From the Institute of Molecular and Cell Biology, 30 Medical Drive,
Singapore 117609, Republic of Singapore
Myelin transcription factor 1 (MyT1) and neural
zinc finger factor 1 (NZF-1) represent the first two members of an
emerging family of neural specific, zinc finger-containing DNA-binding proteins. MyT1 has been shown recently to play a critical role in
neuronal cell differentiation during development. We have cloned the
third member of the NZF/MyT family, referred to as neural zinc finger
factor 3 (NZF-3). The cDNA sequence predicts a protein of 1,032 amino acids which contains two clusters of zinc fingers similar to MyT1
and NZF-1. Unlike MyT1 and NZF-1, NZF-3 does not contain an acidic
domain at the amino terminus or a serine/threonine-rich region between
the two finger clusters. NZF-3 binds to a DNA element containing a
single copy of the previously described AAAGTTT consensus motif for
these factors but exhibits a marked enhancement in relative affinity to
a bipartite element containing two copies of the consensus motif. In
contrast to MyT1 and NZF-1, which are known to activate transcription,
cotransfection experiments revealed that NZF-3 confers repression on
the basal activity of promoters containing the consensus binding
elements. The identification of an additional member of the NZF/MyT
family provides an opportunity to investigate the relative contribution
of members of this family of transcription factors to the complex
regulatory processes in neural development and homeostasis.
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INTRODUCTION |
The role of cell type-specific transcription factors in lineage
specification during invertebrate development (1), vertebrate development (2), vertebrate myogenesis (3), and development of the
immune system (4, 5) is well established. In each of these cases,
critical decisions concerning cell fate involve transcription factors
that are expressed in the earliest precursors of a particular cell
type. If this paradigm also holds true in the generation of specific
neuronal lineages in the developing nervous system, the
identification of transcription factors marking neuronal cell types
from their birth in the neural tubes and throughout their complex
program of differentiation is a critical step in advancing our
understanding of these cell fate decisions.
Zinc-coordinated fingers are one of the most common DNA binding motifs
among eukaryotic transcription factors and are classified according to
the number and position of the cysteine and histidine residues
available for zinc coordination. The Cys-Cys, His-His (C2H2) class, which is typified by the
Xenopus transcription factor IIIA (6), contains the largest
number of members. These proteins contain two or more fingers arranged
in tandem. In contrast, steroid receptors contain only two
zinc-coordinated structures with four (C4) and five
(C5) conserved cysteines. The third class of zinc fingers,
which binds to single-stranded nucleic acids, has a consensus sequence
of
Cys-X2-Cys-X4-His-X4-Cys
(C2HC). Such factors are found in mammals (7),
Drosophila transposable element copia (8), and in
retroviruses (9). Other metal-coordinating proteins have different
structures such as C6 in the yeast GAL4 protein and a
cysteine-rich structure in the E1A oncoprotein (10). In accordance with
their structural diversity, zinc finger proteins play a variety of
important roles in cell growth, differentiation, and development.
Transcription factor IIIA and the ubiquitous transcription factor SP1
are broadly involved in the regulation of transcription, whereas the
Drosophila zinc finger proteins Krüppel and Hunchback
are crucial for proper segmentation of the developing embryo (11-13).
The zinc finger protein REST (RE1-silencing transcription factor) has
been shown to repress neuronal gene expression in non-neuronal tissues
(14, 15).
Two zinc finger proteins, neural specific zinc finger factor 1 (NZF-1)1 (16) and myelin
transcription factor 1 (MyT1, also known as NZF-2) (17), have recently
been identified by virtue of their ability to bind the
cis-regulatory element present in the promoter of the
-retinoic acid receptor and myelin proteolipid protein genes,
respectively. Each of these proteins contains six Cys
X5-Cys-X12-His-X4-Cys (C2HC) type zinc fingers of novel configuration, different
from the previously described C2HC type of zinc fingers
found in proteins present in retroviruses (9). The zinc fingers in
these proteins are arranged in two main clusters. Each of these
clusters can independently bind DNA and recognize similar core
consensus sequences. Other motifs that can be found in these proteins
include an acidic domain at the amino terminus and a
serine/threonine-rich domain situated in the region between the two
zinc finger clusters. These proteins are highly homologous to each
other, suggesting that they belong to a subfamily of zinc
finger-containing transcription factors. In addition, Northern analysis
revealed that they are highly enriched in the brain (16, 17). The
restricted pattern of expression of these proteins indicates that they
may serve important functions in the development and maintenance of the nervous system. Indeed, in the course of this study, an independent report demonstrated that X-MyT1, a Xenopus homolog of MyT1,
serves an important function in the primary selection of neuronal
precursor cells in the developing embryo (18). X-MyT1 is able to
promote ectopic neuronal differentiation in cooperation with the basic helix-loop-helix neural transcription factors (18). Furthermore, normal
neurogenesis and ectopic neurogenesis caused by overexpression of the
neural basic helix-loop-helix factors are inhibited by a dominant
negative form of X-MyT1 (18).
