Role of Zinc Finger Domains of the Transcription Factor
Neuron-restrictive Silencer Factor/Repressor Element-1 Silencing
Transcription Factor in DNA Binding and Nuclear Localization*
Masahito
Shimojo,
Jeong-Heon
Lee, and
Louis B.
Hersh
From the Department of Biochemistry, University of Kentucky,
Lexington, Kentucky 40563-0298
Received for publication, December 12, 2000
 |
ABSTRACT |
The transcription factor neuron-restrictive
silencer factor/repressor element-1 (RE-1) silencing
transcription factor (NRSF/REST) contains nine zinc finger domains and
binds to the DNA element, neuron-restrictive silencer element/repressor
element-1. REST4, a C-terminally truncated form of NRSF/REST, contains
the five N-terminal zinc fingers and binds weakly to DNA yet is
transported into the nucleus. To study the contribution of zinc fingers
6-8 to DNA binding, each was mutated. A mutation in zinc finger 6 or 8 had little effect; however, mutation of zinc finger 7 diminished DNA
binding. Mutations in any two of these zinc fingers eliminated DNA
binding. The contribution of zinc fingers 2-5 to nuclear targeting was
studied. Deletion of zinc finger 5 prevented nuclear targeting. Mutations in zinc finger 2, 4, or 5 did not abolish nuclear targeting. However, a zinc finger 3 mutation together with a zinc finger 2 mutation localized to the nuclear envelope. A zinc finger 3 mutation
alone or in combination with a zinc finger 4 or 5 mutation produced a
punctate nuclear distribution. These results suggest the presence of
signals for nuclear targeting, for nuclear entry, and for release from
the translocation machinery within zinc fingers 2-5 of REST4.
 |
INTRODUCTION |
Recent studies have established that one mechanism for the
maintenance of the neuronal phenotype is through repression of neuronal
gene expression in nonneuronal tissues (1, 2). Thus, a number of
neuron-specific proteins including choline acetyltransferase (3), synapsin I (4), SCG10 (5), the type II sodium channel (6), the
N-methyl-D-aspartate receptor (7) and
neuron-glia cell adhesion molecule (8), to name but a few, contain
within their genes a 21-base pair DNA sequence referred to as the
neuron-restrictive silencer element
(NRSE).1 This element is also
called repressor element-1 (RE-1) (1). The transcription factor that
binds to NRSE/RE-1 is known as the neuron-restrictive silencer factor
(NRSF)/RE-1-silencing transcription factor (REST). It is a 210-kDa
glycoprotein containing nine zinc finger domains whose expression
appears limited to nonneuronal cell types. NRSF/REST can also act as a
silencer of neuron-specific gene expression in undifferentiated
neuronal progenitor cells (1, 2). It has been shown that the absence of
the NRSE/RE-1 sequence (9) or expression of a dominant negative form of
NRSF/REST produces expression of target genes in nonneuronal cells
(10). A mouse model in which the NRSF/REST gene was deleted, although embryonic lethal, showed aberrant expression of SCG10 and tubulin
III in nonneuronal tissues (10).
A number of splice variants of NRSF/REST have been reported (11). Two
of these, REST4 and REST5, were found to be expressed at low levels in
mature neurons of adult brain (11). These splice variants contain an
insertion between zinc finger domains 5 and 6 that leads to truncated
proteins containing only five of the nine zinc finger domains found in
NRSF/REST. We previously suggested that, at least in PC12 cells, one of
these isoforms, REST4, regulates cholinergic gene expression. The
mechanism appears to involve formation of hetero-oligomers between
REST4 and NRSF/REST, which prevents binding of NRSF/REST to the
NRSE/RE-1 sequence (12). To further study the function of NRSF/REST and
REST4, we have prepared and studied deletion constructs as well as
constructs containing point mutations that cause disruption of the
structure of individual Cys2-His2 type zinc
finger domains. The results of these studies suggest that proper zinc
finger structure is important for DNA binding as well as for nuclear localization.
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EXPERIMENTAL PROCEDURES |
Materials--
Deoxyribonucleotides, Dulbecco's modified
Eagle's medium, and fetal bovine serum were obtained from Life
Technologies, Inc. Oligonucleotide primers were synthesized with a
Beckman Oligo1000 DNA synthesizer or purchased from commercial sources.
