(Received for publication, June 13, 1995; and in revised form, July 14, 1995)
From the
To identify genes that can repress the expression of growth
regulatory molecules, a human fetal cDNA library was screened with a
degenerate oligonucleotide that corresponds to the conserved stretch of
6 amino acids connecting successive zinc-finger regions in the
Wilms' tumor suppressor/Egr-1 family of DNA-binding proteins. One
clone, designated zinc-finger protein 174 (ZNF174), corresponds to a
putative transcription factor with three zinc fingers and a novel
finger-associated domain, designated the SCAN box. The three
Cys-His
-type zinc fingers are positioned at the
carboxyl terminus, while the 65-amino acid finger-associated SCAN box
is located near the amino terminus. Chromosomal localization using
somatic cell hybrid analysis and fluorescent in situ hybridization mapped the gene for ZNF174 to human chromosome
16p13.3. The 2.5-kilobase transcript from this gene is expressed in a
variety of human organs, but most strongly in adult testis and ovary.
Fusion of the upstream regulatory region of ZNF174 to the DNA-binding
domain of GAL4 revealed that the gene could confer a repression
function on the heterologous DNA-binding domain. ZNF174 selectively
repressed reporter activity driven by the platelet-derived growth
factor-B chain and transforming growth factor-
promoters and bound to DNA in a specific manner. This member of
the C
H
-type zinc-finger family is a novel
transcriptional repressor.
Cellular responsiveness to environmental signals is mediated, at
least in part, by the induction or modulation of gene transcription.
Relatively detailed models of basal and activated transcription have
been generated in eukaryotes (reviewed by Tjian and Maniatis(1994)).
Repression, an equally important aspect of transcriptional control, is
not nearly as well characterized. Transcriptional repressors can
inhibit gene expression by at least two general mechanisms. Passive
repressors down-regulate the activity of transcription factors by
competing for their DNA binding sites; alternatively, active repressors
have an intrinsic repressing activity and directly inhibit
transcription (reviewed by Cowell(1994) and Johnson(1995)). The
regulation of growth factor gene expression can involve both types of
repressive mechanisms. There is increasing evidence that members of the
Cys-His
zinc-finger class of transcription
factors are involved in the transcriptional repression of growth factor
gene expression. The Wilms' tumor suppressor gene (wt-1)
encodes a zinc-finger DNA-binding protein, which can interact with the
insulin-like growth factor type II promoter to repress expression of
the gene during nephrogenesis (Drummond et al., 1992;
Pritchard-Jones et al., 1990). Similarly, WT-1 can also
repress expression driven by the transforming growth factor
TGF-
(Dey et al., 1994), PDGF-A
chain (Gashler et al., 1992), colony-stimulating factor-1
(Harrington et al., 1993) and the insulin-like growth factor-I
(IGF-I) receptor (Werner et al., 1994) promoters. Recent
studies have revealed that WT-1 expression can be autoregulated through
multiple potential WT-1 binding sites located in its promoter (Fraizer et al., 1994; Malik et al., 1994). The loss of
function of the wt-1 gene product is thought to contribute to
neoplastic transformation. Structural alteration of the wt-1 gene or its abnormal expression have been implicated in the
continued proliferation of embryonic kidney blastemal cells seen in
Wilms' tumors. Mutations in the wt-1 gene have been
detected in 5-10% of Wilms' tumors (Haber et al.,
1990; Gessler et al., 1990; Call et al., 1990; Huff et al., 1991).
Since the expression pattern of WT-1 is
restricted to specific cell types (reviewed by Rauscher(1993)), we
reasoned that there may be additional Cys-His
factors, which could play an important role in negative growth
factor gene regulation. In this report, we have cloned and
characterized a novel zinc-finger-containing gene, ZNF174, from a human
fetal library. ZNF174 corresponds to a putative transcription factor
with three zinc fingers and a novel finger-associated structural
element. When the amino-terminal region of ZNF174 was fused to the
DNA-binding domain of GAL4, the chimera repressed transcription of a
reporter construct containing multiple GAL4 binding sites. Moreover,
overexpression of full-length ZNF174 cDNA with a series of
promoter-reporter constructs revealed that ZNF174 could repress
expression driven by the human PDGF-B chain and TGF-
promoters, but not the PDGF-A chain, Egr-1, c-Fos, or c-Jun
promoters. ZNF174 repression of PDGF-B promoter activity was
dose-dependent, and 5`-deletion analysis mapped the putative negative
element to the proximal promoter. DNase I footprint and gel shift
analysis revealed that recombinant ZNF174 protein binds to an element
near the transcriptional start site, indicating that ZNF174 repression
may be mediated by direct interaction with DNA. These results suggest
that ZNF174 may contribute to the negative regulation of certain growth
factor genes.
