From the Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, Republic of China
Received for publication, January 9, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our previous studies showed that Aie1 (aurora-C),
is a novel testis kinase belonging to the aurora kinase family (1). In this report, we describe a testis zinc finger protein (Tzfp) that binds
to the upstream flanking sequence of the Aie1 gene. The mouse Tzfp gene, mapped to chromosome 7 B2-B3, encodes a
465-amino acid transcription factor containing a conserved N-terminal
BTB/POZ domain and three C-terminal PLZF-like
C2H2 zinc fingers. The zinc finger domain of
Tzfp binds to the TGTACAGTGT motif (Tzfp binding site, termed
tbs) located at the upstream flanking sequence of the
Aie1 gene by gel mobility shift, DNase I footprinting, and competition analyses. When the C-terminal zinc fingers of Tzfp were
fused to the transactivation domain of VP16, the chimera activated
transcription of a reporter construct containing multiple copies of the
tbs. In contrast, the same chimera did not activate the
reporter gene when an essential nucleotide fifth C was mutated to A at
the tbs. Furthermore, we showed that the N-terminal BTB/POZ domain of TZFP has a repressor activity. Taken together, our results indicate that Tzfp recognizes a sequence-specific motif
(tbs) and may play a role in the regulation of the genes
carrying the tbs.
We previously isolated two novel protein kinases, designated as
Aie1 (mouse) and AIE2 (human), which share high amino acid identities
with the serine/threonine (S/T) kinase domain of yeast Ipl1, fly
aurora, and frog Eg2 (1). The central kinase domain of Aie1 revealed
46.8%, 59.2%, and 64.6% identity to that of Ipl1, aurora, and Eg2,
respectively, but much less homology was found in the sequence outside
the kinase domain. Northern blot analysis revealed that Aie1 kinase is
specifically expressed in testis (1) and particularly in meiotic
pachytene spermatocytes, thereby suggesting a possible role of Aie1 in
spermatogenesis (2).
Yeast Ipl1 (3) and fly aurora (4) constitute a new family of
serine/threonine kinases, which have been shown to play roles in the
regulation of chromosome segregation and centrosome function (5, 6).
Currently, three different aurora kinases have been identified in
mammals. Aurora-B, also known as aurora 1 (5), AIK2 (7), ARK2 (8), and
AIM-1 (9), is cell-cycle regulated and could play a role in events that
occur during anaphase and/or telophase. Aurora-A, also known as aurora
2 (5), AIK (10), BTAK (11), and IAK1 (12), is oncogenic and is
overexpressed in many human cancer cell lines and tissues. Mouse Aie1
and human AIE2 (1), also known as STK-13 (13) and AIK3 (14), exhibit a
testis-specific expression pattern (1, 2) and constitute a third type
of aurora-related kinase (aurora-C).
We previously isolated the gene encoding Aie1 and mapped it to mouse
chromosome 7A2-A3 (2). The Aie1 gene spans ~14
kb1 and contains seven exons.
RNA in situ hybridization indicated that the expression of
the Aie1 transcript was restricted to meiotically active
germ cells, with the highest levels detected in pachytene spermatocytes
(2). The biological function and the mechanisms involved in the
regulation of Aie1 expression are poorly understood.
Recently, we isolated a novel human PLZF-related transcription factor,
designated as TZFP, which is predominantly expressed in testis (15).
The human TZFP contains a conserved N-terminal BTB (bric-a-brac,
tramtrack, broad complex) or POZ (poxvirus, zinc finger) domain and
three C-terminal PLZF-like C2H2 zinc fingers (15). A computer search of the protein data base revealed that the zinc
finger domain of TZFP is more closely related to the promyelocytic
leukemia zinc finger (PLZF) protein, which was previously reported to
be a DNA-binding transcriptional repressor (16). The biological
function and the target genes regulated by TZFP remain largely unknown.
In the present study, we isolated the mouse Tzfp gene and
mapped it to chromosome 7 B2-B3. Interestingly, Tzfp binds directly to
the upstream flanking sequence of the mouse Aie1 gene. We
further demonstrated that the C-terminal zinc finger domain of Tzfp
binds to a 10-bp element located at the putative Aie1
promoter with high affinity and specificity. This is the first report
to characterize the binding site for Tzfp and to suggest that
Aie1 may be a candidate gene regulated by Tzfp.
Cloning and Isolation of Mouse Tzfp cDNA and Genomic
Clones--
We previously isolated a full-length cDNA encoding
human TZFP (15). The BamHI cDNA fragment (nucleotides
882-1761) of human TZFP was used as a probe to screen a
mouse testis cDNA library (Stratagene, La Jolla, CA). The screening
and cloning conditions were described previously (1). Sequencing of the
DNA inserts was performed on both strands of the positive cDNA
clones. The sequences of human TZFP and mouse
Tzfp were analyzed using the GCG software program of the
Wisconsin Sequence Analysis Package V9.1. The percentage of similarity
between sequences was analyzed by the GAP program.
The mouse cDNA fragment (~1.7 kb) spanning two-thirds of the
coding region of Tzfp was used as probe to screen a mouse 129/SvJ genomic BAC library (Genome System, St. Louis, MO). One positive BAC
clone, 119/j1 (~150 kb), was detected and isolated. The 119/j1 DNA
was digested with appropriate restriction enzymes, and the resulting
DNA fragments were subcloned and sequenced as described previously (2).
To determine the positions of the exons and the sequences of
exon-intron boundaries, a series of sense or antisense primers derived
from a previously defined sequence of Tzfp cDNA were
used for sequence analysis of individual genomic DNA subclones.
