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INTRODUCTION |
The human and murine MOK2 orthologue genes, which are
preferentially expressed in brain and testis tissues, encode two
different Krüppel/TFIIIA-related zinc finger proteins. The
human and murine genes have been localized to band q13.2-q13.3 of
chromosome 19 and chromosome 6, respectively (1, 2). The human hsMOK2 protein shows substantial differences with the murine MOK2 protein. The
mouse MOK2 protein contains seven tandem zinc finger motifs with only
five additional amino acids at its COOH-terminal end (3). The seven
fingers motifs are highly similar to one another but are distinct from
those of other zinc finger proteins. The structural feature of murine
MOK2 protein is also found at the end of human hsMOK2 protein.
Furthermore, the human protein contains three additional zinc finger
motifs in tandem with the others and a nonfinger acidic domain of 173 amino acids at the NH2-terminal end (2). We have previously
shown that human MOK2 RNA maturation results in three
mRNAs with different 5'-untranslated exons. One of these three
mRNAs encodes a smaller MOK2 protein (hsMOK2
) containing
10 zinc finger motifs and a small NH2-acidic domain made up
of 76 amino acids. We have shown that the human and murine MOK2
proteins are able to recognize both DNA and RNA through their zinc
finger motifs (4). Electron microscopy and specific RNA homopolymer
binding activity showed clearly that the murine and human MOK2 proteins
are RNA-binding proteins that associate mainly with nuclear RNP
components, including nucleoli and extranucleolar structures. Murine
and human MOK2 proteins have been shown to bind the same 18-base pair
(bp)1-specific sequence in
duplex DNA (4). This 18-bp-specific sequence has been identified by two
approaches, randomized oligonucleotide and whole genome PCR techniques.
The 18-bp MOK2-binding site occurs in an intron of two different human
genes. It is interesting to note that these two potential target genes
for MOK2 protein function in the brain, where the MOK2 gene
is preferentially expressed. The first one is the human PAX3
gene, a transcription factor expressed during brain development (5, 6).
Interestingly, in this gene, the MOK2-binding site occurs in the last
intron (7), in which disruption is associated with the translocation in
human alveolar rhabdomyosarcomas (8). The second potential target gene encodes the human interphotoreceptor retinoid-binding
protein (IRBP), which is expressed exclusively in retinal photoreceptor cells and in a subgroup of pinealocytes. IRBP is thought to be involved
in the visual cycle of vertebrate retina (9-11). In developing mice,
IRBP is first expressed at the birth of the photoreceptors, suggesting
that it may also be involved in photoreceptor differentiation (12). The
importance of IRBP in normal photoreceptor development has been
demonstrated by the recent generation of mice with a targeted
disruption of the IRBP gene (13). In the absence of the
Irbp gene, there is a slowly progressive degeneration of
retinal photoreceptors. The MOK2-binding site is located in intron 2 of human and bovine IRBP genes. It has been suggested that the
highly conserved introns 2 and 3 of the IRBP gene might
contain important regulatory elements for IRBP gene
expression (14).
Here we have focused our attention on IRBP as a potential
MOK2 target gene. Sequence comparison and binding studies of the 18-bp
MOK2-binding sites present in intron 2 of human, bovine, and mouse
IRBP genes show that the 3'-half sequence is the essential core element for MOK2 binding. Very interestingly, 8 bp of this core
sequence are found in a reverse orientation, in the IRBP promoter. The results presented here demonstrate that MOK2 can bind to
the 8-bp sequence present in the IRBP promoter and repress its transcription when transiently overexpressed in retinoblastoma Weri-RB1 cells. Furthermore, we show that Mok2 expression in
the developing mouse organism and adult retina seems to be concordant with IRBP expression.
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MATERIALS AND METHODS |
Plasmid Constructions--
All plasmids generated for this study
were confirmed by DNA sequencing. The recombinant GST-hsMOK2 and
CBD-hsMOK2 fusion proteins were obtained by inserting the blunt-ended
XbaI-EcorI fragment (1850 bp) into EcoRI/Klenow
fragment-treated pGEX-3X (Amersham Pharmacia Biotech) and
BamHI/Klenow fragment-treated pET35 (Novagen), respectively.
