closely to the gamma B-box, as is the -46/-38 element in the gamma B promoter, but can also be located at a distance, as is the -10 region in the gamma DB3 construct.


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Fig. 9.   The gamma D-crystallin -10 region acts as an activator. In the gamma D-crystallin constructs indicated (see also Fig. 7), the TATA box and downstream region were exchanged for the gamma F equivalent, and the -375/-73 region was deleted. The gamma D-box is denoted by gamma -box, in gamma DB3 the gamma D-box has been mutated to the gamma B-box sequence, B2 represents the AP-1 consensus sequence as found in the gamma B promoter. Constructs were transfected into newborn rat lens explants, which had been precultured for 10 days in the presence of bFGF. After three additional days of culture, the cells were harvested, and promoter activities were assessed as described under "Experimental Procedures." Activities are shown relative to the wild-type gamma D(-375/+10) construct (100%). The bars indicate the standard deviation.

Differences between the gamma B- and gamma D-Crystallin Silencers-- We have shown above that the gamma -crystallin genes share the gamma -box activator. The question arises whether they all have silencers, as predicted from the in vivo mRNA levels (see Ref. 4) and, if so, whether these silencers are common or specific. To examine the presence of silencer elements within the gamma -crystallin promoters, we determined the silencing activity of the upstream regions of the gamma -crystallin promoters (from position -69) on the heterologous HSV tk promoter. All promoter regions tested repressed activity of the tk promoter when transfected into explanted lens cells, indicating that in all of these promoters, a functional silencing element is present (not shown). Again the gamma B sequence is most divergent and was selected for further analysis.

The -414/-69 gamma B fragment, which showed silencing activity in transient transfections, could be deleted to -110 without loss of silencing activity in lens cells during terminal differentiation (not shown), indicating that the gamma B silencer element must be located between -110 and -69. To test whether the gamma B and the gamma D silencing regions bind the same or different factors, the mobility of the gamma B and gamma D complexes was compared (Fig. 10A). Two gamma B complexes were found, both with mobility higher than that of the single gamma D complex, suggesting the formation of different gamma B and gamma D complexes. The non-identity of the complexes was confirmed by competition assays; the gamma D fragment did not compete for the gamma B complexes nor did the gamma B fragment compete for the gamma D complex (Fig. 10A).


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Fig. 10.   Comparison of the gamma B- and gamma D-crystallin silencers. A, the gamma B- and gamma D-crystallin silencer regions bind different factors in vitro. 32P-labeled fragments of the gamma B- (-110/-69, (BS) and gamma D-crystallin (-93/-76, gamma DS1) promoters were incubated with rat lens nuclear extract and electrophoresed through a native polyacrylamide gel (+). Fragments were bound in the presence of 10 mM KCl and either 50 ng/µl poly(dGdC·dGdC) (in the case of the gamma B probe) or 50 ng/µl poly(dIdC·dIdC) (in the case of the gamma D probe). Specific competitor DNA was added in a 100-fold molar excess. In the first and fifth lanes, no extract was added (-). Free (F) and bound (B) probes are indicated. See "Experimental Procedures" for details. B, functional analysis of the gamma B-crystallin silencer region. The gamma B(-414/-69)-tkCAT and gamma B(-110/-69)-tkCAT constructs were transfected to newborn rat lens explants, precultured with bFGF for the time as indicated. The cells were harvested 3 days later, and promoter activities were determined as described under "Experimental Procedures." Activities are shown relative to that of the tk promoter (pBLCAT2; 100%). The bars indicate the standard deviation.

