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ZNF232: structure and expression analysis of a novel human C2 H2
zinc ¢nger gene1, member of the SCAN/LeR domain subfamily
Lampros A. Mavrogiannis a;2 , Alexandros Argyrokastritis a , Nicholas Tzitzikas a ,
Emmanuel Dermitzakis a;3 , Theologia Sara¢dou a , Philippos C. Patsalis b ,
Nicholas K. Moschonas a;c; *
a
Department of Biology, University of Crete, P.O. Box 2208, 71409 Heraklion, Greece
Cyprus Institute of Neurology and Genetics, P.O. Box 3462, 1683 Nicosia, Cyprus
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, P.O. Box 1527, 71110 Heraklion, Greece
b
c
Received 26 October 2000; received in revised form 18 January 2001; accepted 18 January 2001
Abstract
We have identified a novel zinc finger gene, ZNF232, mapped to human chromosome 17p12. The coding region of the gene is organized in
three exons corresponding to a 417 amino acid long polypeptide containing a SCAN/LeR domain and five C2 H2 -type zinc fingers. ZNF232
is possibly a nuclear protein, as suggested by expression analysis of GFP/ZNF232 chimeric constructs. ZNF232 transcripts were detected in
a wide collection of adult human tissues. The gene is possibly subjected to tissue-specific post-transcriptional regulation by means of
alternative splicing. ß 2001 Elsevier Science B.V. All rights reserved.
Keywords : Zinc ¢nger protein ; SCAN/LeR domain; Human erythroleukemia K562 cell; Normalized cDNA library ; Chromosome 17; Nucleus
During the past decade, partial cDNA sequences (expressed sequence tags, ESTs) corresponding to novel human genes, obtained through systematic library screening,
have been rapidly accumulated and exploited for the construction of an integrated framework genome map [1,2]. In
addition to ordinary cDNA libraries, screening of normalized libraries greatly facilitated the identi¢cation of relatively rare transcripts and signi¢cantly contributed to the
enrichment of the EST database [3]. Currently, determination of novel full-length cDNAs and sequence comparisons with already characterized genes frequently reveal
well-known protein motifs, thus enabling essential gene
annotation, i.e. classi¢cation of novel genes to various
* Corresponding author, at address c. Fax: +30-81-394-408;
E-mail : [email protected]
1
The cDNA sequence of ZNF232 (name approved by the HUGO/
GDB Nomenclature Committee) has been submitted to GenBank and
assigned accession No. Y15067; the corresponding genomic DNA sequences are available under accession Nos. AF080169^171.
2
Present address: Clinical Genetics Group, Institute of Molecular
Medicine, John Radcli¡e Hospital, Oxford OX3 9DS, UK.
3
Present address: Institute of Molecular Evolutionary Genetics, Department of Biology, Pennsylvania State University, University Park, PA
16802, USA.
gene/protein families [4,5] and incorporation of analyzed
genes to the emerging high resolution human genic map.
Human erythroleukemia K562 cells, considered as pluripotent hematopoietic progenitors, have been proven to
be useful in studying regulated gene expression. Numerous
studies on important biological functions such as developmental stage-speci¢c gene switching, signal transduction
and transcriptional regulation have been based on the
analysis of genes active in K562 cells [6^9]. In order to
systematically search for novel genes expressed in these
cells, we generated a normalized cDNA library [10] and
isolated a number of partial cDNA clones. Among several
ESTs of unknown nature, EST HSIMBB246 (GenBank
accession No. X93860), displaying similarities to genes
coding for C2 H2 -type zinc ¢nger proteins, was considered
for further analysis. In order to obtain a full-length cDNA
and avoid possible sequence ambiguities due to normalization, we screened the corresponding native K562 library
[10]. A 114 bp 32 P-labeled PCR product, synthesized by
using a pair of EST-deduced primers (5P-AGATGGGGTGCTCATCTTG-3P and 5P-CCGATGCTGACTTAGATATG-3P, nucleotide (nt) positions 1149^1167 and 1243^
1262, respectively), was applied as a probe. PCR conditions included 35 cycles with denaturation at 94³C (1 min),
annealing at 57³C (1 min) and extension at 72³C (2 min).