Given the high possibility of members of the NZF/MyT family being
involved in neural development and homeostasis, it would be valuable to
identify and characterize novel members of this family. Thus, a
strategy was devised to isolate new members of the NZF/MyT gene family.
Degenerate oligonucleotides representing all possible codons for two
stretches of 11 amino acid residues conserved between NZF-1 and MyT1
were used as primers in the polymerase chain reaction (PCR). DNA
complementary to poly(A)-selected RNA from human brain, thymus, liver,
spleen, kidney, placenta, and rat brain were used as templates. Here,
we describe the cloning and characterization of a novel member of the
NZF/MyT family, referred to as neural zinc finger factor 3 (NZF-3).
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EXPERIMENTAL PROCEDURES |
cDNA Cloning and Isolation--
Degenerate primers were
designed based on two conserved regions (non-zinc finger) between human
MyT1 and rat NZF-1 (Fig. 1A). The amino acid sequence and degenerate nucleotide sequence of the
5'-primer were EILAMHENLVK and
5'-GA(G/A)AT(C/T)(C/T)T(T/A)GCCATGCATGA(A/G)AA(C/T)GT(A/T/G)CT(C/G)AAG-3', respectively. Those of the 3'-primer were EVDENGTLDLS and
5'-GCT(A/G)A(A/G)GTCCA(A/G)(C/G)CA(A/C)GG(C/T)AA(A/G)AG(C/T)AG(A/G)TG(A/G)AG-3', respectively.

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Fig. 1.
Cloning of NZF-3. Panel A, cloning
strategy for identification of a novel member of the NZF/MyT family. On
the schematic diagram of NZF-1 and hMyT1 the positions of zinc fingers
are indicated by hatched boxes. Amino acid sequences chosen
for designing degenerate primers for PCR are as indicated. Panel
B, cloning of a full-length cDNA of NZF-3. Full-length
cDNA encoding NZF-3 was assembled from three overlapping clones
(K1, K2, and K3) obtained from screening a gt11 rat brain cDNA
library. nt, nucleotides. Panel C, primary DNA
and deduced amino acid sequences of NZF-3. The six Cys-Cys, His-Cys
zinc fingers are boxed. Arrows indicate the
conserved amino acid region corresponding to the primers used in PCR.
The polyadenylation signal at the 3'-end is
underlined.
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cDNA from the following tissues was used as a template in the PCR:
human thymus, liver, spleen, kidney, placenta, and brain (all purchased
from CLONTECH, Palo Alto, CA) and rat brain
(CLONTECH). 40 PCR cycles were performed:
denaturation at 94 °C for 30 s, annealing at 42 °C for 2 min, and extension at 72 °C for 30 s. PCR products were
amplified from human and rat brain cDNA. The ends of the PCR
products were repaired with the Klenow fragment of DNA polymerase I
(New England Biolabs), phosphorylated with T4 polynucleotide kinase
(New England Biolabs), and subcloned into the EcoRV site of
the plasmid vector pBKSII(
) (Stratagene, La Jolla, CA) according to
standard procedures (19). These products were subsequently
sequenced.
A random primed 32P-labeled probe was prepared from a novel
fragment amplified from rat brain cDNA and used to screen a rat brain
gt11 cDNA library by DNA hybridization. Phages were
plated, transferred to nitrocellulose filters, and denatured according to standard procedures (19). After being baked, the filters were
prehybridized for 2 h at 55 °C. A solution composed of 0.5 M sodium phosphate buffer (pH 7.0), 7% SDS, 15%
formamide, and 1 mM EDTA was used for prehybridization as
well as hybridization. The denatured radiolabeled probe was then added
directly to the prehybridization solution (1 × 106
cpm/ml), and the hybridization was continued at 55 °C overnight. After hybridization, the filters were washed twice in 50 mM
sodium phosphate buffer (pH 7.0) and 0.1% SDS at 55 °C for 0.5 h and subsequently subjected to autoradiography. Positive clones were isolated, and the cDNA inserts from these clones were subcloned into the EcoRI site of pBKSII(
) vector and characterized.
The sequences from three overlapping clones were used to obtain the full-length clone.
Nucleotide sequencing was determined by the dideoxynucleotide chain
termination procedure on double-stranded DNA templates using a
Sequenase 2.0 kit (U. S. Bichemical Corp.).
RNA Analysis--
A poly(A) RNA Northern blot of rat tissue was
purchased from CLONTECH, and Northern analysis was
carried out according to the manufacturer's protocol. The blot
contained approximately 2 µg of rat tissue mRNA/lane. The blot
was probed with a random primed 32P-labeled
SacI-BglI fragment from NZF-3. This 0.55-kb
fragment (nucleotides 336-892) corresponds to the 5'-region of the
NZF-3 cDNA upstream from the sequence coding for the first cluster
of two zinc fingers and is unique to NZF-3. After hybridization at 68 °C overnight, the blot was rinsed in 2 × SSC (1 × SSC
is 0.15 M NaCl and 15 mM sodium citrate) and
0.5% SDS several times at room temperature for 30 min followed by
washes in 0.1 × SSC and 0.2% SDS at 50 °C for another 30 min
before autoradiography. The blot was later stripped and rehybridized
with a
-actin probe.
