Báo cáo khóa học: Isolation and characterization of the Xenopus HIVEP gene family ppt

Isolation and characterization of the
Xenopus
HIVEP gene family
Ulrike Du¨rr
1
, Kristine A. Henningfeld
1
, Thomas Hollemann
1
, Walter Kno¨ chel
2
and Tomas Pieler
1
1
Abteilung Entwicklungsbiochemie, Universita
¨
tGo
¨
ttingen, Germany;
2
Abteilung Biochemie, Universita
¨
t Ulm, Germany
The HIVEP gene family encodes for very large sequence-
specific DNA binding proteins containing multiple zinc
fingers. Three mammalian paralogous genes have been
identified, HIVEP1,-2 and -3, as well as the closely related
Drosophila gene, Schnurri. These genes have been found
to directly participate in the transcriptional regulation of a
variety of genes. Mammalian HIVEP members have been
implicated in signaling by TNF-a and in the positive selec-

tion of thymocytes, while Schnurri has been shown to be an
essential component of the TGF-b signaling pathway. In this
study, we describe the isolation of Xenopus HIVEP1,aswell
as partial cDNAs of HIVEP2 and -3. Analysis of the tem-
poral and spatial expression of the XHIVEP transcripts
during early embryogenesis revealed ubiquitous expression
of the transcripts. Assays using Xenopus oocytes map-
ped XHIVEP1 domains that are responsible for nuclear
export and import activity. The DNA binding specificity of
XHIVEP was characterized using a PCR-mediated selection
and gel mobility shift assays.
Keywords: DNA binding; Schnurri; Xenopus; zinc finger.
The HIVEP family of zinc finger proteins regulates a diverse
array of developmental and biological processes through
direct DNA binding, as well as interaction with other
transcription factors and components of signal transduction
pathways [1,2]. Representative members include three
human genes: HIVEP1 (also called ZAS1/Shn1/MBP1/
PRDII-BF1) [3–6], HIVEP2 (ZAS2/Shn2/Mbp2) [7,8] and
HIVEP3 (ZAS3/Shn3) [7,9], as well as the corresponding
mouse homologues aACRYBP1 [10,11], MIBP1 [12] and
KRC [13]. Schnurri (Shn), a distantly related ortholog from
Drosophila, which is most closely related to HIVEP1,has
also been isolated and characterized [14–16].
Typically, the large zinc finger (Znf) DNA binding
proteins have a molecular mass greater than 250 kDa and
contain two ZAS domains (N and C) that are widely
separated in the primary sequence [2,9]. Each ZAS domain
harbors a pair of DNA binding C
2

H
2
type zinc fingers
followed by an acidic domain located in close proximity to
a serine/threonine-rich sequence. Mammalian members of
the HIVEP family have been implicated in transcriptional
regulation via direct binding to cis-regulatory elements of
several genes, including p53 [17], IRF-1 [17], c-myc [12],
aA-crystallin [11], human immunodeficiency virus type1 long-
terminal repeat [4], somatostatin receptor type II [18] and the
metastasis-associated gene S100A4/mts1 [19].
The HIVEP family also has cellular regulatory activities
not associated with DNA binding. KRC was shown to
regulate the response of the TNF receptor to proinflamma-
tory stimuli via the interaction with the adapter TRAF2 [1].
In addition, knockout studies in mouse have demonstrated
that Shn2 plays a pivotal role in the positive selection of
thymocytes [20,21]. However, the molecular mechanism
for this observation remains undefined.
Drosophila Shn is the most functionally characterized
HIVEP member and has been shown to be essential for
signaling by the TGF-b superfamily ligand, decapentaplegic
(dpp), during anterior–posterior patterning of the wing [22].
Shn mutants mimic a large number of dpp loss-of-function
phenotypes and mutations in the Dpp-receptors tkv and
punt [15,16]. Cells that lack Shn do not respond to ectopic
Dpp [14,15,23]. In response to Dpp, Shn was found to form
a complex with Mad and Medea, the intracellular trans-
ducers of Dpp signaling [23,24]. Taken together, these
results suggest that Shn acts as a Mad/Medea coactivator

for Dpp-responsive genes. However, genetic studies have
demonstrated that the primary function of Shn is to repress
the transcription of brinker (brk), which serves as a repressor
for many Dpp-target genes [25,26]. Shn may also cooperate
with Mad/Medea to regulate additional Dpp-responsive
target genes [23]. A Dpp-regulated silencer element has
been identified that controls the expression of brk [27]. This
silencer is regulated directly by a complex consisting of
Mad/Medea and Shn. While the fundamental aspects of
TGF-b signaling are highly conserved and the requirement
of this pathway in embryonic patterning in both inverte-
brates and vertebrates is well established, a role for
vertebrate Shn related transcription factors in TGF-b
signaling is currently unknown. Moreover, it is also unclear
whether vertebrate HIVEPs regulate cellular events through
the repression of brk transcription, as vertebrate brk
homologs have not yet been identified.
Presently, we describe the isolation of one complete and
two partial cDNAs corresponding to three different HIVEP
Correspondence to T. Pieler, Abt. Entwicklungsbiochemie, Universita
¨
t
Go
¨
ttingen, Justus-von-Liebig Weg 11, 37077 Go
¨
ttingen, Germany.
Fax: + 49 551 3914614, Tel.: +49 551 395683,
E-mail:
Abbreviations: BMP, bone morphogenetic protein; BRE, BMP-4