Fluorescence in Situ Hybridization--
FISH was performed at
the laboratory of Genome Systems, Inc., using a method previously
described (2). Briefly, the mouse Tzfp DNA (~150 kb)
isolated from the BAC clone (119/j1) was labeled with digoxigenin dUTP
by nick translation. Metaphase chromosomes prepared from mouse embryo
fibroblast cells were hybridized with the labeled DNA probe and sheared
mouse DNA. The hybridization signals were detected using
anti-digoxigenin antibodies, and mouse chromosomes were counterstained
with 4',6-diamidino-2-phenyl-indole.
Electrophoretic Mobility Shift Assay (EMSA)--
The
BamHI cDNA fragment encoding the C-terminal zinc finger
domain (residues 271-465) of mouse Tzfp (Tzfp-ZF) was inserted in-frame into a pGEX-3X expression vector (Amersham Pharmacia Biotech,
Uppsala, Sweden) to generate glutathione S-transferase (GST)/Tzfp-ZF fusion proteins. Overexpression and affinity purification of GST fusion proteins were performed as previously described (17).
The DNA fragment used for EMSA was PCR amplified from the upstream
flanking sequence (UFS) of the mouse Aie1 gene using a primer set, 103pF4 and 103pR1 (see Fig. 4B). This
PCR-amplified fragment was then subcloned into a PCRII vector. Two DNA
fragments, UFS1 and UFS2 (see Fig. 4B), which were generated
by EcoRI and PstI digestion of the originally
PCR-amplified DNA in the PCRII vector, were treated with calf intestine
alkaline phosphatase and labeled with [
EMSA was carried out by incubating the GST/Tzfp-ZF with
32P-labeled UFS1 or UFS2 (~20,000 cpm) in EMSA buffer
containing 20 mM Tris-HCl, pH 7.5, 50 mM KCl, 3 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 1 µg of poly(dI-dC)·poly(dI-dC), and
10% glycerol. After 20-min incubation at room temperature, the mixture
was loaded on a 5% polyacrylamide nondenaturing gel (see Fig.
5A). For antibody supershift assays (see Fig.
5B), the appropriate antiserum was added to the reaction
mixture 20 min after the addition of the probe. Polyclonal antibodies
against Tzfp were raised in rabbits as previously described (18).
For the EMSA competition assay (see Fig. 7), unlabeled oligo-DNA
competitors (in 0 to 800-fold molar excess) were preincubated with
GST/Tzfp-ZF for 20 min on ice, followed by the addition of labeled UFS1
or UFS2 probes and incubation on ice for 20 min. The resulting
DNA·protein complexes were separated on a polyacrylamide nondenaturing gel as described above. The oligonucleotide competitors used in the EMSA competition were: oligo-N,
5'-GATCCAAAAATATGTACAGTGTTATG-3" and 3'-G TT
TTTATACATGTCACAATACCTAG-5'; oligo-M1, 5'-GATCCAAAAATATGTAAAGTGTTATG-3' and 3'-GTTTTTATACATTTCACAATACCTAG-5'; oligo-M2,
5'-GATCCAAAACGATGTACAGTGTTATG-3' and 3'-GTTTTGCTACATGTCACAATACCTAG-5';
oligo-M3, 5'-GATCCAAAAATATGTACAGTTTTATG-3' and
3'-GTTTTTATACATGTCAAAATACCTAG-5'; oligo-M4,
5'-GATCCAAAAATATGTACATTGTTATG-3' and
3'-GTTTTTATACATGTAACAATACCTAG-5'.
DNase I Footprint Analysis--
The
PstI-EcoRI fragment of UFS2 containing the
tbs sequence (see Fig. 4) was labeled at the
EcoRI site using the Klenow fragment and
[ Plasmid Constructions, Cell Transfection, CAT, and Luciferase
Assay--
To test the DNA binding activity of Tzfp in transfected
cells (see Fig. 8), the FLAG-tagged cDNA spanning the entire
coding region of mouse Tzfp was subcloned into a CAGGS expression
vector, which was driven by a chicken
To examine the transactivation properties of VP/Tzfp-ZF hybrids in
transfected cells (see Fig. 9), the BamHI-XbaI
DNA fragment that covers the entire three zinc fingers (residues
271-465) of mouse Tzfp was fused in-frame into the C terminus of HSV
VP16 activation domain in a pVP16 plasmid
(CLONTECH, Palo Alto, CA); the resulting vector was
designated as pVP-Tzfp-ZF. Oligonucleotides containing one to seven
copies of the Tzfp binding site (tbs) were cloned into a
pG5CAT reporter plasmid (CLONTECH) in which the
five GAL4 binding sites had been replaced with Oligo-N; the resulting
plasmid was designated as pTbsCAT (see Fig. 9). The pTbs7MCAT mutant plasmid was constructed by insertion of
seven copies of oligo-M1 (in which the fifth C had been mutated to A at
the tbs) upstream of the E1b promoter (see Fig.
9A).
To examine the repression activity of Gal4/TZFP-N hybrids in
transfected cells (see Fig. 9B), the cDNA fragment that
spans the N-terminal BTB/POZ domain (residues 1-373) of human TZFP was fused in-frame into the C terminus of the Gal4 DNA-binding domain in a
pM vector (CLONTECH); the resulting vector was
named pM-TZFP-N. The reporter plasmid, pGal4TK-Luc, carries five copies
of the Gal4 binding site before the TK promoter, driving a luciferase reporter gene.
The transient transfection assay was carried out using LipofectAMINE
(Life Technologies, Rockville, MD) as previously described (18).