The pET35 vector contains the cellulose-binding domain tag (CBD). The
XbaI-EcorI fragment, which contains the entire coding
sequence of hsMOK2, was obtained from pBhsMOK2. The pBhsMOK2 plasmid
and the eukaryotic expression CMV-hsMOK2 vector were obtained by
inserting the blunt-ended AcsI fragment from cDNA1 (2)
into the SmaI site of pBluescript KS+
(Stratagene) and NotI/Klenow fragment-treated pCMV,
respectively. The plasmids encoding recombinant maltose-binding
(MBP)-MOK2 fusion proteins and the eukaryotic expression vector
CMV-MOK2 were described previously (4). In this article, the eukaryotic
expression vector referred to as pCMV-hsMOK2, which encodes an isoform
containing a smaller NH2-acidic domain, was renamed
CMV-hsMOK2
(4).
The plasmid phsIRBPCAT contains a 332-bp fragment from the human
IRBP gene (from
291 to +41 relative to the transcription start site) inserted upstream of the CAT reporter gene (10). The
fragment of the human IRBP gene promoter was obtained from genomic DNA by PCR using a 5' primer containing an added
StuI site and a 3' primer containing an added Tth111I site.
The product was digested by StuI-Tth111I and cloned into the
corresponding sites of the PSV2CAT vector. In the phsIRBPCAT vector,
the SV40 promoter was replaced by the human IRBP promoter.
A 276-bp fragment from intron 2 of the bovine IRBP gene (nt
7130-7406 (15)) was obtained from Bos taurus genomic
DNA by PCR and cloned into EcoRV pZero-2 (Invitrogen).
Murine intron 2 of the Irbp gene was obtained from
C3H mouse genomic DNA by PCR amplification with a human 5' primer that
localizes to exon 2 (nt 5045-5065) and a 3' reverse human primer that
localizes to exon 3 (nt 7017-7037 (14)). The fragment (~2000 bp,
EMBL/AJ294749) was cloned into the SmaI site of the
pBluescript KS+ vector and sequenced.
Cells, Transient Transfections, and CAT Assays--
For
transient transfection assays, HeLa cells were plated at
106 cells on a 100-mm Petri dish for 24 h prior to the
addition of the recombinant plasmids by the calcium phosphate method as
described previously (16). For nuclear extracts, the HeLa cells were
transfected with 15 µg of MOK2 expression vectors. For CAT assays,
Weri-RB1 retinoblastoma cells were transfected in 6-well plates with
GenePORTER transfection reagent (Gene Therapy System) according to the
manufacturer's recommendations. About 106 cells were
cotransfected with 2 µg of IRBP promoter-CAT reporter vector (phsIRBPCAT) and 2 µg of MOK2 expression vector or
parental pCMV plasmid. The cells were harvested 36 h later for the
reporter gene assay using the freeze/thaw method (Promega). The
activity of the resulting extracts was determined using the CAT assay
protocol (Promega). Protein concentrations were determined by the
Coomassie protein assay (Pierce). For data interpretation, CAT activity was normalized to the protein concentration of the extracts. All transfection experiments were repeated at least five times with different CsCl-DNA preparations.
DNA Probes--
The plasmids containing human, bovine, or mouse
intron 2 of IRBP genes (1.5 µg) were cut once with the
appropriate restriction enzymes, treated with intestine phosphatase
alkaline, and end-labeled with T4 polynucleotide kinase. After a second
digestion with appropriate restriction enzymes, each end-labeled
fragment was purified on a 6% polyacrylamide gel. The double strand
oligonucleotides were labeled with T4 polynucleotide kinase in the
presence of [
-32P]ATP and purified on a 15%
polyacrylamide gel. For DNA probes containing dITP, dUTP, or
deaza-dATP, a 189 bp-fragment of intron 2 of human
IRBP (nt 6660-6849) was amplified by PCR using two primers,
one of which was 5' end-labeled by treatment with T4 polynucleotide
kinase in the presence of [
-32P]ATP. The PCR products
were purified on a 6% polyacrylamide gel. The 92-bp fragment of the
human IRBP promoter (
88 to +4) was labeled by PCR using
[
-32P]CTP and purified on a 6% polyacrylamide gel.