[
-32P]ATP was from ICN (Irvine, CA). The BCA protein
assay kit was obtained from Pierce. Poly(dI-dC)·poly(dI-dC) was
obtained from Amersham Pharmacia Biotech. The ECL Western blotting
detection system and HybondTM-P membranes were purchased
from Amersham Pharmacia Biotech. Effectene Transfection Reagent and
Qiagen plasmid kit were obtained from Qiagen Inc. (Valencia, CA). The
pcDNA3 expression vector was obtained from Invitrogen (Carlsbad,
CA), while pEGFP and pCMV
were obtained from
CLONTECH (Palo Alto, CA). All other reagents were
from Sigma and were of the highest quality available.
Construction of Plasmids--
Constructs containing green
fluorescent protein (GFP) fused to REST4 were generated by subcloning
the HindIII-BamHI fragment of corresponding
FLAG-REST4 constructs into pEGFP. Mutations were generated by the
QuickChangeTM site-directed mutagenesis kit (Stratagene, La
Jolla, CA) using NRSF/REST or GFP-REST4 as the DNA template and the
following oligonucleotide pairs as primers: 5'-C AAA GGC CCC ATC CGC
CGT GAC CGC TGT GGC TAC-3' and 5'-GTA GCC ACA GCG GTC
ACG GCG GAT GGG GCC TTT G-3' (for Z2mut), 5'-GAG CGC ATC
TAC AAG CGT ATC ATC TGC ACG TAC-3' and 5'-GTA CGT GCA GAT
GAT ACG CTT GTA GAT GCG CTC-3' (for Z3mut), 5'-GG AAA GTC
TAC ACC CGT AGC AAG TGC AAC TAC-3' and 5'-GTA GTT GCA CTT
GCT ACG GGT GTA GAC TTT CC-3' (for Z4mut), 5'-GAA CGC CCG
TAT AAA CGG GAA CTT TGT CCT TAC-3' and 5'-GTA AGG ACA AAG TTC CCG TTT ATA CGG GCG TTC-3' (for Z5mut), 5'-GAG AAG CCA
TTT AAA CGG GAT GAG TGC AAT TAT G-3' and 5'-C ATA ATT GCA
CTC ATC CCG TTT AAA TGG CTT CTC-3' (for Z6mut), 5'-CCT AAA
CCT CTT AAT CGT CCG CAC TGT GAC TAC-3' and 5'-GTA GTC ACA
GTG CGG ACG ATT AAG AGG TTT AGG-3' (for Z7mut), 5'-CCA CGG
CAG TTC AAC CGT CCC GTG TGT GAC TAC-3' and 5'-GTA GTC ACA
CAC GGG ACG GTT GAA CTG CCG TGG-3' (for Z8mut). Underlined
nucleotides denote changes introduced (Cys to Arg). The identities of
the plasmid constructs were verified by the cycle sequencing method
using Sequenase (U.S. Biochemical Corp.). For the preparation of
-galactosidase fused to zinc finger domains 2-5 (Z2,3,4,5) or to
zinc finger domains 2-4 (Z2,3,4), appropriate fragments were amplified
by the polymerase chain reaction from an NRSF/REST template. Primers
used were as follows: Z2S, 5'-GAT GGA CTA CAA GGA CGA CGA CGA CAA GCC
CAT CCG CTG TGA CCG CTG-3'; Z4AS, 5'-GGG CGT TCT CCT GTG TGA GT-3';
Z5AS, TCA CCT GAA TGA GTC CGC AT-3'. The amplified products were cloned into the SmaI site of pBSSK. A
HindIII/BamHI fragment was cloned into
-galactosidase-pcDNA3. The latter was created by inserting the
-galactosidase gene from pCMV
(CLONTECH) into
the NotI site of pcDNA3. The preparation of deletion
mutants was as described in Lee et al. (13).
Cell Culture and Transfection--
HEK293 cells were maintained
in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine
serum (Life Technologies, Inc.). These cells were maintained at
37 °C in a humidified atmosphere of 10% CO2.