Briefly, human 293 or mouse testis sertoli TM4 cells (3 × 105 cells/35-mm culture dish) were transfected with
reporter (0.6 µg) and expression (0.6 µg) plasmids as indicated
below in Fig. 9 along with 0.05 µg of a Isolation of Mouse Tzfp cDNA--
We previously isolated a
novel human zinc finger protein, TZFP (testis zinc finger protein),
which is predominantly expressed in testis (15). To further investigate
the molecular features and biological functions of TZFP, the
BamHI cDNA fragment of human TZFP encoding
all three zinc finger domains was used as a probe to screen a mouse
testis cDNA library (see "Experimental Procedures"). Several
positive clones were obtained. Compiling all the sequences from
cDNA as well as from genomic clones (described below), a full-length cDNA was assembled that encodes a 465-amino acid
protein with a calculated molecular mass of ~51 kDa. Comparative
amino acid analysis between mouse Tzfp and human TZFP revealed 73%
identity and 75% similarity (Fig. 1).
Interestingly, the highest conserved regions between mouse and human
TZFP were restricted in the N-terminal BTB/POZ domain (80% identity)
and the C-terminal zinc fingers (97% identity).
Genomic Organization and Chromosomal Assignment of the Mouse Tzfp
Gene--
Using the mouse Tzfp cDNA as a probe, a
positive BAC clone (119/j1; ~150 kb) was isolated from a mouse
129/SvJ genomic library. The 119/j1 DNA was digested with appropriate
restriction enzymes, and the resulting DNA fragments were subcloned and
sequenced. The assembled DNA sequence (9377 bp) that spans the entire
coding region and part of the 5' upstream flanking sequence of mouse Tzfp gene was deposited in GenBankTM under accession number
AY015272. The precise locations of individual exons and the sequences
of each exon-intron junction were determined as described under
"Experimental Procedures". The restriction enzyme map and genomic
organization of the mouse Tzfp gene are shown in Fig.
2. The mouse Tzfp gene spans
~10 kilobases long and contains at least six exons, which are
interrupted by five introns. The donor and acceptor splice site
sequences match the consensus sequences for the exon-intron boundaries
of most eukaryotic genes (Fig. 2B). Interestingly, three
C2H2 zinc fingers located at the C terminus of
Tzfp are encoded by two separated exons. The first zinc finger is
encoded by exon 5, whereas the second and the third zinc fingers are
encoded by exon 6. The BTB/POZ domain of Tzfp is encoded by exon 2.
Assignment of the Tzfp Locus to Mouse Chromosome 7 B2-B3--
The
chromosomal localization of the mouse Tzfp locus was
determined by fluorescence in situ hybridization (FISH) as
described under "Experimental Procedures." A total of 80 metaphase
cells were analyzed with 69 exhibiting specific labeling. On the basis of FISH analysis, Tzfp appears to be located on mouse
chromosome 7.
To more specifically define the chromosome location, a second probe
specific for the telomeric region of chromosome 7 was cohybridized with
the Tzfp clone (119/j1). As shown in Fig.
3, positive signals were detected at the
telomere as well as at the proximal portion of chromosome 7. Measurements of specifically labeled chromosome 7 indicated that
Tzfp is located at a position corresponding to band 7 B2-B3.
The Zinc Finger Domain of Tzfp Binds to the Upstream Flanking
Sequence of the Aie1 Gene in Vitro--
The high degree of sequence
homology between the zinc finger domains of TZFP and PLZF suggests that
TZFP may also bind to the cognate binding site of PLZF with a core
consensus sequence (A(T/G)(G/C)T(A/C)(A/C)AGT) (16). Interestingly,
this core consensus motif (ATGTACAGT) and a putative TATA box were also
found in the upstream flanking sequence (UFS) of the Aie1
gene (Fig. 4B; GenBankTM accession number AF195272 (2)).
We previously reported that Aie1 (aurora-C) is a novel testis-specific
serine/threonine kinase belonging to a growing aurora/Ipl1 kinase
family (1). To investigate whether the C-terminal zinc finger domain of
Tzfp (Tzfp-ZF) binds directly to the UFS of the Aie1 gene,
we performed a gel mobility shift assay using the purified recombinant
GST/Tzfp-ZF proteins and two PCR-amplified fragments (UFS1 and UFS2)
derived from the UFS region of the Aie1 gene (Fig. 4B). Incubation of GST/Tzfp-ZF with the UFS2 region
containing the core motif resulted in the formation of DNA·protein
complexes (C1 and C2) in a dose-dependent manner (Fig.
5A, lanes 2-4). In contrast, GST/Tzfp-ZF did not bind to UFS1, which lacks this core motif
(Fig. 5A, lanes 5-8). Similar results were
obtained when the C-terminal zinc finger domain of human TZFP was
incubated with UFS2 (data not shown). The C1 and C2 complexes were
competed away by adding an excess amount of the unlabeled UFS2 but not by an unrelated UFS1, showing that this binding was specific (data not
shown). The direct interaction between Tzfp-ZF and UFS2 was further
confirmed by an antibody-induced super shift experiment. As shown in
Fig. 5B, we detected a super-shift complex in the experimental reactions containing GST/Tzfp-ZF, UFS2, and anti-Tzfp antibody (5B, lanes 3-5). In contrast, no such
complex was detected in the presence of increasing amounts of control
antibody (5B, lanes 6-8). Taken together, these
results indicate that the C-terminal zinc finger domain of Tzfp binds
to a target sequence located at the UFS2 region of the Aie1
gene.