Total RNA was also prepared from selected rat tissues and cell lines
using UltraspecTM RNA solution (Biotecx Inc., Houston) and
used in ribonuclease protection assays (Ambion) according to the
manufacturer's instructions. Hybridization reactions contained 25 µg
of total RNA. Antisense ribonucleotide probes were transcribed in
vitro in the presence of [
-32P]dCTP from a pBKS
construct containing a 351-base pair PstI-StuI fragment (nucleotides 1711-2062) and were gel purified. This fragment corresponded to a portion of the region between the two zinc finger clusters of NZF-3. RNase-resistant fragments were resolved by electrophoresis on an 8 M urea, 6% polyacrylamide gel.
Protein Expression and Antibodies--
Fragments of NZF-3
cDNA were subcloned into the pGEX-KG bacterial expression vector
(20) to produce glutathione S-transferase (GST) fusion
proteins. The GST- 2ZF construct consisting of the first two fingers of
the four-zinc finger cluster with additional upstream sequences (amino
acids 601-796) was generated using a PCR-based strategy. The GST-4ZF
construct, which contained the entire four-zinc finger cluster, was
generated by first subcloning a 0.8-kb StuI-AccI
(coding for amino acids 688-962) fragment into the EcoRV
and AccI sites of pBKSII(
). A fragment was then excised using EcoRI and XhoI and cloned into the
corresponding sites in pGEX-KG. The recombinant plasmids or vector
alone was introduced into the Escherichia coli BL21 (DE3)
strain (21). Transformed bacteria were grown to an
A600 of 0.8 and then induced with 0.5 mM isopropyl-1-thio-
-D-galactopyranoside.
After 3 h the bacterial cells were harvested and centrifuged.
Bacterial pellets containing insoluble GST-2ZF fusion protein were
resuspended in SDS sample buffer (2% SDS, 100 mM Tris (pH 7.5), 280 mM
-mercaptoethanol, and 20% glycerol). After
heating the samples and centrifugation, the supernatant was
electrophoresed in preparative SDS-polyacrylamide gels. The expressed
GST-2ZF protein was visualized with Coomassie Blue and excised from the gel. Subsequently, the protein was eluted from the gel and used to
immunize rabbits to raise antibodies. Bacterial pellets containing the
GST-4ZF fusion protein were resuspended in lysis buffer (0.4 M NaCl, 50 mM Tris (pH 7.6), 1 mM
EDTA, 1 mM dithiothreitol, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, and 5 mg/ml leupeptin) and sonicated
followed by centrifugation. The supernatant that represented the
bacterial extract was analyzed using SDS-polyacrylamide gel
electrophoresis and Western blot. This extract was used in DNA binding
experiments.
Immunoblot Analysis--
Protein samples (bacterial and
mammalian cell extracts) were mixed with SDS sample buffer,
fractionated on a 10% SDS-polyacrylamide gel, and then transferred to
a 0.2-nm pore size nitrocellulose membrane (Amersham). Membranes were
blocked in phosphate-buffered saline (pH 7.4) with 0.2% Tween 20 (PBS-T) containing 5% non-fat milk for 1 h before incubation with
rabbit polyclonal antibody (preimmune and anti-GST-2ZF immune sera)
diluted 1:10,000 in PBS-T containing 1% non-fat milk. The blots were
washed three times with PBS-T, incubated with a 1:15,000 dilution of
horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham) for
1 h, washed three times, and visualized using the Enhanced
Chemiluminescence (ECL) system (Amersham).
Whole Cell, Nuclear, and Cytoplasmic Extract
Preparation--
Full-length NZF-3 cDNA was cloned into the
BamHI-XhoI sites of the pXJ40 mammalian
expression vector, which is under the control of the cytomegalovirus
(CMV) promoter/enhancer (22). Transfections were carried out in 293 human embryonic kidney cells that were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum
(Hyclone). The cells were transfected with either pXJ40 or pXJ40 NZF-3
using Lipofectin reagent (Life Technologies, Inc.) and harvested
24 h later. Whole cell extracts were prepared by resuspending the
transfected cells in binding buffer used for electrophoretic mobility
shift assays (EMSAs), freezing them at
80 °C, thawing them over
ice, and centrifuging at full speed for 15 min at 4 °C. The
supernatant was used in EMSAs. Nuclear extracts were prepared as
described previously (23) except that the cytoplasmic fraction was
retained for analysis. The fractions were separated by
SDS-polyacrylamide gel electrophoresis and subjected to Western blot
analysis.
Immunofluorescence Staining--
293 cells were seeded onto
glass coverslips and transfected with either pXJ40 or pXJ40-NZF-3
expression plasmid. 24 h after transfection, the cells were washed
in ice-cold PBS, fixed in ice-cold methanol for 5 min at
20 °C,
washed in PBS, and incubated in blocking buffer (5% goat serum, 5%
fetal calf serum, 2% bovine serum albumin in PBS) for 1 h. The
cells were then incubated with primary antibody (preimmune sera and
anti-GST-2ZF immune sera) diluted 1:150 in blocking buffer for 1 h, washed three times with PBS, and then incubated with the fluorescein
isothiocyanate-conjugated goat anti-rabbit secondary antibody
(Molecular Probes Inc., Eugene, OR) for another hour. After three
washes in PBS for 10 min each, the coverslips were mounted in 80%
glycerol in PBS containing 1 mg/ml p-phenylenediamine and
examined with a Zeiss Axioplan microscope.
