From the Friedrich Miescher Institute, P. O. Box 2543, CH-4002 Basel and the § Institute of Plant Sciences, Federal Institute of Technology, Universitätstrasse 2, CH-8092 Zürich, Switzerland
Received for publication, July 25, 2000, and in revised form, October 11, 2000
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ABSTRACT |
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Nuclear proteins from rice (Oryza
sativa) were identified that bind specifically to a rice tungro
bacilliform virus promoter region containing a vascular bundle
expression element (VBE). One set of proteins of 29, 33, and 37 kDa, present in shoot and cell suspension extracts but hardly
detectable in root extracts, bound to a site containing the sequence
AGAAGGACCAGA within the VBE, which also contains two CpG and one CpNpG
potential methylation motifs. Binding by these proteins was determined
to be cytosine methylation-independent. However, a novel protein
present in all analyzed extracts bound specifically to the methylated
VBE. A region of at least 49 nucleotides overlapping the VBE and
complete cytosine methylation of the three Cp(Np)G motifs was required for efficient binding of this methylated
VBE-binding protein
(MVBP).
Promoters for genes transcribed by RNA polymerase II are composed
of different cis elements that provide binding sites for trans-acting factors (1-2). The interactions of these
trans-acting factors with each other, with associated
cofactors, and with the basal transcription machinery determine the
expression characteristics of a promoter. Promoter activity is also
influenced by epigenetic control mechanisms that may modulate the
accessibility of promoter sequences for the transcription machinery in
various ways. Plant promoter studies have elucidated several molecular
components involved in tissue-specific gene expression (1-2). These
include cis-acting DNA elements and trans-acting
protein factors involved in regulating gene expression in vascular
tissue (3-8).
The rice tungro bacilliform virus
(RTBV)1 promoter (Fig.
1) has been analyzed in both transformed
rice plants (9-11) and transfected rice protoplasts (12-15). It was
found that sequences downstream of the transcription start site are
required for promoter activity in protoplasts (13-15). In transgenic
plants, the RTBV promoter containing the first 250 transcribed
nucleotides showed activity in the epidermis and in all cells of the
vascular bundle, not just the phloem (9). In the latter study, the most
important promoter element for this vascular bundle activity was
localized to the region between
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
100 and
165 (9), whereas previous
analyses indicated sequences termed box II (
53 to
39), ASL box
(
98 to
79), and GATA-like motif (
143 to
135) as phloem
specificity elements (10, 11, 16). Proteins binding to all these
regions were detected in nuclear extracts from rice. It was also found that the vascular bundle activity was suppressed in some transgenic rice lines with methylated promoter
sequences,2 indicating that
methylation directly or indirectly alters the assembly of transcription
factors on the promoter.
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Fig. 1.
Schematic presentation of the RTBV
promoter. AE, activator element; DPS,
dps1, dps2, downstream promoter elements
(15).
In this report, we have further analyzed DNA-protein interactions of
nuclear proteins with the vascular bundle expression element located in
the region between 165 and
100 with a detailed mutagenesis analysis
and have compared factor-binding to methylated and nonmethylated
sequences. Our results show that independent of the methylation status,
at least three rice nuclear proteins (29, 33, and 37 kDa) specifically
interact with this element, and a motif of twelve residues
(AGAAGGACCAGA) within this element is critical for protein binding.
Binding activity is high in extracts from shoots and low in extracts
from roots, correlating with the promoter activity in the respective
tissues. It is also high but slightly different in extracts from cell
suspensions, where the element has a negative effect on promoter
activity. The methylated DNA sequence bound in addition or
alternatively to a novel protein present in all extracts. This binding
was highly sequence- and methylation-specific and may be related to the
specific inactivation of the methylated promoter in vascular tissue.