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared from five 100-mm Petri dishes of transfected
HeLa cells by a modified method of Dignam et al. (17). The crude nuclei were resuspended in 250 µl of
extraction buffer (20 mM HEPES, pH 7.9, 150 mM KCl, 0.05 mM ZnCl2, 0.1%
Nonidet P-40, 0.5 mM dithiothreitol, 20% glycerol) with a
protease inhibitor mixture without EDTA (Roche Molecular
Biochemicals) and disrupted by sonication. After centrifugation
at 15,000 × g for 10 min, the nuclear extract was
stored at
70 °C. For bacteria expressing fusion proteins, the
crude protein extracts were prepared as described previously (4). The
standard DNA binding reaction (20 µl) contained 20.000 cpm of
32P-labeled probe, 2.5 µg of crude MBP fusion protein or
10 µg of nuclear extract, and 2 µg of poly(dI-dC) in binding buffer
(20 mM HEPES, pH 7.9, 100 mM KCl, 0.05 mM ZnCl2, 0.1% Nonidet P-40, 0.5 mM dithiothreitol, 20% glycerol). Complexes were analyzed by electrophoresis on a nondenaturing premigrated 4% polyacrylamide gel (acrylamide/bis ratio of 19:1) or 1% agarose gel in 0.5× TB buffer (45 mM Tris borate, pH 8.3) at 4 °C at 200 or 120 V, respectively. EDTA was omitted in all binding and electrophoresis
buffers to avoid denaturing MOK2. All probes were gel-purified.
Antibody Preparation--
The purified GST-hsMOK2 fusion protein
was injected into a New Zealand White rabbit. Affinity-purified hsMOK2
antibodies were obtained by elution of immunoglobulins bound to the
human CBD-hsMOK2 protein as described previously (4).
Northern Blot and S1 Nuclease Analysis--
Total cellular RNAs
were extracted from normal tissues of 1-month-old mice by the
guanidinium thiocyanate procedure (18). Polyadenylated RNAs were
prepared using oligo(dT) cellulose (Type III, Collaborative Research)
columns (19). Five µg of poly(A)+ RNAs from each tissue
was used for Northern blot analysis, using nucleotides 1848-2276 of
the mouse Mok2 cDNA as probe (3). For S1 nuclease
analysis, 10 µg of poly(A)+ RNAs from mouse embryos were
hybridized with a single strand of the mouse genomic fragment
(nt
563 to +51 (16)). 5' end-labeled with 32P at nt +51.
5' end-labeling and strand separation were carried out by standard
techniques. The S1 nuclease-resistant products were resolved by
electrophoresis on 10% polyacrylamide denaturing gels and
detected by autoradiography.
In Situ Reverse Transcriptase-PCR Experimental
Procedure--
In situ reverse transcriptase-PCR was
performed as described by Thaker (20). The eyes of 1-month-old mice
were enucleated, immediately frozen in OCT (TissueTek, Sakura,
Netherlands) and sectioned (10 µm). Cryosections were collected on
silane-coated glass slides. The sections were fixed for 1 h
with a 10% Formalin/PBS for 1 h, washed three times in a
0.1% Triton X-100 in PBS, permeabilized for 10 min at
20 °C in an ethanol/acetic acid solution, washed three times in
PBS, and dehydrated in graded alcohols. mRNAs were reverse-transcribed for 1 h at 42 °C with 200 units of Moloney murine leukemia virus (M-MLV) reverse transcriptase RNase H minus, using random hexamers (Promega). Sections were washed twice in PBS and dehydrated in graded alcohols. PCR reactions were carried out
with the 5' primer (5'-TCTAACTGTCTCCACTTCCCA-3') and the 3' primer
(5'-AAGGCACATAATTTCAGAGGA-3') located in the 3'-untranslated region of
Mok2 mRNA at nucleotides 1848-1869 and 2276-2297 in the presence of Biotin16-dUTP (Roche Molecular Biochemicals) with a
1:19 ratio of dTTP. The amplification program was as follows: 94 °C
for 1 min, 54 °C for 1 min, and 72 °C for 1 min repeated for 40 cycles. The slides were subsequently washed three times in PBS and
incubated for 1 h in 1/100 extravidin alkaline phosphatase conjugate (Sigma) in PBS solution. The signal was revealed using a
nucleic acid detection kit (Roche Molecular Biochemicals) containing levimasol and a subsequent phosphatase alkaline-catalyzed color reaction with Xphosphate and nitro blue tetrazolium salt, which produces a precipitate. Control sections were done in the same way
except that the reverse transcriptase reaction was omitted.