Transfections were performed with the use of the Effectene Transfection
Reagent (Qiagen) according to the manufacturer's instructions.
Detection of GFP and
-Galactosidase--
NIH3T3 cells were
cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine
serum at 37 °C in 5% CO2. For transfection with GFP or
-galactosidase-containing constructs, cells were plated on
100-mm2 glass coverslips at a density of 105
cells/coverslip on the day before transfection. The cells were transfected with GFP-tagged or
-galactosidase-tagged expression constructs using the Effectene Transfection Reagent. For visualizing, GFP cells were grown for 24 h and then fixed in dry ice-methanol for 5 min. Cells were then washed three times in PBS, counterstained with 4',6-diamidino-2-phenylindole in PBS (1:5000 dilution) for 5 min
and then washed with PBS. Coverslips were mounted in Vectashield H-1000
(Vector Laboratories, Inc.). GFP was observed using a Nikon E600
epifluorescent microscope (Melville, NY). For
-galactosidase staining, cells were fixed in 1% glutaraldehyde for 5 min; washed three times in PBS; soaked in 100 mM sodium phosphate, pH
7.5, 10 mM KCl, 1 mM MgCl2, 3 mM K4(Fe(CN)6), 3 mM K3(Fe(CN)6), 0.1% Triton X-100,
and 1 mM X-gal at 37 °C for 30 min; and then washed with
PBS.
Electrophoretic Mobility Shift Assays (EMSA)--
Nuclear
extracts were prepared from cells as described previously (12). The
probe for EMSA was made by polymerase chain reaction using the human
vesicular acetylcholine transporter/choline acetyltransferase
NRSE/RE-1 as a template (see Fig. 2A). DNA fragments were
end-labeled with [
-32P]ATP using T4 polynucleotide
kinase. For binding, 10 µg of nuclear protein was preincubated on
ice, with or without a 100-fold excess of unlabeled competitor DNA, for
10 min in 20 µl of 20 mM HEPES (pH 7.6), 0.1% Nonidet
P-40, 10% glycerol, 1 mM dithiothreitol, 2.5 mM MgCl2, 250 mM KCl, and 2 µg of
poly(dI-dC)·poly(dI-dC). Labeled oligonucleotide (100 fmol) was mixed
with nuclear protein, and the mixture was incubated for 10 min at
25 °C. For supershift assays, 10 µg of nuclear protein was
preincubated on ice with or without a monoclonal antibody to NRSF/REST
(12C11-1) for 30 min before adding the labeled probe. The reaction
mixture was loaded onto a 4% nondenaturing polyacrylamide gel with
0.25× TBE buffer and electrophoresed for 60 min at 120 V.
Western Blot Analysis--
Nuclear extracts (10 µg) were
solubilized in Laemmli sample buffer. After separation on a reducing
SDS-polyacrylamide gel, proteins were transferred onto
HybondTM-P membrane as described previously (12). The
membrane was then incubated with anti-NRSF/REST (12C11-1), anti-FLAG,
or anti-Myc monoclonal antibody followed by horseradish
peroxidase-labeled goat anti-mouse antiserum, and visualized using the
ECL detection kit per the manufacturer's instructions.
 |
RESULTS |
As illustrated in Fig. 1, NRSF/REST
is a transcription factor containing nine zinc finger domains; the
first of these is near the N terminus and is followed by a cluster of
seven zinc finger domains, with the last zinc finger domain being near
the C terminus of the molecule. REST4, which is a truncated version of
NRSF/REST containing only zinc finger domains 1-5, binds weakly to the
NRSF/REST target sequence NRSE/RE-1 (13). This finding as well as other studies (1) suggests that zinc finger domains 2-5 are required for DNA
binding, while zinc finger domains 6-8 appear to contribute significantly to the strength of the binding interaction (13). To study
further the contributions of zinc finger domains 6-8, NRSF/REST
constructs with combinations of point mutations in these zinc finger
domains were constructed, Fig. 1. Each zinc finger structure was
disrupted by introducing a point mutation where the amino acid Cys was
converted to Arg in the zinc finger domain. This mutation has been
shown to disrupt the structure of Cys2-His2 type zinc finger domains (14). Each construct was transfected into
HEK293 cells, and nuclear extracts were prepared and used for EMSA with
a probe containing the human vesicular acetylcholine transporter/choline acetyltransferase NRSE/RE-1 sequence (Fig. 2A). As can be seen in Fig.