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EXPERIMENTAL PROCEDURES |
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Plasmid Constructions--
Standard cloning techniques were used
for all plasmid constructions (17). Plasmid R-218 contains RTBV
promoter sequences up to position 218, the full-length RTBV leader
sequences, and a chloramphenicol acetyltransferase (CAT) gene fused to
RTBV open reading frame I (12).
To generate plasmid R-218Vd, a DNA fragment covering the region from
218 to
165 was amplified from the R-218 template by polymerase
chain reaction with a 5'-end primer (P5) located at vector sequences
upstream of the RTBV promoter and a primer corresponding to nucleotides
180 to
165 flanked with an EcoRV site. A second DNA
fragment (from
100 to the first 20 nucleotides of the CAT open
reading frame) was amplified with a primer corresponding to nucleotides
100 to
85 flanked with an EcoRV site and a 3'-end primer
(P3) covering the first 20 nucleotides of the CAT open reading frame.
These two polymerase chain reaction fragments were digested with
EcoRV and then ligated. The resulting DNA fragments were
digested with XbaI and XhoI and were cloned into
the corresponding sites of R-218 to yield R-218Vd.
To obtain R-218Vm, two overlapping fragments, one amplified with primer
P5 and a 3'-end primer of the antisense strand with mutations of
nucleotides GGTCCTT (from positions 144 to
150) to CCAGGAA, and the
other with a 5'-end primer corresponding to the sense strand with
mutations of nucleotides AAGGACC (from positions
150 to
144) to
TTCCTGG and primer P3, were used as templates to amplify a mutated
promoter fragment using primer P5 and P3. The resulting products were
digested with XbaI and XhoI and introduced between the corresponding sites of R-218.
To construct plasmids VR-218Vd(+) and VR-218Vd(-), double-stranded
oligonucleotides corresponding to sequences from 165 to
100 were
introduced into the XbaI site (filled in with Klenow fragment of DNA polymerase) of R-218Vd. The resulting constructs were
designated VR-218Vd(+) and VR-218Vd(-), with the insertion in sense and
antisense orientation, respectively.
Double-stranded oligonucleotides covering the sequences from 165 to
100 were phosphorylated and ligated. Dimers were purified from a 2%
agarose gel. The dimers were cloned into the EcoRV site of
R-218Vd to generate R-218V2 and into the XbaI site to yield 2VR-218d(+) with the insertion in sense orientation and 2VR-218Vd(-) with the insertion in antisense orientation.
To create plasmid mP165 for preparation of the DNA probe used in DNA UV
cross-linking assays, double-stranded oligonucleotides corresponding to
sequence from 165 to
100, flanked by an XbaI site at the
5'-end and HindIII site at the 3'-end, were introduced between the corresponding sites of M13mp18.
All plasmids were confirmed by restriction digestions and DNA
sequencing. Plasmids were isolated from Escherichia coli
(strain DH5) using a Qiagen plasmid kit.
Nuclear Extract Preparation and Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear extracts from cell suspension cultures of Oryza sativa line Oc were prepared as described (13). Nuclear extracts from 2-week-old O. sativa plant shoots and roots were prepared as described previously (12).
Methylated or nonmethylated double-stranded oligonucleotides
corresponding to sequences from 169 or
165 to
100 with a
5'-protruding end in the antisense strand, were labeled with
[
-32P]dCTP using the Klenow fragment of DNA
polymerase. The labeled probe was purified on a 5% native
polyacrylamide gel. EMSAs were performed as described (12). Typical
mixtures (15 µl) for in vitro binding reactions contained
5 µg of poly(dI-dC), 1× binding buffer (10 mM HEPES, pH
7.6, 8 mM MgCl2, 1 mM
dithiothreitol, 4 mM spermidine, and 5% (v/v) glycerol),
10 µg of nuclear extracts, and 15,000 cpm of labeled DNA probe
(around 0.04 pM DNA). For the competition experiments
variable amounts of competitor as indicated were included. Binding
reaction mixture was first preincubated for 10 min at room temperature
before the addition of the labeled DNA probe and incubated for a
further 20 min at room temperature after the addition of the labeled
DNA probe and the competitors.