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RESULTS |
Conservation of the MOK2-binding Site in Intron 2 of the Murine
IRBP Gene--
The high homology found in introns 2 and 3 of the human
and bovine IRBP genes suggested that these sequences might
contain important regulatory elements for IRBP gene
expression (14). One of these elements might be the MOK2-binding site,
which is highly conserved between the intron 2 regions of human and
bovine IRBP genes. In bovine intron 2, we observed a
deletion of one nucleotide at position 7 in the 18-bp MOK2-binding site
(Fig. 1A). To determine
whether this sequence was also conserved in murine intron 2, a fragment
(~2000 bp) obtained from mouse genomic DNA by PCR using
human-specific primers, which localized to exon 2 and 3, was sequenced.
Intron 2 of the murine Irbp gene shows a similar high level
of homology with intron 2 of human and bovine IRBP genes
(62.3 and 58.2%, respectively). Sequence comparison of the 18-bp
MOK2-binding sites of the human, bovine, and murine genes showed that
the MOK2-binding site of mouse intron 2 was the most divergent. Four
nucleotides present in the first 9 bp did not agree with the previously
determined consensus sequence for MOK2 binding (4) or with the human or
bovine MOK2-binding sites in intron 2 (Fig. 1A).
Nevertheless, EMSAs showed that the truncated human MBP-hsMOK
and
mouse MBP-MOK2 fusion proteins were still able to interact with the
murine MOK2-binding site (Fig. 1B). These results suggested
that the nucleotides at positions 3, 5, 7, 8, and 9 within the 18-bp
MOK2-binding site were not crucial for MOK2 protein binding.

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Fig. 1.
A, comparison of 18-bp MOK2-binding
sites found in intron 2 of human, bovine, and murine IRBP
genes. The unconserved nucleotides are indicated by lowercase
letters. The 8-bp sequence found in the human and mouse
IRBP promoters is underlined. B, EMSA
analyses using human, mouse, or bovine probes with MBP or MBP-MOK2
fusion proteins were performed as described under "Material and
Methods." MBP-hsMOK2 and MBP-MOK2 fusion proteins correspond to
fusion protein of the human MOK2 protein truncated for the
NH2-acidic domain and the mouse MOK2 protein. The 201-bp
human probe contains 103 bp from intron 2 of the human IRBP
gene (nt 6722-6825, GenBankTM accession no.
J05253). The 265-bp mouse probe contains 220 bp from intron 2 of the
mouse Irbp gene (nt 1439-1659, GenBankTM
accession no. AJ294749). The 289-bp bovine probe contains 276 bp from
intron 2 of the IRBP bovine gene (nt 7130-7406,
GenBankTM accession no. J04441). Complexes were analyzed by
electrophoresis on 1% agarose gel in 0.5× TB buffer.
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We performed EMSAs using a series of probes containing a modified
nucleotide to determine which bases were crucial for MOK2 binding. The
probes were obtained by PCR using dITP, deaza-dATP, or dUTP instead of
dGTP, dATP, or dTTP, respectively. Fig. 2
shows that the human and murine MBP-MOK2 fusion proteins were only
unable to bind to the probe containing deaza-adenines instead of
adenines. This result suggested that the adenines located in the 18-bp
DNA-binding site were essential for the binding of human and murine
MOK2 proteins.

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Fig. 2.
EMSA analysis with different probes
containing one modified nucleotide or not (N), as
described under "Materials and Methods." Complexes were
analyzed by electrophoresis on a nondenaturing 4% premigrated
polyacrylamide gel.