Fluorescent in situ hybridization to metaphase chromosomes was used to localize an ZNF174 genomic clone to the p13.3 region of chromosome 16 (Cherif et al., 1989; Fan et al., 1990). Metaphase chromosomes were prepared from 5-bromodeoxyuridine-synchronized lymphocyte cultures. The probe was biotinylated, hybridized to the chromosome spreads, and detected by fluorescein-conjugated avidin (Vector Laboratories). Slides were evaluated with a Nikon florescence microscope. Forty-one metaphases were examined. Q (DAPI counterstaining) and R banding (propidium iodide counterstaining) were used to confirm the identity of the chromosome. The idiogram demonstrates the localization of an ZNF174 genomic clone to 16p13.3 by double fluorescent signals, one on each chromatid.
Conversion of radiolabeled acetyl-coenzyme A to acetylated
chloramphenicol was assayed by the two-phase fluor diffusion technique
(Sambrook et al., 1989). Assays for -galactosidase
activity were performed in a 100-µl volume containing 10 µl of
extract, 0.1 M sodium phosphate, 0.01 MgCl
, 0.1 M 2-
-mercaptoethanol, and 3 mMO-nitrophenyl-
-D-galactopyranoside (Sigma).
Reactions were incubated at 37 °C for 1 h and stopped by the
addition of sodium bicarbonate to 0.6 M. Assays were compared
to a standard curve on a Dynatech Lab MR700 microtiter plate reader at
410 nm.
Sequence analysis of the cDNA clones revealed a single open reading frame of 1221 nucleotides (Fig. 1A). The open reading frame is closed by a termination codon located 93 bp upstream of the translational start site. The polypeptide predicted from the open reading frame has a calculated relative molecular mass of 46,414 Da and an estimated isoelectric point of 10.1. A long 5`-untranslated region containing multiple translational start and stop codons flanks the open reading frame. The cDNA has a relatively short 3`-untranslated region with a consensus polyadenylation signal (AAUAAA) and poly(A) tail. A schematic representation of ZNF174 cDNA appears in Fig. 1B.
Figure 1: Structure of ZNF174 cDNA. A, nucleotide and predicted amino acid sequence of the corresponding polypeptide. The complete nucleotide sequence of the 2265-bp ZNF174 cDNA is shown above with the encoded protein sequence below in single-letter code. The putative SPLK phosphorylation site and nuclear localization signals have been underlined. Conserved amino acids in the zinc-finger domain are in boldprint and underlined. The polyadenylation signal has been underlined. B, schematic representation of ZNF174 open reading frame and flank. The spotted region represents the SCAN box. The location of the zinc fingers is indicated by a diagonallystripedbox. The singlestraightlines depict untranslated regions of the cDNA. UT denotes untranslated regions.
Figure 2:
Amino acid alignment of zinc fingers and
finger-associated domains of ZNF174 with those of other zinc-finger
proteins. A, zinc-finger sequence alignment. B,
alignment of zinc-finger-associated domains. Regions of identity with
ZNF174 are highlighted. The putative -helical region is indicated (H) below the sequence. C, helical wheel
representation of the SCAN box amino acid sequence from ZNF174. Each
residue is offset from the preceding one by 100°, the typical angle
of rotation for an
-helix. Analysis was done with the assistance
of the GCG Helical Wheel graphics program. Boxed residues indicate hydrophobic amino acids.
