Báo cáo khoa học: Novel repressor of the human FMR1 gene ) identification ¨ of p56 human (GCC)n-binding protein as a Kruppel-like transcription factor ZF5 ppt

Novel repressor of the human FMR1 gene
)
identification
of p56 human (GCC)
n
-binding protein as a Kru
¨
ppel-like
transcription factor ZF5
Sergey V. Orlov
1,2
, Konstantin B. Kuteykin-Teplyakov
1
*, Irina A. Ignatovich
3
, Ella B. Dizhe
1
,
Olga A. Mirgorodskaya
3
, Alexander V. Grishin
2
, Olga B. Guzhova
1
, Egor B. Prokhortchouk
4
,
Pavel V. Guliy
1,2
and Andrej P. Perevozchikov
1,2

1 Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, St Petersburg, Russia
2 Department of Embryology, St Petersburg State University, Russia
3 Institute of Cytology, Russian Academy of Sciences, St Petersburg, Russia
4 Department of Molecular Basis of Medicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia
A variety of human diseases have been associated with
expansion of CGG ⁄ CCG triplet repeat tracts within
the human genome [1–3]. To date, five human chromo-
somal folate-sensitive fragile sites associated with
expansion of 5¢-(CGG)
n
-3¢ trinucleotide repeats have
been characterized at the molecular level: FRAXA,
FRAXE, FRAXF, FRA16A, and FRA11B. Expansion
at the FRAXA locus results in fragile X-mental retar-
dation syndrome, whereas expansion at the FRAXE
locus leads to mild mental retardation, and the
FRA11B locus has been implicated in Jacobsen’s
syndrome [3,4]. In the case of the FRAXA locus,
the (GCC)
n
triplet repeat amplification has occurred
within the 5¢-UTR of the fragile X-mental retarda-
tion 1 (FMR1) gene [5]. Expansions of n > 200
(n < 70 is normal) followed by cytosine methylation
inactivate the FMR1 gene. The FMR1 gene product
is an RNA-binding protein that associates with
Keywords
FMR1; fragile X syndrome; (GCC)
n
; triplet

repeats; ZF5; zinc finger transcription factors
Correspondence
S. V. Orlov, Department of Biochemistry,
Institute of Experimental Medicine, Russian
Academy of Medical Sciences, 197376,
Acad. Pavlov Street 12, St Petersburg,
Russia
Fax: +7 812 234 0310
Tel: +7 812 346 0644
E-mail:
Present address
*Department of Molecular Neurobiochemistry,
Ruhr-University, Bochum 44801,
Germany
(Received 1 May 2007, revised 20 July
2007, accepted 24 July 2007)
doi:10.1111/j.1742-4658.2007.06006.x
A series of relatively short (GCC)
n
triplet repeats (n ¼ 3–30) located within
regulatory regions of many mammalian genes may be considered as puta-
tive cis-acting transcriptional elements (GCC-elements). Fragile X-mental
retardation syndrome is caused by an expansion of (GCC)
n
triplet repeats
within the 5¢-untranslated region of the human fragile X-mental retarda-
tion 1 (FMR1) gene. The present study aimed to characterize a novel
human (GCC)
n
-binding protein and investigate its possible role in the regu-

lation of the FMR1 gene. A novel human (GCC)
n
-binding protein, p56,
was isolated and identified as a Kru
¨
ppel-like transcription factor, ZF5, by
MALDI-TOF analysis. The capacity of ZF5 to specifically interact with
(GCC)
n
triplet repeats was confirmed by the electrophoretic mobility shift
assay with purified recombinant ZF5 protein. In cotransfection experi-
ments, ZF5 overexpression repressed activity of the GCC-element contain-
ing mouse ribosomal protein L32 gene promoter. Moreover, RNA
interference assay results showed that endogenous ZF5 acts as a repressor
of the human FMR1 gene. Thus, these data identify a new class of ZF5
targets, a subset of genes containing GCC-elements in their regulatory
regions, and raise the question of whether transcription factor ZF5 is impli-
cated in the pathogenesis of fragile X syndrome.
Abbreviations
CAST, cyclic amplification and selection of targets; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; NFjB, nuclear factor kappa B; siRNA, small interfering RNA.
4848 FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works
polyribosomes as part of large messenger ribonucleo-
protein complex, modulating the translation of its
RNA ligands [6]. FMR1 protein is thought to be
involved in RNA interference machinery [7–9].
The molecular mechanisms responsible for the insta-
bility of (GCC)
n
triplet repeats remain largely

unknown. Several models have been suggested, includ-
ing DNA polymerase slippage [10–12], formation of
unusual secondary structures by contiguous (GCC)
n
tracts [13,14], and interactions of (GCC)
n
triplet
repeats with DNA-binding proteins [15,16]. (GCC)
n
triplet repeats are not restricted to folate-sensitive frag-
ile sites in mammalian genomes [4,17]. There are rela-
tively short (GCC)
n
triplet repeats (n ¼ 3–30) located
within regulatory regions of many mammalian genes
that may operate as cis-acting transcriptional elements
(GCC-elements) [17]. We previously characterized
GCC-elements within the promoter of the gene that
encodes mouse ribosomal protein L32 (rpL32) [18–20]
and within the 5¢-UTR of the human very low density
lipoprotein receptor gene (VLDLR) [21]. Such GCC-
elements are transcriptionally active and can interact
specifically with human and mouse nuclear proteins.
To date, only one (GCC)
n
-binding protein, p20, has
been characterized [16,18,22]. The p20 protein was first
detected in HeLa cells as a protein interacting with the
GCC-element of rpL32 [18], and then purified accord-
ing to its ability to interact specifically with (GCC)

n
triplet repeats within the 5¢-UTR of the human FMR1
gene; the corresponding gene was cloned [16,22]. Puri-
fied p20 (20 kDa) was found not to be homologous to
any known DNA-binding proteins and was designated
CGG triplet repeat binding protein 1 (CGGBP1). Sub-
sequently, in cotransfection experiments, CGGBP1 has
been demonstrated to repress the activity of the FMR1
[23], VLDLR [21], and rpL32 (K. B. Kuteykin-Teplya-
kov, E. B. Dizhe, S. V. Orlov & A. P. Perevozchikov,
unpublished results) genes. Thus, DNA-binding pro-
teins that interact with (GCC)
n
repeats may be
involved in stabilization ⁄ destabilization of the triplet
repeats [16,22] and ⁄ or transcriptional regulation of
GCC-triplet containing genes [19–21]. Hence, in our
previous studies, we proposed the existence of other
yet uncharacterized mammalian (GCC)
n
-binding pro-
teins [19–21]. In the present study, we report the iso-
lation of a novel (GCC)
n
-binding protein p56 from
human hepatoma HepG2 cells that interacts specifi-
cally with the GCC-elements of the rpL32 and FMR1
genes. This protein was identified herein to be a
Kru
¨

