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

The HIVEP family of zinc finger proteins regulates a diverse array of developmental and biological processes through direct DNAbinding, as well as interaction with other transcription fac

Isolation and characterization of the Xenopus HIVEP gene family Ulrike Du¨rr1, Kristine A Henningfeld1, Thomas Hollemann1, Walter Kno¨chel2and Tomas Pieler1

1 Abteilung Entwicklungsbiochemie, Universita¨t Go¨ttingen, Germany; 2 Abteilung Biochemie, Universita¨t Ulm, Germany

The HIVEP gene family encodes for very large

sequence-specific DNAbinding 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, as well

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 DNAbinding specificity of XHIVEPwas characterized using a PCR-mediated selection and gel mobility shift assays

Keywords: DNAbinding; Schnurri; Xenopus; zinc finger

The HIVEP family of zinc finger proteins regulates a diverse

array of developmental and biological processes through

direct DNAbinding, 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) DNAbinding

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 DNAbinding C2H2 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

type1long-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 DNAbinding 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] Shnmutants 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] ADpp-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 verteinverte-brates 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: tpieler@gwdg.de

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)

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 DNAbinding specificity

Materials and methods

Isolation and cloning ofXenopus XHIVEP1, -2 and -3

Screening of amplified cDNAlibraries was performed by

PCR screening as described previously [28] Approximately

1.9· 106 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¢) to amplify a 1.5 kb product

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

cDNAphage library with primers derived from XHIVEP1

(5¢-TACTGGGGCATTAGAACAACCTT-3¢ and 5¢-GA

CATTTCACTTCCACTCTTTCTTG-3¢) resulted in the

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 RNAfrom embryos and tissues was isolated by

phenol/chloroform extraction and LiCl precipitation [30]

The Qiagen RNeasy Kit was used for RNAisolation from

dissected gastrula stage embryos All RNA samples were

treated with DNAse I (Boehringer Mannheim) and checked

by PCR for DNAcontamination RT-PCR was carried out

using the Gene Amp RNA PCR kit (Perkin Elmer), and

1 lCi of [32P]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¢,

60C, 28 cycles; Gsc, 5¢-ACAACTGGAAGCACT GGA-3¢ and 5¢-TCTTATTCCAGAGGAACC-3¢, 60 C,

28 cycles; XWnt8, 5¢-TGTGGCCGGGTCTGAACTTA TTTT-3¢ and 5¢-GTCATCTCCGGTGGCCTCTGTTCT-3¢,

60C, 28 cycles

Microinjection ofXenopus oocytes and analysis

of nuclear transport [35S]Methionine radiolabelled proteins were expressed from cDNAs using the coupled transcription/translation (TNT) 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 coliBL21, 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 DNAduplexes were labeled on the upper strands with [32P]ATP[cP] and T4 polynucleotide kinase The labeled oligomers were annealed by heating to 90C 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 AATGATAGGCTTCACAATAGC-3¢; mut2, 5¢-AGAG AGAATGATAGGCTTCCCAATAGC-3¢; mut3, 5¢-AGA GAGAATGAGAGGCTTCACAATAGC-3¢

Binding reactions were performed in a total volume of

50 lL containing 50 mM Tris/HCl, pH 8.0, 30 mM KCl,

10 mM MgCl2, 30 lM ZnCl2, 1 mM 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 4C and analyzed on a 12% native polyacrylamide gel contain-ing 0.5· Tris-borate buffer (run at 300 V at 4 C)

In vitro selection

PCR-based site selection was performed essentially as

described [33] with bacterially expressed ZAS-N Znf and a

16 nucleotide degenerate DNAduplex Binding reactions

were performed as described above After seven rounds of

binding, recovery of shifted DNAand PCR, the targets

were cloned and sequenced

Results

Isolation ofHIVEP genes in Xenopus

Xenopusembryonic 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 Three cDNA s of

Xenopus HIVEPrelated genes XHIVEP1, -2 and -3 were

isolated Overlapping clones covering 8578 bp of XHIVEP1

cDNAwere obtained by RT-PCR on total embryonic

RNAusing combinations of degenerate and specific primers

and by rescreening the cDNAlibraries 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

AGenBank 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 cDNAsequence 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 C2H2 type Znf containing ZAS domains and a single C2HC 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 (http://www.expasy.org)

Expression ofXenopus HIVEP transcripts

To determine if the different XHIVEP genes are differen-tially expressed, their temporal mRNAexpression patterns

Fig 1 Structural organization of the XHIVEP proteins Black bars

indicate the position of the ZAS C 2 H 2 zinc fingers (N and

ZAS-C) and the isolated C 2 HC zinc finger (I-Znf) The ZAS zinc finger

DNAbinding 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, amino acids.

Fig 2 Sequence comparison of conservedzinc 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 DNAbinding are boxed Identical amino acids are represented by dashes.

were analyzed by semiquantitative RT-PCR analysis using

total RNAisolated 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

has been implicated in TGF-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 RNAisolated 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 ofXenopus 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 [35S]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 acidsequence 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.

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 ofXenopus HIVEP Schnurri proteins are known to have DNAbinding activity; therefore, preferential DNAbinding 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 Aduplex DNAlibrary, 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–DNAcomplexes 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 andspatial expression of XHIVEP mRNAs

Semi-quantitative RT-PCR analysis was performed with RNAisolated 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, fat tissue; 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.

(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

HIVEPrelated 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 C2H2Znf and, with the exception of the HIVEP2 family, a conserved C2HC-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 DrosophilaSchnurri proteins Alarger 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-philaSchnurri 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 andexport 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.

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 DNAcontaining 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 DNAduplex degenerate in 16 positions, flanked by sequences for PCR amplification After seven cycles, the DNAsequences 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 DNAelectrophoretic 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.

reproducible in vivo interaction data between XHIVEP1

and Smad proteins, we observed an in vitro interaction

with 35S-labeled XHIVEP1 and bacterially expressed

GST-Smads (data not shown)

The DNAbinding 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-rich sequences related to the NFjB related enhancer

motifs with the consensus target sequence GGG(N)4)5CCC

[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 DNAbinding

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

XHIVEP1for use in microinjection experiments (data not

shown) Unfortunately, premature in vitro transcription

termination events at several distinct sites within the 8 kb

synthetic mRNAled to the production of predominately

truncated mRNAs Thus, there was an insufficient quantity

of full length mRNAtranscripts 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). References

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