Outside the putative zinc-finger DNA-binding domain, the predicted protein contains proportionately high numbers of serine (9.3%), glycine (8.9%), proline (8.6%), and glutamine (8.2%) residues (Fig. 1A). Outside the strikingly high content and stretches of these residues, characteristics of the other transcription factors in the WT-1/Egr/Sp1 family were not seen. Although amino acid sequence homology within these enriched regions is not generally observed among transcription factors, the high content of these amino acids in the non-zinc-finger segment of the predicted protein fits the motif of a number of known and putative transcription factors (Mitchell and Tjian, 1989). A single consensus phosphorylation site (Ser*/Thr*-Pro-X-Lys/Arg) for Cdc2 kinase was identified in the open reading frame (SPLK, Fig. 1A). An adjacent region rich in basic amino acid residues (LKKSKGGKR) (Fig. 1A) suggests the existence of a nuclear localization signal within the protein.
Figure 3:
Expression pattern of the ZNF174 gene.
Northern blots containing 2 µg of poly(A) RNA/lane
from 16 different human adult tissues (Clontech) were hybridized with
an EcoRI fragment of ZNF174 that did not contain the
zinc-finger region. Lane1 contains RNA size markers
(not shown). Lanes 2-17 contain mRNA from human heart,
brain, placenta, lung, liver, skeletal muscle, kidney, pancreas,
spleen, thymus, prostate, testis, ovary, small intestine, colon, and
peripheral blood leukocytes, respectively. The Northern blots were also
hybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to normalize for loading
differences.
To map the ZNF174 gene more precisely, we performed fluorescent in situ hybridization to metaphase chromosomes. An idiogram
indicates the distribution of fluorescent signal at 16p13.3 on both
chromatids of a single chromosome 16 in 21 out of the 41 cells (51%) (Fig. 4A). Double signals also appeared once at 7q22
and twice at 16q12.1; however, the frequency of the
appearance of these signals suggests false hybridization sites.
Figure 4:
Chromosomal location of the ZNF174 gene. A, idiogram of the distribution of ZNF174 signals on both
chromatids of chromsome 16. Each dot represents two fluorescent
signals, one on each chromatid of a chromosome 16. 51% of the cells
examined (21/41) had signals on both chromatids of a single chromosome
16 at p13.3. B, mapping of the ZNF174 gene within the PKD1 region of chromosome 16. ZNF174 does not hybridize to a somatic
cell hybrid with a breakpoint at 23HA, but is able to hybridize to a
different somatic cell hybrid whose breakpoint extends to N-OH1. C, schematic representation of chromosome 16p13.3 indicating
the location of the somatic cell hybrid breakpoints. Also shown are the
approximate locations of the globin gene, MS205.2 (a
microsatellite marker), TSC-2 (tuberous sclerosis gene), and PKD-1
(polycystic kidney disease gene). ZNF174 maps to the region between the
N-OH1 and 23HA breakpoints.
The ZNF174 gene was further localized to the polycystic kidney disease (PKD1) region of chromosome 16 by analysis of selected somatic cell hybrids. The breakpoints of two der(16) chromosomes isolated in rodent-human somatic cell hybrids (N-OH1, 23HA) approximate the telomeric and centromeric boundaries of the PKD1 region (Fig. 4C) (Germino et al., 1992). Southern blot analysis revealed that the gene for ZNF174 was contained within the breakpoint interval defined by these hybrids (Fig. 4B).
Figure 5: Repression by GAL4-ZNF174 chimeras. A, schematic representation of various plasmid constructs used to assess the function of upstream domains of ZNF174. B, CAT activities from cotransfections with GAL4-ZNF174 fusion constructs and promoter-CAT reporter plasmids. The left portion of the figure shows transfections with the GAL4x5 E1B CAT reporter, which contains five copies of the GAL4 binding site placed in front of the minimally active E1B promoter and CAT. The plasmid pE1B CAT does not contain GAL4 binding sites and is used as a control. As a positive control, a cotransfection was done with a known activator GAL4-Egr-1 (amino acids 3-281) (Gashler et al., 1993) The right portion of the figure depicts transfections with the reporter GAL4x5 TK-CAT, which is a pBL-CAT2-based plasmid with 5 copies of the GAL4 binding site placed in front of the basally active TK promoter. The histograms represent the mean of four replicative assays. The error bars indicate standard deviation from the mean. Where the error bars are not visible, this indicates that the standard deviation was smaller than the bar data point.