Protein clipping band shift assays were performed as described (18). One µl of protease was added to the binding reaction mixture at the amounts indicated 20 min after the addition of the labeled DNA probe and allowed to react for 5 min at room temperature. Upon incubation, the reaction products were separated on a 5% native polyacrylamide gel.
DNA UV Cross-linking Assays--
Single-stranded mP165 DNA was
prepared as described previously (17). A uniformly labeled DNA probe
with thymidine residues substituted by bromodeoxyuridine (BrdUrd) was
prepared with [-32P]dCTP as described previously (19).
The labeled DNA probe was digested with XbaI and
HindIII and purified on a 5% native polyacrylamide gel.
Binding reactions and UV cross-linking were performed as described
previously (12).
Transient Expression Assays--
Protoplasts from O. sativa line Oc were isolated from cell suspension cultures and
transfected by polyethylene glycol-mediated transfection (20, 21).
Routinely, 10 µg of test plasmid DNA was used to transfect 0.6 × 106 protoplasts, and an internal control plasmid
expressing -glucuronidase under the control of the cauliflower
mosaic virus 35S promoter was cotransfected in all experiments. Protein
extracts were prepared after overnight incubation of protoplasts and
were analyzed for CAT (CAT ELISA kit, Roche Diagnostics Ltd) and
-glucuronidase activities (22, 23). CAT activity was normalized to
-glucuronidase activity in the same extract in all cases. Relative
expression levels did not vary more than ±15% with the given
constructs in this study. All constructs were tested in at least three
independent experiments with at least three independent colonies from
each transformation.
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RESULTS |
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Nuclear Proteins Interact with the Vascular Bundle Expression
Element--
In our previous study of the RTBV promoter in transgenic
rice plants, a promoter element required for gene expression in the vascular tissue was identified between nucleotide positions 165 and
100 (9). Here, we refer to this region as the vascular bundle expression element (VBE).
To further characterize nuclear proteins interacting with the VBE,
EMSAs were performed using labeled DNA probes from 165 to
100 of
the RTBV promoter and nuclear extracts prepared from rice shoots
(S), roots (R) and cell suspension cultures
(C). Multiple DNA-protein complexes were detected upon
incubation of each of these extracts with the labeled DNA probe (Fig.
2). However, these complexes were barely
detected in root nuclear extracts even if the amount of nuclear
extracts in the assay was increased, indicating that the DNA-binding
proteins responsible for complex formation are much less abundant in
roots. DNA-protein complexes VBP1 and VBP2 show a slightly lower
mobility in cell suspension nuclear extracts than in shoot nuclear
extracts. Additional DNA-protein complexes (VBP3, VBP4, and VBP5) were
seen in nuclear extracts from cell suspensions. The observed gel shift
patterns were qualitatively reproducible; however, the intensity of
individual bands varied with different batches of extracts and
sometimes complexes were resolved in two bands (e.g. VBP1 in
Fig. 2 and VBP2 in Fig. 3). Whether this
is caused by the presence of different proteins or by natural or
artificial protein modifications will have to await protein
characterization.
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Determination of Minimal Sequences Required for Protein
Binding--
Initially, the specificity of DNA-protein interactions
was analyzed using specific unlabeled DNA fragments as competitors in
EMSA, and it was found that all observed complexes are equally well
competed by DNA fragments covering the regions 165 to
140 or
150
to
125 (data not shown). Therefore, the sequence common to both
fragments was analyzed in greater detail by introduction of mutations
into a fragment covering the region from
165 to
125, which we
designated as fragment V (Fig. 3A). These mutants (Vm1 to
Vm10) were used as competitors in EMSA for protein binding to the
labeled fragment V. From these, only the competitors Vm9 and Vm10
abolished the formation of complex VBP2 (Fig. 3, B,
lanes 21-24 and C) as effectively as wild-type V
(lanes 3 and 4). Vm2 and Vm10 competed complex
VBP1 most efficiently (Fig. 3, B, lanes 7, 8,
23, 24 and C), followed by Vm1 and Vm4
(Fig. 3, B, lanes 5, 6, 11, 12 and C).