0167-4781 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 7 - 4 7 8 1 ( 0 1 ) 0 0 1 7 7 - 4
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Fig. 1. Nt and aa sequences derived from the analysis of three largely overlapping ZNF232 cDNA clones, i.e. No. 3a (nt 1^1473), No. 6a (nt 131^1445)
and No. 7b (nt 370^1473). The proposed translation start codon (nt 126), the corresponding stop codon (nt 1376) and the SCAN/LeR domain (aa 49^
130) are indicated in bold. The ¢ve consecutive Cys2 His2 zinc ¢nger repeats (numbered 1 to 5) and the unique polyadenylation signal are underlined.
An internal nine amino acid long peptide (aa 182^190) corresponding to a region of ZNF232 transcript that is subjected to alternative splicing and the
putative nuclear localization signal (RHRR) are framed. Vertical arrows on the cDNA sequence denote the exon/intron boundaries.
Five positive clones with insert sizes ranging from 1.1 to
1.35 kb were isolated, subcloned into pBluescript II KS+
(Stratagene), sequenced (Sequenase V.2 kit, Amersham)
and evaluated in silico. A 1473 bp long cDNA corresponding to a novel human zinc ¢nger gene, designated
ZNF232, was assembled (Fig. 1). An internal 27 bp long
coding sequence, nt positions 670^696, corresponding to
nine amino acid (aa) residues, was absent in two of the
analyzed clones, possibly re£ecting an alternative splicing
event (Figs. 1 and 2). ZNF232 cDNA and genomic DNA
(a ZNF232-speci¢c PAC No. 202L17, see below) sequencing revealed the presence of three potential translation
start codons at the 5P end of the gene. AUG at nt position
126 (Fig. 1) was considered the most suitable, as being in
optimum accordance to the Kozak rule [11], whereas the
others, located 81 (Fig. 1) and 390 bp (not shown) upstream from it, were considered less possible. In addition,
an in frame translation stop codon was determined 504 bp
upstream from the putative translation start codon (not
shown). Moreover, our sequence data combined with recent data from GenBank (No. AC074339) and EST database (EST No. AI344299; EST No. AW952625), suggested a ZNF232 transcription unit of at least 2.0 kb
with a proposed 5P untranslated region of about 0.5 kb.
The actual size of the transcript was con¢rmed by Northern analysis (see below). The putative 417 aa long polypeptide has a predicted molecular mass of 47.6 kDa and
may be divided into two distinct regions (Figs. 1 and 2A):
the amino terminal, where a ¢nger associated motif, the Khelical leucine-rich SCAN/LeR domain [12,13] is recognized (aa 49^130), and the carboxy terminal zinc ¢nger
region, characterized by ¢ve consecutive repeats of the
C2 H2 type (aa 278^410). A putative nucleus localization
signal (NLS; aa sequence: -RHRR-) [14] is embedded in
the ¢fth repeat (Fig. 1).
The C2 H2 zinc ¢nger protein family exempli¢ed by the
Drosophila melanogaster Kru«ppel gene product [15], the
Xenopus laevis TFIIIA [16,17] and the human transcription factor Sp1 [18], constitutes one of the largest protein
families in the mammalian genomes [19]. The ZNF232
zinc ¢nger motifs are closely related; they ¢t the Kru«ppel-type consensus [20] and are joined by the conserved
S/TGEKPY/FX linker peptide. Application of the proposed DNA-zinc ¢nger recognition rules [21] suggests
that all ZNF232 ¢ngers, and especially the second, the
fourth and the ¢fth, can, in principle, participate in optimal DNA binding. The SCAN/LeR domain placed about
50 aa downstream from the amino terminus, lies in a region which is conserved in the majority of the members
belonging to the respective subfamily. This domain, initially identi¢ed in human p18 [12] and ZNF174 [13] proteins, is shared by a rapidly growing number of mammalian C2 H2 zinc ¢nger proteins. Although ZNF232 appears
to be overall neutral (deduced pI = 6.73), the non-¢nger
portion is markedly acidic (pI = 4.85; aspartate and glutamate content: 17.3%), reminiscent of several transcription
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regulators [22]. However, transient expression experiments
in COS-7 cells, designed to test the possible in£uence of a
GAL4-ZNF232 chimeric protein on the transcriptional
activity of a chloramphenicol acetyltransferase (CAT) reporter gene driven by a thymidine kinase (tk) promoter
containing a GAL4 binding site [23], did not result in
any signi¢cant change of CAT activity (data not shown).