Defining the Binding Sequence for Tzfp--
To define the target
sequence of Aie1 that interacts with Tzfp, DNase I footprint
analysis was performed. Fig. 6 shows that recombinant GST/Tzfp-ZF bound to a defined sequence (5'-TGTACAGTGT-3'), here termed tbs (Tzfp binding site), located at the UFS2
region of the Aie1 gene (lanes 2 and
3). The same UFS2 region was not protected by control GST
proteins (Fig. 6, lanes 4 and 5). Furthermore, a
ubiquitous footprint protected region that contains an A-rich sequence
located just before the tbs (6A) was present in
all lanes (Fig. 6B) and was possibly generated by a
nonspecific protection due to unknown reasons. This footprint region
was found in all control experiments (6B, lanes 4 and 5), even in the absence of recombinant proteins (Fig.
6B, lane 1).
To more precisely define the essential nucleotides of tbs
for Tzfp binding, we synthesized four mutant oligonucleotides, M1 to M4
(see "Experimental Procedures"; Fig. 6A), and used them as unlabeled competitors in a gel mobility shift assay. Fig.
7 shows that oligo-M1 and oligo-M4, which
contain a C Tzfp Binds to the tbs Motif in Transfected Cells--
To determine
whether the entire Tzfp molecule, including both the BTB/POZ and zinc
finger domains also exhibit sequence-specific binding, we transfected
the FLAG-tagged full-length Tzfp cDNA into 293 cells. Nuclear
extracts prepared from transfected or mock-transfected cells were
incubated with a 32P-labeled oligo-N probe carrying a wild
type tbs motif. As shown in Fig.
8, the Tzfp-DNA binding complexes were
detected in the transfected nuclear extracts (lanes 2 and
7). However, no such complexes were detected in the
reactions with or without the addition of mock-transfected extracts
(lanes 1 and 6; data not shown). Furthermore, the
Tzfp-DNA binding complexes were competed away by adding an excess
amount of unlabeled oligo-N probes (lanes 3-5) but not by
oligo-M1 probes in which the fifth C in the tbs sequence was
changed to A (lanes 8-10). Taken together, these results
further confirmed that Tzfp binds to the tbs site in a sequence-specific manner.
Transactivation Properties of VP/Tzfp Hybrids in Transfected
Cells--
To examine whether Tzfp is able to regulate transcription
via interaction with the tbs site in mammalian cells, we
constructed a reporter plasmid (pTbsCAT) in which the CAT
gene is driven by an E1b promoter with one to seven copies of the
tbs inserted upstream of the promoter (Fig.
9A). The TM4 cells were
cotransfected with the pTbsCAT reporter plasmid and a pVP-Tzfp-ZF
expression plasmid in which the C-terminal zinc finger domain of mouse
Tzfp was fused with the transcription activation domain of HSV VP16
protein. As shown in Fig. 9A, significant transactivation of
the CAT reporter gene was observed in transfected cells in a
tbs copy number-dependent manner. Cotransfection
of the pTbs7MCAT plasmid, which contains seven copies of a
mutated tbs, had no effect on activity, demonstrating that
this activation was specific. In contrast, no CAT activity was detected
in negative control cells, which had previously been cotransfected with
pVP16 and pTbsCAT containing various copies of the tbs.
These results indicate that the Tzfp zinc-finger domain, when fused to
a heterologous transactivation domain, may act as a specific
transcriptional transactivator in mammalian cells.
The N-terminal BTB/POZ Domain of TZFP Has a Repressor
Activity--
To determine the biological function of the BTB/POZ
domain, we fused the N-terminal BTB/POZ domain of human TZFP into the C
terminus of Gal4 DNA-binding domain in a pM vector. The resulting construct (pM-TZFP-N) was cotransfected with a reporter DNA
(pGal4TK-Luc) carrying the luciferase gene linked to a TK promoter
containing five copies of a Gal4 binding site into 293 cells. Fig.
9B shows that TZFP-N significantly repressed expression of
this reporter gene, but not the parental tk-luciferase reporter
(pTK-Luc). The vector (pM) alone did not affect the expression of the
pGal4TK-Luc reporter gene. These results suggest that the N-terminal
BTB/POZ domain of TZFP possesses a repressor activity.
Using human TZFP cDNA as a probe, we isolated the
mouse homologue (Tzfp) of human TZFP from a testis cDNA library.
Both TZFP and Tzfp carry a conserved N-terminal BTB/POZ domain and
three C-terminal PLZF-like C2H2 zinc fingers.
The predicted amino acid sequences of the BTB/POZ domain and the zinc
finger domain of TZFP revealed high homology with those of PLZF (15).
Recently, two novel PLZF-like transcription factors, FAZF (21) and ROG (22), were isolated from a human and a mouse lymphocyte cDNA library. Interestingly, our human TZFP (15) and mouse Tzfp (this report) showed identical amino acid sequences with that of FAZF (Fanconi anemia zinc finger) and ROG (repressor of GATA), respectively. FAZF was reported to be a BTB/POZ transcriptional factor that interacts
with the Fanconi anemia group C protein (21). ROG was demonstrated to
be a GATA-3-interacting protein, which might play a role in regulating
the differentiation and activation of helper T (Th) cells (22). It has
been shown that the TZFP/FAZF/ROG transcript is dominantly expressed in
testis (15, 21, 22) and is rapidly induced in Th cells upon stimulation
with anti-CD3 (22), however, the nature of target genes for
TZFP/FAZF/ROG remain largely unknown. In this report, we define the
binding sequence for Tzfp and describe for the first time a candidate target gene (Aie1) for Tzfp.