2B, the EMSA shows that the binding of NRSF/REST with
mutations in zinc finger domains 6, 7, and 8 (Z6,7,8mut) diminished the
gel mobility shift to levels undetectable at the sensitivity of these
assays. Diminished binding was also seen in double mutants containing
point mutations in zinc finger domains 6 plus 7 (Z6,7mut), 7 plus 8 (Z7,8mut), and 6 plus 8 (Z6,8mut). The presence of a point mutation in
zinc finger domain 6 alone (Z6mut) or zinc finger domain 8 alone
(Z8mut) had little or no effect on DNA binding; however, a point
mutation in zinc finger domain 7 alone (Z7mut) greatly diminished DNA
binding. As shown in Fig. 2C, the expression level of each
construct was nearly the same as confirmed by Western blot analysis,
and if anything the construct with the point mutation in zinc finger
domain 7 was expressed at the highest levels. As shown in Fig.
2D, the electrophoretic mobility band was supershifted with
an anti-NRSF monoclonal antibody but not by an irrelevant monoclonal
antibody, confirming that the electrophoretic mobility band contained
NRSF/REST. Taken together, these results suggest that in addition to
zinc fingers 2-5, two adjacent zinc fingers (zinc fingers 6 plus 7 or
7 plus 8) are required for maximal DNA binding.

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Fig. 1.
Schematic diagram of NRSF/REST constructs
with point mutations in zinc finger domains. NRSF/REST is
schematically represented, with striped boxes,
indicating zinc finger domains and filled boxes
indicating zinc finger domains with a Cys to Arg point mutation.
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Fig. 2.
Electrophoretic mobility shift assays of
NRSF/REST constructs with point mutations in zinc finger domains.
A, the probe used for EMSA corresponds to the human
NRSE/RE-1 sequence upstream of the vesicular acetylcholine
transporter and choline acetyltransferase genes in the human
cholinergic gene locus. The 21-base pair NRSE/RE-1 sequence is
underlined. B, EMSA of nuclear extracts (10 µg)
from HEK293 cells containing NRSF/REST with point mutations.
C, immunoblot analysis of each nuclear extract with
anti-NRSF monoclonal antibody. D, supershifting of NRSE/RE-1
gel shift band. Nuclear extracts with NRSF (+NRSF) or
without NRSF ( NRSF) from HEK293 cells were applied to
EMSA. For the supershift assay, nuclear extracts were preincubated for
30 min on ice before adding the probe with anti-NRSF monoclonal
antibody or control monoclonal antibody against mouse choline
acetyltransferase where indicated.
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Although the above studies demonstrate the importance of zinc finger
domains 6-8 in NRSF/REST binding to DNA, previous studies have shown
that the neuron-specific isoform REST4, which contains only zinc finger
domains 1-5, is targeted to the nucleus (15). This was a bit
surprising, since it had previously been proposed that a nuclear
localization domain of NRSF/REST resides in the C-terminal part of the
molecule (1, 16), and this signal is clearly absent from REST4. We
first tested the effect of deleting the N-terminal region of REST4,
which is known to bind the transcriptional repressor mSin3 (16-18),
and then a larger fragment of the N-terminal region, which contains the
first zinc finger domain. Fig.
3A shows a schematic
representation of these and other truncated forms of REST4 that were
studied, each containing an N-terminal FLAG epitope for ease of
identification. These constructs were transiently expressed in HEK293
cells followed by fractionating the cells into nuclear and cytosolic
extracts and locating the REST4 mutant by Western blot analysis with an
anti-FLAG antibody. Western blots employing an anti-Myc antibody were
used as positive controls to detect endogenous Myc protein in the
nucleus. As shown in Fig. 3B, cell fractionation analysis
showed that deletion of the mSin3 binding region (
N152) or this
region plus the first zinc finger domain (
N209) did not affect
nuclear targeting. On the other hand, constructs containing deletions
of zinc finger domains 3-5 (
C89), zinc finger domains 2-5
(
C151), or zinc finger domains 1-5 (
C173) were detected in the
cytosol but not in the nucleus. These results suggest that zinc finger
domains 2-5 are required for nuclear targeting of REST4 and presumably
NRSF/REST.