EMSA--
Complementary oligonucleotides for the SIN element,
containing a single copy of the AAAGTTT core motif, were synthesized to carry out analysis on the DNA binding properties of NZF-3. In addition,
a series of complementary oligonucleotides based on a direct repeat of
the AAAGTTT core motif was synthesized. The spacing between the repeats
in the oligonucleotides DR2, DR5, DR7, DR9, and DR11 were 2, 5, 7, 9, and 11 nucleotides, respectively. The BamHI site at the
5'-end and a BglII site at the 3'-end were added for cloning
purposes.
The sequences of oligonucleotides used in EMSA are as follows:
Bacterial extracts containing GST-4ZF fusion protein and whole
cell extracts from transfected mammalian cells were used in EMSAs as
described previously (24).
Competition and supershift reactions were performed by preincubating
the reaction mixture with unlabeled oligonucleotide or immune sera for
15 min at room temperature before addition of the radiolabeled probe.
Quantitative competitive EMSAs were used to compare relative affinities
of different DNA-binding elements for NZF-3. In these experiments, the
DR5 element was end labeled and used as a probe. Increasing amounts of
unlabeled competitor were added to the sample 15 min before the
addition of the radiolabeled DR5 probe. After electrophoresis, the
quantity of shifted probe of each sample was determined using a
PhosphorImager (Molecular Dynamics). The amount of shifted probe in the
absence of competitor was defined as the amount of maximal binding. The
fraction of maximal binding at each competitor concentration was then
calculated as the ratio of bound probe in the presence of competitor to
that in the absence of competitor. To assess the effect of the
competitors visually, the fraction of maximal binding was plotted
against the amount of unlabeled competitor used. This allowed us to
estimate the IC50 or the concentration of competitor which
inhibited maximal binding by 50%. The IC50 was taken as an
indication of the relative binding affinity of a particular
oligonucleotide for NZF-3.
Mammalian Cell Transfection--
Oligonucleotides
containing single or double (bipartite) copies of the consensus motif
(described earlier) were inserted upstream of a minimal herpes simplex
virus thymidine kinase promoter driving a luciferase reporter gene
(designated TK-Luc) to generate reporter plasmids. In addition,
reporter plasmids containing unrelated DNA sequences were also
generated. The sequences are as follows:
Deletion mutants of NZF-3 were generated using a PCR-based
protocol and cloned into the BamHI and KpnI sites
of the pXJ40 mammalian expression vector.
All transfections were carried out in 293 human embryonic kidney cells,
which were plated out 24 h before transfection. In the deletion
mutant analysis, 1.5 µg of reporter plasmid and 1 µg of the pXJ40
vector, pXJ40 NZF-3, or deletion mutant expression plasmids was used in
transient transfections using Lipofectin. In addition, 0.2 µg of a
-galactosidase expression vector, CMV-
Gal, was also cotransfected
in each dish. 48 h after transfection, the cells were rinsed with
PBS and lysed in the following buffer: 50 mM potassium
phosphate (pH 7.8), 1 mM dithiothreitol, and 1% Triton
X-100. The lysate was cleared of cellular debris by centrifugation and
analyzed using a Monolight 2010 Luminometer (Analytical Bioluminescence Laboratories, Ann Arbor, MI). Experiments were performed at least three
times in duplicate, and results were normalized with
-galactosidase activity to correct for transfection efficiency.
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RESULTS |
Amplification of cDNA for NZF/MyT Proteins--
Degenerate
primers corresponding to two stretches of conserved sequences in MyT1
and NZF-1 (Fig. 1A) were used in PCR. Because it is unclear
whether novel members of this family are expressed in tissues other
than brain, cDNAs from several other tissue sources, including
kidney, liver, spleen, thymus, and placenta, were also used. PCR
products were cloned and sequenced. NZF-1 and MyT1 cDNA fragments
were amplified readily from samples derived from both human and rat
brain but not from any other tissues. In addition, a novel cDNA
clone containing an open reading frame encoding a polypeptide that
showed substantial homology to the zinc finger as well as the
inter-zinc finger region of NZF-1 and MyT1 were isolated from the
brain. The encoded protein thus represents a putative novel member of
the NZF/MyT family and is referred to as NZF-3.
Cloning of a Full-length cDNA of NZF-3--
The rat whole
brain cDNA library was probed with a mixture of random primed
radiolabeled fragments from the unique PCR fragment encoding the
putative novel member. Multiple clones were obtained and analyzed by
restriction mapping and in several cases, by partial sequence
determination. Three of the overlapping clones, spanning a region of
approximately 3.5 kb, were sequenced completely on both strands (Fig.
1B). The assembled cDNA sequence contains a single open
reading frame encoding a protein of 1,032 amino acids (Fig.