response element; Dpp, decapentaplegic; NLS, nuclear localization
signal; Shn, Schnurri gene from Drosophila;TGF-b, transforming
growth factor-beta; ZAS, zinc finger, acidic, serine/threonine-rich;
Znf, zinc finger.
(Received 21 November 2003, revised 12 January 2004,
accepted 30 January 2004)
Eur. J. Biochem. 271, 1135–1144 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04017.x
related genes in Xenopus (XHIVEP1,-2 and -3). The
Xenopus XHIVEPs are characterized with respect to
temporal and spatial expression, nuclear import/export
activity and DNA binding specificity.
Materials and methods
Isolation and cloning of
Xenopus XHIVEP1,
-
2
and -
3
Screening of amplified cDNA libraries was performed by
PCR screening as described previously [28]. Approximately
1.9 · 10
6
plaque-forming units were screened. PCR was
performed in a final volume of 22.5 lL with 2.5 lL of phage
lysate as template, using the Gene Amp PCR Kit (Perkin
Elmer). Degenerate oligonucleotides initially used as pri-
mers were created by comparing the ZAS-C Znf from
different members of the HIVEP family: (upper primer
5¢-AARTAYATHTGYGARGARTGYGGIATHCG-3¢ and
lower 5¢-CAYTTYTTCATTRGIGCYTTIGYYTTCAT

RTG-3) resulting in the amplification of a 173 nucleotide
product. Individual positive clones were identified by serial
dilution of the positive phage fractions. The initial
XHIVEP1,-2 and -3 clones contained 2.3, 5.9 and 3.2 kb
of cDNA, respectively, in the pBKCMV vector.
The full length XHIVEP1 sequence was obtained by a
combination of additional phage screening and RT-PCR
amplification affording five partially overlapping cDNA
fragments of XHIVEP1. In the first amplification, a
degenerate primer (Shn amino acids 1552–1560) and a
XHIVEP1 gene specific primer set were used (5¢-GAR
GAYTGYTTYGCNCCNAARTAYCA-3¢ and 5¢-TCCA
CGGATGTACACATAC-3¢)toamplifya1.5kbproduct
from stage 34–38 Xenopus cDNA. In the second amplifica-
tion, the degenerate primer (HIVEP1 amino acids 971–980)
and a XHIVEP1 gene specific primer set derived from the
additional sequence obtained in the first amplification
(5¢-GARAAYTTYGARAAYCAYAARAARTTYTAYTG-3¢
and 5¢-AGTTCTAATGCTATGTTTGGATGC-3¢)affor-
ded a product of 1.7 kb. Additional screening of a Xenopus
cDNA phage library with primers derived from XHIVEP1
(5¢-TACTGGGGCATTAGAACAACCTT-3¢ and 5¢-GA
CATTTCACTTCCACTCTTTCTTG-3¢)resultedinthe
identification of two partially overlapping clones containing
3.5 kb and 3.9 kb of the 5¢ sequence of XHIVEP1. PCR-
amplified deletion mutants for transport experiments were
subcloned into pCS2+NLS-MT vector [29].
Semi-quantitative RT-PCR analysis
Total RNA from embryos and tissues was isolated by
phenol/chloroform extraction and LiCl precipitation [30].