The PLZF gene encodes a Kruppel-like
transcription factor containing nine C-terminal
C2H2 zinc fingers and a conserved N-terminal BTB/POZ domain involved in protein·protein interactions (23). Recently, Li et al. (16) proposed a potential binding motif (A(T/G)(G/C)T(A/C)(A/C)AGT) for PLZF on the basis of sequence comparison. A similar motif was also found in the upstream flanking sequence of our Aie1 gene (Fig. 4). Because the zinc finger
domain of Tzfp revealed high sequence homology with that of PLZF and both Tzfp and Aie1 are dominantly expressed in testis, these
observations suggest the possibility that Aie1 may serve as
a candidate target gene for Tzfp.
In this study, we found that the zinc finger domain of Tzfp exhibits
sequence-specific binding to a 10-bp DNA element (tbs) located at the upstream flanking sequence of the Aie1 gene.
DNase I footprinting analysis defined the tbs binding site,
5'-TGTACAGTGT-3' (Fig. 6). Interestingly, the GTACAGT sequence starting
from the nucleotide position 2-8 of the tbs (here termed
tbs core motif) was 100% identical to the sequences located
at the lex A operator, as well as at the IL-3
receptor (IL-3R) promoter (Fig. 4C). Based on sequence identity, it is reasonable to speculate that Tzfp may also
bind to the lex A operator and the IL-3R
promoter. Indeed, our unpublished
data2 showed that Tzfp binds
to the lex A operator, and Hoatlin et al. (21)
reported that FAZF binds to the IL-3R promoter core sequence. Furthermore, our competition analyses demonstrated that the
fifth C and seventh G of tbs sites are important residues for interacting with the C-terminal zinc finger domain of Tzfp (Fig.
7). Taken together, we conclude that this 7-bp DNA element (GTACAGT) is
a tbs core motif, which acts as the target sequence for Tzfp binding.
The PLZF target gene reported so far is cyclin A2. The human
cyclin A2 promoter has two potential binding sites for PLZF, a distal site (5'-AGCTAAAGG-3') and a proximal site (5'-ACGTCAAGG-3'), which are located at nucleotides PLZF not only can bind to the cyclin A2 promoter, but also
to the lex A operator (25). However, no sequence-specific
motifs were found. The lack of a dominant consensus for a zinc finger protein is not a surprise. For example, the WT1 zinc finger protein binds to two DNA sequences that have little in common (26, 27). Similarly, PLZF recognizes many sequences with a less conserved core
motif. This could be partially attributed to the fact that PLZF
recognizes different DNA sequences by utilizing various subsets of its
nine zinc finger motifs. In our current study, we found that Tzfp binds
to the tbs located at the upstream flanking sequence of the
Aie1 gene. This binding is sequence-specific as evidenced by
gel mobility shift (Fig. 5), DNA footprinting (Fig. 6), and competitive
gel mobility shift (Fig. 7) assays. Because the zinc finger domains of
Tzfp and PLZF share high amino acid identities and both bind to the
lex A operator,2 it may be of considerable
interest to examine the binding ability and transcriptional regulation
of Tzfp to the cyclin A2 promoter.
Recent studies showed that the N-terminal POZ domain of PLZF mediates
its binding to several nuclear corepressors (including SMRT, mSin3A,
and HDAC1) and represses gene transcription (28, 29). Like PLZF, human
TZFP is localized to the nucleus2 and contains a BTB/POZ
domain. Transient transfection experiments showed that TZFP may act as
a transcriptional repressor when the N-terminal domain (residues
1-373) of human TZFP is tethered to a heterologous DNA-binding domain
of Gal4 protein (Fig. 9B). The presence of a BTB/POZ domain
in TZFP suggests that TZFP may repress gene transcription via a common
repression pathway similar to that found in PLZF, e.g. by
recruiting nuclear corepressor SMRT and the histone deacetylase complex
(HDAC). Recently, Miaw et al. (22) reported that ROG, which
shows identical sequence to our Tzfp, is a repressor of GATA-3-induced
transactivation. Interestingly, the repressor activity of ROG did not
appear to occur through the recruitment of the SMRT·HDAC complex,
because the addition of Trichostatin A (a potent inhibitor of
HDAC), even at a concentration as high as 1000 nM, did not
inhibit ROG repressor activity. Although both PLZF and ROG/Tzfp
proteins exhibit a repressor activity, the differential ability in the
recruitment of the SMRT·HDAC complex between the BTB/POZ domain of
PLZF and ROG/Tzfp is an interesting topic worthy of further analysis.
The WT1 tumor suppressor transcript is a subject of alternative
splicing. One alternative spliced form of WT1 with a 17-amino acid
insertion N-terminal to the zinc finger domain binds to the EGR1/WT1
site (30), whereas another form, which contains a 3-amino acid
insertion between zinc fingers 3 and 4 of the protein, binds to a
sequence distinct from the EGR1/WT1 site (31). Interestingly, multiple
isoforms of TZFP/Tzfp with diverse structures were also identified in
mammalian cells (15). An alternative spliced form of mouse Tzfp without
the BTB/POZ domain was also identified in mouse testis.2
Because most proteins that carry the BTB/POZ domain possess a transcription repressor activity, this short isoform may play a role
different from that of the large isoform. The differential functions of
these diverse Tzfp isoforms need to be further analyzed.
Finally, the interaction between Tzfp and the target genes carrying the
tbs core sequence may also occur in cell types other than
testis. Although TZFP (15) and Aie1 (1) transcripts are predominantly
expressed in testis, some TZFP transcripts have been detected in
ovarian tumors, gastric cancer cells (15), and activated lymphocytes
(22). The observations that the expression of TZFP is not restricted to
testis and that the BTB/POZ domain of PLZF is involved in
protein·protein interaction and corepressors recruitment raise the
possibility that multiple TZFP isoforms may play different roles in
different cell types.