ppel-like Zn-finger transcription factor ZF5.
Mammalian ZF5 protein was first identified in
mouse as a transcriptional repressor of the murine
c-myc gene with a molecular mass of approximately
52 kDa. It was subsequently shown to repress the her-
pes simplex virus thymidine kinase gene promoter [24].
ZF5 homologues have since been cloned in humans
and in chicken [25–27]. Vertebrate ZF5 proteins are
highly conserved and contain five Kru
¨
ppel-like Zn-fin-
gers at the C-terminus. They also have a hydrophobic
BTB ⁄ POZ domain (124 N-terminal amino acid resi-
dues), which is responsible for protein–protein inter-
actions, and a nuclear localization signal [25,26]. The
DNA-binding domain of ZF5 consists of the third and
forth Zn-fingers [28]. Cyclic amplification and selection
of targets (CAST) assays performed independently by
two groups confirmed the GC-rich content of ZF5
binding sites with a common core sequence
5¢-GCGCG-3¢ [28,29]. Interestingly, none of the ZF5
sequences isolated by CAST assays contained (GCC)
n
triplet repeats.
Several novel ZF5 targets have been described
[29,30]. Transcription factor ZF5 has been reported to
have both positive and negative effects on target gene
transcription. Moreover, in silico content analysis of
the core promoter regions of human genes revealed the
presence of ZF5 consensus sites within approximately

60% of human gene core promoters [31]. In spite of
significant over-representation of these sites in pro-
moter regions relative to nonpromoter background
data, these results must be verified experimentally,
especially in terms of the relative low complexity of
the ZF5 consensus sequence. In the present study, we
have shown for the first time that endogenous ZF5
protein acts as a repressor of the FMR1 gene in
HepG2 cells.
Results
Characterization of human nuclear proteins that
interact with a composite cis-acting element of
rpL32 ()24 +11)
We have previously shown that the mouse rpL32 gene
contains a composite cis-acting element that spans the
transcription start site ()24 +11) [19]; this composite
element consists of a GCC-element and a pyrimidine
block (Fig. 1A). Prior tissue distribution studies of
mammalian nuclear proteins interacting with the
rpL32 fragment ()24 +11) revealed similarities
between the DNA–protein complexes formed by the
rpL32 fragment ()24 +11) with nuclear proteins
from human embryonic fibroblasts and HepG2 cells
[19]. Electrophoretic mobility shift assay (EMSA) com-
petition experiments in the present study indicated that
the complete rpl32 fragment ()24 +11) has a greater
affinity to nuclear proteins from human fibroblasts
Sergey V. Orlov et al. A novel human (GCC)
n
-binding protein

FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works 4849
than does either the GCC-element or pyrimidine block
alone (Fig. 1B,C). Similar results were obtained with
nuclear proteins from HepG2 cells (data not shown).
These results suggest that this rpL32 fragment is a syn-
ergetic composite cis-acting element.
EMSA was conducted with 1,10-phenanthroline (a
chelator that specifically removes Zn
2+
from metallo-
proteins) [32] to examine whether Zn-finger DNA-bind-
ing human proteins are involved in DNA–protein
complexes formed by the rpL32 fragment. The presence
of 1,10-phenanthroline in the reaction mixture inhibited
formation of DNA–protein complexes (Fig. 1D) and the
addition of Zn
2+
partially restored complex formation.
The relatively weak Zn-chelator EGTA produced less
inhibition of DNA–protein interactions than 1,10-phe-
nanthroline. These results suggest that the DNA-binding
activity of human nuclear proteins interacting with
rpL32 fragment ()24 +11) depends on Zn
2+
, which is
consistent with the supposition that those proteins con-
tain Zn-finger DNA-binding domains.
Although there are similarities between the DNA–
protein complexes formed by the rpL32 fragment
Fig. 1. Characterization of human nuclear

proteins interacting with the rpL32 fragment
()24. . .+11). (A) Composite cis-acting ele-
ment of mouse ribosomal protein L32 gene.
The numbers indicate position relative to
the rpL32 transcription start point. The
sequences corresponding to the GCC-ele-
ment and pyrimidine tract are underlined.
(B,C) Results of EMSA competition experi-
ments with nuclear proteins from human
embryonic fibroblasts. The competitors and
their molar excesses are shown above the
lanes: (GCC)
3
-element; rpL32 ()6. +11)-
pyrimidine part of the rpL32 fragment
()24. . .+11); K
–
-negative control (without
nuclear proteins); K
+
-binding reaction with
no competitors; CI and CII refer to the spe-
cific DNA-protein complexes I and II formed
by the rpL32 fragment ()24. +11); F refers
to the free rpL32 fragment ()24. +11). (D)
EMSA experiments with chelators specific
to bivalent cations: 1, free rpL32 fragment
()24. . .+11) without human embryonic fibro-
blast nuclear proteins (negative control);
2, binding reaction of the rpL32 fragment

()24. . .+11) with human embryonic fibro-
blast nuclear proteins with no chelators;
3, binding reaction with addition of 5 m
M
1,10-phenanthroline; 4, binding reaction with
addition of 5 m
M 1,10-phenanthroline and
5m
M ZnCl
2
; 5, binding reaction with addi-
tion of 5 m
M EGTA. CI and CII refer to the
specific DNA-protein complexes I and II
formed by the rpL32 fragment ()24. +11).
(E) Southwestern assay of HepG2 nuclear
proteins with the rpL32 fragment
()24. . .+11) revealing the p56 and p68
bands (arrowheads).
A novel human (GCC)
n
-binding protein Sergey V. Orlov et al.
4850 FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works
()24 +11) with human fibroblast nuclear proteins
and those formed with HepG2 cell nuclear proteins,
the latter were found to be much more abundant.
Therefore, nuclear proteins from HeG2 cells were
employed in the subsequent Southwestern assays. Two
major putative (GCC)
n