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Fig. 3.
Cellular localization of truncated NRSF/REST
mutants in HEK293 cells. A, schematic representation of
N- or C-terminally truncated mutants of REST4. A FLAG tag was fused to
the N terminus of all constructs so that they could be detected with
anti-FLAG antibody. Striped boxes represent zinc
finger domains. B, Western blot analysis of nuclear extracts
and the cytosolic fraction from transfected HEK293 cells. Each
truncated NRSF/REST mutant was expressed in HEK293 cells, and nuclear
(N) and cytosolic (C) extracts were prepared.
Each extract (10 µg) was subjected to immunoblot analysis with
anti-FLAG or anti-Myc antibodies, respectively. Myc was used as an
endogenous nuclear protein marker.
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To demonstrate the importance of zinc finger domains 2-5 in nuclear
targeting in vivo, GFP was fused to REST4; to REST1 (11), which is a natural isoform lacking zinc finger domain 5; and to a
C-terminally truncated mutant lacking zinc finger domains 3-5 (
C89). These constructs were transiently expressed in NIH3T3 cells,
and their localization was determined by GFP fluorescence microscopy.
As shown in Fig. 4, although REST4 was
localized in the nucleus, the deletion of zinc finger domain 5 was
sufficient to prevent nuclear localization. We next confirmed whether
zinc finger domains 2-5 would localize a larger protein to the nucleus by fusing this region to
-galactosidase. As shown in Fig.
5, the attachment of zinc finger domains
2-5 to the N terminus of
-galactosidase (Z2,3,4,5) conferred
nuclear localization in NIH3T3 cells as judged by histochemical
staining of the cells with X-gal. Neither
-galactosidase alone nor a
construct containing zinc finger domains 2-4 (Z2,3,4) fused to
-galactosidase was localized to the nucleus.

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Fig. 4.
Localization of GFP-truncated REST4 mutants
in NIH3T3 cells. A, schematic of constructs of
GFP-truncated REST4 mutants. Each construct was fused C-terminally to
GFP. Striped boxes indicate zinc finger
domains. B, fluorescence microscopy of GFP-truncated REST4
mutants. NIH3T3 cells were transfected with expression constructs
encoding the GFP-truncated REST4 mutants shown above. After fixation,
cells were examined by fluorescence microscopy. Green and
blue staining represent GFP and nuclear
(4',6-diamidino-2-phenylindole) staining, respectively.
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Fig. 5.
Cellular localization of
-galactosidase-REST4 zinc finger domain
chimeras. A, schematic representation of
-galactosidase-REST4 zinc finger domain chimeras. B,
histochemical staining of NIH3T3 cells transfected with the
-galactosidase-REST4 zinc finger domain chimeras shown above.
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To further dissect the importance of zinc finger domains 2-5 for
nuclear localization of REST4, a series of constructs were generated
that contained a point mutation that disrupted the conformation of the
zinc finger domain. Constructs containing one, two, three, or four of
these point mutations fused to GFP were prepared and are schematically
represented in Fig. 6A. As
shown in Fig. 6B, a single point mutation in any one zinc
finger domain permitted nuclear targeting. However, as noted below, the
zinc finger domain 3 mutant, although localized to the nucleus, showed
an abnormal staining pattern. Similarly, combinations of double mutants
involving zinc finger domains 2 and 4 (Z2,4mut), zinc finger domains 2 and 5 (Z2,5mut), and zinc finger domains 4 and 5 (Z4,5mut) all showed normal nuclear localization, as did the triple mutant involving zinc
finger domains 2, 4, and 5 (Z2,4,5mut). However, all constructs containing a mutation in zinc finger domain 3 (Z3mut, Z3,4mut, Z3,5mut,
Z2,3,5mut, and Z3,4,5mut), although associated with the nucleus,
appeared abnormal. The zinc finger domain 3 single mutant as well as
the zinc finger domain 3 plus 4 double mutant and the zinc finger
domain 3 plus 5 double mutant showed nuclear staining that was punctate
rather than uniform across the nucleus. A more dramatic pattern was
seen with a zinc finger domain 3 mutant in combination with a zinc
finger domain 2 mutation. Mutant Z2,3mut, Z2,3,4mut, or Z2,3,5mut or
the construct containing mutations in all four zinc finger domains
(Z2,3,4,5mut) exhibited GFP fluorescence that appeared on the surface
of the nucleus rather than inside the nucleus.