VBP1 competition was also clear for Vm9 (Fig. 3, B, lanes 21, 22 and C), whereas other competitors reduced complex formation only slightly (Fig. 3, B, lanes 9, 10,
13-20 and C).
These data indicate that sequences from at least 149 to
145 are
required for formation of complex VBP2, but binding was modulated by
sequences closely upstream of them. Mutations of the same DNA region in
some cases also abolished the formation of complexes VBP1; however, the
binding requirements for VBP1 and VBP2 may be different because
e.g. the mutations in Vm2 and Vm4 only affected VBP2
formation and Vm9 mainly VBP1 formation. These differences also suggest
that the formation of the complexes occurs independently because
abolishing one complex did not alter the gel shifts caused by the
other. All fragments still efficiently competing for complex VBP1
formation contain two intact AGA motifs flanking the sequence important
for VBP2 formation.
Mutation of the GATA motif, implicated by Yin et al. (11) in phloem specificity to AGCG in fragment Vm10, did not impair the competitivity (Fig. 3, B, lanes 23, 24 and C) and thus excludes this sequence as a binding site for the factors observed in our assay.
Characterization of Nuclear Proteins Bound to the VBE by DNA UV
Cross-linking Assays--
To identify nuclear proteins interacting
with the VBE, we performed DNA UV cross-linking assays with a
BrdUrd-substituted, uniformly radiolabeled DNA probe from 165 to
100 and nuclear extracts from rice shoots and cell suspension
cultures. As shown in Fig. 4, three
proteins with apparent sizes of 29, 33, and 37 kDa were cross-linked to
the labeled probe in both shoot and cell suspension nuclear extracts
(lanes 2 and 9), whereas other bands marked with
asterisks (also present in reactions without added protein
extract, lanes 1 and 8) resulted from incomplete
digestion of the DNA probe. The 37-kDa protein predominates in both
nuclear extracts, whereas the 29-kDa protein is much less abundant. We did not detect additional proteins in the suspension cell extract that
could form the DNA-protein complexes VBP3, VBP4, and VBP5 observed in
EMSA. It might be that these proteins may function as accessory
proteins that interact with other DNA-binding proteins via
protein-protein interactions instead of directly binding to the DNA
element. Competition experiments proved that the detected proteins bind
specifically to the
165 to
125 DNA fragment and not to other DNA
fragments (lanes 3, 4, 10,
11).
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The VBE Acts as an Inhibitor Element in Transiently Transfected
Rice Protoplasts--
The VBE is very important for regulating gene
expression in the vascular bundle (9) and interacts with nuclear
proteins VBP1 and VBP2 prepared from all tissues tested (Fig. 2).
Additional DNA-protein complexes (VBP3, VBP4, and VBP5) were detected
predominantly with nuclear extracts from cell suspension cultures (Fig.
2). Therefore, it was of interest to assess the function of this
element in a transient-expression system. To this end, we deleted or
mutated the element in the context of the plasmid R-218 (Fig.
5) and tested the activity of the
resulting plasmids in transfected rice protoplasts. In repeated
transfection assays, deletion of this element resulted in a 2-fold
increase in promoter activity compared with wild-type R-218 (Fig. 5,
R-218Vd). The negative effect of the VBE element was
position independent and slightly increased by VBE duplication (Fig.
5). Mutation of nucleotides AAGGACC (from nucleotide positions 150 to
144) to TTCCTGG abolished protein binding to VBE (Fig. 3, lanes
15, 16) and resulted in alleviation of the negative
effect of VBE on expression in protoplasts (Fig. 5,
R-218Vm).