-binding proteins, p56 and p68,
were detected by Southwestern assays. (Fig. 1E).
Purification of human nuclear proteins that
interact with a composite cis-acting element of
rpL32 ()24 +11)
We designed a four-step purification procedure to iso-
late proteins that interact with the rpL32 fragment
()24 +11) (Fig. 2A). This approach included a con-
sequent preparative EMSA stage, two-step purification
by DNA-affinity chromatography with the rpL32 frag-
ment ()24 +11) immobilized on magnetic beads as a
substrate, and DNA-affinity chromatography on a
nonspecific substrate to remove the remaining nonspe-
cific DNA-binding proteins. Figure 2B shows the elu-
tion profile of the preparative EMSA of the rpL32
fragment ()24 +11) with nuclear proteins from
HepG2 cells. The first peak corresponds to unbound
labeled DNA probe, the second and third peaks corre-
spond to DNA–protein complexes formed by the
rpL32 fragment ()24 +11) with studied proteins.
Fractions 21–27, corresponding to latter two peaks,
were collected, concentrated by ultrafiltration and
checked for DNA-binding activity by analytical EMSA
(Fig. 2C). The slower migrating complex (Complex I)
spontaneously converted into the faster migrating one
(Complex II) (Fig. 2C). Therefore, fractions 22–25
with high DNA-binding activity were pooled, analyzed
by SDS electrophoresis (Fig. 2E, lane 1), and subjected
to purification by DNA-affinity chromatography.
In the final purification step, the proteins were eluted

from immobilized rpL32 fragment ()24 +11) onto
immobilized human lactoferrin binding sites (nonspe-
cific DNA substrate). The DNA-binding activity analy-
sis results are summarized in Fig. 3D. The final fraction
containing unbound last stage proteins was analyzed by
SDS electrophoresis and compared with proteins bound
to nonspecific sorbent (negative control) (Fig. 2E, lanes
3 and 2, respectively). Only two proteins, p56 (approxi-
mately 56 kDa) and p68 (approximately 68 kDa), that
were not in the negative control fraction remained in a
final fraction. There were also two bands at 65 kDa and
72 kDa that were detected in both the negative control
and final fractions. The p56 and p68 proteins in the
final fraction fit with the rpL32 fragment ()24 +11)-
binding proteins detected by Southwestern assay (see
above). Together, these findings indicate that p56 and
p68 are DNA-binding proteins that interact specifically
with composite cis-acting element of rpL32.
Identification of rpL32-binding protein p56 by
MALDI-TOF assay as a Kru
¨
ppel-like transcription
factor ZF5
MALDI-TOF assays were employed to identify the
p56 protein. After SDS electrophoresis, the corres-
ponding bands from silver-stained gel were cut out,
digested by trypsin and subjected to analysis. A typical
representative spectrum fragment containing several
peaks corresponding to p56-derived peptides is shown
in Figure 3A. Identification of p56 protein was per-

formed using the Mascot search engine. Despite the
presence of substantial noise, we were able to detect
among the potential candidates one Zn-finger protein
with a molecular weight of approximately 52 kDa:
human Kru
¨
ppel-like Zn-finger transcription factor ZF5
(Fig. 3B). The cDNA gene encoding human ZF5
protein has been cloned by two-step RT-PCR and
subcloned into a pBluescript vector. Experiments to
identify the p68 protein are currently in progress.
Recombinant fusion protein GST-ZF5-ZF5 purified
from bacterial cells specifically interacts with
(GCC)
n
repeats
EMSA experiments with a recombinant fusion protein
containing ZF5 Zn-finger domains linked to bacterial
glutathione transferase (GST-ZF5) were performed to
test the capacity of ZF5 to interact with the rpL32
fragment ()24 +11) and to examine the possible
(GCC)
n
-binding activity of ZF5. The recombinant pro-
tein was isolated from bacterial cells by glutathione
affinity chromatography (Fig. 4). Recombinant GST-
ZF5 protein efficiently recognized the ZF5 consensus
sequence (Fig. 4A, lanes 2 and 6). Molar excesses of
unlabeled ZF5 binding site depleted the corresponding
bands in a dose-dependent manner (Fig. 4A, lanes

3–5), indicating a good specificity of the DNA–protein
interactions. Interestingly, the unlabeled rpL32 frag-
ment ()24 +11) depleted the corresponding band
even more efficiently (Fig. 4A, lanes 7–9).
Similar results were obtained in reciprocal EMSA
experiments (Fig. 4B). Recombinant GST-ZF5 pro-
tein specifically interacted with the rpL32 frag-
ment ()24 +11). Moreover, the rpL32 fragment
()24 +11) was a more effective competitor than the
ZF5 binding site (Fig. 4B, lanes 2–4 and 6–8, respec-
tively). The irrelevant lactoferrin binding site (negative
control) did not disrupt the corresponding DNA–pro-
tein complex (Fig. 4B, lanes 10–12). Therefore, these
Sergey V. Orlov et al. A novel human (GCC)
n
-binding protein
FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works 4851
Fig. 2. Purification of HepG2 nuclear proteins interacting with the rpL32 fragment ()24. . .+11). (A) Summary of purification scheme. (B) Pre-
parative EMSA elution profile. (C) Testing of the rpL32 fragment ()24. +11)-binding activity of 21st to 27th fractions obtained on a prepara-
tive EMSA purification stage. (D) EMSA testing of the rpL32 fragment ()24 +11)-binding activity in the samples after DNA affinity
chromatography: 1, negative control (without proteins); 2, positive control (EMSA with HepG2 crude nuclear extracts); 3, the proteins eluted
from the immobilized rpL32 ()24 .+11) fragment (first round of DNA affinity chromatography); 4, the proteins eluted after second round of
DNA affinity chromatography; 5, proteins not bound to immobilized lactoferrin binding site (final fraction). (E) Analysis of fractions obtained
from the preparative EMSA and DNA affinity chromatography stages by SDS electrophoresis: 1, pooled fractions 22–25 obtained from the
preparative EMSA stage; 2, the proteins bound by the lactofferin binding site in the last purification stage; 3, unbound proteins obtained from
the last stage of purification (final fraction). CI and CII, DNA-protein complexes I and II, respectively. C¢, Nonspecific DNA–protein complexes;
F, free rpL32 fragment ()24. +11), p56 and p68- purified proteins specifically interacting with the rpL32 fragment ()24. +11).
A novel human (GCC)
n
-binding protein Sergey V. Orlov et al.