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MOK2 Binds to the TAAAGGCT Sequence of the Human IRBP
Promoter--
Transgenic and other studies have identified regulatory
regions in the promoter important for the expression of the IRBP
protein (10, 21-25). The IRBP upstream region has been
found to have a 156-bp sequence that is well conserved between human,
bovine, and mouse (26). The short promoter fragment from
123 to + 18 (relative to the transcription start site) was found to confer photoreceptor-specific expression in transgenic mice (22). In the
123-bp sequence, we found 8 bp corresponding to the 3' end of
MOK2-binding consensus sequence. The TAAAGGCT sequence is present in a
reverse orientation compared with that of intron 2 (Fig. 3A). To determine whether this
sequence binds MOK2, we performed an EMSA. The nuclear extract from
control HeLa cells (transfected with the pCMV vector alone) and cells
overexpressing human or murine MOK2 were used for comparison. Nuclear
extracts were mixed with a labeled 92-bp fragment from the human
IRBP promoter (
88 to +4) and then electrophoresed. The
weak bands with different mobilities obtained with control HeLa cell
extract correspond to endogenous binding proteins that were not
affected by anti-hsMOK2 antibody (Fig. 3B, left panel,
lanes 1 and 2). A retarded complex was detected
with nuclear extract from hsMOK2-overexpressing HeLa cells (lane
3). The anti-hsMOK2 antibody clearly supershifted the DNA-protein
complex, showing that this complex contains human hsMOK2 protein
(lanes 4 and 5). In lane 6, a faster
migrating complex produced by murine MOK2 protein can be seen. The
difference in the mobilities of murine and human MOK2 proteins-DNA
complexes can be accounted for by the difference in the molecular mass
of these two proteins (22.8 and 51.5 kDa, respectively). To test whether MOK2 interacts directly with the 8-bp partial MOK2-binding site, we used two different mutated oligonucleotides as probes. These
two oligonucleotides contained 6- and 5-bp modifications of the
potential MOK2-binding site and the previously described AP-4-binding
element, respectively (21). The DNA-protein complex was abolished when
we used the mutant MOK2 probe but not the mutant AP-4 probe (Fig.
3B, right panel). These data illustrate that MOK2 proteins
are able to bind to the 8-bp partial MOK2-binding site present in the
IRBP promoter.

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Fig. 3.
EMSA analysis of the human IRBP
promoter (nt 13 to 76) with nuclear extracts of HeLa cells
transfected with MOK2 expression vectors. A, sequences
of the human IRBP promoter tested in the binding assays. The
Ret1/PCE-I-, CRX-, and AP-4-binding sites are indicated. The bold
letters show the 8-bp sequence present in the MOK2-binding site.
Mutations are represented by lowercase letters. B,
left panel, Anti-hsMOK2 supershift EMSA with nuclear extracts from
control HeLa cells (lanes 1 and 2) and
overexpressing hsMOK2 (lanes 3-5) or murine MOK2
(lane 6) HeLa cells. The indicated amount (in microliters)
of the affinity-purified anti-hsMOK2 antibody was added to EMSA
reactions containing the wild-type probe (WT) and 2.5 µg
of nuclear extracts. SS indicates the super-shifted bands.
Right panel, EMSA with wild-type, mutant MOK2
(MOK2mut), or mutant AP-4 (AP4mut) probes and 2.5 µg of nuclear extracts from normal or overexpressing hsMOK2 cells.
Complexes were analyzed by electrophoresis on a nondenaturing 4%
premigrated polyacrylamide gel.
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MOK2 Represses the Human IRBP Promoter Activity--
Having shown
that human and murine MOK2 proteins bind the 8-bp partial MOK2-binding
site in the IRBP promoter, we analyzed whether MOK2 proteins
were able to repress or transactivate the IRBP promoter. The
retinoblastoma cell line Weri-RB1, which expresses IRBP as
well as other photoreceptor genes (27), was used for transient
transfections to assay the effect of MOK2 on the IRBP promoter. Cells were transiently cotransfected with the reporter construct phsIRBPCAT, which contains the
291 to +41 region of the
IRBP promoter, and the human or murine MOK2 expression
vectors or the corresponding empty plasmid (pCMV). In the presence of MOK2 protein, a significant reduction in transcription activity was
observed (Fig. 4). The most important
reduction, about 70%, was found with the human hsMOK2 protein. Both
the hsMOK2
isoform, which is truncated for its
NH2-acidic domain, and the murine MOK2 protein repressed
CAT activity by about 50%. No effect was seen on the SV40
early viral promoter (data not shown). This result directly
demonstrates the ability of MOK2 to act as a transcriptional repressor
of the IRBP promoter.