The Qiagen RNeasy Kit was used for RNA isolation from
dissected gastrula stage embryos. All RNA samples were
treated with DNAse I (Boehringer Mannheim) and checked
by PCR for DNA contamination. RT-PCR was carried out
using the Gene Amp RNA PCR kit (Perkin Elmer), and
1 lCi of [
32
P]dCTP[aP] was included in each PCR. One-
tenth of the PCR products were separated on 6% polyacryl-
amide gels under denaturing conditions and analyzed using
a PhosphorImager (Molecular Dynamics). Primers and
conditions used for RT-PCR were as follows: XHIVEP1,
5¢-ATCCAGAGGCAGAAGCAG-3¢ and 5¢-CTGCATT
CAGAGTAAGCC-3¢,60°C, 29 cycles; XHIVEP2,
5¢-AAGCAGAGGAATGCAGTAG-3¢ and 5¢-AATGTC
TTTCTCTCCATGG-3¢,60°C, 29 cycles; XHIVEP3,
5¢-GCAGCACTATCCCTGCTAAG-3¢ and 5¢-TCCCTC
GTCCACGGCCTCTTACAT-3¢,60°C, 29 cycles. Further
oligonucleotides: Histone H4, 5¢-CGGGATAACATTCA
GGGTATCACT-3¢ and 5¢-ATCCATGGCGGTAACTG
TCTTCCT-3¢,60°C, 22 cycles; Xbra, 5¢-GGATCGTTAT
CACCTCTG-3¢ and 5¢-GTGTAGTCTGTAGCAGCA-3¢,
60 °C, 28 cycles; Gsc, 5¢-ACAACTGGAAGCACT
GGA-3¢ and 5¢-TCTTATTCCAGAGGAACC-3¢,60°C,
28 cycles; XWnt8, 5¢-TGTGGCCGGGTCTGAACTTA
TTTT-3¢ and 5¢-GTCATCTCCGGTGGCCTCTGTTCT-3¢,
60 °C, 28 cycles.
Microinjection of
Xenopus
oocytes and analysis

of nuclear transport
[
35
S]Methionine radiolabelled proteins were expressed from
cDNAs using the coupled transcription/translation (T
N
T)
system (Promega). In vitro translation products were
analyzed by SDS/PAGE and phosphoimaging (Molecular
Dynamics). Preparation of oocytes and microinjection assays
were performed as described in [31]. Immunoprecipitation
was performed as described [32]. Phosphatase treatment of
immunopellets was performed with 100 U of k-phosphatase
(NEB) per pellet for 1 h in the appropriate buffer.
In vitro
protein preparation
The Znf pair derived from the XHIVEP2 ZAS-N domain
(234 bp, amino acids GGFK…KCLE) was cloned in-frame
with the N-terminal His-tag of the pRSET vector (Invitro-
gen). Hexa-His-tagged ZAS-N Znf was expressed in
Escherichia coli BL21, induced with CE3 lysogen according
to manufacturer’s instructions (Stratagene). The fusion
protein was purified under native conditions using Ni/
nitrilotriacetic acid/agarose (Qiagen) according to the
manufacturer’s protocol. The purified protein was quanti-
fied by the Bradford method.
Electrophoretic mobility shift assays
DNA duplexes were labeled on the upper strands with
[
32

P]ATP[cP] and T4 polynucleotide kinase. The labeled
oligomers were annealed by heating to 90 °C an equimolar
mixture of the upper and lower strands in reaction buffer
and cooling slowly to ambient temperature (1 h). Sequences
of the upper strand of the duplexes are listed below.
Sites of mutation are underlined: wt, 5¢-AGAGAGAA
TGAGAGGCTTCCCAATAGC-3¢;mut1,5¢-AGAGAG
AATGA
TAGGCTTCACAATAGC-3¢;mut2,5¢-AGAG
AGAATGA
TAGGCTTCCCAATAGC-3¢;mut3,5¢-AGA
GAGAATGAGAGGCTTC
ACAATAGC-3¢.
Binding reactions were performed in a total volume of
50 lL containing 50 m
M
Tris/HCl, pH 8.0, 30 m
M
KCl,
10 m
M
MgCl
2
,30l
M
ZnCl
2
,1m
M
dithiothreitol, 10%

glycerol, 1 lg poly(dI-dC) and 100 lg BSA. The hexa-His-
tagged ZAS-N Znf concentration used was 84 or 214 ng.
The reactions were allowed to proceed for 30 min at 4 °C
and analyzed on a 12% native polyacrylamide gel contain-
ing 0.5· Tris-borate buffer (run at 300 V at 4 °C).
1136 U. Du
¨
rr et al.(Eur. J. Biochem. 271) Ó FEBS 2004
In vitro
selection
PCR-based site selection was performed essentially as
described [33] with bacterially expressed ZAS-N Znf and a
16 nucleotide degenerate DNA duplex. Binding reactions
were performed as described above. After seven rounds of
binding, recovery of shifted DNA and PCR, the targets
were cloned and sequenced.
Results
Isolation of
HIVEP
genes in
Xenopus
Xenopus embryonic tailbud stage head and tailtip cDNA
libraries [34] were screened for HIVEP related genes in a
PCR-approach using degenerate primers deduced from the
second Znf pair within Drosophila Shn.ThreecDNAsof
Xenopus HIVEP related genes XHIVEP1,-2 and -3 were
isolated. Overlapping clones covering 8578 bp of XHIVEP1
cDNA were obtained by RT-PCR on total embryonic
RNA using combinations of degenerate and specific primers
and by rescreening the cDNA libraries. The partial clones