4852 FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works
Fig. 3. Identification of p56 polypeptide as Kru
¨
ppel-like transcription factor ZF5 by MALDI-TOF assay. (A) Fragment of MALDI-TOF spectrum of
p56-derived peptides. (B) Mascot search results. Peptides with matched mass values are listed with their locations in the total ZF5 protein.
Sergey V. Orlov et al. A novel human (GCC)
n
-binding protein
FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works 4853
data provide a robust demonstration of the capacity of
ZF5 to interact specifically with rpL32 composite cis-
acting element. Furthermore, the affinity of recombi-
nant GST-ZF5 for the rpL32 fragment ()24 +11) is
higher than its affinity for a classic ZF5 binding site.
EMSA experiments with a labeled GCC-element
probe or pyrimidine motif probe were conducted to
determine whether the ZF5 binds to the GCC-element
or pyrimidine motif of rpL32 composite cis-acting ele-
ment. The pyrimidine part of the rpL32 fragment
bound nuclear proteins from HepG2 cells, but did not
interact with recombinant GST-ZF5 (Fig. 4C). Mean-
while, a (GCC)
9
sequence corresponding to a 5¢-UTR
fragment of the human FMR1 gene (normal allele)
bound specifically to GST-ZF5. Molar excesses
of unlabeled rpL32 fragment ()24 +11), (GCC)
9
sequence, or ZF5 binding site, but not lactoferrin bind-
ing site, efficiently disrupted (GCC)

9
⁄ ZF5 complexes
(Fig. 4D,E). These data demonstrate unequivocally
that ZF5 transcription factor interacts specifically with
Fig. 4. Interactions of recombinant GST-ZF5 with GCC-element of the rpL32 promoter. (A) EMSA using the ZF5 binding site as a probe. (B)
EMSA using the rpL32 fragment ()24. +11) as a probe. (C) EMSA using the pyrimidine site of rpL32 ()6 +11) as a probe. (D,E) EMSA
using a (GCC)
9
-element as a probe. K
–
, without any proteins; K
+
, EMSA with purified recombinant GST-ZF5 without competition; HepG2,
EMSA with nuclear proteins from HepG2 cells. Competitors are shown above each image. Triangles above images represent the increasing
amounts of competitor applied. C, Specific DNA–protein complex; F, free DNA probe.
A novel human (GCC)
n
-binding protein Sergey V. Orlov et al.
4854 FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works
(GCC)
n
triplet repeats and, hence, may be considered
a novel (GCC)
n
-binding protein.
ZF5 overexpression leads to repression of rpL32
promoter that contains GCC-element
The capacity of ZF5 transcription factor to interact
with GCC-elements suggests that it may regulate rpL32
gene expression. To investigate this possibility, we con-

structed a eukaryotic expression vector containing the
ZF5 coding region under the control of the human
cytomegalovirus early gene promoter (pCMVZF5) and
performed cotransfection experiments. When HepG2
cells were transiently cotransfected with a pCMVZF5
expression vector and pL32luc plasmid containing a
firefly luciferase reporter gene under the control of
rpL32 promoter, ZF5 overexpression strongly down-
regulated rpL32 promoter activity in dose-dependent
manner (Fig. 5A) relative to its impact on the activity
of a synthetic minimal promoter containing five tandem
cis-acting elements for transcription factor nuclear fac-
tor kappa B NFeˆ B (negative control) (Fig. 5B). These
results demonstrate that human ZF5 can regulate tran-
scription of genes that contain (GCC)
n
triplet repeats
within their regulatory regions.
Endogenous ZF5 down-regulates expression of
the FMR1 gene in HepG2 cells
RNA interference was used to test whether ZF5 is
involved in regulation of the human FMR1 gene.
Three small interfering RNAs (siRNAs) matching dif-
ferent regions of ZF5 mRNA were selected based on
general rules for siRNA design [43]. The efficiency of
ZF5 down-regulation in HepG2 cells by siRNA trans-
fection was estimated by semiquantitative RT-PCR.
HepG2 cells were transfected with the most active
siRNA (matched bases 945–963 of human ZF5
mRNA; accession number D89859). The amount of

FMR1 mRNA in siRNA
ZF5
transfected cells har-
vested after 48 h was accessed by semiquantitative
RT-PCR and compared with the mRNA from non-
transfected cells and cells transfected with control
siRNA that was not homologous to any human
mRNAs (Fig. 6A,B). ZF5 down-regulation increased
FMR1 mRNA levels in HepG2 cells; transfection of
HepG2 cells with control siRNA did not influence
FMR1 mRNA levels. It was suggested previously that
FMR1 protein may be involved in the RNA interfer-
ence machinery [7–9]. Thus, accumulation of FMR1
mRNA may be due to stimulation of FMR1 gene
expression in response to activation of RNA interfer-
ence machinery in the cells trasfected by siRNA
ZF5
but not to down-regulation of ZF5. To test this
assumption, we transfected HepG2 cells by siRNA
for human grp58 gene [44]. siRNA
grp58
down-regu-
lated expression of grp58 gene but did not affect
FMR1 mRNA (Fig. 6C). This, these data indicate
that endogenous transcription factor ZF5 is a repres-
sor of the human FMR1 gene.
Discussion
CGGBP1 is the only DNA-binding protein described
to date that specifically interacts with (GCC)
n

repeats
[16,22]. Two observations led us to hypothesize that
other (GCC)
n
-binding proteins exist and to search for
Fig. 5. Effect of ZF5 overexpression on activity of the rpL32 pro-
moter relative to the synthetic NFjB-dependent promoter. (A)
HepG2 cells were cotransfected with pL32luc (5 lg), pCMVL
(2.5 lg) and different amounts of pCMVZF5: 1, without pCMVZF5;
2, with 50 ng pCMVZF5; 3, with 150 ng pCMVZF5; 4, with 375 ng
pCMVZF5. (B) HepG2 cells were cotransfected with pNFjBluc
(5 lg), pCMVL (2.5 lg) and different amounts of pCMVZF5: 1, with-
out pCMVZF5; 2, with 50 ng pCMVZF5; 3, with 150 ng pCMVZF5;
4, with 375 ng pCMVZF5.
Sergey V. Orlov et al. A novel human (GCC)
n
-binding protein
FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works 4855
possible novel human (GCC)
n
-binding transcription
factors. First, the DNA-binding activity of nuclear
proteins that interact with the rpL32 fragment
()24 +11) is Zn
2+
-dependent (Fig. 1D), although
the CGGBP1 protein does not contain any Zn-bind-
ing domains [22]. Second, the affinity of purified
CGGBP1 to (GCC)
n