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An Additional Protein Binds to Methylated VBE--
Previously, we
observed in some transgenic rice lines harboring a -glucuronidase
gene under the control of the RTBV promoter, specific inactivation of
-glucuronidase expression in the vascular tissue.2 The
resulting expression pattern was identical to that obtained with a VBE
deletion and was correlated with cytosine methylation of the promoter
region including the VBE. We therefore investigated whether methylation
of the VBE region interferes with protein binding by testing several
methylated and unmethylated DNA fragments (Fig.
6) in the EMSA.
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Methylation of fragment V did not interfere with binding of proteins
from rice nuclear extracts that also bound to the unmethylated fragment. However, a novel, additional shift-band appeared (Fig. 7, MVBP). Formation of this
novel complex required DNA methylation and consequently methylated but
not unmethylated DNA fragments could compete for binding. All other
VBE-binding proteins from rice cell suspensions, shoot, or root nuclear
extracts bind to the DNA irrespective of the degree of VBE methylation.
Chymotrypsin sensitivity proved that the novel complex was caused by a
protein (Fig. 8), which we consequently
named methylated VBE-binding protein (MVBP).
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Simultaneous binding of MVBP and VBPs is unlikely because no supershifts were observed. Rather, the different proteins may compete for the DNA fragments. This may be the reason why the VBP1 complexes appear less pronounced when DNA fragments are used that can also bind MVBP. VBP1 complexes are better detectable in cell-suspension extracts, which seem to contain a lower amount of active MVBP than the shoot extracts (Fig. 7).
MVBP Is a Sequence-specific DNA-binding Protein--
The sequence
specificity of MVBP binding was determined by competition with mutated
DNA fragments (Fig. 6). All tested mutations between 164 and
116
abolished competitivity for MVBP binding, whereas mutations outside of
this region did not affect binding (Fig.
9). Even small changes of the sequence
context of two methylated CpG motifs or a change of one CpNpG to a
CpGpN were detrimental for binding. MVBP binding was also abolished by
mutations of the region important for formation of a DNA-protein
complex with the unmethylated V fragment (described above). These data
indicate that the binding site(s) for MVBP is contained in a DNA region covering 49 base pairs, which overlaps the binding sites for the other
VBE-associated proteins.
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MVBP Binding Requires Complete Cytosine Methylation--
It has
been shown that hemi-methylated DNA is a poor substrate for the binding
of the mammalian methyl-CpG-binding protein 1 and 2 (MeCP1 and MeCP2;
Refs. 24, 25). On the other hand, an Ac transposon-derived protein
preferentially binds to hemi-methylated DNA (26). To test whether MVBP
can bind to hemi-methylated DNA, EMSA was performed using fully
methylated DNA as substrate and hemi-methylated DNA as competitor
(methylation on either the top or bottom strand as shown in Fig. 6,
Hemi-U and Hemi-L). Methylation on only one of
the strands resulted in only weak competition for MVBP (Fig.
10, lanes 7-10)
when compared with symmetrically methylated DNA (lanes
3-4). This shows that hemi-methylated DNA is a less efficient substrate for MVBP.
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The number of methylated CpGs required for binding of specific proteins can vary considerably from case to case. For example, mammalian MeCP1 requires at least 12 symmetrically methylated CpGs for binding (25), whereas MeCP2 can bind to DNA with a single methyl-CpG pair (24). Experiments were designed to assess the individual role of the two methylated CpG sites and the one CpNpG site in MVBP binding. For this purpose site-specific methylated oligonucleotides were synthesized (Fig. 6, Vm 11-15) and used as competitors in EMSA. The results show that cytosine methylation at only one of these sites (Vm11, Vm12, or Vm13) was incapable of competing the formation of MVBP (Fig. 9), suggesting that MVBP cannot bind to DNA containing a single methyl-CpG or CpNpG pair. Further mutants were used to determine whether the binding of MVBP requires both CpG pairs (Vm14) and whether the CpApG pair could be replaced by a CpG pair (Vm15). None of these mutants competed with the wild-type element for MVBP binding.