of XHIVEP2 and -3 covered 5.9 and 3.2 kb of the respective
3¢ ends and included 3¢-UTRs and poly(A)-tails.
A GenBank search revealed highest homology of the
three deduced proteins, XHIVEP1, -2 and -3, with the
mammalian zinc finger proteins HIVEP1, -2 and -3,
respectively (Fig. 1). Similar to other vertebrate HIVEP
related proteins, the XHIVEP proteins lack the C-terminal
Znf triad found in Drosophila Shn, but exhibit between 75
and 92% homology to Znf pairs within the two ZAS
domains of Shn (Fig. 2). Compared to the corresponding
vertebrate proteins, the Xenopus ZAS Znf DNA binding
domains and the isolated Znf has between 96 and 100%
identity (Fig. 2). The regions outside these domains exhibit
lower sequence identities in a comparison of the three
vertebrate proteins (40–60%), although regions of higher
sequence conservation are distributed over the proteins,
including several serine-rich stretches [9].
The 8578 bp cDNA sequence of XHIVEP1 contains
242 bp of the 5¢-UTR, an open reading frame of 7734 bp
and 602 bp of the 3¢-UTR. The deduced 2578 amino acid
protein has two C
2
H
2
type Znf containing ZAS domains
and a single C
2
HC type Znf (Fig. 3). The reported start and
stop codons, as well as the Znf sequences of XHIVEP1,
correspond to those of mammalian HIVEP1. Overall amino

acid sequence identity between XHIVEP1 and the corres-
ponding human and mouse sequences is 50% and 70%,
respectively. XHIVEP1 is likely to be post-translationally
modified, as 10% of all amino acids constitute putative
target sites for a wide array of different Ser-, Thr- and
Tyr-kinases ().
Expression of
Xenopus HIVEP
transcripts
To determine if the different XHIVEP genes are differen-
tially expressed, their temporal mRNA expression patterns
Fig. 1. Structural organization of the XHIVEP proteins. Black bars
indicate the position of the ZAS C
2
H
2
zinc fingers (ZAS-N and ZAS-
C) and the isolated C
2
HC zinc finger (I-Znf). The ZAS zinc finger
DNA binding domains are followed by acidic domains indicated by
white bars. The Znf triad unique to Schnurri is indicated in the figure
by an asterisk. The level of sequence conservation between the
respective domains within Xenopus proteins and their human ortho-
logs are indicated as percentages. Within the Drosophila protein, the
relative positions of the oligomerization domain and a Mad interaction
domain are indicated. dm, Drosophila melanogaster;hs,Homo sapiens;
xl, Xenopus laevis;aa,aminoacids.

Fig. 2. Sequence comparison of conserved zinc fingers within HIVEP
type proteins. The predicted primary sequences of the Xenopus (xl)
HIVEP ZAS domain zinc fingers (ZAS-N and ZAS-C) and the iso-
lated zinc finger (I-Znf) are shown in comparison with the corres-
ponding domains from human (hs), mouse (mm), rat (rn) and fly (dm).
Amino acids involved in complex formation with zinc ions are marked
in bold and amino acids within zinc fingers that are likely to be
involved in DNA binding are boxed. Identical amino acids are
represented by dashes.
Ó FEBS 2004 Xenopus HIVEP gene family (Eur. J. Biochem. 271) 1137
were analyzed by semiquantitative RT-PCR analysis using
total RNA isolated from various stages of Xenopus
embryos. As shown in Fig. 4A, XHIVEP1,-2 and -3
transcripts are maternal and continue to be detected at
similar levels until the onset of gastrulation (egg until stage
10). Throughout late gastrula and neurula stages (stages 11–
20), expression decreases temporarily and increases again at
stage 24. In adult tissue, XHIVEP transcripts were detected
at comparable levels in all tissues examined (Fig. 4B).
Attempts to analyze the spatial expression of XHIVEP
mRNAs by whole mount in situ analysis on Xenopus
embryos revealed only a weak expression suggesting low
abundance of the transcripts (data not shown). As Schnurri
hasbeenimplicatedinTGF-b signaling, we further
investigated the spatial expression of the XHIVEPs during
gastrulation, when these signals play an essential role in
patterning of the mesoderm. Early gastrula stage embryos
were dissected and total RNA isolated from pools of seven
defined regions shown in Fig. 4C. Semiquantitative RT-
PCR analysis revealed that the three XHIVEP transcripts