1C). The 5'-region of the assembled cDNA clone contains stop codons in all three reading frames preceding the predicted translation start site. The 3'-untranslated region encompasses a
putative polyadenylation signal (Fig. 1C). Analysis of the
protein sequence of NZF-3 revealed that it contains six zinc fingers of the
Cys-X5-Cys-X12-His-X4-Cys
type. The fingers are arranged in two clusters of two and four, most
similar to MyT1 (Fig. 2A). The
amino acid sequence of the zinc fingers among members of this family
displays a very high degree of conservation, suggesting that they may
recognize similar cis-regulatory elements (Fig. 2B). In addition to the zinc finger domain, the carboxyl
terminus of NZF-3 also displays a high degree of homology to MyT1 and
NZF-1 (Fig. 2A). However, in contrast to MyT1 and NZF-1,
NZF-3 does not contain an acidic domain at the amino terminus or a
serine/threonine-rich motif in the region between the two finger
clusters (Fig. 2A).

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Fig. 2.
Comparison of members of the NZF/MyT family.
Panel A, schematic representation of the protein sequences
of various members of the NZF/MyT family. Percentages of amino acid
identities of various regions of NZF-1 and MyT1 to NZF-3 are shown.
Zinc fingers are represented by hatched boxes.
Spotted regions represent acidic domains, and black
boxes denote Ser/Thr-rich domains. Panel B, alignment
of zinc finger sequences of NZF-3 and human (h)MyT1. The conserved
cysteine and histidine residues are boxed. Dashes represent identity to NZF-3.
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Expression of NZF-3 mRNA--
Northern blot analysis was
performed to determine whether NZF-3 has a restricted pattern of
expression. We screened a rat multiple tissue Northern blot and
observed that a NZF-3 transcript of 7.5 kb was expressed in the brain
and was completely absent in the heart, spleen, lung, liver, muscle,
and kidney (Fig. 3A). A
smaller and less abundant transcript was also detected in the testis.
This may represent an alternatively spliced form of NZF-3 or other
homologous transcripts. RNase protection assay utilizing a specific
probe corresponding to the inter-zinc finger clusters region of the
cDNA confirmed the results from the Northern analysis that the
NZF-3 message was highly restricted to the brain (Fig. 3B).
Furthermore, the NZF-3 message was detected in the neuronal type cell
line, PC12, but not in a panel of non-neuronal cell lines tested (Fig.
3B).

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Fig. 3.
Tissue distribution of the NZF-3 transcript.
Panel A, Northern blot analysis of NZF-3. A multiple tissue
Northern blot containing 2 µg of poly(A) RNA/lane was probed with a
specific cDNA fragment corresponding to the 5'-region of NZF-3
cDNA. The blot was subsequently stripped and rehybridized with a
-actin probe. Panel B, RNase protection analysis of total
RNA obtained from various tissues and cell lines. A riboprobe
(complementary to nucleotides 1711-2062 in Fig. 1C) was
hybridized to total RNA. A protected fragment of the expected size (351 nucleotides) was observed in the brain, testis, and in the PC12 cell
line.
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Subcellular Localization of NZF-3--
Polyclonal antibodies
raised against the bacterially expressed GST fusion proteins containing
the first two fingers (GST-2ZF) of the four-finger cluster of NZF-3
were used to determine the subcellular localization of NZF-3. The
antibody was shown to be specific as it recognized the GST-2ZF fusion
protein in Western blot analysis but not the GST or any other unrelated
proteins in the bacterial extracts (Fig.
4A). The antibody, however,
failed to detect the endogenous NZF-3 in PC12 cells by Western
immunoblot as well as immunohistochemical analysis (data not shown).
The inability of the antibody to detect NZF-3 protein in PC12 cells may
be attributed to a low level of expression of the protein in these
cells. We therefore decided to overexpress the NZF-3 protein by
transient transfection in mammalian cells. 293 human embryonic kidney
cells, devoid of any NZF-3 mRNA as determined by RNase protection
experiments, were transfected with an expression plasmid containing the
cDNA encoding the full-length NZF-3. Nuclear and cytoplasmic
fractions were obtained from the transfected cells and subjected to
Western blot analysis. The specific antibody was able to recognize a
protein of the expected molecular mass in the nuclear fraction (Fig.
4B). A much weaker band was also detected in the cytoplasmic
fraction, possibly representing a minor contamination with nuclear
proteins during the separation process (Fig. 4B). In
addition, immunohistochemistry experiments were performed on the
transfected cells. Overexpressed NZF-3 was found to localize to the
nucleus of the transfected cells (Fig. 4C). Based on these
results, we conclude that NZF-3 is essentially a nuclear protein.

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Fig. 4.
Subcellular localization of NZF-3.