repeat sequences is dramatically
decreased if there are fewer than eight repeats [16].
However, a few GCC repeats are sufficient to
regulate an artificial herpes viral thymidine kinase
promoter (three repeats) or the rpL32 promoter (four
repeats) [19,20].
Purification of transcription factors can be challeng-
ing due to their low abundance in cells and their weak
affinity to bait sequences. Indeed, (GCC)
n
triplet
repeats with n > 10 are needed to bind mammalian
nuclear proteins with high affinity [15,16]. However the
regulatory regions of many mammalian genes contain
shorter (GCC)
n
motifs (n ¼ 3–10) that are adjacent to
binding sites of other transcription factors (i.e. Sp1,
Egr1, WT1) [17]. Thus, low affinity binding of short
(GCC)
n
repeats to (GCC)
n
-binding proteins may be
compensated for by cooperative protein–protein inter-
actions with transcription factors that bind to adjacent
sequences.
We previously characterized a fragment of mouse
rpL32 promoter ()24 +11) containing an adjacent
GCC-element ()19 )6) and pyrimidine motif

()5 +11) as a composite cis-acting element that
interacts with nuclear proteins from mammalian cells
[18–20]. The present data (Fig. 1B,C) suggest that
there may be cooperation between DNA–protein and
protein–protein interactions within complexes formed
by the rpL32 fragment ()24 +11). We proceeded to
detect, by Southwestern assay, only two polypeptides
in nuclear extracts from HepG2 cells that interact with
the rpL32 fragment ()24 +11) (Fig. 1E). These find-
ings indicate that the rpL32 fragment ()24 +11)
would serve as an effective bait for purifying GCC-ele-
ment binding proteins.
The rpL32 fragment ()24 +11) formed two major
complexes with nuclear proteins from HepG2 cells and
human embryonic fibroblast cells (Fig. 1B,C). In our
attempt to isolate these complexes, an exploratory pre-
parative EMSA revealed that the slower migrating
Complex I converted spontaneously to the faster
migrating Complex II (Fig. 2C). This observation sug-
gests that the difference between the DNA-binding
complexes might be caused by reversible post-transla-
tional modifications of rpL32-binding proteins. Alter-
natively, those complexes may differ by unstable
proteins that were lost during the purification process.
Additional experiments are needed to distinguish
between these possibilities.
We detected two purified DNA-binding proteins spe-
cifically interacting with the rpL32 fragment ()24 +
11) that coincided with the rpL32-binding proteins
revealed by Southwestern assay. The results of the

MALDI-TOF analysis indicated that one of these, p56,
is a Kru
¨
ppel-like transcription factor ZF5. Although all
ZF5-binding sites found previously by CAST assay are
GC-rich, none are within (GCC)
n
triplet repeats [28,29].
Our EMSA experiments using recombinant protein
Fig. 6. Down-regulation of endogenous ZF5 in HepG2 cells by RNA
interference. (A) Effects of HepG2 cell transfection by siRNAs on
ZF5 expression (semiquantitative RT-PCR): lanes 1–6, RT-PCR of
GAPDH mRNA (18 cycles, control); lane 7, marker; lanes 8–13,
RT-PCR of ZF5 mRNA (27 cycles); 1, 8, negative control; 2, 9, trans-
fection of HepG2 cells by siRNA
ZF5
with oligofectamine; 3, 10,
transfection of HepG2 cells by control siRNA with oligofectamine;
4, 11, transfection of HepG2 cells by siRNA
ZF5
with RNAFect; 5,
12, transfection of HepG2 cells by control siRNA with RNAFect; 6,
13, untreated cells. (B) Effects of HepG2 cell transfection by
siRNA
ZF5
on FMR1 expression (semiquantitative RT-PCR, 34 cycles):
1, marker; 2, negative control; 3, transfection by siRNA
ZF5
with oli-
gofectamine; 4, transfection by control siRNA with oligofectamine;

5, transfection by siRNA
ZF5
with RNAFect; 6, transfection by control
siRNA with RNAFect; 7, untreated cells. (C) Effects of HepG2 cell
transfection by siRNA
grp58
on FMR1 expression (semiquantitative
RT-PCR): 1–3, RT-PCR of GAPDH mRNA (18 cycles, control); 4, mar-
ker; lanes 5–7, RT-PCR of grp58 mRNA (27 cycles); 8–10, RT-PCR of
FMR1mRNA (34 cycles); 1, 5, 8, negative control; 3, 7, 10, untreated
cells; 2, 6, 9, transfection by siRNA
grp58
with RNAFect.
A novel human (GCC)
n
-binding protein Sergey V. Orlov et al.
4856 FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works
containing the DNA-binding domain of ZF5 fused with
GST confirmed that recombinant GST-ZF5 specifically
interacts with its canonical binding site, the rpL32
fragment ()24 +11), and the GCC-element alone
(Fig. 4). Moreover, competition experiments showed
that the affinity of ZF5 for the rpL32 fragment
()24 +11) is greater than its affinity for its own
consensus sequence.
Although the rpL32 fragment ()24 +11) contains
a core part of the ZF5 consensus sequence GCGC
immediately before the GCC-element (Fig. 1A), a
(GCC)
9