are ubiquitously present at this stage. In comparison, the
mesodermal marker genes XWnt8, Xbra and Gsc showed
the expected restricted expression patterns (Fig. 4C) [35].
Mapping of
Xenopus
HIVEP1 import and export domains
Several members of the HIVEP family have been shown to
be nuclear transcriptional regulators [6,23,24]. In order to
analyze the in vivo subcellular localization of the 300 kDa
XHIVEP1 protein, we used the Xenopus oocyte system.
Myc-tagged fragments of the XHIVEP1 protein were
translated in vitro in the presence of [
35
S]methionine and
the radiolabelled protein fragments were microinjected
into Xenopus oocytes (stage V and VI). The XHIVEP1
fragments (F1–F5) that were used are shown schematically
in Fig. 5A. To evaluate import and export activity, the
protein fragments were injected into the cytoplasm or the
nucleus, respectively. At different time points, nuclear and
Fig. 3. Amino acid sequence of the XHIVEP1
protein. The protein sequence of XHIVEP1
was predicted from five overlapping cDNA
fragments (GenBank Accession number
AY363297). Zinc finger domains are shaded in
gray and acidic-rich regions are boxed in
black. The serine-rich regions are underlined
with dashed lines. Putative nuclear localiza-
tion signals are underlined in black and the
putative nuclear export signal is underlined

with a dotted line.
1138 U. Du
¨
rr et al.(Eur. J. Biochem. 271) Ó FEBS 2004
cytoplasmic fractions were prepared, the labeled proteins
immunoprecipitated, resolved by SDS/PAGE and visual-
ized by autoradiography.
To evaluate for import activity, the labeled proteins were
injected into the cytoplasm (Fig. 5B, Import). As shown
in the control at time 0 h, labeled protein fragments are
detected only in the cytoplasm, demonstrating appropriate
targeting. F4 and F5 were maintained exclusively in the
cytoplasm, even after 24 h. In contrast, the amino-terminal
protein F1 was strongly imported to the nucleus. The
internal fragments F2 and F3 were also imported to the
nucleus, albeit weakly, with both fragments detected
predominately in the cytoplasm even after 24 h. Moreover,
the F3 protein band appeared as a blurred band after
isolation from the Xenopus oocyte, suggesting post-trans-
lational modification such as phosphorylation of the
fragment. Correspondingly, treatment of the immuno-
precipitated proteins with k-phosphatase prior to loading
on the gel resolved the blur into a sharp band.
To identify fragments containing nuclear export activity,
the labeled proteins were injected into the nucleus (Fig. 5B,
Export). The F2 and F3 proteins, which exhibited weak
import activity, were not exported from the nucleus. The
strongly imported N-terminal F1 protein displayed weak
export activity. In contrast, the C-terminal F4 and F5 were
strongly exported from the nucleus, with 50% of the labeled

protein becoming cytoplasmic after 6 h and exclusively
located in the cytoplasm after 24 h.
DNA binding specificity of
Xenopus
HIVEP
Schnurri proteins are known to have DNA binding
activity; therefore, preferential DNA binding sites of a
Znf pair derived from the XHIVEP2 ZAS-N domain was
determined in a PCR based in vitro site selection assay
[33]. As the amino acids that confer DNA binding
specificity are conserved among the XHIVEPs (Fig. 2), it
is therefore anticipated that they have similar DNA
binding activities. A duplex DNA library, containing 16
base pairs of degenerate sequence flanked by known
sequences that contained restriction sites and served as
primer binding sites, was used as a substrate for the
bacterially expressed ZAS-N Znf in electrophoretic mobi-
lity shift assays. The protein–DNA complexes were
recovered from the gel and used in successive rounds of
amplification and selection.
After seven rounds, the selected DNA duplexes were
subcloned and 49 clones were sequenced (Fig. 6A). In all of
the clones analyzed, a CCC trinucleotide was present. Many
sequences also contained a TG or TT dinucleotide imme-
diately upstream from the invariant CCC sequence and
displayed a preference for GC-rich sequences upstream of
this motif. The isolated pool was also enriched in sequences
having a GAGA or GACCG. These motifs were often
overlapping and the GAGA and GACCG sequences were
located with a variable distance of 6–8 nucleotides and 3–4

nucleotides, respectively, upstream of the invariant CCC
trinucleotide. In addition, one sequence was represented five
times (Fig. 6A).
The finding that HIVEP members directly interact with
members of the Smad family, led us to investigate TGF-b
responsive elements for Shn binding sites [21,23,24]. One
well characterized TGF-b responsive promoter is that of
Xvent-2B [36,37]. In vitro DNase I footprinting experi-
ments demonstrated that ZAS-N Znf protected the region
between )280 and )260 located at the 5¢-end of the bone
morphogenetic protein (BMP)-4 response element (BRE)
(data not shown). This BRE has previously been shown
to contain Smad1 and Smad4 binding sequences and is
sufficient to drive expression in the early Xenopus embryo
in a similar manner to that of the endogenous gene
[36,37]. The protected region within the characterized
BRE of the Xvent-2B promoter (Fig. 6B) resembles
preferred sequences identified by in vitro selection. This
sequence has, at its core the invariant CCC and an
upstream GAGA box. To evaluate the contribution of
these elements to ZAS-N Znf binding, mutations were
created in either the GAGA box or the trinucleotide CCC
sequences in a 27-mer duplex spanning the protected
region. The binding of the ZAS-N Znf to the mutated and
the corresponding wild type duplexes was evaluated in
electrophoretic mobility shift experiments (Fig. 6B). While
ZAS-N Znf bound strongly to the wild type duplex,
binding was completely abolished in the duplex containing
mutations in both the GAGA and the CCC sequences
Fig. 4. Temporal and spatial expression of XHIVEP mRNAs. Semi-