In ewborn explants, the gamma D silencer is active only in terminally differentiated fiber cells (Ref. 7; see also Fig. 5B). To determine whether the gamma B silencer shows the same differentiation-dependent expression, both the gamma B(-414/-69)-tkCAT and gamma B(-110/-69)-tkCAT fusion genes were transfected to in vitro differentiating lens fiber cells. Like the gamma D element, the gamma B silencer demonstrated differentiation-dependent recognition, as silencing activity was present only during a restricted period of differentiation (Fig. 10B). However, this silencing activity was apparent already between days 4-7 of differentiation and continued through the terminal stage of differentiation. The -110/-69 fragment demonstrated silencing activity only during terminal differentiation, similar to the gamma D silencer. Although the extent of silencing by the -110/-69 fragment is similar to that of the larger -414/-69 fragment during the course of differentiation, surprisingly, in early differentiation it strongly activated the tk promoter. Hence the -414 to -69 region of the gamma B-crystallin gene must contain additional enhancers and silencing elements.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

A simple mechanism for developmental regulation of the promoter activity of the gamma -crystallin genes would be that the level of activity is determined by the affinity of a common activating factor for the proximal activator, the gamma -box. One would then predict that a gamma -crystallin promoter that is shut down early in development (e.g. gamma E, gamma F) would have a low affinity gamma -box, whereas a gene of which expression continues until later in development would have a high affinity gamma -box. Our data clearly show that this hypothesis is not correct; we find that the gamma -crystallin gene with the most sustained expression during development, the gamma B gene, has the gamma -box with the lowest affinity.

Comparison of the gamma B-box sequence with that of the gamma D-box shows that in the gamma B-box the C at -54, which shows a protein contact in vivo (7), has been replaced by a T. This suggests that it is the 5'-half of the binding site in the gamma B-box that is responsible for the low affinity. However, our results show that even such a scrambled site is sufficient to target the corresponding cognate factors to the promoter, providing that additional activating elements are present. The additional activating element can be either a closely linked AP-1 site, as in the gamma B promoter, or the more distant up- and downstream elements of the gamma D promoter. Hence, there is no constraint on either the nature or the distance of the additional activating element.

The gamma -box resembles a MARE in sequence, and binding of a cognate factor in lens extracts is competed by a consensus MARE sequence. Furthermore, the gamma D promoter is slightly activated by cotransfection of an expression vector for Maf-2. Hence, the in vivo activator of the gamma -crystallin promoter might well belong to the Maf family of transcription factors. Interaction of the mouse gamma F-box with a (chicken) zinc finger protein has also been reported (11). However, this protein acts as a transcriptional repressor rather than as an activator.

The Maf family is a diverse one with small and large members (for recent reviews, see Refs. 43-45). The large members, which include the founding member of this family, v-Maf, have an N-terminal acidic activation domain. The small members, such as MafF, MafG, and MafK, lack this activation domain and can activate transcription only as heterodimers with, for example, a large Maf member or a member of the AP-1 family (e.g. see Refs. 22, 23, 45). Two large Maf family members have thus far been shown to be present in the rat lens. Maf-1 is the rat homologue of the mouse MafB, and Maf-2 may be the homologue of the chicken c-Maf (41, 42). Maf-1 mRNA is primarily found in the lens epithelial layer; Maf-2 mRNA is prominent in the fiber cell mass, the site of expression of the gamma -crystallin genes. Expression of Maf-2 is not limited to the lens, since Maf-2, as well as Maf-1, is also found in many other tissues of the body such as kidney, spleen, and liver. Hence, a role for Maf-2 in the expression of the gamma -crystallin promoters is seemingly at odds with the lens specificity of these promoters in transgenic mice (13, 14) or even in transgenic Xenopus laevis (46). However, Maf-2 could partner a lens-specific protein, and the role of Maf-2 in directing lens-specific expression could be analogous to that of MafK in erythroid-specific transcription; MafK acts as the partner of the erythroid-specific transcription factor NF-E2 (47). Alternatively, the "true" activator of the gamma -crystallin promoters could be another Maf protein. A possible candidate would be the rat homologue of L-Maf, a lens-specific member of the Maf family found in the chicken lens (40).3 However, it is not yet known whether such a rat homologue indeed exists. Clearly, further experiments are required to elucidate the role of Maf proteins in the regulation of gamma -crystallin gene expression.