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Fig. 4.
Effect of MOK2 on IRBP
promoter-reporter gene expression in Weri-RB1 cells. Cells
were cotransfected as described under "Materials and Methods." The
activity of the reporter gene in the absence of MOK2 was set at 100%.
The data represent the mean of at least five independent transfections
with different CsCl-DNA preparations.
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Mok2 Is Expressed in Mouse Embryos and in Photoreceptor
Cells--
It has been shown that IRBP protein and mRNA appear at
early developmental stages (12, 28, 29). Therefore, we investigated Mok2 expression during mouse embryogenesis. In previous
work, we found that MOK2 mRNAs are present at low levels
in cells and tissues proficient for the expression of the murine or
human MOK2 gene (2, 3). Expression of the Mok2
gene during mouse embryogenesis was consequently determined by S1
nuclease, a more sensitive method than Northern blot analysis, as
described under "Experimental Procedures." As shown in Fig.
5A, S1 nuclease products were
detected at all developmental stages tested. The presence of
Mok2 mRNA at embryonic day 9.5 shows that the
Mok2 gene is expressed early during mouse
embryogenesis.

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Fig. 5.
Expression of Mok2
in mouse embryos and eyes. A, S1 nuclease
analysis of poly(A)+ RNAs (10 µg) extracted from mouse
embryonic day: 9.5 (lane 1), 11.5 (lane
2), 12.5 (lane 3), 14.5 (lane 4), and 15.5 (lane 5). B, Northern blot analysis of
Mok2 mRNA. Poly(A)+ RNAs (5 µg) from
liver, brain, and eye normal tissues of 1-month-old mice were analyzed
by hybridization with a fragment localized in the 3'-untranslated
region (nt 1848-2276) of the mouse Mok2 cDNA. The blot
was then stripped and rehybridized with a -actin probe.
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We have previously shown that murine and human MOK2 genes
are preferentially expressed in the brain (2, 3). First, we investigated MOK2 expression in the eyes. Northern blot
analyses showed that the murine Mok2 gene is faintly
expressed in the eye of 1-month-old mice (Fig. 5B). To know
whether Mok2 is expressed in the retina, we used the
in situ polymerase chain reaction (PCR) technique, which
provides a very powerful tool for the study of the in situ
expression of rare genes. The results showed that Mok2
expression in the retina of 1-month-old mice was restricted to the
outer nuclear layer, which corresponds to the photoreceptors cells of
the retina (Fig. 6). However, we observed
that not all of the cells were stained within the outer nuclear layer
(ONL, green or white arrow in Fig. 6).
The outer nuclear layer of the mouse retina is composed of two kinds of
photoreceptors, rods and cones, but contains a majority of rods. One
possible explanation could be that only one kind of photoreceptor was
stained within the outer nuclear layer.

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Fig. 6.
In situ reverse
transcriptase-PCR analysis of Mok2 mRNA expression in
1-month-old mouse retinal sections. A and B,
magnification x240. Controls (A) correspond to PCR without
reverse transcriptase. C, magnification ×375; D,
magnification ×937 to the region with the green star in
panel C. The retinal cell layers are indicated as follows:
GCL, ganglion cell layer; IPL, inner plexiform
layer; INL, inner nuclear layer; OPL, outer
plexiform layer; ONL, outer nuclear layer. Similar results
were obtained in six independent experiments in which photoreceptors
cells were always stained.
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DISCUSSION |
In our previous studies, we showed that human and murine MOK2 bind
to an 18-bp sequence (A/G)CCTT(A/G)TCAG(A/G)GCCTTTA in duplex DNA (4).
This MOK2-binding site was found within introns 7 and 2 of human
PAX3 and IRBP genes, respectively. As these two genes are expressed in the brain as MOK2, we suggested that
PAX3 and IRBP genes are two potentially important
target genes for the MOK2 protein. In this study, we focused our
attention on IRBP as a potential MOK2 target gene. First, we
compared the sequences of the MOK2-binding site found in intron 2 of
human, bovine, and murine IRBP genes. The sequence
comparison showed that the 3' half-site of MOK2 is strictly conserved
between human, bovine, and mouse. The most divergence was observed on
the 5'-side of the MOK2-binding site of murine intron 2 compared with
those of the corresponding regions of the bovine and human MOK2-binding sites. EMSAs showed that MOK2 proteins could interact with the human,
bovine, and murine MOK2-binding site, suggesting that nucleotides at
positions 3, 5, 7, 8, and 9 were not crucial for MOK2 protein binding
to DNA. Furthermore, missing base contact probing suggested that MOK2
interacts with the adenines of the MOK2-binding site. The conserved 3'
half-site of the MOK2-binding site contains a triplet of adenines.