Since the presence of the zinc ¢ngers may interfere with
chimeric protein folding, thus making a potentially
ZNF232 activation domain inaccessible to the transcriptional machinery, we also designed a zinc ¢nger deletion
construct, by omitting 614 bp from the 3P end of ZNF232
cDNA. Similarly, no signi¢cant change in CAT activity
was observed. Moreover, transfection in mouse NIH-3T3
cells, under optimized conditions [13], also demonstrated
no signi¢cant e¡ects. Our results are in agreement with
previous observations [12,13] and may indicate that
ZNF232 is a weak transcriptional regulator or that the
SCAN/LeR domain cannot act on a heterologous DNA
binding site [12,13], suggesting that it is not an independent transcriptional regulatory element. Interestingly, a human gene encoding an isolated SCAN/LeR domain was
reported recently [24]. Although the function of the domain is still unknown in depth, it was demonstrated that
SCAN/LeR mediates hetero- and/or homotypic protein
associations and thus modulates the function of transcriptional factors [24^26].
In order to investigate whether ZNF232 exists in a single or multiple copies in the human genome, we performed
genomic DNA blot hybridization analysis. The simple hybridization pattern detected in several restriction digests
(data not shown) is consistent with the existence of a
unique ZNF232 gene. To elucidate the exon/intron organization of ZNF232, we screened the gridded RPCI-1 human genomic PAC library (a generous gift of UK HGMPResource Centre, Hinxton, UK). This was performed by
probing with a 32 P-labeled 730 bp EcoRI fragment derived
from the 5P end of a ZNF232 cDNA clone spanning nt
positions 370^1100. A positive clone, No. 202L17, was
isolated. Structural integrity of the clone was con¢rmed
by DNA blot hybridization analysis in parallel with a
human genomic DNA sample (data not shown). Subcloning of appropriate PAC clone fragments, sequencing, and
PCR using a series of ZNF232 cDNA-speci¢c primers,
enabled us to elucidate the genomic organization of the
gene (Fig. 2). Our analysis suggested that ZNF232 encompasses a genomic region of at least 6 kb and the translated
portion of the gene spans three exons, encoding 139, 42
and 236 amino acids, respectively (Figs. 1 and 2). The
putative 5P UTR of the gene is interrupted by an intron
Fig. 2. Exon/intron organization and genomic sequence of ZNF232. (A) Schematic illustration of ZNF232 exon/intron organization. Boxes represent
ZNF232 exons. Relative exon sizes are on scale, while the approximate intron sizes are indicated in kb. The SCAN/LeR and the zinc ¢nger domains as
well as the alternatively spliced part of exon 4 are depicted by ¢lled boxes. The translation start and the stop codon are marked on exon 2 and 4, respectively. (B) Genomic sequences £anking ZNF232 exons. Exons are indicated by uppercase letters; lowercase letters denote intron sequences. The
splice sites, all complying with the GT/AG consensus, and the alternative acceptor site in exon 4, are in bold. Intron sizes were estimated by either
PCR using primer pairs corresponding to exon sequences (introns 2 and 3), or by Southern analysis exploiting appropriate cDNA fragments as probes
(intron 1). Intron 2 was sequenced to its entity.