Certain zinc-finger
transcription factors, such as Egr-1, have the capacity to regulate
transcription in a bimodal manner (Gashler et al., 1993). To
determine whether ZNF174 also encodes an activator function, the
GAL4-ZNF174 chimera was cotransfected with a reporter containing five
GAL4 binding sites in front of the E1b minimal promoter
(pGAL4E1bCAT) (Fig. 5A). This construct,
however, failed to stimulate the expression of the reporter gene (Fig. 5B). In contrast, a similarly constructed Egr-1
(amino acids 3-281) chimera (Gashler et al., 1993)
stimulated transcription by approximately 100-fold. Thus, ZNF174 (amino
acids 3-320) does not confer positive transcriptional activity on
a heterologous DNA-binding domain.
Figure 6: Effect of ZNF174 on the expression of various promoter-CAT constructs in COS cells. Fifteen µg of the various promoter-CAT constructs were transiently cotransfected into COS cells with 2 µg of ZNF174 expression construct (pcZNF174) or 2 µg of pcDNA using calcium phosphate technique. CAT activity was assessed as described under ``Experimental Procedures.'' Transcriptional repression is expressed as a percentage of the reporter activity obtained using the empty expression vector (pcDNA) alone. The values (% repression) represent the mean of between two and five separate experiments.
Figure 7: Effect of ZNF174 on expression driven by PDGF-B chain promoter fragments in BAEC. Fifteen µg of PDGF-B 6a-CAT or d77-CAT constructs were transiently cotransfected into COS cells with 0, 0.5, 2, and 8 µg of ZNF174 expression construct (pcZNF174), the backbone alone (pcDNA), or a construct containing the amino-terminal region of ZNF174 without the zinc fingers fused to an irrelevant DNA binding site (GAL4), using a calcium phosphate technique. CAT activity was assessed as described under ``Experimental Procedures.'' The data are representative of two independent experiments.
To examine whether ZNF174 protein interacts directly with the PDGF-B promoter, DNase I footprint and gel shift assays were performed with a recombinant form of ZNF174. Since ZNF174 could suppress expression of d77-CAT as effectively as 6a-CAT (Fig. 7), a footprint probe encompassing the minimal promoter was used to allow visualization of bases protected from DNase I digestion. Bacterially expressed ZNF174 bound to a defined region in the PDGF-B promoter in a dose-dependent manner (Fig. 8A). To demonstrate the specificity of the interaction, another zinc-finger protein, Egr-2, failed to interact with this site (Fig. 8A).
Figure 8:
Interaction of recombinant ZNF174 with the
PDGF-B chain promoter. A, in vitro DNase I footprint
analysis of PDGF-B core promoter. The single-end P-labeled
PDGF-B core promoter fragment was incubated with increasing amounts of
bacterially expressed ZNF174 or Egr-2 for 60 min at 4 °C and
digested with 0.02 units of DNase I for 5 min at this temperature.
Binding was assessed as described under ``Experimental
Procedures.'' B, electrophoretic mobility shift assay
using an oligonucleotide spanning the PDGF-B transcriptional start
site. [
P]B-prom was incubated with approximately
1 µg of recombinant ZNF174 for 30 min at 4 °C in the absence or
presence of unlabeled oligonucleotide. Oligonucleotide sequences are:
B-prom, 5`-GAAAGGGTGGCAACTTCTCCTCC-3`; PEA-3,
5`-GATCTCGAGCAGGAAGTTCGACTAG-3`. Binding was assessed as described
under ``Experimental
Procedures.''
In support of these
observations, a single nucleoprotein complex was observed when a P-labeled oligonucleotide (B-prom) spanning the region
bound by ZNF174 was used in electrophoretic mobility shift assay with
the recombinant protein (Fig. 8B). The shift was
abolished by the presence of a 50-fold molar excess of the unlabeled
cognate (Fig. 8B), whereas 100-fold excess of an
unrelated oligonucleotide, PEA-3, failed to compete (Fig. 8B). The region bound by ZNF174 spans the major
transcriptional start site in the human PDGF-B gene (Rao et
al., 1986). These findings suggest that ZNF174 may serve as a
competition-type transcriptional repressor (reviewed by Levine and
Manley(1989)). Thus, ZNF174 can interact with the PDGF-B chain promoter
in a specific manner and suppress the activity of B chain reporter
constructs.