As a control experiment, we used Vm8, Vm11, Vm12, Vm13, Vm14, and Vm15 as labeled probes in the EMSA. When tested with the nuclear extracts from shoots and roots, no binding of MVBP to any of the probes was observed (data not shown). These findings confirm the results obtained with the competition assays and taken together show the astonishing specificity of MVBP for sequence and methylation status of the target sequence.
MVBPs from Shoot and Root Nuclear Extracts Have an Identical
Binding Specificity--
The comparison of root and shoot extracts in
Fig. 9 indicates that in root nuclear extracts, MVBP is as abundant as
in shoot extracts, whereas proteins binding to the unmethylated
fragment such as VBPs are much less abundant. The methylation specific DNA-protein complex from root nuclear extracts was located at the same
position as that from shoot extracts and the complex formation was
competed or not by the same oligonucleotides. This indicates that MVBPs
from shoot and root extracts are identical.
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DISCUSSION |
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Promoter specificity is determined by the complement of factors interacting on a given sequence, often in competition with other factors with similar or overlapping binding sites. Protein binding can be modulated by epigenetic DNA modifications like chromatin condensation or cytosine methylation. In vertebrates, cytosine methylation can result in the attraction to the DNA of nonspecific, methyl cytosine-binding proteins like MeCP1 or MeCP2, which may suppress promoter activity directly (27-28) or tether corepressor complexes to the DNA that turn off promoters in a stable manner (29-34). In mammalian systems, there is evidence indicating that DNA methylation directly prevents the binding of some trans-acting factors to DNA (34-41). However, other transcription factors, such as Sp1, were found to be insensitive to DNA methylation (42) or affected at only some of their binding sites (Sp1, Ref. 43; E2F, Ref. 35). Therefore, DNA methylation could be involved not only in the general suppression of promoter activity but also in tissue- or development-specific regulation (35). The existence of a transcription factor family with sequence and methylation specificity, which is involved in the regulation of a number of mammalian genes, supports this possibility (44-46). In plants, DNA methylation as yet has been connected mainly to the stable transcriptional silencing of transgenes (47-48), but it is certainly also involved in developmental control of gene activity (49). Only a few methyl cytosine-binding proteins from plants are known (50), and only a few protein-DNA interactions have been investigated with respect to the influence of DNA methylation. Some CREB-like factors, present in nuclear extracts of several plant species, have been shown to be sensitive to cytosine methylation within their binding sites (51). In contrast, binding of the maize O2 protein to its target sequence, TGACGTTG, was not impaired by CpG methylation in vitro (52), and the tobacco factor CG-1 could at least bind to hemi-methylated DNA (53).
The promoter of RTBV contains within about 280 base pairs around the
transcription initiation site the information for activity in all cells
of the vascular bundle and the epidermis, and several protein binding
sites in this region have been detected by EMSA. An element important
for activity in the vascular bundle has been localized in the region
between 100 and
165. This region probably interacts with others,
and there may be some redundancy with elements closer to the TATA box
(11). However, deletion of this region abolished expression in the
vascular bundle (9), and the same expression pattern was obtained
consistent with specific promoter methylation.2 Protein
binding sites in the region from
125 to
165 have been detected (9,
11) and particularly a GATA motif has been implicated in protein
binding (11). We show here that several proteins bind to a AGAAGGACCAGA
motif upstream of the GATA sequence designated here as VBE, but that
GATA is not required for this binding. The binding activity was
abundant in extracts from rice shoots but not in root extracts,
correlating with the promoter activity in the respective tissue
(9-10). In cell suspensions, a similar binding activity was found.