These results suggest that the essential core element of the
MOK2-binding site consists of the 9-bp sequence G(A/G)GCCTTTA. Very
interestingly, 8-bp of this core sequence are found in a reverse
orientation in the IRBP promoter.
In addition, we demonstrated that MOK2 binds to the TAAAGGCT sequence
and represses transcription from reporter constructs carrying the
291
to +41 regulatory sequence from the human IRBP promoter when
transiently overexpressed in Weri-RB1 retinoblastoma cells. This
repressive effect of MOK2 appeared to depend on the presence of a
TAAAGGCT sequence; no effect was seen on viral promoters such as the
SV40 early region promoter (data not shown). The
IRBP upstream region has been found to have a 156-bp
sequence that is well conserved between human, bovine, and mouse (26).
The short promoter fragment from
123 to +18 relative to the
transcription start site was found to confer photoreceptor-specific
expression in transgenic mice (22). Previous studies have identified
several functional cis-acting elements in the 123-bp proximal promoter of the IRBP gene (21, 23). These cis-acting elements
included two photoreceptor-specific consensus sequence, Ret1/PCEI
(AATTAG) and its reverse repeat, GATTAA. Ret1/PCEI has been identified as a functionally active cis element in several photoreceptor-specific genes (30, 31) including IRBP (22, 32, 33). Ret1/PCEI is
potentially one of the target sites of Rx, Rax, and Erx proteins of the
paired-type homeobox family (34-36). The reverse GATTAA sequence
(CRXE) is recognized by a photoreceptor-specific pair-related homeobox protein, cone-rod homeobox protein (CRX), which acts as a
transcriptional activator (37, 38). It has been shown that CRX
bound to and transactivated weakly from the Ret1/PCEI sequence.
These observations have suggested that CRX is not likely to be the
factor that most tightly binds to the Ret1/PCEI site (21, 38). The
TAAAGGCT MOK2-binding motif, which is conserved between human, bovine,
and mouse, overlaps with the CRX-binding element (CRXE, Fig.
3). MOK2 may repress transcription mediated by CRX by competing for CRX
binding to DNA, thereby decreasing activation. CRX has been shown to
bind and regulate several photoreceptor-specific promoters such as the
rhodopsin, IRBP,
-PDE, and arrestin
promoters (37). Unlike the CRX-binding element, the 8-bp MOK2-binding site is found only in the photoreceptor-specific IRBP
promoter. Our results show that the complete human hsMOK2 protein,
which contains the NH2-acidic domain, represses
transcription to a greater extent than the mouse MOK2 or human hsMOK
truncated isoforms, which essentially contain only the zinc
finger-binding domains. The higher repression could be due to the
additional steric hindrance of the hsMOK2 and other positively acting factors.
Our results did not directly demonstrate MOK2 regulation of the
IRBP promoter in vivo. However, in addition to
the results of binding assays and transient transfections, we showed
that Mok2 expression in the developing mouse and adult
retina seems to be concordant with IRBP expression. First,
we found that Mok2 is expressed in the photoreceptor cells
of the mouse retina where IRBP is synthetized. Mok2
mRNA was not detected in all photoreceptor cells. The morphological
similarity of rods and cones in the rodent retina does not allow one to
discriminate between rods and cones, but it is known that the
photoreceptor cells of the mouse retina are composed of a majority of
rods. IRBP is synthetized by both rod and cone photoreceptors. Rod
cells synthetize 4-fold higher amounts of IRBP (39). It is possible
that the photoreceptor cells expressing Mok2 might
correspond to cones cells that express lower levels of IRBP.