quantitative RT-PCR analysis was performed with RNA isolated from
staged embryos (A), adult organs and tissues (B) and dissected regions
of stage 10 embryos (C) making use of primers specific for either
XHIVEP1, XHIVEP2, XHIVEP3, Wnt8, Gsc, Xbra or Histone H4.
Abbreviations: a, animal pole; bl, bladder; br, brain; d, dorsal; E,
embryo;e,egg;ey,eye;fa,fattissue;gu,gut;he,heart;in,intestines;
ki, kidney; l, lateral; li, liver; lu, lung; mu, muscle; ov, ovary; ph,
pharynx; -RT, without reverse transcriptase; sc, spinal chord; sk, skin;
sp, spleen; te, testis; v, ventral.
Ó FEBS 2004 Xenopus HIVEP gene family (Eur. J. Biochem. 271) 1139
(Fig. 6B, compare lanes 2 and 3 with 5 and 6). Mutation
of the GAGA motif only slightly altered the ZAS-N Znf
binding compared with the wild type duplex (Fig. 6B;
compare lanes 2 and 3 with 8 and 9). In contrast, the
mutation of the CCC alone significantly disrupted binding
demonstrating the essential contribution of this motif for
binding (Fig. 6B; lanes 11 and 12).
Discussion
The central components of the TGF-b pathway, including
ligands, receptors and intracellular signaling molecules, are
highly conserved. In vertebrates as well as in insects, TGF-b
signaling is crucial during early patterning of the embryonic
mesoderm. The finding that in Drosophila, the large nuclear
multizinc finger transcription factor Schnurri, related to
the vertebrate HIVEP family, functions to interpret the
intracellular signaling of Dpp, prompted us to analyze a
functional conservation of Schnurri related proteins in
vertebrates [26].
In a homology screen, we identified three Xenopus laevis
HIVEP related cDNAs, XHIVEP1,-2 and -3, which show

high similarity with the corresponding mammalian HIVEP
genes. The overall structure of the three Xenopus proteins
with their respective orthologs from vertebrates is well
conserved (Fig. 1), while sequence conservation outside the
Znf domains and in a number of other regions is much
lower, even among the mammalian orthologous proteins.
HIVEP and the Drosophila Schnurri proteins contain two
pairs of C
2
H
2
Znf and, with the exception of the HIVEP2
family, a conserved C
2
HC-type Znf. In addition, Drosophila
Schnurri contains a conserved carboxyl-terminal Znf triplet
that is not found in the vertebrate members. Sequence
conservation between vertebrate HIVEP and the Drosophila
Schnurri proteins is generally low with the exception of the
two ZAS domains, which are highly conserved (Fig. 2). An
additional stretch of 31 amino acids in XHIVEP1, located
between the ZAS-N and isolated zinc fingers (amino acids
703–733), is also weakly conserved between HIVEP1/2 and
Drosophila Schnurri proteins. A larger protein fragment of
Schnurri that contains this sequence element was shown to
form homo-oligomers in vitro [24]. Our data indicate that
the HIVEP/Shn protein family has retained remarkable
conservation in their overall structure as well as in the
sequence of specific domains in different vertebrate species.
Accumulating experimental evidence supports that the

HIVEP proteins are nuclear transcription factors. Droso-
phila Schnurri was localized in the nucleus after transfection
of COS cells [23,24], and the endogenous human HIVEP
(PRDII-BF1) protein was detected in the nucleus of MG63
cells [6]. Visual inspection of the full length XHIVEP1
protein revealed the presence of five classical nuclear
localization signal sequences (NLS1–5) of the SV40
type with the basic core sequence K(K/R)X(K/R) [38]
(Fig. 5A,C). All of the classical NLSs, with the exception of
NLS1, are conserved between mammalian and Xenopus
Fig. 5. Delineation of nuclear import and export domains within XHI-
VEP1. (A) Schematic representation of the full length XHIVEP1 and
the Myc-tagged (MT) deletion mutants. Black and white boxes indi-
cate the position of the Znf domains and the serine-rich stripe,
respectively. The relative positions of putative nuclear localization
signals (NLSs) are indicated by an asterisk and the nuclear export
signal by a plus symbol. The amino acid residues contained in each
fragment are indicated to the right of each mutant. (B) To map nuclear
transport regulatory domains within XHIVEP1,
35
S-labeled XHI-
VEP1 deletion mutants were produced in vitro and microinjected into
the nucleus or the cytoplasm of Xenopus oocytes. Immediately, or after
an incubation of 6 or 24 h, nuclear (N) and cytoplasmic (C) fractions
were manually separated and analyzed for XHIVEP1 protein content
by immunoprecipitation and SDS/PAGE. (C) Sequence comparison
of five putative NLS sequences of the SV-40 type within the XHIVEP1
protein. Consensus sequences for the NLS are shaded and basic amino
acids are indicated in bold. NLS1–5 contain classical NLS character-
ized by a K(K/R)X(K/R) consensus sequence. The bipartite sequence