AP-1 elements are targets of the Fos and Jun family of transcription factors, well known for their role in transmitting growth factor signals to the transcriptional apparatus. The involvement of an AP-1 element in the activity of the gamma B promoter suggests that the activity of this promoter is subject to regulation by extracellular factors. This could play a role in the developmental regulation of the activity of this gene and could further explain our rather puzzling observation that the level of gamma B mRNA reached during in vitro differentiation is significantly lower than that found in vivo.2 Apparently, bFGF is not capable of providing the proper signals for maximal activation of the gamma B promoter during in vitro differentiation.

The gamma -crystallin promoters contain an invariant sequence (CCCTTTTGTG) located -35 base pairs upstream from the TATA box (-73 to -63 in the gamma D promoter). The TTTG region in this sequence has been shown to be a binding site for Sox-2, a member of the Sry family of transcription factors (Ref. 48; see Fig. 11). The CCC are contacted by a factor in vivo, as they are detected on the in vivo footprint of the gamma D promoter (7). In that paper, the assumption was made that these CCC formed part of the silencer element. However, we show here that the silencer is located further upstream and contacts the bases -90 to -78. The close proximity of the CCC footprint at -73/-71 to the Sox-2 target site suggests that this footprint belongs to a factor that forms a heterodimeric complex with Sox (note that Sox binds in the minor groove, whereas in vivo DMS footprinting detects only major groove G contacts). We have not studied the effect of this site on gamma -crystallin promoter activity here as the sequence is invariant. We have previously shown that deletion of -77/-71 in the gamma F promoter caused a 60% drop in activity (35).


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Fig. 11.   Regulatory elements in the gamma D- and gamma B-crystallin promoters. The transcription start sites are shown by open arrows. The gamma -box elements and surrounding sequences are fully shown, whereas for the consensus binding sites of known transcription factors only differences are indicated. Nucleotide sequences and numbering are according to Den Dunnen et al. (9). The nucleotide sequence of the gamma -box elements resembles the phorbol-12-O-tetradecanoate-13-acetate-responsive element-type MARE (T-MARE; consensus sequence TGCTGACTCAG; see Refs. 44 and 45). Also, the consensus binding sequences of Sry/Sox-2 (48-50) and AP-1 (e.g. Refs. 51 and 52) are shown.

Together, our studies show that there is a plethora of positive and negative factor interactions in the proximal gamma -crystallin promoter (see Fig. 11). During development, the balance between these factors shifts toward repression. At least for the gamma D-crystallin promoter, it is the developmental change in the pattern of expression of the silencing factor that is the primary cause of promoter repression. Lack of demethylation is probably a secondary cause. However, the rate of promoter demethylation progressively decreases during development, and at even later developmental stages the rate of promoter demethylation may well be too slow to allow transcriptional activation, even if the cognate transactivating factors are present. Note that demethylation of the gamma D promoter region cannot be passive, i.e. due to lack of maintenance methylation following DNA replication, but must be active as there is no cell division coincident with promoter demethylation in differentiating lens explants.2 The sequence elements that signal gamma D promoter demethylation are unknown. Mapping these elements has thus far been precluded by the low transfection efficiency of lens explants at early stages of differentiation. Our efforts are now directed at overcoming this practical problem.

    ACKNOWLEDGEMENTS

We thank Dr. M. Sakai for the kind gift of the Maf-1 and Maf-2 expression constructs, and Lilian Hendriks and Cécile Lesturgeon for excellent technical assistance.

    FOOTNOTES

* This work has been carried out under the auspices of the Netherlands Foundation for Chemical Research and with financial aid from the Netherlands Organization for the Advancement of Pure Research and the Dutch Diabetes Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

dagger Deceased.

Dagger To whom correspondence should be addressed. Tel.: 31-24-3652911; Fax: 31-24-3652938; E-mail: nhl{at}sci.kun.nl.

1 The abbreviations used are: bFGF, basic fibroblast growth factor; MARE, Maf recognition element; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; EMSA, electrophoretic mobility shift assay; tk, thymidine kinase; HSV, herpes simplex virus.

2 E. J. Klok, S. T. van Genesen, A. Civil, J. G. G. Schoenmakers, and N. H. Lubsen, unpublished results.

3 Yasuda, K., and Ogino, H. (1998) Science 280, 115-118

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Abstract
Introduction
Procedures
Results
Discussion
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