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Fig. 3. Expression pattern of ZNF232 in human tissues. (A) Each of the indicated MTC panel cDNA was used as a PCR template with a ZNF232-speci¢c primer pair (see text). After 35 ampli¢cation cycles (annealing at 55³C), samples were subjected to 2.2% agarose/EtBr electrophoresis. Lanes: 1^8,
heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, respectively; 9^16, spleen, thymus, prostate, testis, ovary, small intestine, colon, leukocytes, respectively; a, PCR product from a ZNF232 cDNA containing the 27 bp region at nt positions 670^696; b, PCR product from a ZNF232
cDNA lacking the 27 bp region at nt positions 670^696; a/b, co-electrophoresis of a mixture of the PCR products of lanes a and b. (B) The same as
above cDNA samples (lanes 1^8 and 9^16) used as PCR templates with a control glyceraldehyde-3-phosphate dehydrogenase gene (G3PDH) primer
pair (Clontech) after 27 ampli¢cation cycles (annealing at 55³C). Lanes M (both A and B panels), pBluescript/HinfI size marker.
of about 2.0 kb. The sequence encoding for the SCAN/
LeR domain covers the second half of exon 2, while all
¢ve zinc ¢nger repeats are clustered in exon 4. An AG
dinucleotide located 27 bp downstream from the 5P end
of exon 4 (nt position 696, Figs. 1 and 2B), may represent
an appropriate alternative acceptor site, able to be utilized
interchangeably by the splicing machinery in vivo. This
observation correlates perfectly with the absence of a 27
bp long coding sequence (nt positions 670^696) determined in two of the analyzed ZNF232 cDNA clones.
RNA blot hybridization analysis using 2^3 Wg of poly(A)‡ RNA from K562 cells, detected a single ZNF232-speci¢c transcript of about 2.1 kb (data not shown). This is
comparable with the size of the suggested ZNF232 overall
cDNA sequence. Furthermore, in order to explore the
ZNF232 expression pro¢le in various human tissues and
simultaneously investigate the possibility of ZNF232 in
vivo alternative splicing, we employed a multiple tissue
panel of normalized ¢rst-strand cDNAs (MTC, Clontech)
in PCR experiments using a pair of primers (5PACTGTGCTGGAGGATTTAGAG-3P and 5P-ATGAACTCTCTGGTGGACAAC-3P) £anking cDNA nt positions 670^696. PCR performed under non-saturating conditions (Clontech MTC manual) permitted semiquantitative estimation of relative ZNF232 transcript levels. The gene is expressed in all tissues tested (Fig. 3);
however, slightly higher expression was observed in liver,
testis and ovary. Furthermore, two transcript variants
with a size di¡erence comparable to the PCR products
obtained by two ZNF232 cDNA templates di¡ering internally by 27 bp at nucleotide positions 670^696, were detected in all samples (Fig. 3). This suggests that ZNF232 is
subjected to alternative splicing in vivo, a feature shared
by other members of the SCAN/LeR subfamily [27^29].
Fig. 4. Fluorescence in situ hybridization on human male metaphase spreads using PAC clone RPCI-1 No. 202L17 as a probe. The unique signal at
17p12 is denoted by an arrow.
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Interestingly, the relative abundance of the two splice variants di¡ered among tested samples (Fig. 3), indicating a
tissue-speci¢c post-transcriptional control for ZNF232. It
remains to be seen whether these alternatively spliced transcripts suggesting two ZNF232 isoforms, di¡ering internally by nine amino acids, are of functional signi¢cance.
Chromosomal assignment of ZNF232 was initially performed using a monochromosomal human-rodent somatic
cell hybrid panel (Coriell Institute of Medical Research).