Panel A, specificity of the polyclonal antibody against
NZF-3. From Western blot analysis of lysates from bacteria expressing
GST (lanes 1 and 3) and GST-2ZF (lanes
2 and 4) the fusion protein was detected with
anti-GST-2ZF immune sera (I, lane 4) but not with
preimmune sera (P, lane 2). Panel B,
Western blot analysis of cytoplasmic (C, first
and third lanes) and nuclear extracts (N,
second and fourth lanes 2) from 293 cells
transfected with an expression plasmid containing the full-length NZF-3
cDNA. NZF-3 was detected by immune sera in the nuclear extract of
cells transfected with NZF-3 expression plasmid (fourth
lane). Panel C, immunofluorescence analysis of cells
transfected with either the vector or expression plasmid containing the
full-length NZF-3 cDNA. Cells were incubated with anti-GST-2ZF
immune serum followed by fluorescein isothiocyanate-conjugated anti-rabbit IgG and visualized using fluorescence microscopy.
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DNA Binding Properties of NZF3--
NZF-1 was shown to recognize a
single copy of the AAGTT sequence within the
-retinoid acid response
element and proteolipid protein enhancer element (16). However, random
oligonucleotide selection experiments performed with bacterial fusion
proteins containing the second zinc finger cluster of X-MyT1 revealed
that all of the selected binding sites are bipartite elements
containing two copies of an AAA(G/C)TTT motif arranged in tandem with a
variable spacing from 1 to 11 nucleotides (18).
To characterize the in vitro binding property of NZF-3,
whole cell extracts of 293 cells transiently expressing NZF-3 were tested in EMSAs. NZF-3 recognizes the AAAGTTT core motif and displayed strong binding to a bipartite element consisting of a direct repeat of
the AAAGTTT motif separated by five nucleotides (DR5 element) (Fig.
5A). Two DNA-protein
complexes, C1 and C2, were detected. However, only C1 appeared to be
specific because formation of the complex was eliminated in the
presence of a 50-fold molar excess of unlabeled specific
oligonucleotide (Fig. 5A, lane 3) or immune sera
against NZF-3 (Fig. 5A, lane 6). At higher
concentrations, a DNA element containing a single copy of the consensus
AAAGTTT motif (SIN) was able to abolish almost completely the formation of the C1 complex (Fig. 5A, lane 4), but the
consensus binding sequence for p53 protein had no effect on the binding
(Fig. 5A, lane 5). The radiolabeled SIN element,
however, was unable to form a detectable DNA-protein complex with the
whole cell extract (data not shown). This may be explained by the low
affinity of NZF-3 for the SIN element and because higher concentrations
of NZF-3 are required for the binding to be detected. Indeed, when a
bacterially expressed GST fusion protein containing the four-zinc finger cluster of NZF-3 (GST-4ZF) was used in the binding analysis, specific DNA-protein complexes with the SIN element were formed (Fig.
5B). Consistent with the observation from binding analysis with the full-length protein, more avid binding was detected with the
DR5 than the SIN element when identical amounts of the bacterial protein were used (Fig. 5B, left and center
lanes).

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Fig. 5.
DNA binding properties of NZF-3. Panel
A, specific binding of the full-length NZF-3 protein to the core
consensus sequence. Whole cell extracts from mammalian cells
transfected with expression plasmid containing full-length NZF-3
cDNA were tested in EMSAs with the DR5 element. Unlabeled
competitor oligonucleotides and antisera were added as indicated. Two
DNA-protein complexes, C1 and C2, were detected. F, free
probe. Panel B, binding of bacterially expressed NZF-3 to
SIN and DR5. Bacterially expressed GST fusion proteins containing the
four-zinc finger cluster of NZF-3 were tested for specific binding
against the indicated radiolabeled probes. Equivalent amounts of probe
and bacterial extract were used in each binding reaction.
Arrows indicate specific bands. F, free probe.
Panels C and D, quantitative analysis of NZF-3 binding to the DR5 probe. Panel C, comparison of relative
binding affinities of NZF-3 for the DR5 and SIN elements. Relative
binding affinities of DR5 and SIN were determined by comparing their
effectiveness as binding competitors. Various concentrations of
unlabeled oligonucleotides were preincubated with equal concentrations
of NZF-3 protein before the addition of a constant amount of
radiolabeled DR5 element. The extent of competition was analyzed after
gel electrophoresis as described under "Experimental
Procedures." The IC50 determined for each DNA element
allows the quantitative comparison of their relative affinities for
NZF-3. Data shown are representative of at least three independent
determinations, which gave similar results. Panel D,
representative experiment of competitive EMSA using unlabeled DR5
oligonucleotide. Binding was inhibited by increasing concentrations of
unlabeled oligonucleotides.
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Because an apparent difference was noted in the affinity of NZF-3
binding to the single (SIN) versus the double copy (DR5) site, we decided to evaluate this difference quantitatively by carrying
out competitive DNA binding experiments. The relative effectiveness of
increasing concentrations of various unlabeled oligonucleotides in
competing with a constant concentration of radiolabeled DR5 for binding
to NZF-3 was evaluated. The fraction of maximal binding (defined as the
ratio of probe shifted in the presence of competitor to that in the
absence of competitor) was plotted against the concentration of
unlabeled competitor (Fig. 5C). The concentration of each
competing unlabeled oligonucleotide required to reduce the quantity of
the shifted protein-DNA complex by 50%, i.e.
