From the Department of Molecular Biology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
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ABSTRACT |
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The six closely related and clustered rat
-crystallin genes, the
A- to
F-crystallin genes, are
simultaneously activated in the embryonic lens but differentially shut
down during postnatal development with the
B-crystallin gene, the
last one to be active. We show here that developmental silencing of the
D-crystallin promoter correlates with delayed demethylation during
lens fiber cell differentiation. Methylation silencing of the
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
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
-crystallin promoters
tested contain upstream silencers, but at least the
B-crystallin
silencer is distinct from the
D-crystallin silencer. The
-crystallin promoters were found to share a proximal activator (the
-box; around
50), which behaves as a MARE. The
B-box is
recognized with much lower avidity than the
D-box. By swapping
elements between the
B- and the
D-crystallin promoter, we show
that activation by the
B-box requires a directly adjacent
46/
38
AP-1 consensus site. These experiments also uncovered another positive
element in the
D-crystallin promoter, around
10. In the context of
the
D-crystallin promoter, this element is redundant; in the context
of the
B-crystallin promoter, it can replace the
46/
38
element.
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INTRODUCTION |
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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 -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
-crystallin gene family (the
A- to
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
B-crystallin mRNA is still present at 90% of
the level at birth, whereas the transcript level from the
D-crystallin gene has dropped to 60%, and those of the
E- and
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
-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 -crystallin
gene expression is not known. There is a strong negative correlation
between the methylation state of a
-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
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
-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
F-crystallin
-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
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 -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
-crystallin mRNA and protein
(18, 21); in differentiating explants from 10-day-old rats, the
-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
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
-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
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
D-crystallin promoter with the
B-crystallin promoter, the promoter with the most
extended developmental expression. We show that the
-boxes of the
D- and
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
B-box for this
factor is much lower than that of the
D-box. Activity of the
B-crystallin promoter requires interaction with an AP-1 binding site
directly downstream of the
B-box. Like the
D-crystallin promoter,
the expression of the
B-crystallin promoter is subject to silencing,
but the silencing factor differs from the one that represses the
D-crystallin gene.
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EXPERIMENTAL PROCEDURES |
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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
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
DB1,
DB2, and
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
D-crystallin promoter and the
mutant
DB3 promoter to obtain
D4 and
DB4, respectively, and
oligonucleotide BD1 (5'-TGG AGG CAG CAG ACC TCC TGC TAT ATA TAC CAG-3'
at position
55/
21) to obtain
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
D
10 region by its
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
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
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).
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 pBLCAT2
DS1. The ApaI (
73)/BamHI
fragment of pBLCAT2
(8) was exchanged with that of pBLCAT2
DS1 to
obtain pBLCAT2
DS. The
B- XhoI (
414)/ApaI
(
73),
C- NheI (
183)/ApaI (
69),
D- PvuII (
193)/ApaI (
73), and
F-crystallin
SpeI (
280)/ApaI (
70) blunt-ended/sticky
fragments were inserted into pBLCAT2
by replacement of its
HindIII (blunt)/ApaI fragment to obtain
pBLCAT2
B, pBLCAT2
C, pBLCAT2
D, and pBLCAT2
F, respectively.
Construct pBLCAT2
BS was obtained by deletion of the
414/
110
fragment from pBLCAT2
B using the SacI site at position
10.
DNA Transfection, Chloramphenicol Acetyltransferase (CAT) Assay,
and -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/
-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/
-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
-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.
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RESULTS |
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Demethylation of the D-Crystallin Promoter Region--
In
explants from newborn rats, the
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
D promoter is about 50-fold lower, the
state of methylation of the genomic ThaI site at position
13 of the
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.
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Appearance of Trans-acting Factors during in Vitro Lens Cell
Differentiation--
We have previously proposed that the
differentiation stage-specific expression of the 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
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
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
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
D activating factor in these explants is very
similar to that in explants from newborn rats (see Ref. 7). However,
the activity of the
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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The D-Crystallin Silencing Factor--
The
D silencer was
originally found by chance, when testing the effect of the
84/
71
G/C-rich region conserved among the
-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
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
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
D-crystallin
promoter.
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The Common Proximal Activator of the -Crystallin
Promoters--
The alignment of the proximal promoters of the
-crystallin genes shows that the
-box is a well conserved element
(Fig. 1) and predicts that all
-crystallin promoters, with the
possible exception of the
B promoter, bind the same factor. Indeed,
nuclear factor binding to the
D promoter is efficiently competed for by the
C and the
F promoter (data not shown). The
B promoter fragment, however, competed poorly for binding (Fig.
6A). As the
B promoter is
also the one with the most extended developmental expression, we
analyzed the
B promoter element in more detail.
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The D-Box Is a MARE--
Kataoka et al. (22) first
suggested that the
-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
D-box also binds the MARE consensus sequence. As
shown in Fig. 8, in an EMSA using lens
nuclear extract, a
D promoter fragment competes efficiently with
binding to a consensus MARE. In addition, the mobility of the
D-box
complex is the same as that of a MARE complex (data not shown),
suggesting that the
D-box does indeed bind a Maf, at least in
vitro.
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The D
10 Element--
The data presented above show that the
B-box can function as an activator only in conjunction with the
downstream
46/
38 element. Yet, the
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
D promoter, which, in cooperation with the
low affinity
DB3-box, drives promoter activity in this mutant.
Genomic footprinting of the rat
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
D
promoter and absent from the otherwise nearly identical
E and
F promoters. To test the effect of the
D
10 element, we
constructed
D/
F promoter chimeras by replacing the
D TATA box
and downstream region with the
F equivalent, causing the mutations
21(T
C),
18(C
T), and
15 to
12(CGCG
T
). 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.
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Differences between the B- and
D-Crystallin
Silencers--
We have shown above that the
-crystallin genes share
the
-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
-crystallin promoters, we determined the silencing activity of the
upstream regions of the
-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
B sequence is
most divergent and was selected for further analysis.
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DISCUSSION |
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A simple mechanism for developmental regulation of the promoter
activity of the -crystallin genes would be that the level of
activity is determined by the affinity of a common activating factor
for the proximal activator, the
-box. One would then predict that a
-crystallin promoter that is shut down early in development (e.g.
E,
F) would have a low affinity
-box, whereas
a gene of which expression continues until later in development would have a high affinity
-box. Our data clearly show that this
hypothesis is not correct; we find that the
-crystallin gene with
the most sustained expression during development, the
B gene, has
the
-box with the lowest affinity.
Comparison of the B-box sequence with that of the
D-box shows
that in the
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
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
B promoter, or the more distant up- and
downstream elements of the
D promoter. Hence, there is no constraint
on either the nature or the distance of the additional activating
element.
The -box resembles a MARE in sequence, and binding of a cognate
factor in lens extracts is competed by a consensus MARE sequence. Furthermore, the
D promoter is slightly activated by cotransfection of an expression vector for Maf-2. Hence, the in vivo
activator of the
-crystallin promoter might well belong to the Maf
family of transcription factors. Interaction of the mouse
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 -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
-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
-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
-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 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
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
B promoter during
in vitro differentiation.
The -crystallin promoters contain an invariant sequence (CCCTTTTGTG)
located
35 base pairs upstream from the TATA box (
73 to
63 in the
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
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
-crystallin promoter activity here as the sequence is invariant. We
have previously shown that deletion of
77/
71 in the
F promoter
caused a 60% drop in activity (35).
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Together, our studies show that there is a plethora of positive and
negative factor interactions in the proximal -crystallin promoter
(see Fig. 11). During development, the balance between these factors
shifts toward repression. At least for the
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
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
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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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