In summary, we have isolated and characterized the gene structure,
chromosome localization, and the DNA binding property of Tzfp, a novel
PLZF-like transcription factor. Our results show that Tzfp binds
specifically to the tbs site located at the upstream flanking sequence of the Aie1 gene, suggesting that Tzfp may
regulate the expression the gene carrying tbs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP using
T4 polynucleotide kinase. The 32P-end-labeled DNA fragments
with (UFS2) or without (UFS1) the Tzfp binding site (tbs)
were then purified by a push column and used as target probes for
EMSA.
-32P]dATP. The probe was incubated with GST or
GST/Tzfp-ZF in EMSA buffer. After incubation for 15 min at room
temperature, RNase-free DNase I (Promega, Madison, WI) was added for 1 min at room temperature. The reaction was then stopped by addition of a
stop solution containing 200 mM NaCl, 30 mM
EDTA, and 1% sodium dodecyl sulfate. The DNA was extracted with
phenol-chloroform, ethanol-precipitated, and analyzed on a denaturing
8% polyacrylamide gel. Sequencing lanes of the same probe were
generated by the Sequenase (Amersham Pharmacia Biotech. Inc.,
Piscataway, NJ) and Maxam-Gilbert procedures (19).
-actin promoter. The resulting plasmid (FLAG-Tzfp) was transfected into human 293 cells (a human embryonic kidney cell line). Forty-eight hours after transfection, nuclear extracts were prepared from transfected or nontransfected cells
as previously described (20). For EMSA, 2 µg of nuclear extracts and
32P-labeled oligo-N probes were incubated in the binding
buffer (20 mM HEPES-KOH, pH 7.5, 10% glycerol, 50 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol) on ice for 20 min. The Tzfp·DNA
binding complexes were resolved on a 5% nondenaturing polyacrylamide
gel. For competition assays (see Fig. 8), unlabeled oligo-N or oligo-M1
competitor (in 0 to 1000-fold molar excess) were preincubated with the
nuclear extracts and analyzed as described above.
-galactosidase internal
control plasmid. At 48 h post-transfection, cell extracts were
prepared in lysis buffer (CAT ELISA kit), and aliquots were normalized
for transfection efficiency by assay of
-galactosidase activity
(Promega). The CAT and luciferase activity were analyzed by a CAT ELISA
kit (Roche Molecular Biochemicals, Mannheim, Germany) and a luciferase
assay system (Promega), respectively. Transfection was performed in duplicate for each experiment, and the experiments were repeated at
least three times.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (59K):
[in a new window]
Fig. 1.
Alignment of amino acid sequences of human
TZFP (hTZFP) and mouse Tzfp
(mTzfp). The position of three
C2H2 zinc fingers are boxed, and
cysteine and histidine residues are highlighted in gray. A
potential BTB/POZ domain is underlined. Vertical
bars, identical residues; colons, similar
residues.
View larger version (31K):
[in a new window]
Fig. 2.
A, the restriction enzyme map and
genomic organization of the mouse Tzfp gene (derived from
part of BAC clone 119/j1). B, exon-intron arrangement and
boundary sequences of the mouse Tzfp gene (GenBankTM
AY015272).
View larger version (14K):
[in a new window]
Fig. 3.
The localization of the Tzfp
gene on the mouse chromosome by FISH. A,
metaphase chromosome spreads were hybridized with a
digoxigenin-labeled Tzfp DNA probe (arrow) and a
probe (arrowhead) specific to the telomeric region of mouse
chromosome 7. B, idiogram of mouse chromosome 7. The
Tzfp gene is located at mouse chromosome 7 B2-B3.
View larger version (29K):
[in a new window]
Fig. 4.
A, schematic representation of the
genomic organization of the mouse Aie1 gene. Solid
boxes indicate exons of the coding region, and the open
box indicates a Tzfp binding site (tbs). The
translation initiation codon (ATG) and stop codon
(TGA) are located at exons 2 and 7, respectively. The
translation initiation site is denoted by +1. The complete
nucleotide sequence of Aie1 gene (14,045 bp) was deposited
in GenBankTM under accession number AF195272. B, nucleotide
sequence of a portion of the 5' upstream flanking region of the mouse
Aie1 gene. The tbs sequence is boxed,
and the putative TATA is underlined. UFS1 and UFS2 are two
DNA fragments (see "Experimental Procedures") that were used for
DNA·protein binding analyses. C, the Tzfp binding
site (tbs) located at the upstream flanking region of
Aie1 gene was compared with sites found within the lex
A operator and IL-3R promoter.
View larger version (39K):
[in a new window]
Fig. 5.
The zinc finger domain of Tzfp protein binds
to the upstream flanking region containing the tbs of
the Aie1 gene. A, the
32P-labeled UFS2 (lanes 1-4) or UFS1
(lanes 5-8) probe derived from the Aie1 gene was
incubated without (lanes 1 and 5) or with of
increasing amounts of bacterially synthesized GST-Tzfp-ZF (0.2, 0.4, and 0.8 µg). The C1 and C2 DNA·protein complexes and free UFS1 and
UFS2 probes are indicated. B, antibody-induced supershift
experiments. GST-Tzfp-ZF (0.5 µg) was incubated with
32P-labeled UFS2 probe in the absence (lane 2)
or presence of anti-Tzfp (lanes 3-5), or control preimmune
antiserum (lanes 6-8). Each antiserum was added in the
amounts of 3, 6, and 12 µg per reaction. F, free UFS2
probe; *, antibody-induced supershift band; C1,
GST-Tzfp-ZF·UFS2 complex.
View larger version (36K):
[in a new window]
Fig. 6.