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Fig. 6.
Cellular localization of GFP-REST4
constructs containing zinc finger mutations. A,
schematic representation of GFP-REST4 constructs containing point
mutations in zinc finger domains. The striped
boxes represent zinc finger domains, while the
filled boxes represent zinc finger domains with a
Cys to Arg point mutation. B, fluorescence microscopy of
NIH3T3 cells expressing GFP-REST4 point mutants. Green and
blue staining represent GFP and nuclear
(4',6-diamidino-2-phenylindole) staining, respectively.
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 |
DISCUSSION |
NRSF/REST is a transcription factor that contains nine
Cys2-His2 type zinc finger domains. It has
previously been suggested that the zinc finger domains of NRSF/REST
contribute to its binding to the DNA sequence known as NRSE/RE-1.
REST4, which is a C-terminally truncated neuron-specific isoform of
NRSF/REST, contains only the first five of the nine
Cys2-His2 type zinc finger domains. Although
the physiological function of REST4 remains unclear, the binding
affinity of REST4 to NRSE/RE-1 is lowered dramatically to
~
to
of NRSF/REST (13). These data suggest
that other zinc finger domains, notably zinc finger domains 6-8,
contribute to DNA binding (13). To study the contributions of these
zinc finger domains to DNA binding, a point mutation was introduced
into each in which the amino acid Cys was changed to Arg. It has
previously been established that this mutation disrupts the
conformation and function of Cys2-His2 type
zinc finger domains (14). As expected introducing this mutation into
zinc finger domains 6-8 together (Z6,7,8mut) decreased DNA binding to
an undetectable level. Binding of the mutant could only be detected by
adding higher concentrations of the nuclear extract. Similarly,
combinations of mutations in any two of the three zinc finger domains
(Z6,7mut, Z7,8mut, or Z6,8mut) greatly diminished binding to the
NRSE/RE-1 probe. Single mutations in zinc finger domains 6 or 8 had
little or no effect on DNA binding; however, mutation of zinc finger
domain 7 alone greatly reduced DNA binding. These data suggest that all
three zinc finger domains can contribute to DNA binding; however, two
adjacent zinc fingers, either zinc finger 6 plus 7 or zinc finger 7 plus 8, are required to produce maximal DNA binding.
Although zinc finger domains 6-8 contribute to DNA binding, REST4,
which lacks these domains as well as zinc finger domain 9, appears to
be efficiently targeted to the nucleus (Figs. 3 and 4). REST4 also
lacks the putative nuclear localization signal found in the C-terminal
region of NRSF/REST (1, 16). Therefore, either there are two nuclear
localization signals in NRSF/REST, or the nuclear localization signal
found in REST4 is the functional one. Deletion mutagenesis previously
indicated that the nuclear targeting signal of REST4 resides within the
C-terminal region containing zinc finger domains 2-5 (15), and this
was confirmed in this study by both cell fractionation and in
vivo fluorescence measurements using GFP-REST4 constructs. We
further demonstrated that zinc finger domains 2-5, but not 2-4, could
target
-galactosidase to the nucleus. In addition, REST1, which is
similar to REST4, but lacks zinc finger domain 5, was not targeted to
the nucleus. Although these results would suggest that zinc finger 5 is
required for nuclear localization, a point mutation in zinc finger 5 did not prevent nuclear localization. We can conclude that there is a
nuclear targeting signal, other than the zinc finger domain, located
within amino acids 296-328, which comprise the region preceding and
through zinc finger 5. There are several basic residues in this region
that could participate in a nuclear targeting signal. Alternatively,
deletion of amino acids 296-328 changes the structure of the protein
such that a critical conformation needed for nuclear targeting is disrupted.