contains two adjacent basic amino acids followed by a spacer con-
taining 10 amino acids and at least three basic residues in the subse-
quent five positions (NLS6 and 7). Position of the terminal amino acid
for each of the depicted sequences is indicated to the right of each
sequence. (D) Amino acid sequence of a hydrophobic putative nuclear
export signal sequence within XHIVEP1. Hydrophobic amino acids
are indicated in bold. The position of the terminal amino acid for the
depicted sequence is indicated to the right.
1140 U. Du
¨
rr et al.(Eur. J. Biochem. 271) Ó FEBS 2004
HIVEP1 proteins. Also found within the XHIVEP1
sequence, are two bipartite NLS motifs that are not present
in the corresponding mammalian XHIVEP1 proteins
(NLS6 and 7). This NLS motif is characterized by a stretch
of DNA containing two adjacent basic amino acids (K or
R) followed by a spacer of 10 residues and at least three
basic residues in the five subsequent positions [39].
Using labeled XHIVEP1 protein fragments in nuclear
import and export assays in the Xenopus oocyte, we were
able to gain further insights into the regulation of
HIVEP1 subcellular localization. While an amino-terminal
fragment (F1) containing four putative NLSs was strongly
imported into the nucleus, the internal fragments F2 and
F3, which harbored one and two putative NLSs, respect-
ively, were only weakly imported (Fig. 5B). Interestingly,
NLS4 is located adjacent to a serine-rich sequence element
that may have caused phosphorylation of protein frag-
ment F3 in the oocyte (Fig. 5B). The close proximity of
the NLS to the serine-rich region suggests that it may be

regulated by phosphorylation. Site-directed mutagenesis of
the putative NLS should unambiguously identify the
motifs that are responsible for XHIVEP1 nuclear local-
ization. It is however, apparent from the deletion studies
that multiple motifs are capable of localizing XHIVEP1 to
the nucleus.
Experiments in which the protein fragments were injected
into the nucleus revealed that two overlapping fragments
of the carboxyl terminus of XHIVEP1 (F4 and F5)
were strongly exported from the nucleus. Nuclear export
signals are frequently composed of hydrophobic leucine-
rich sequences [40–42]. Within the carboxyl terminus of
XHIVEP1 (F4 and F5), a hydrophobic stretch of 21 amino
acids length could be identified that contains a high content
of leucine and isoleucine residues (Fig. 5D). This region is
also conserved in the mouse and human HIVEP1 proteins.
The presence of import as well as export activity, located
at opposite ends of HIVEP1, could enable the protein
to undergo nucleocytoplasmic shuttling. Post-translational
modification at numerous phosphorylation sites may also
regulate the localization of the protein.
At the mid-blastula transition, TGF-b ligands, their
receptors and Smad mRNAs are ubiquitously expressed,
and their expression patterns are refined during gastrulation
in those regions where the corresponding pathways are
active [44,45]. We found XHIVEP mRNAs to be expressed
maternally and maintained until the onset of gastrulation,
at which point they are distributed equally throughout
the embryo. The corresponding proteins can therefore be
expected to be present at the right time and place to function

as mediators of TGF-b signaling during mesoderm pat-
terning events. Consistent with a function of HIVEP
members in regulating TFG-b signaling is the finding that
both vertebrate and invertebrate proteins can associate with
the Smads [21,23,24]. While we were not able to obtain
Fig. 6. Sequence-specific binding by XHIVEP. (A) Comparison of target sites for the Znf pair derived from the XHIVEP2 ZAS-N domain, as
determined by PCR site selection using a DNA duplex degenerate in 16 positions, flanked by sequences for PCR amplification. After seven cycles,
the DNA sequences were cloned. In total, 49 clones were sequenced and aligned in reference to the CCC trinucleotide that was found in all
sequences (left). The bars indicate the frequency of the nucleotides at each position. On the right, the abundance of specific sequences upstream of
the CCC is indicated. In addition, one sequence that was identified five times is shown. (B) Specific binding of ZAS-N Znf to the BMP-4 response
element of the Xvent-2B promoter. Nucleotides of the Xvent-2B promoter protected by ZAS-N Znf in DNase I footprinting experiments are
underlined in the wild type (wt) duplex and the GAGA and CCC sequences are boxed in gray. The nucleotides that were mutated are indicated by
unfilled boxes. DNA electrophoretic mobility shift analysis comparing ZAS-N Znf binding to a wild type 27 bp duplex spanning the protected
region of the Xvent-2B promoter and with that of the same duplex containing a mutation in either the GAGA box or CCC trinucleotide motifs are
shown on the right.
Ó FEBS 2004 Xenopus HIVEP gene family (Eur. J. Biochem. 271) 1141
reproducible in vivo interaction data between XHIVEP1
andSmadproteins,weobservedanin vitro interaction
with
35
S-labeled XHIVEP1 and bacterially expressed
GST-Smads (data not shown).
The DNA binding specificity of the XHIVEPs was
evaluated in a PCR site selection experiment using a Znf
pair derived from the XHIVEP2 ZAS-N domain (ZAS-N
Znf). All 49 clones that were sequenced contained a CCC
trinucleotide. We also observed a preference for a TG or TT
dinucleotide immediately upstream of the invariant CCC
sequence. The isolated pool was also enriched in sequences
having a GACCG or GAGA motif at a variable distance