Furthermore, like IRBP mRNA, Mok2 mRNAs are detected at early developmental stages. Mok2 mRNAs
are found at embryonic day 9.5 before the beginning of IRBP
expression. Previous studies have shown that Irbp expression
begins on embryonic day 13 in the mouse, which is similar to the
photoreceptor trans-acting factor CRX (12, 37, 38). Mok2 is
not only expressed before the beginning of IRBP expression
but at all of the developmental stages tested. Increasing evidence
suggests that the regulation of many genes is the result of a fine
balance between positive and negative regulatory proteins. The
IRBP regulation could be, among other things, the result of
a balance between the repressor MOK2 and the activator CRX. Unlike
Crx, the Mok2 gene is not only a
photoreceptor-specific gene. Mok2 is more highly expressed
in the adult mouse brain than in the whole eye. The finding
that Mok2 is expressed early in mouse embryonic development
and in the adult brain suggests that Mok2 might play an
important role in development and particularly in neuronal development.
IRBPgene expression is highly regulated. It is known that
IRBP gene transcription can be modulated by light (40) and
by agents such as cAMP (41), indicating that both activation and
repression of IRBP activity are required for fine regulation of IRBP
levels. Some factor seems to be responsible for regulating
IRBP mRNA levels. For example, when developing or adult
mice were deprived of normal light, there was a decrease in the
IRBP mRNA level. Furthermore, it has been postulated
that aberrant expression of IRBP may be implicated in
certain genetically mediated retinal degenerations of the cat
(42, 43) and mouse (44). Abyssinian cats homozygous for a slowly
progressive form of hereditary rod and cone degeneration show a 50%
reduction in IRBP mRNA and protein as early as 4 weeks
of age, well before the onset of significant changes in retinal
structure. The repressor activity of MOK2 might play a role in the
reduction of IRBP mRNA levels. It seems worth mentioning
that the human hsMOK2 gene maps to chromosome 19q13.2--
q13.3
near the disease locus for autosomal dominant cone-rod distrophy
(CORDII, 19q13.3 (45, 46)). The trans-acting factor CRX has been
located to 19q13.3. In the human, several clinical phenotypes have been
associated with CRX mutations, including cone-rod
dystrophy, Leder congenital amaurosis, and retinitis pigmentosa
(47-51). The locus for dominant retinitis pigmentosa also lies near
the location of the CRX gene (RP11, 19q13.4 (52)).
The IRBP gene contains two MOK2-binding elements, a complete
18-bp MOK2-binding site located in intron 2 and the essential core
MOK2-binding site (8 bp of conserved 3'-half site) located in the
IRBP promoter. The results presented here demonstrate that MOK2 can bind to the 8-bp present in the IRBP promoter and
repress transcription. Actually, we do not know the role of the 18-bp MOK2-binding site present in intron 2 of the IRBP gene. This
site could allow MOK2 to repress transcription in another way by
blocking transcriptional elongation. Interestingly, it has been
suggested that a negative regulatory element affecting mRNA
elongation might be involved in controlling IRBP gene
expression during fetal retinal development (53). The arrangement of
the two MOK2-binding sites, the conserved 3'-half site in the promoter
and the 18-bp binding site in the intron, is reminiscent of that of
another potential MOK2 target gene, PAX3. In this gene, the
18-bp MOK2-binding site is located in the last intron. A search for
MOK2-binding sites in the proximal promoter region of human
PAX3 reveals the presence of a TAAAAGGCT sequence that could
bind to MOK2. Therefore, MOK2 might regulate the transcriptional
activity of target genes at different levels. The six other potential
genes isolated by whole genome technique are still unknown (4). We
previously showed that MOK2 is also an RNA-binding protein associated
mainly with nuclear RNP components (4). Numerous examples of
multifunctional proteins that bind to both DNA and RNA have emerged
(reviewed in Refs. 54-56). The best known members in the zinc finger
family are TFIIIA and WT1. MOK2 was shown here to be a transcriptional repressor, but, in other circumstances, it might also be an activator as has been shown for many other DNA-binding transcription factors. For
example, WT1 has been shown to repress and activate transcription depending on the promoter and the physiological context (reviewed in
Ref. 57). This hypothesis is supported by the fact that the human
isoform hsMOK2 contains an NH2-acidic domain that has
frequently been shown to act as an activation domain in many
transcription factors.