To determine whether the zinc finger domain structures are important
for nuclear targeting, REST4 with combinations of Cys to Arg point
mutations in the zinc finger domains was constructed and fused
C-terminally to GFP. None of the combinations of point mutations in
zinc finger domains 2-5 changed the localization of REST4 from nuclear
to cytoplasmic; thus, a functional structure of the zinc finger domains
is not required to target REST4 to the nucleus. On the other hand,
disruption of zinc finger domain 3 alone or in combination with other
zinc finger domain mutations produced abnormal nuclear localization. A
punctate staining of the nucleus was seen with the zinc finger domain 3 single mutant as well as with the zinc finger domain 3 and 4 and zinc
finger domain 3 and 5 double mutants. These GFP-REST4 constructs
appeared to enter the nucleus but were not dispersed uniformly
throughout the nucleus. A different pattern was observed with the zinc
finger domain 3 mutant in combination with a zinc finger domain 2 mutation. In these cases, the GFP-REST4 constructs appeared to localize to the edge of the nucleus, as if they were attached to the nuclear envelope.
It has been suggested that zinc finger domains not only function in DNA
recognition (19), but also can be involved in protein-protein interactions (20, 21) and in determining subcellular localization (22,
23). Nuclear protein import including that of transcription factors
such as NRSF/REST or REST4 is a key control point in regulating gene
expression. Nuclear localization signal-mediated transport into the
nucleus probably involves at least two processes: targeting to the
nucleus and translocation with the nuclear pore complexes. The first
step is suggested to be an energy-independent recognition of the
targeting signal of the transport substrate and subsequent docking at
the nuclear pore complexes. The second step is
energy-dependent and involves translocation through the
pore and into the nucleus (24, 25). Taken together, our results suggest
the presence of three signals in REST4 that contribute to its nuclear
localization. One signal, which does not require a functional zinc
finger domain, is necessary to target REST4 to the nucleus. This signal
lies within amino acids 296-328, since REST1 and the REST4 zinc finger 5 deletion mutant, both of which lack these amino acids, are not targeted to the nucleus. A second signal appears to be required to
translocate REST4 across the nuclear membrane, and this signal appears
to require both zinc finger domain 2 and zinc finger domain 3. Third, a
signal residing in zinc finger domain 3 releases REST4 from the
translocation machinery so that it can disperse throughout the nucleus.
A less likely but alternative explanation is that the mutation in zinc
finger domain 3 introduces a cryptic signal, which causes association
of REST4 with nuclear membranes.
 |
FOOTNOTES |
*
This work was supported in part by NIA, National Institutes
of Health, Grant AG05893.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: Dept. of Biochemistry,
University of Kentucky, Chandler Medical Center, 800 Rose St.,
Lexington, KY 40536-0298. Tel.: 859-323-5549; Fax: 859-323-1727; E-mail: lhersh@pop.uky.edu.
Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M011193200
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ABBREVIATIONS |
The abbreviations used are:
NRSE, neuron-restrictive silencer element;
RE-1, repressor element-1;
NRSF, neuron-restrictive silencer factor;
REST, RE-1 silencing
transcription factor;
X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside;
GFP, green fluorescent protein;
PBS, phosphate-buffered saline;
EMSA, electrophoretic mobility shift assay(s).
 |
REFERENCES |
1.
|
Chong, J. A.,
Tapia-Ramirez, J.,
Kim, S.,
Toledo-Aral, J. J.,
Zheng, Y.,
Boutros, M. C.,
Altshuller, Y. M.,
Frohman, M. A.,
Kraner, S. D.,
and Mandel, G.
(1995)
Cell
80,
949-957[Medline]
[Order article via Infotrieve]
|
2.
|
Schoenherr, C. J.,
and Anderson, D. J.
(1995)
Science
267,
1360-1363[Medline]
[Order article via Infotrieve]
|
3.
|
Lonnerberg, P.,
Schoenherr, C. J.,
Anderson, D. J.,
and Ibanez, C. F.
(1996)
J. Biol. Chem.
271,
33358-33365[Abstract/Free Full Text]
|
4.
|
Li, L.,
Suzuki, T.,
Mori, N.,
and Greengard, P.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
1460-1464[Abstract]
|
5.
|
Mori, N.,
Schoenherr, C. J.,
Vandenbergh, D. J.,
and Anderson, D. J.