sequence that does not contain any ZF5
consensus core was recognized specifically by ZF5
(Fig. 4C). Thus, the high affinity of ZF5 for the rpL32
fragment may be due to cooperation interactions of
ZF5 with both its consensus core sequence and the
GCC-element. Hence, ZF5 appears to bind GCC-ele-
ments with an affinity that is similar to that for its
consensus sequence. This finding may explain why pre-
vious CAST assays did not reveal GCC-elements to be
among ZF5-binding sites. The affinity of ZF5–DNA
interactions may depend on the length of the binding
site. Random sequences used for CAST assays are
approximately 20 bp in length [28], and the selection
procedure is based on single site affinities to the
protein of interest. In the case of the rpL32 fragment
or the (GCC)
9
sequence, there are several repeated
ZF5-binding sites within a single oligonucleotide.
Therefore, the affinity of those sequences for ZF5
revealed in the present study may be the result of
cooperative interactions between several ZF5 molecules
and adjacent ZF5-binding sites. Such a cooperative
mechanism is especially interesting with respect to the
role of (GCC)
n
triplet repeat amplification in inactiva-
tion of the FMR1 gene. That is, ZF5 affinity for
(GCC)
n

repeat tracts in the FMR1 gene would be
expected to be greatly enhanced in alleles with very
extended (GCC)
n
repeats.
Mammalian ZF5 protein was first identified as a
transcriptional repressor of the murine c-myc gene.
Although ZF5-mediated transcriptional activation has
been reported [29,30], ZF5 is most often observed to
down-regulate target genes. Here, we show that ZF5
overexpression led to down-regulation of the rpL32
promoter. Moreover, down-regulation of ZF5 by
RNA interference caused up-regulation of the FMR1
gene. These data clearly support the assertion that
endogenous ZF5 acts as a transcriptional repressor of
the human FMR1 gene. Given that FMR1 inactivation
in FMR patients has been attributed to long (GCC)
n
triplet repeats within the 5¢-UTR of the FMR1 gene,
further investigations to clarify the role of ZF5 in
FMR pathogenesis are warranted.
In conclusion, we report that p56 protein purified
from HepG2 cells interacts with GCC-elements of the
rpL32 promoter and the 5¢-UTR of the human
FMR1 gene. We further identify p56 as the Kru
¨
ppel-
like transcription factor ZF5. We show, for the first
time, that recombinant mammalian ZF5 protein
interacts specifically with (GCC)

n
triplet repeats, and
further show that endogenous ZF5 acts to down-reg-
ulate the FMR1 gene. The present study has revealed
a novel class of ZF5 target genes that have GCC-ele-
ments within their regulatory regions and implicates
ZF5 in the pathogenesis of fragile X-mental retarda-
tion syndrome.
Experimental procedures
Materials
Chemicals were purchased from Sigma (St Louis, MO,
USA), Amersham Biosciences (Piscataway, NJ, USA),
Roche Applied Science (Mannheim, Germany), Invitrogen
(Carlsbad, CA, USA), Promega (San Luis Obispo, CA,
USA) and from local Russian manufacturers (analytical or
high purity grade). Enzymes used in gene engineering were
purchased from Fermentas (Vilnius, Lithuania) and the Sci-
entific Industrial Corp. SibEnzyme (Novosibirsk, Russia).
Genetic constructions
pL32luc plasmid containing the reporter gene encoding
firefly luciferase under the control of rpL32 promoter
()155 .+195 relative to the transcription start point) was
constructed as follows. First, rpL32 promoter was cut out
from pL3A plasmid containing the genomic rpL32 gene (a
gift from Dr N. V. Tomilin, Institute of Cytology, Russian
Academy of Sciences) by AccI and SmaI and inserted into
pUC19 vector. Next, it was digested from pUC19 by
EcoRI, blunt ended by Klenow fragment of Escherichia coli
DNA polymerase I, digested by HindIII and inserted into a
pGL3basic plasmid containing the luciferase gene (Pro-

mega). The pCMVL plasmid containing the reporter gene
lacZ and encoding bacterial b-galactosidase driven by a
promoter of early human CMV genes have been described
previously [19]. pNFjBluc plasmid contains luciferase gene
driven by synthetic minimal promoter carrying five binding
sites for transcription factor NFjB was purchased from
Stratagene (La Jolla, CA, USA).
Cell culture
HepG2 cells were obtained from the Cell Culture Bank of
the Institute of Cytology, Russian Academy of Sciences.
Human embryonic fibroblast cells were obtained from the
Institute of Influenza, Russian Academy of Medical
Sergey V. Orlov et al. A novel human (GCC)
n
-binding protein
FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works 4857
Sciences. HepG2 cells and human embryonic cell lines were
grown in Dulbecco’s modified Eagle’s medium supple-
mented with 10% fetal bovine serum (Life Technologies,
Inc., Gaithersburg, MD, USA). All cultures were main-
tained at 37 ° C in a humidified 5% CO
2
atmosphere.
Nuclear extract preparation and EMSA
Nuclear extracts were prepared from cultured cells as
described previously [33] with slight modifications [19]. The
following synthetic oligonucleotides were purchased from
MWG Biotech (Ebersberg, Germany) (upper strands only):
rpL32 ()24 +11) fragment, 5¢-CTTGCGCGCCGCC
GCCGCCTCTTCCTTCTTCCTCG-3¢; ZF5 binding site,

5¢-CAGGTACCGCGCCTTCGCTGCCA-3¢; (GCC)
3
ele-
ment, 5¢ -AGCTTGCCGCCGCCTTCGA-3¢; (GCC)
9
ele-
ment, 5¢-AGCTTGCCGCCGCCGCCGCCGCCGCCGCC
GCCTTCGA-3¢; rpL32 pyrimidine motif ()6 +11),
5¢-CTCTTCCTTCTTCCTCG-3¢; lactoferrin binding site,
5¢-CTAGTGCAAGTGCCA-3¢. The synthetic oligonucleo-
tides were end-labeled with [c
32
P]ATP using T4 polynucleo-
tide kinase (Scientific Industrial Corp. SibEnzyme) and
annealed to form double stranded probes. EMSA was
performed as described previously [34]. Reaction mixtures
contained 100 mm NaCl, 10.5 mm Hepes, pH 7.9, 0.8 mm
Na
2
-EDTA, 0.15 mm Na
2
-EGTA, 0.8 mm dithiothreitol,
0.15 mm polymethanesulfonyl fluoride, 6 mm MgCl
2
,
0.25 mm ZnCl
2
, 150 lgÆmL
)1
of sonicated salmon sperm