DNA samples (50^250 ng) from each hybrid and comparable amounts from the respective human and rodent parental cell lines were used as templates in PCR ampli¢cations with a ZNF232-speci¢c primer pair. The experiment
suggested that ZNF232 maps to chromosome 17 (data not
shown). Subsequent FISH analysis with £uorescence labeled PAC No. 202L17 as a probe, performed to normal
metaphase chromosome spreads according to standard
high resolution procedures [30,31], resulted in a strong
hybridization signal at 17p12 (Fig. 4). Database searches
revealed that a partially sequenced BAC clone (GenBank
accession No. AC012146) roughly mapped to chromosome
17, encompasses ZNF232. Our FISH data place this BAC
to 17p12. The chromosomal distribution of human genes
encoding for zinc ¢nger proteins is clearly non-random
[32,33]. A disproportionate density characterizes chromosomes 1, 3, 6, 7, 10, 11, 22 and an excessive abundance
marks
chromosome
19
(Genome
Database ;
www.gdb.org). Moreover, available mapping data for
members of the human SCAN/LeR group reveal an apparently clustered genomic organization. The majority of
the reported members are mapped to certain chromosomal
regions, i.e. 3p21-22, 6p21-22 and 16p13 [34^37]. Apart
from ZNF232, at least four other zinc ¢nger genes of
the C2 H2 type map to 17p12-p13, i.e. ZNF18/KOX11,
ZNF29/KOX26, ZNF62 and ZFP3, thus pinpointing another zinc ¢nger gene-rich region in the human genome.
Structural abnormalities of C2 H2 zinc ¢nger genes have
been found in a variety of genetic diseases. In particular,
deletions at 17p12-13 have been associated with gliomas,
astrocytomas, colon, and lung tumors [32,38].
Finally, in order to investigate experimentally the sub-
cellular localization of the ZNF232 polypeptide and the
functional role of the predicted NLS, we proceeded in
transient expression of a green £uorescent proteinZNF232 chimera, in african green monkey COS-7 cells.
The construct, pEGFP/ZNF232, contained the enhanced
green £uorescent protein (GFP) open reading frame combined with the ZNF232 coding region. This was prepared
by co-ligation of the linearized pEGFP-C1 vector (Clontech) with a PCR product (primers : 5P-AGGATGGCTGTATCACTAAC-3P and 5P-GATCCAGTCCTAAAGTAGATTAGAC-3P, nt positions 123^142 and 1418^1444,
respectively) representing the entire ZNF232 translated region; the ampli¢ed DNA was inserted into the bluntended HindIII site of the vector polylinker. A deletion
derivative, pEGFP/delZNF232, lacking the NLS region
was prepared by excision of a KpnI fragment de¢ned by
the unique internal (nt position 830, Fig. 1) and the vector
KpnI sites (expressed ZNF232 polypeptide : 1^235 aa).
About 106 COS-7 cells were transfected with 2 Wg of either
pEGFP/ZNF232 or pEGFP/delZNF232 DNA using the
calcium phosphate co-precipitation method [39]. Twentyfour to 36 h after transfection, the cells were examined
with £uorescence microscopy. The chimeric GFP/
ZNF232 polypeptide was detected speci¢cally in the nucleus; on the contrary, expression of pEGFP/delZNF232
resulted in the exclusion of the truncated polypeptide from
the nucleus (Fig. 5). These ¢ndings suggest that ZNF232 is
a nuclear protein and demonstrate the role of the predicted NLS.
To summarize, this study reports the detailed analysis of
a novel chromosome 17p12 zinc ¢nger gene, ZNF232,
member of the SCAN/LeR subfamily. ZNF232 is widely
expressed in man, encodes a nuclear protein and is subjected to tissue-speci¢c post-transcriptional regulation.
The latter may lead to two structural variants di¡ering
in relative abundance among tissues. Like other members
of the group, ZNF232 may be associated with the transcriptional machinery in a wide variety of human tissues.
It would be interesting to investigate the potential of
ZNF232 as a transcriptional modulator as well as to explore its interaction repertoire.
Fig. 5. Fluorescence photomicrographs showing the subcellular localization of GFP-ZNF232 chimeras in transiently transfected COS-7 cells. Plasmid
DNA used: (A) pEGFP-C1 vector (control) ; the GFP product exhibited passive nucleocytoplasmic di¡usion, as expected [40]; (B) pEGFP/ZNF232;
(C) pEGFP/delZNF232.
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The authors wish to thank S. Vrontou for her contribution in the initial screening of the cDNA library, A. Pasparaki for technical support and Dr. G. Chalepakis for
critical reading of the manuscript. This work was supported by grants of the University of Crete and IMBBFORTH.
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