A, the Tzfp binding site
(tbs) defined by DNase I footprint analysis. Competition
analysis revealed that the fifth C and the seventh G residues are
important for Tzfp binding. Synthetic normal (N) or mutant
(M1-M4) oligonucleotides (for details, please see
"Experimental Procedures") used for competition assays are
described in Fig. 7. B, in vitro DNase I
footprint analysis. The single-end 32P-labeled UFS2
fragment was incubated without (lane 1) or with increasing
amounts of bacterially expressed GST-Tzfp-ZF (2 µg, lane
2; 3 µg, lane 3) or GST (2 µg, lane 4; 3 µg, lane 5). Binding was assessed as described under
"Experimental Procedures." Sequencing lanes of the same probe were
generated by Sequenase (United States Biochemical) and the
Maxam-Gilbert procedure (data not shown).
A and a G
T substitution, respectively (Fig.
6A), were not able to compete with the UFS2 probe for Tzfp
binding. By contrast, the DNA·protein complexes (C1) were easily
competed away by the addition of excess normal (oligo-N; Fig. 7) or two
other mutant oligonucleotides (oligo-M2 and oligo-M3; Fig. 7). Taken
together, we defined the target sequence (tbs),
TGTACAGTGT, for Tzfp and identified that the
fifth C and seventh G residues within the tbs are important for interacting with the C-terminal zinc finger domain of Tzfp.
View larger version (48K):
[in a new window]
Fig. 7.
Mapping of the essential residues for Tzfp
binding by competition EMSA. EMSAs were performed without
(lanes 1 and 20) or with (other
lanes) the addition of purified GST-Tzfp-ZF and a
32P-labeled UFS2 fragment. The binding reactions were
performed in the absence or presence of increasing amounts of unlabeled
competitors, which included an identical tbs-containing
oligo (oligo-N, lanes 2-7), a mutant oligo-M1
(lanes 8-13), a mutant oligo-M2 (lanes 14-19),
a mutant oligo-M3 (lanes 21-26), or a mutant oligo-M4
(lanes 27-32). Unlabeled competitors were added at 50-, 100-, 200-, 400-, and 800-fold molar excesses.
View larger version (48K):
[in a new window]
Fig. 8.
DNA binding activity of Tzfp in transfected
cells. Nuclear extracts were prepared from human 293 cells
transfected with (+) or without ( ) FLAG-tagged Tzfp
cDNA and analyzed for Tzfp-DNA binding activity by EMSA. The
extracts were incubated with 32P-labeled oligo-N probes in
the presence of 10-, 100-, and 1000-fold excess unlabeled DNA
competitors, oligo-N (carrying a wild type tbs site;
lanes 2-5) or oligo-M1 (carrying a tbs mutant
site; lanes 7-10).
View larger version (21K):
[in a new window]
Fig. 9.
A, transactivation properties of
VP-Tzfp-ZF hybrids in transfected cells. The pTbs-CAT reporter plasmid
contains the CAT gene driven by adenovirus E1b minimum
promoter with one (1×), four (4×), or seven (7×) copies of the Tzfp
binding site (tbs). The pTbsM-CAT mutant plasmid contains a
mutation in which the fifth C was changed to A at the tbs.
The BamHI DNA fragment of Tzfp containing three
zinc fingers was fused to the HSV VP16 activation domain driven by the
SV40 promoter (pVP-Tzfp-ZF). The expression plasmids were transfected
into TM4 cells with the reporter plasmids as indicated. CAT activity
was determined 48 h after transfection. B, the
N-terminal BTB/POZ domain of TZFP possesses a repressor activity. The
N-terminal BTB/POZ domain of TZFP was fused into the C terminus of the
Gal4 DNA-binding domain in a pM vector (pM-TZFP-N). Human 293 cells
were cotransfected with a TK-luciferase reporter gene containing five
copies of the Gal4 DNA-binding site (pGal4TK-Luc) or the parental
TK-Luc along with the indicated expression plasmids. At 48 h after
transfection, the cells were assayed for luciferase activity. All
transfections were normalized by -galactosidase activity as
described under "Experimental Procedures." The error
bars indicate standard derivations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
342 and
75, respectively, 5' to
the major transcription initiation site (24). No such tbs core motif was found in the cyclin A2 promoter. Although the
sequences of these two sites are not identical, PLZF can specifically
bind to two sites within the cyclin A2 promoter and repress
the promoter activity (24).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Jiann-Shiun Lai for providing the expressing vector (pGal4TK-Luc) and Dr. I.-Cheng Ho for providing the ROG sequence information.
![]() |
FOOTNOTES |
---|
* This work was supported by an Institutional grant from Academia Sinica, Taiwan, ROC.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AY015272 and AF195272.
To whom correspondence should be addressed: Institute of
Biomedical Sciences, Academia Sinica, 128 Yen-Chiu-Yuan Rd., Sec. 2, Taipei 115, Taiwan. Tel.: 886-2-2652-3901; Fax: 886-2-2782-5573; E-mail: tktang@ibms.sinica.edu.tw.