This paper describes the isolation and characterization of a novel zinc-finger protein, ZNF174, located on human chromosome 16p13.3 with the apparent ability to repress expression driven by the promoters of a number of pathophysiologically relevant genes. ZNF174 was also found to have a number of interesting structural features. The primary sequence of the putative ZNF174 polypeptide revealed a potential single phosphorylation site and nuclear translocation sequence. The SPLK sequence (Fig. 1A) fits the consensus phosphorylation sequence recognized by Cdc2 kinase (Ser*/Thr*-Pro-X-Lys/Arg). Cdc2 is a highly conserved cell cycle-regulatory protein serine kinase that has been reported to phosphorylate nuclear transcription factors (reviewed by Hunter and Karin(1992)). Located near the phosphorylation site, and just upstream of the zinc-finger domain, is a region that contains a high proportion of basic amino acid residues (LKKSKGGKR) (Fig. 1A). Nuclear localization sequences often share a sequence of four predominantly basic amino acids (Lys-Arg/Lys-X-Arg/Lys) flanked by acidic and proline residues (reviewed by Powers and Forbes(1994)). The core of this short stretch of basic residues in ZNF174 could constitute a nuclear localization signal or may be part of more complex bipartite signal involving another region of the protein, as observed with Egr-1 (Gashler et al., 1993). Phosphorylation at the SPLK site may affect the function of the adjacent nuclear localization signal and regulate import of the protein into the nucleus (reviewed by Silver (1991)).
The ZNF174 gene contains a novel finger-associated element located
upstream of the zinc-finger domain. This domain, or SCAN box, is also
found in a number of other zinc-finger proteins including SRE-ZBP,
CTfin-51, #18 cDNA, ZNF165, and 3c3 (Fig. 2B). This
domain may contain amphipathic -helices (Fig. 2C),
which would permit interactions with other proteins containing similar
domains. CTfin-51 is a mouse protein with seven tandemly repeated
carboxyl-terminal zinc fingers, which has been functionally associated
with meiosis in both male and female gametogenesis (Noce et
al., 1992) and has also been shown to be a strong transcriptional
activator (Chowdhury et al., 1992). SRE-ZBP is a human
transcription factor that binds to the c-fos serum response
element. This protein also contains seven tandemly repeated
carboxyl-terminal zinc fingers and is thought to function as a
repressor of c-fos transcription (Attar and Gilman, 1992).
ZNF165 (
)is a zinc-finger gene located on chromosome 6p21,
which is expressed specifically in the testis. The element was also
identified in two novel partial cDNAs termed #18 cDNA (Pengue et
al., 1993) and 3c3 (Calabro et al., 1995), both of which
correspond to transcribed sequences from chromosome 3p21. The 3p region
is of particular interest because of its frequent involvement in
rearrangements and/or deletions associated with various human tumors,
including lung and renal carcinomas (Naylor et al., 1987;
Bergenhein et al., 1989). The conserved SCAN box does not
correspond with previously reported modules linked to zinc-finger
domains such as the 75-amino acid Kruppel-associated box
(KRAB), which occurs in one-third of the Kruppel-type finger
genes (Bellefroid et al., 1991; Rosati et al., 1991)
and mediates transcriptional repression (Witzgall et al.,
1994; Margolin et al., 1994), or the finger-associated box
(FAX), which has been found in Xenopus finger proteins
(Knochel et al., 1989). Although the function of the SCAN box
has not yet been elucidated, the conservation of this module and its
-helical structure suggests that it may serve ZNF174 as a
dimerization domain or a site that interacts with components of the
transcriptional machinery resulting in the repression of gene
expression.