DNA, 8000–80000 cpm of labeled probe (1–10 ng), and
2–20 lg of nuclear extracts. In the competition experiments,
unlabeled competitors were added to reaction mixture
together with labeled probe as indicated in the figure legends.
Southwestern assay
The procedure was performed using a previously described
protocol [35]. Briefly, crude nuclear protein extracts were
separated by electrophoresis on 10% SDS polyacrylamide
gels [36] and transferred onto nitrocellulose membrane
(0.4 lm; Amersham) via a semidry transfer unit. The pro-
teins on the membrane were denatured with 6 m guanidine
hydrochloride and then renatured by incubation in decreas-
ing concentrations of guanidine hydrochloride. The nitro-
cellulose membranes were then soaked in a Southwestern
buffer (100 mm NaCl; 10 mm Hepes, pH 7.9; 1 mm Na
2
-
EDTA; 1 mm dithiothreitol; 6 mm MgCl
2
; 0.25 mm ZnCl
2
;
15 lgÆmL
)1
of sonicated salmon sperm DNA; 5% nonfat
dry milk) for 1 h at room temperature. Labeled probe
(10
5
cpmÆmL
)1

) was added and incubated for 1 h at room
temperature. The membrane was washed to remove nonspe-
cifically bound DNA and subjected to autoradiography.
The relative molecular sizes of the proteins bound by
the probe were determined by comparison with
Kaleidoscope
TM
prestained protein standards (Bio-Rad,
Hercules, CA, USA).
Purification of the rpL32 fragment ()24 +11)-
binding proteins
Protein isolation was performed using a four-step purifica-
tion procedure consisting of preparative EMSA followed
by three-step DNA affinity chromatography. Crude
nuclear extract obtained from 5 · 10
12
HepG2 cells was
subjected to preparative EMSA. The binding reactions
were performed under the same conditions as an analytical
EMSA (see above), but contained 1–3 · 10
6
cpm of
32
P-labeled rpL32 fragment ()24 +11) as a probe. Elec-
trophoretic separation of DNA-protein complexes was car-
ried out in 5% nondenaturating polyacrylamide gels using
V. P. Kalinovski’s device for preparative gel electrophore-
sis [37]. Two-milliliter fractions were eluted in flowing
Tris-borate buffer (with a flow rate of approximately
12 mLÆh

)1
). Fractions containing DNA–protein complexes
were detected by measuring their radioactivity on a scintil-
lation counter (Beckman Coulter Inc., Fullerton, CA,
USA), concentrated and equilibrated in the EMSA bind-
ing buffer by ultrafiltration in Centricon YM-10 centrifu-
gal filter units (Millipore, Bedford, MA, USA; catalog
number 4205). Sorbent for DNA affinity chromatography
was prepared as follows. The rpL32 fragment ()24 +11)
was extended by oligo(T), biotin end-labeled with terminal
transferase (Roche Applied Science) and immobilized on
streptavidin-coated magnetic beads (Roche Applied Sci-
ence, catalog number 1641778) in accordance with the
manufacturer’s guidelines. Partially purified proteins
obtained in the previous stage were incubated with the
rpL32 fragment-coated beads in binding buffer (100 mm
NaCl, 10.5 mm Hepes, pH 7.9, 0.8 mm Na
2
-EDTA,
0.15 mm Na
2
-EGTA, 0.8 mm dithiothreitol, 0.15 mm poly-
methanesulfonyl fluoride, 6 mm MgCl
2
, 0.25 mm ZnCl
2
,
150 lgÆmL
)1
of sonicated salmon sperm DNA) for 1 h at

4 °C, washed three times in binding buffer, and the rpL32
binding proteins were eluted from the beads in 1 m NaCl
binding buffer. Next, eluted fractions were equilibrated in
binding buffer by ultrafiltration, as described above, and a
second round of DNA affinity chromatography was per-
formed. Finally, the double stranded oligonucleotides con-
taining the lactoferrin binding site were immobilized on
magnetic beads as described for the rpL32 fragment and
used as a nonspecific substrate to remove contaminant
nonspecific DNA-binding proteins. The unbound proteins
were considered to be the final fraction enriched with the
rpL32 fragment ()24 .+11) binding proteins. That frac-
tion was analyzed by SDS electrophoresis and compared
with the fraction captured by the lactofferin binding site
containing substrate (negative control).
MALDI-TOF assay
Proteins separated by one-dimensional gel electrophoresis
were in-gel digested as described elsewhere [38]. Samples
A novel human (GCC)
n
-binding protein Sergey V. Orlov et al.
4858 FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works
were prepared using the dried-droplet method. Peptide
samples (0.2–1 mL) were mixed with an equal volume of
2,5-dihydroxybenzoic acid solution (20 mgÆmL
)1
, Sigma)
containing 20% acetonitrile and 0.1% trifluoroacetic acid,
and the resulting droplets were air dried. Mass spectra of
trypsin-digested proteins were obtained for the mass

range of 500–2000 Da using a Reflex III MALDI-TOF
mass spectrometer (Bruker, Ettlingen, Germany). The
mass spectrometer was operated in the reflector mode
using previously described settings [39]. Mass spectra were
calibrated using known masses of internal standards [40].
Peptide peak lists were composed using bruker data
analysis software (Bruker Daltonik, Bremen, Germany).
Proteins were identified by the sets of proteolytic peptide
masses using the Peptide Fingerprint option in
Mascot software ( />html). The accuracy of MH
+
mass determination was
0.02% and the possible modification of cysteine residues
by acrylamide and methionine oxidation was taken into
consideration.
Isolation of human ZF5 cDNA and construction
of ZF5 expression vectors
cDNA containing the coding region of human ZF5 tran-
scription factor (1425 bp) was cloned by two step RT-
PCR. Total cellular RNA was isolated from HepG2 cells
using the guanidine isothiocyanate method [41]. After
digestion with RNase-free DNase I (Roche Applied
Science), RNA was reverse transcribed using an oligo(dT)
primer and reverse transcriptase (Invitrogen) to make the
first cDNA strand. Two pairs of primers, which amplify
overlapping 5¢- and 3¢-parts of ZF5 cDNA, were designed
using Primer software developed by V. Prutkovsky and
O. Sokur of the Institute of Influenza, Ministry of Health
of the Russian Federation. The first pair of primers
(5¢-GGCCTTCAAGGCATTAAG-3¢,5¢-AAACAAATGG