IC50, was estimated from the graph and was taken as an
indication of the relative binding affinity. The IC50 for the DR5 and SIN was estimated to be 2 nM and 90 nM, respectively. An example of a competition experiment
used to determine the relative binding affinity of NZF-3 for the DR5
oligonucleotide is shown (Fig. 5D).
Remarkably, the relative affinities of NZF-3 to direct repeat elements
with different spacing (DR2, DR5, DR7, DR9, and DR11) were essentially
identical, with less than a 2-fold difference among the elements,
regardless of spacing configuration (data not shown).
Transrepression Activity of NZF-3--
Both X-MyT1 and NZF-1 were
shown to possess transactivation activity from consensus elements in
transient transfection analysis (16, 18). To determine the effect of
NZF-3 on transcription, transient transfection analysis was carried out
in 293 cells with luciferase reporter constructs containing either the
single copy (SIN) or the various bipartite elements fused upstream to a
herpes simplex virus thymidine kinase promoter. Reporter constructs
containing the various DNA elements exhibited basal promoter activity
similar to that containing only the thymidine kinase promoter,
suggesting the absence of an endogenous source of activation activity
working through these elements in 293 cells. Surprisingly,
overexpression of NZF-3 did not result in transactivation of the
reporter constructs tested. Expression of the full-length protein was
confirmed by Western blot analysis (data not shown). In contrast,
overexpression of NZF-3 resulted in a repression of the basal promoter
activity (ranging from 40 to 70%) from the reporter constructs
containing the bipartite elements with various spacing (data not
shown). The most significant inhibition (69%) of the basal promoter
activity was observed from the DR9 element. The repression by NZF-3
occurred in a dose-dependent manner (Fig.
6A) and appeared to be binding site-dependent as it did not repress the thymidine kinase
promoter alone or the thymidine kinase promoter fused to the p53 or the thyroid hormone response elements (Fig. 6A). The basal
promoter activity of the reporter construct containing only a single
copy of the consensus motif was repressed as well but to a lesser
extent (20-25%) (data not shown).

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Fig. 6.
Element-dependent
transcriptional repression by NZF-3. Panel A, an
oligonucleotide containing two copies of the core consensus AAAGTTT
motif (DR9) was fused 5' to thymidine kinase promoter
driving the luciferase reporter cDNA (TK-Luc). The
reporter construct was cotransfected with increasing amounts
of NZF-3 expression plasmid or vector into 293 cells. The
results, normalized for transfection efficiency by measuring the
-galactosidase activity, are expressed as a percentage of the levels
obtained in the presence of vector alone. The results represent the
mean (± S.E.) of three to five independent experiments and are
presented as fold transcriptional activation/repression produced by
transfection of NZF-3. Transfections were also carried out using
control reporter constructs containing unrelated DNA elements such as
the p53 consensus binding site (p53-TK-Luc) and the thyroid
hormone response element (TRE-TK-Luc). Panel B,
mapping of the region required for the repressor activity. Horizontal bars represent the sequence of NZF-3, with the
zinc fingers indicated by hatched boxes. NZF-3 deletion
mutants were tested in transient transfection assays. The repressor
activity of different mutants is shown as a percentage of the reporter activity obtained using the vector alone.
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To define the region of the protein required for mediating the
repressor activity, a series of deletion mutants was constructed and
used in transient transfection analysis. All of the mutants were
expressed to a similar extent as determined by Western blot analysis
(data not shown). Binding analysis revealed that all of the mutants
were competent for DNA binding with the exception of the mutant M6
(data not shown). Deletion of the amino and carboxyl terminus of NZF-3
(mutants M1 and M2) had no significant effect on its repressor
activity. Further deletion of either one of the two zinc finger
clusters (M3 and M4) also failed to affect the repressor activity.
However, the deletion of the inter-zinc finger region (mutant M5)
abolished the repressor activity of NZF-3 completely. Mutants M6 and
M7, consisting of only the inter-zinc finger region and the distal zinc
finger cluster, respectively, also failed to exhibit repressor
activity. Inactive mutants that lacked the repressor activity could
still be detected in the nucleus (data not shown), suggesting that the
loss of repressor function is not caused by changes in subcellular
localization. These data indicated that neither the amino nor the
carboxyl-terminal domain of NZF-3 is important for the repressor
activity. The inter-zinc finger region in combination with either one
of the two zinc finger clusters appears to represent the minimum
requirement for the activity.
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DISCUSSION |
We have identified and characterized NZF-3, the third member of
the emerging NZF/MyT gene family. The full-length cDNA of NZF-3
encodes a protein of 1,032 amino acids with six zinc fingers of the
C2HC type. Similar to other members of this family, NZF-3 is expressed primarily in the nervous system. PCR studies failed to
identify any other novel member in non-neuronal tissues, which offers
support to the idea that this is a neural specific gene family. The
most conserved motif found among members in this family is the zinc
finger. The spacing between the cysteine and histidine residues within
a finger predicts that the zinc fingers in NZF-3 would be likely to
fold into a structure closely resembling that of the
C2H2 Krüppel type of zinc finger rather
than the C2HC fingers of the gag-derived
retroviral proteins (9). However, unlike the Krüppel zinc fingers
where the most highly conserved feature is the inter-zinc finger linker
region, the most conserved amino acids in the finger domain of this
family of proteins are those between the first two invariant cysteines
and those between the histidine and last cysteine.