The ability of ZNF174 to function as a transcriptional repressor is similar to the activities of other zinc-finger transcription factors such as Egr-1 and WT-1 (Madden et al., 1991; Gashler et al., 1993). Repression domains, like activation domains, have been demonstrated to function as independent modular elements in transcription factors (reviewed by Levine and Manley(1989) and Johnson (1995)). Only a small number of repression domains have been well characterized. These include the Drosophila proteins Kruppel (Licht et al., 1990; Zuo et al., 1991), Engrailed (Han et al., 1989; Jaynes and O'Farrell, 1991), and Even-Skipped (Han and Manley, 1993) and the mammalian DNA-binding proteins WT-1 (Madden et al., 1991), YY1/NF-E1 (Shi et al., 1991), and Kid-1 (Witzgall et al., 1994). Like ZNF174, these proteins can confer their repression function onto heterologous DNA-binding domains. The specific region(s) within ZNF174 that repress transcription of other genes is not yet clear, although the GAL4 chimera studies were able to show that the SCAN box alone can not confer repression. This implies that either the repression domain of ZNF174 is contained within another portion of the gene, or that for conformational reasons a more extensive region of the gene is required in order to confer repression. The suppression domain in Kruppel was mapped to an alanine-rich domain (Zuo et al., 1991), and that of Egr-1 was mapped to a 34-amino acid element (Gashler et al., 1993). ZNF174, however, does not contain an alanine-enriched element or a region that resembles the repression element of Egr-1 (Fig. 1A). A small number of factors contain modular domains capable of regulating transcription both positively and negatively. These include Egr-1 (Gashler et al., 1993), WT-1 (Wang et al., 1993), Kruppel (Zuo et al., 1991), YY1/NF-E1 (Park et al., 1991), and the immediate-early proteins c-Fos and c-Jun (Abate et al., 1991). The inability of ZNF174 to activate transcription in the context of promoter-reporter constructs examined (Fig. 5B, 6, and 7) does not exclude the possibility that the zinc-finger protein has a positive regulatory function with other genes.
Gel retardation and DNase I footprint studies established the ability of the purified zinc-finger domain of ZNF174 to interact with promoter elements that appear in certain reporter constructs it could repress. These assays shed light on a possible mechanism with which ZNF174 suppressed expression driven by the PDGF-B promoter. That ZNF174 bound at the transcriptional start site of the PDGF-B gene (Rao et al., 1986) suggests that it may impair the assembly of various components of the general transcriptional machinery into a functional complex at the initiation site. Thus, ZNF174 could function as a competition-type transcriptional repressor (Levine and Manley, 1989) as recently observed with another zinc-finger protein (Werner et al., 1994). Transcriptional repression of the IGF-I receptor gene by WT-1 is thought to be mediated, at least in part, by direct interaction with the initiator site (Werner et al., 1994).
The gene encoding ZNF174 was mapped to human chromosome 16p13.3. Because two disease loci have been identified in this region near the distal end of the short arm of chromosome 16, tuberous sclerosis and polycystic kidney disease, it has been intensely studied and is known to be rich in transcribed sequences. Autosomal dominant polycystic kidney disease is a common genetic defect in humans that frequently results in renal failure (Gabow, 1993). The candidate gene for this disease occurs within a duplicated region on the chromosome and it encodes a large novel protein of unknown function (European Polycystic Kidney Disease Consortium, 1994). Tuberous sclerosis complex (TSC) is a dominantly inherited disorder that causes mental retardation, seizures and tumors in multiple organs (Gomez, 1988). Linkage studies have demonstrated that TSC has at least two possible genetic loci within the human genome with disease loci on chromosomes 9 (Fryer et al., 1987) and 16 (Kandt et al., 1992). A recently described locus, designated TSC-2, generates a transcript that is widely expressed, and its protein product, tuberin, has a region of homology to the GTPase-activating protein GAP3. Loss of heterozygosity for alleles at 16p has been observed in hamartomatous lesions seen in TSC patients (Green et al., 1994). Often, an alteration in one allele, such as a point mutation or small deletion, is followed by a more extensive loss of sequence from the homologous chromosome. If ZNF174 lies in close proximity to the TSC-2 gene, it may be lost in the second mutational event. While not the causative gene for TSC, the inclusion or exclusion of the ZNF174 gene in subsequent chromosomal deletions may account for some of the variation of symptoms and complications seen in the disorder.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U31248[GenBank].