CCTGTCCG-3¢) spanned a 958 bp 5¢-part of the ZF5
coding region; the second pair (5¢-CCCCTCAAGCCTT
AACAT-3¢,5¢-TCTCCACTTTCCAGGCAA-3¢) spanned a
686 bp 3¢-part of the ZF5 coding region. Those two over-
lapping parts of ZF5 cDNA were amplified separately,
digested by BglI, ligated and reamplified by outer primers
(5¢-primer from first pair and 3¢-primer from second
pair). The resulting fragment was subcloned into pBlue-
script vector, and the ZF5 sequence was verified by
sequencing to exclude mutations that could have occurred
during amplification. The ZF5 eukaryotic expression vec-
tor with a full coding region of ZF5 driven by a CMV
promoter (pCMVZF5) was then constructed. ZF5 cDNA
was digested from pBluescript by XhoI, NotI, and
inserted into a pDSRed-N1 plasmid (ClonTech, Palo
Alto, CA, USA) digested by SalI and NotI in place of
the red fluorescent protein gene.
Purification of recombinant GST-ZF5 fusion
protein
Escherichia coli strain BL21 bearing the prokaryotic
expression vector pGEXZF5 encoding recombinant fusion
protein, which consists of glutathione S-transferase
(N-terminal part) and the DNA-binding domain of ZF5
(C-terminal part), was kindly provided by Dr S. Yamam-
oto, Hiroshima University. Bacteria were grown at 37 °C
to the late logarithmic phase (D
600 nm
¼ 1) and GST-ZF5
expression was induced by addition of 1 mm isopropyl-b-
d-galactothiopyranoside. The cells were harvested after

additional incubation for 5 h, and the recombinant
protein was purified by standard glutathione-agarose bead
binding procedures.
Cell transfection, b-galactosidase and luciferase
assays
HepG2 cells were seeded on 35 mm culture dishes at a
density of 10
4
cellsÆcm
)2
and grown to a subconfluent
layer in Dulbecco’s modified Eagle’s medium containing
10% fetal bovine serum, 5% CO
2
at 37 °C. Ca-phosphate
tranfection was performed as described previously [42].
Ten micrograms of DNA per dish was used in all experi-
ments. b-galactosidase assays were performed following
standard protocols, using O-nitrophenyl-b-d-galactopyr-
anoside as a substrate. Relative b-galactosidase activity
was calculated as D
420 nm
optical density per mg of total
protein in cell lysates per hour. Luciferase activity was
measured in a scintillation counter (Beckman Coulter,
Fullerton, CA, USA) employing a Luciferase Assay Sys-
tem (Promega, catalog number E4030) in accordance with
manufacturer’s guidelines. Luciferase activity is indexed in
relative light units that correspond to counts per min per
mg of total protein in cell lysates. Protein concentration

in cell lysates was measured by the Bradford assay.
RNA interference assay
Three siRNAs for human ZF5 mRNA were selected based
on general rules for siRNA selection [43]. The first
siRNA
ZF5
5¢-GGUUGAGGAUGUGAAAUUCUU-3¢ and
5¢-GAAUUUCACAUCCUCAACCUU-3¢) matched bases
192–210; the second siRNA
ZF5
(5¢-GAGGAAGCAUGA
GAAACUCUU-3¢ and 5¢-GAGUUUCUCAUGCUUCCU
CUU-3¢) matched bases 945–963; and the third siRNA
ZF5
(5¢-GGUCCUGAACUACAUGUACUU-3¢ and 5¢-GUAC
AUGUAGUUCAGGACCUU-3¢) matched bases 333–351
of the ZF5 mRNA sequence (accession number D89859).
siRNA
grp58
sequence (5¢-UAGUCCCAUUAGCAAAGG
UUU-3¢ and 5¢-ACCUUUGCUAAUGGGACUAUU-3¢)
was published previously [44] and used as a control. The
control siRNA sequence did not match any human mRNAs
Sergey V. Orlov et al. A novel human (GCC)
n
-binding protein
FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works 4859
(5¢-GACGCGGGAAAAAUUAAGCUU-3¢ and 5¢-GCUU
AAUUUUUCCCGCGUCUU-3¢). All RNAs were ordered
from Syntol Corp. (Moscow, Russia) as single stranded oli-

gonucleotides and annealed in 300 mm NaCl by heating at
85 °C for 15 min followed by gradual cooling to room tem-
perature. Transfection of HepG2 cells by siRNAs was per-
formed by RNAiFect reagent (Qiagen, Hilden, Germany)
or Oligofectamine reagent (Invitrogen Corp., Carlsbad, CA,
USA) in accordance with the manufacturer’s instructions.
Thirty nanomolar concentrations of siRNAs were used in
all experiments. Cells were harvested 48 h after transfection
and total RNA was isolated as described above. Semiquan-
titative RT-PCR was used to compare ZF5 and FMR1
mRNA levels in transfected cells. Reverse transcription was
performed as described above. The optimal numbers of
PCR cycles for the glyceraldehyde-3-phosphate dehydroge-
nase (GAPDH) housekeeping gene (primers 5¢-TCAC
CATCTTCCAGGAGCGA-3¢ and 5¢-TACTCCTTGGAG
GCCATGT-3¢) (18 cycles), ZF5 (primers 5¢ -CCCCTC
AAGCCTTAACAT-3¢ and 5¢-TCTCCACTTTCCAGGC
AA-3¢) (27 cycles), FMR1 (primers 5¢-GGGTGAGGATT
GAGGCTGA-3¢ and 5¢-GCCGTGCCCCCTATTTCT-3¢)
(34 cycles), and grp58 (primers 5¢-ATCTCCGACACG
GGCTCT-3¢ and 5¢-TGCTGGCTGCTTTTAGGAA-3¢
(27 cycles) were determined by preliminary PCR experi-
ments. All probes were normalized relative to GAPDH.
Statistical analysis
The results are presented as the mean ± SEM. The
statistical analyses of group differences were performed using
a nonpaired t-test. P<0.05 was considered statistically sig-
nificant. All statistical analyses were performed using the
program statistica 5.0 (StatSoft, Inc., Tulsa, OK, USA).
Acknowledgements

We thank D. Issakov for helpful discussion and proof-
reading, and S. Yamamoto for supplying bacteria
expressing the recombinant GST-ZF5 protein. This
work was supported by the Russian Fund for Basic
Research (grants 00-04-49426, 04-04-48691, 06-04-
48714) and by the Higher Education Ministry of the
Russian Federation (grant PD02-1.4-385).
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