Published, JBC Papers in Press, March 12, 2001, DOI 10.1074/jbc.M100170200
2 C.-J. C. Tang, C.-K. Chuang, H.-M. Hu, and T. K. Tang, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: kb, kilobase(s); BTB, bric-a-brac, tramtrack, broad complex domain; POZ, poxvirus, zinc finger domain; PLZF, promyelocytic leukemia zinc finger; TZFP, testis zinc finger protein; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; PCR, polymerase chain reaction; UFS, upstream flanking sequence; HSV, herpes simplex virus; CAT, chloramphenicol acetyltransferase; bp, base pair(s); FAZF, Fanconi anemia zinc finger; ROG, repressor of GATA; Th cells, helper T cells; IL-3R, interleukin-3 receptor; HDAC, histone deacetylase complex; tbs, Tzfp binding site.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Tseng, T. C., Chen, S. H., Hsu, Y. P., and Tang, T. K. (1998) DNA Cell Biol. 17, 823-833[Medline] [Order article via Infotrieve] |
2. | Hu, H.-M., Chuang, C.-K., Lee, M.-J., Tseng, T.-C., and Tang, T. K. (2000) DNA Cell Biol. 19, 679-688[CrossRef][Medline] [Order article via Infotrieve] |
3. | Francisco, L., Wang, W., and Chan, C. S. (1994) Mol. Cell. Biol. 14, 4731-4740[Abstract] |
4. | Glover, D. M., Leibowitz, M. H., McLean, D. A., and Parry, H. (1995) Cell 81, 95-105[Medline] [Order article via Infotrieve] |
5. | Bischoff, J. R., and Plowman, G. D. (1999) Trends Cell Biol. 9, 454-459[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Giet, R.,
and Prigent, C.
(1999)
J. Cell Sci.
112,
3591-3601 |
7. | Kimura, M., Matsuda, Y., Yoshioka, T., Sumi, N., and Okano, Y. (1998) Cytogenet. Cell Genet. 82, 147-152[CrossRef][Medline] [Order article via Infotrieve] |
8. | Shindo, M., Nakano, H., Kuroyanagi, H., Shirasawa, T., Mihara, M., Gilbert, D. J., Jenkins, N. A., Copeland, N. G., Yagita, H., and Okumura, K. (1998) Biochem. Biophys. Res. Commun. 244, 285-292[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Terada, Y.,
Tatsuka, M.,
Suzuki, F.,
Yasuda, Y.,
Fujita, S.,
and Otsu, M.
(1998)
EMBO J.
17,
667-676 |
10. |
Kimura, M.,
Kotani, S.,
Hattori, T.,
Sumi, N.,
Yoshioka, T.,
Todokoro, K.,
and Okano, Y.
(1997)
J. Biol. Chem.
272,
13766-13771 |
11. | Sen, S., Zhou, H., and White, R. A. (1997) Oncogene 14, 2195-2200[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Gopalan, G.,
Chan, C. S.,
and Donovan, P. J.
(1997)
J. Cell Biol.
138,
643-656 |
13. | Bernard, M., Sanseau, P., Henry, C., Couturier, A., and Prigent, C. (1998) Genomics 53, 406-409[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Kimura, M.,
Matsuda, Y.,
Yoshioka, T.,
and Okano, Y.
(1999)
J. Biol. Chem.
274,
7334-7340 |
15. | Lin, W., Lai, C. H., Tang, C. J., Huang, C. J., and Tang, T. K. (1999) Biochem. Biophys. Res. Commun. 264, 789-795[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Li, J. Y.,
English, M. A.,
Ball, H. J.,
Yeyati, P. L.,
Waxman, S.,
and Licht, J. D.
(1997)
J. Biol. Chem.
272,
22447-22455 |
17. |
Tang, C. J.,
and Tang, T. K.
(1998)
Blood
92,
1442-1447 |
18. |
Hung, L. Y.,
Tang, C. J.,
and Tang, T. K.
(2000)
Mol. Cell. Biol.
20,
7813-7825 |
19. | Maxam, A. M., and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 560-564[Abstract] |
20. | Andrews, N. C., and Faller, D. V. (1991) Nucleic Acids Res. 19, 2499[Medline] [Order article via Infotrieve] |
21. |
Hoatlin, M. E.,
Zhi, Y.,
Ball, H.,
Silvey, K.,
Melnick, A.,
Stone, S.,
Arai, S.,
Hawe, N.,
Owen, G.,
Zelent, A.,
and Licht, J. D.
(1999)
Blood
94,
3737-3747 |
22. | Miaw, S. C., Choi, A., Yu, E., Kishikawa, H., and Ho, I. C. (2000) Immunity 12, 323-333[Medline] [Order article via Infotrieve] |
23. | Bardwell, V. J., and Treisman, R. (1994) Genes Dev. 8, 1664-1677[Abstract] |
24. | Yeyati, P. L., Shaknovich, R., Boterashvili, S., Li, J., Ball, H. J., Waxman, S., Nason-Burchenal, K., Dmitrovsky, E., Zelent, A., and Licht, J. D. (1999) Oncogene 18, 925-934[CrossRef][Medline] [Order article via Infotrieve] |
25. | Sitterlin, D., Tiollais, P., and Transy, C. (1997) Oncogene 14, 1067-1074[CrossRef][Medline] [Order article via Infotrieve] |
26. | Rauscher, F. J. d., Morris, J. F., Tournay, O. E., Cook, D. M., and Curran, T. (1990) Science 250, 1259-1262[Medline] [Order article via Infotrieve] |
27. | Wang, Z. Y., Qiu, Q. Q., Enger, K. T., and Deuel, T. F. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8896-8900[Abstract] |
28. | David, G., Alland, L., Hong, S. H., Wong, C. W., DePinho, R. A., and Dejean, A. (1998) Oncogene 16, 2549-2556[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Hong, S. H.,
David, G.,
Wong, C. W.,
Dejean, A.,
and Privalsky, M. L.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
9028-9033 |
30. | Madden, S. L., Cook, D. M., Morris, J. F., Gashler, A., Sukhatme, V. P., and Rauscher, F. J., III (1991) Science 253, 1550-1553[Medline] [Order article via Infotrieve] |
31. | Bickmore, W. A., Oghene, K., Little, M. H., Seawright, A., van Heyningen, V., and Hastie, N. D. (1992) Science 257, 235-237[Medline] [Order article via Infotrieve] |