DNA binding studies showed that each zinc finger cluster can interact
with DNA independently. Members of many zinc finger gene families are
found to bind to identical or highly similar nucleotide elements.
Examples are members of the NGF1-A family (25), the GATA family of
transcription factors (26), and also the family of proteins homologous
to erythroid Krüppel-like factor (27). Given that the zinc finger
domains represent the most conserved region among members of the
NZF/MyT family, it is not surprising that they show striking
similarities in their DNA binding profile. Like MyT1 and NZF-1, NZF-3
was found to recognize the consensus AAAGTTT element. However, a
bipartite element demonstrated a markedly higher binding affinity to
NZF-3. The cooperativity in binding was observed with the full-length
protein as well as the isolated zinc finger cluster (GST-4ZF). The
mechanism underlying the cooperative binding of NZF-3 to the bipartite
element is currently unknown.
That members of this family share similar DNA recognition profiles and
expression patterns raises the question of whether they regulate a
similar set of target genes. Several possibilities exist. Although
these proteins recognize identical sites, differences in their optimal
binding affinity for closely related recognition sequences may allow
them to regulate genes differently in vivo. In association
with differential binding affinity, the DNA binding affinity of these
proteins can also be modulated by post-transcriptional modification
such as phosphorylation. Examples of proteins that exhibit differential
binding activity as a result of their phosphorylation status are the
cAMP response element binding protein (CREB) (28) and the serum
response factor (SRF) (29). Furthermore, phosphorylation is known to
affect the transactivation activity of certain proteins as in the case
of Oct-2, which transactivates preferentially from certain promoters on
which Oct-1 has no effect even though both proteins apparently
recognize the same consensus sequence (30). Lastly, individual members
may each interact with different accessory factors that can control
their activities in a cellular or developmentally regulated
fashion.
Under the conditions tested, NZF-3 appeared to have an inhibitory
effect on the basal transcription activity of reporter constructs containing the bipartite DNA-binding elements. It is possible that
NZF-3 was displacing a nuclear factor that is capable of binding to and
transactivating from the same consensus site as NZF-3. Indeed, NZF-3
competed with NZF-1, another member of the NZF/MyT family of zinc
finger proteins, in vitro for binding to the DR9 element
(data not shown). Furthermore, overexpression of NZF-3 antagonizes the
2-3-fold activation of the DR9 reporter rendered by overexpression of
NZF-1 (data not shown). In principle, NZF-3 can mediate its repression
effect by simply displacing NZF-1. However, because both MyT1 and NZF-1
are unlikely to be expressed to any appreciable level in 293 cells, the
observed repressor activity is unlikely to be accounted for on the
basis of competition with MyT1 and NZF-1 by NZF-3. Moreover, the lack
of activation of the basal promoter activity from the bipartite element
in the transfection experiments argues against the presence of
endogenous activator molecules working through the DR9 element in 293 cells. Hence, a simple displacement model is inadequate to account for the repression activity of NZF-3. In addition, the repressor activity of NZF-3 required the inter-zinc finger cluster domain as well as the
DNA binding domain. It is likely that both protein-protein interaction
and DNA binding mechanisms are involved in mediating the repressor
activity. Because NZF-3 is expressed mainly in neuronal tissues, a
fuller understanding of its role in transcription can only be achieved
when further studies can be carried out in appropriate neuronal
models.
Amino acid sequence analysis of MyT1 and NZF-1 revealed that both
proteins contain acidic and serine/threonine-rich domains that are
absent in NZF-3. The acidic domains in GAL4 (31) and VP16 (32) and
serine/threonine-rich regions in Pit-1/GHF-1 (33) and Egr1 (34) have
been demonstrated to serve transactivation functions. Thus, there is a
strong likelihood that these domains may contribute to the
transactivation activity associated with NZF-1 and MyT1. The absence of
these motifs in NZF-3 coupled with the observation that NZF-3 possessed
repressor activity in a site-dependent manner permit us to
hypothesize that NZF-3 may represent a negative regulator in pathways
served by members of this family.
The cloning of the third member of the NZF/MyT gene family thus
provides an additional opportunity to examine the relative contributions of members of this family of neural specific
transcription factors in the development and homeostasis of the nervous
system.
We are grateful to Dr Michael G. Rosenfeld
for support in this study and Dr. Youhang Jiang for the NZF-1
expression plasmid. We thank Dr Shing-Leng Chan for critically reading
the manuscript.
While this work was in progress, a rat cDNA clone (r-MyT3) was
deposited in the GenBankTM (accession no. U67080). The
nucleotide and amino acid sequences of this clone are essentially
identical to NZF-3 except for the first 17 codons in NZF-3 and the
first 9 codons in r-MyT3. It is likely that the mRNA messages of
r-MyT3 and NZF-3 represent two alternatively spliced products derived
from the same gene.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF031942.