However additional shift-bands were observed, indicating association
with other proteins most likely by protein-protein interaction. In cell
suspensions, the VBE has no positive effect on expression but rather
reduces promoter activity, suggesting that the detected different
protein interaction is repressive. Negative elements have been found in
several promoters with differential regulation of expression in the
different cell types of the vascular bundle (5-6). VBE duplication
further represses promoter activity in protoplasts. How the VBE
represses transcription from the RTBV promoter remains unknown.
Possibly, VBE interacts with other regulatory elements located between
70 and
35 (12) or the basal transcriptional machinery via
protein-protein interactions that will exert a negative effect in the
transient expression system.
The VBE lacks C- or CA-rich elements that have been associated with the
binding of regulatory factors to other vascular bundle-specific promoters (16). However, the VBE shows some homology to the VSF-1
binding site in the xylem-specific promoter of the bean grp1.8 gene (Fig. 11 and
Ref. 4). This factor has a strong sequence similarity to the rice bZIP
protein RF2a, which is expressed in the same cells where the RTBV
promoter is active (16). RF2a has been characterized by its binding to
another RTBV promoter region termed box II, which overlaps (or is
identical to) the sequence identified by us as an activator element
(12). The binding of RF2a to box II was not very efficient, and it
therefore is of interest to determine whether RF2a alone or as part of
a heterodimer can also bind to VBE.
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DNA methylation did not have a negative effect on the binding of
proteins to VBE or to other promoter regions (data not shown). The only
alteration in the interaction pattern was the appearance of a novel
binding activity, MVBP. This activity was present in all extracts
tested and unlike most previously characterized, methylated DNA-binding
proteins displayed a high specificity for the DNA sequence and the
degree of methylation. Sequence alterations over a region covering 49 nucleotides abolished binding, and when the two CpG and one CpNpG sites
were only partially methylated, binding was strongly reduced. To our
knowledge, no protein with a comparable binding requirement has as yet
been characterized for any organism. The closest would be the MDPB/RFX
protein family whose members recognize a variety of 14-base pair
motifs, some of which have to be methylated at specific sites for
recognition by the factor (44-46, 54). On the other hand, MeCP1
requires at least 12 methylated CpG pairs, i.e. it most
likely interacts with an extended DNA region, although in a
sequence-nonspecific manner (25). It is tempting to associate MVBP
binding with the tissue-specific promoter inactivation observed in
plant lines with a methylated promoter sequence. This could be caused
by competition between MVBP and one or the other VBP for DNA binding.
Replacement of transcription factors by methyl cytosine-binding
proteins has been observed for some methylated animal promoters (36,
55). Evidence for competition could be deduced from our experiments with extracts from suspension cells with apparently lower MVBP activity
in which formation of the VBP1 complex with methylated oligonucleotides
appeared to be efficient, whereas in extracts from rice shoots with
higher MVBP activity, it was reduced. Alternatively, MVBP may be
directly involved in promoter repression. The presence of such a
protein in rice raises the question of its normal function. Isolation
and further characterization of the protein will be required to address
this question.
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ACKNOWLEDGEMENTS |
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We thank Matthias Müller for expert technical assistance, Ingo Potrykus for excellent support, Andreas Klöti for interesting discussions, and Jean-Pierre Jost and Helen Rothnie for thorough critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Novartis Research Foundation, a part of the Friedrich Miescher Institute.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.
Present address: Dept. of Molecular Biology and Microbiology,
Tufts University Medical School, 136 Harrison Ave., Boston, MA 02111.
¶ To whom correspondence should be addressed. Tel.: 41 61 697 7266; Fax: 41 61 697 3976; E-mail: thomas.hohn@fmi.ch.
Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M006653200
2 A. Klöti, X. He, I. Potrykus, T. Hohn, and J. Fütterer, submitted manuscript.
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ABBREVIATIONS |
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The abbreviations used are: RTBV, rice tungro bacilliform virus; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; BrdUrd, bromodeoxyuridine; VBE, vascular bundle expression element; VBP, VBE-binding protein; MVBP, methylated VBP; MeCP, methyl-CpG-binding protein.
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