(1992)
Neuron
9,
45-54[Medline]
[Order article via Infotrieve]
|
6.
|
Kraner, S. D.,
Chong, J. A.,
Tsay, H. J.,
and Mandel, G.
(1992)
Neuron
9,
37-44[Medline]
[Order article via Infotrieve]
|
7.
|
Bai, G.,
Norton, D. D.,
Prenger, M. S.,
and Kusiak, J. W.
(1998)
J. Biol. Chem.
273,
1086-1091[Abstract/Free Full Text]
|
8.
|
Kallunki, P.,
Jenkinson, S.,
Edelman, G. M.,
and Jones, F. S.
(1995)
J. Biol. Chem.
270,
21291-21298[Abstract/Free Full Text]
|
9.
|
Kallunki, P.,
Edelman, G. M.,
and Jones, F. S.
(1997)
J. Cell Biol
138,
1343-1354[Abstract/Free Full Text]
|
10.
|
Chen, Z.-F.,
Paquette, A. J.,
and Anderson, D. J.
(1998)
Nat. Genet.
20,
136-142[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Palm, K.,
Belluardo, N.,
Metsis, M.,
and Timmusk, T.
(1998)
J. Neurosci.
18,
1280-1296[Abstract/Free Full Text]
|
12.
|
Shimojo, M.,
Paquette, A. J.,
Anderson, D. J.,
and Hersh, L. B.
(1999)
Mol. Cell. Biol.
19,
6788-6795[Abstract/Free Full Text]
|
13.
|
Lee, J.-H.,
Shimojo, M.,
Chai, Y.-G.,
and Hersh, L. B.
(2000)
Brain Res. Mol. Brain Res.
80,
88-98[Medline]
[Order article via Infotrieve]
|
14.
|
Tapia-Ramirez, J.,
Eggen, B. J.,
Peral-Rubio, M. J.,
Toledo-Aral, J. J.,
and Mandel, G.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
1177-1182[Abstract/Free Full Text]
|
15.
|
Lee, J.-H.,
Chai, Y-G.,
and Hersh, L. B.
(2000)
J. Mol. Neurosci.
80,
88-98
|
16.
|
Grimes, J. A.,
Nielsen, S. J.,
Battaglioli, E.,
Miska, E. A.,
Speh, J. C.,
Berry, D. L.,
Atouf, F.,
Holdener, B. C.,
Mandel, G.,
and Kouzarides, T.
(2000)
J. Biol. Chem.
31,
9461-9467[CrossRef]
|
17.
|
Huang, Y.,
Myers, S. J.,
and Dingledine, R.
(1999)
Nat. Neurosci.
2,
867-872[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Naruse, Y.,
Aoki, T.,
Kojima, T.,
and Mori, N.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
13691-13696[Abstract/Free Full Text]
|
19.
|
Wolfe, S. A.,
Nekludova, L.,
and Pabo, C. O.
(2000)
Annu. Rev. Biophys. Biomol. Struct.
3,
183-212
|
20.
|
Lee, J. S.,
Galvin, K. M.,
and Shi, Y.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
6145-6149[Abstract]
|
21.
|
Milne, C. A.,
and Segall, J.
(1993)
J. Biol. Chem.
268,
11364-11371[Abstract/Free Full Text]
|
22.
|
Bruening, W.,
Moffett, P.,
Chia, S.,
Heinrich, G.,
and Pelletier, J.
(1996)
FEBS Lett.
393,
41-47[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Matheny, C.,
Day, M. L.,
and Milbrandt, J.
(1994)
J. Biol. Chem.
269,
8176-8181[Abstract/Free Full Text]
|
24.
|
Newmeyer, D. D.,
and Forbes, D. J.
(1988)
Cell
52,
641-653[Medline]
[Order article via Infotrieve]
|
25.
|
Richardson, W. D.,
Mills, A. D.,
Dilworth, S. M.,
Laskey, R. A.,
and Dingwall, C.
(1988)
Cell
52,
655-664[Medline]
[Order article via Infotrieve]
|
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