from the CCC trinucleotide. Most vertebrate HIVEP
proteins and Drosophila Schnurri were also shown to bind
GC-richsequencesrelatedtotheNFjB related enhancer
motifs with the consensus target sequence GGG(N)
4)5
CCC
[13]. Such sequences are present in cis-regulatory regions of
promoters involved predominantly in immune response,
and the HIVEP1 protein has been shown to activate
transcription of the human immunodeficiency virus
enhancer in human [46] and the aA-crystallin gene in the
mouse [11]. However, many of the HIVEP Znfs have
also been shown to bind additional unrelated sequences.
HIVEP3/KRC Znf has been shown to exhibit dual DNA
binding specificity, binding to both the NFjB related
enhancer and to the V(D)J recombination signal sequence
elements [47–49].
With the intention to search TGF-b responsive promoter
elements in Xenopus for XHIVEP binding sites, we could
identify an optimal target site for the ZAS-N Znf that
closely resembles the known mammalian consensus sites.
An element that is similar to the one identified in vitro was
found within the BRE of the Xvent-2B promoter and is
located adjacent to the immediate BMP-responsive region
of the 5¢ flanking region of Xvent-2B [37]. DNase I footprint
analysis and gel shift assay with the wild type and a mutated
duplex confirmed the specific binding of ZAS-N Znf to this
sequence and demonstrated the essential nature of the CCC
sequence for DNA binding.
The physiological relevance of the interaction of

XHIVEP with the Xvent-2B promoter is not known.
Luciferase reporter assays with the BRE and the corres-
ponding mutations that disrupt ZAS-N Znf binding dem-
onstrated that during gastrulation the reporter was still
responsive to BMP signaling (data not shown). While the
mutations were sufficient to disrupt binding of the zinc
finger pair, they may not be capable of inhibiting binding of
the full length protein. Additionally, it has been shown in
Drosophila, that the Dpp-mediated early patterning of the
dorsal-ventral axis is independent of Schnurri activity [26].
To analyze the function of XHIVEP transcription factors
in BMP signaling in the Xenopus embryo in more detail, we
performed in vitro transcription of the full length 300 kDa
XHIVEP1 for use in microinjection experiments (data not
shown). Unfortunately, premature in vitro transcription
termination events at several distinct sites within the 8 kb
synthetic mRNA led to the production of predominately
truncated mRNAs. Thus, there was an insufficient quantity
of full length mRNA transcripts for microinjection experi-
ments. Attempts to eliminate the termination sites by silent
mutations in the affected regions were not successful. We
have also performed injection experiments with mRNA
encoding fusions of ZAS-N Znf to VP16 activator and En
repressor domains to analyze the function of XHIVEP in
the context of Xenopus embryogenesis (data not shown).
However, the interpretation of the in vivo role of XHIVEP
was not conclusive as the activator and repressor fusion
constructs gave similar effects in various functional assays.
Therefore, to gain further understanding of the function
of the extremely large XHIVEP1 by over expression in

Xenopus embryos, it may be necessary to create specific
dominant negative and constitutively active constructs by
the generation of deletion mutants containing discrete
functional domains of XHIVEP1. Thus, the cloning and
characterization of the XHIVEP interacting factors would
be of interest and should also provide additional insight into
the function of this protein in early development and further
elucidate its role in TGF-b signaling in the vertebrate
embryo.
Acknowledgements
The authors would like to thank Susanne Loop for assistance in the
transport experiments, Dr Sepand Rastegar for performing promoter
reporter assays, and acknowledge the technical assistance of Y. Harbs.
This work was supported by funds from the Deutsche Forschungs-
gemeinschaft to T. P. (SFB 523-A1) and W. K. (SFB 497-A1).
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