Gene, 116 (1992) 195-203 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

195

GENE 06476

DNA-binding activity of the murine homeodomain protein Hox-2.3 produced by a hybrid phage T7/vaccinia virus system (Homeobox; DNA-protein interaction; transcription factor; Southwestern blots; footprint analysis; autoregulation)

Ron de Jong a, Lia de Laaf a, Harry Vennema b and Frits M e i j l i n k a u Hubrecht Laboratory. 3584 CT Utrecht (The Netherlands): and h Institute for Virology. Yalelaan 1. University of Utrecht. 3508 TD Utrecht (The Netherlands) Tel. (31-30)532 486

Received by H. van Ormondt: ! 5 November 199 i, Revised/Accepted: 8 January/! 6 January 1992; Receiveed at publishers: 2 March 1992

SUMMARY

Homeobox-containing genes encode transcription factors that, via the homeodomain, bind specifically to DNA. To study the DNA-binding properties of the murine homeodomain-containing protein, Hox-2.3, a hybrid expression system was used, combining gene expression by recombinant vaccinia virus (reW) with bacteriophage T7 transcription. Expression was achieved by co-infecting HeLa cells with two reWs, one expressing the T7-RNA polymerase-encoding gene directed by the W promoter, P7.5, and another containing the Hox-2.3 coding sequence under control of a T7 promoter [Fuerst et al., Mol. Cell. Biol. 7 (1987) 2538-2544]. Co-infected HeLa cells produced large amounts of full-length Hox-2.3 protein. Cytoplasmic and nuclear extracts from these cells were used to examine DNA-binding specificity in vitro, reW-produced Hox-2.3 protein bound to oligos that contained one or several copies of the common homeodomain-binding site, 5'-TCAATTAAAT, and to a lesser extent to multiple (TAA) repeats. Using Southwestern blot analysis, no Hox-2.3-binding sites were detected in a region of the Hox-2 cluster containing the Hox-2.3, Hox-2.4 and Hox-2.5 genes.

INTRODUCTION

Many regulatory genes in Drosophila melanogaster that are involved in the processes underlying the laying down of the bodyplan, including embryonic axis formation and var-

Correspondence to: D~r. F. Meijlink, Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht ('l'he Netherlands)Tel. (31-30)510 21 I; Fax (31-30)516 464. Abbreviations: aa, amino acid(s): Amp, Antennapedia: BSA, bovine serum albumin: bp. base pair(s): cDNA, DNA complementary to RNA; dATP. deoxyATP: DNase l. deox)ribonuclease !: D'I'L dithiothreitol, Exolll, E. co/i exonuclease I|l: FCS. fetal calf serum: Hepes. 4-(2-hydroxyethyl)l-piperazine-ethanesulfonic acid: Hox-2.3, protein product of the mouse Hox-2.3 gene; kb. kilobase(s) or 1000 bp: NCS. normal calf serum: NP-

ious aspects of segmentation, have a conserved 180-bp sequence, the homeobox, in common (Gehring, 1987; Ingham, 1988; McGinnis etal., 1984; Scott and Weiner, 1984). Many homologs of these Drosophila homeoboxcontaining genes have been found in higher eukaryotic ge-

binding site (NP site), see section e and Table !!; NP-40, Nonidet P-40; nt. nucleotide(s); ofigo, oligodeoxyribonucleotide; P, promoter; PAGE, polyacrylamide-gel electrophoresis; PBS. phosphate-buffered saline (10 mM Na2HPO4/1.8 mM KH,PO4 pH 7.4/0.137 M NaCI/2.7 mM KCI): PMSF, phenylmethylsulfonyl fluoride; Poilk, Klenow (large) fragment of E. coil DNA pol~merase !; pfu. plaque-forming unit(s); p.i.. post infection: re. recombinant; SDS, sodium dodecyl sulfate; TBE, 0.089 M Tris. HCI/ 0.08 M boric acid/0.002 M Na:.EDTA; TK, thymidine kinase; ~sp. transcription start point(s); W . vaccinia ~irus; ~1, wild type.

196 nomes and may be equally important in controlling developmental events (Deschamps and Meijlink, 1991; Scott et al., 1989). The homeodomain, encoded by the homeobox, has sequence similarity with domains in certain yeast mating-type proteins and prokaryotic DNA-binding proteins that interact specifically with DNA via a helix-turn-helix motif (Laughon and Scott, 1984). DNA-binding studies in vitro using bacterially expressed Drosophila homeodomain proteins confirmed that the homeodomain binds DNA in a sequence-specific manner (Beachy et al., 1988; Desplan et al., 1985, 1988; Driever et al., 1989; Hoey and Levine, 1988; Hoey et al., 1988; MOiler et al., 1988; Nelson and Laughon, 1990), while formal proof for the existence of the helix-turn-helix motif was obtained by NMR studies performed with Antennapedia (Antp) homeodomain (Otting et al., 1988) and by X-ray crystallography of engrailed homeodomain-DNA complexes (Kissinger et al., 1990). Several Drosophila homeodomain proteins transiently expressed in cells in culture have been shown to act as trans-acting factors, regulating target gene transcription either positively or negatively (reviewed in Hayashi and Scott, 1990). Binding sites of several vertebrate homeodomain proteins including XIHboxl (Cho et al., 1988), Hox-l.3 (Odenwald et al., 1989), Chox-l.4 and Chox-a (Sasaki et al., 1990) were characterized by in vitro DNA-binding experiments. In addition, a homeodomain is present in several vertebrate tissue-specific transcription factors including some proteins sharing another conserved sequence, the POU-specific domain (Herr and Sturm, 1988). The Hox-2.3 gone (Meijlink et al., 1987) belongs to the family of Antp-related homeobox genes, known as HOX genes. HOX genes resemble Drosophila homeotic genes in their sequence and genomic arrangement. Both gone families have similar expression patterns characterized by overlapping domains along the anteroposterior axis, suggesting an analogous function in embryogenesis (Akam. 1989; Deschamps and Meijlink, 1991; Duboule and Doll~, ~ 1989; Graham et al., 1989). Our goal was to study the DNA-binding activity of the Hox-2.3 protein, Since transcription factors are lowabundance proteins, their study requires an expression system to provide enough protein. The vaccinia virus has been proven to be a suitable eukaryotic expression vehicle, capable of expressing a spectrum of foreign proteins that are correctly folded, processed, post-translationally modified, and are biologically active (Mackett et al., 1985; Piccini et al., 1987). Here we report the use of the hybrid phage TT/reVV transient expression system described by Fuerst et al. (1987) as a method to synthesize large amounts of Hox-2,3 protein. Furthermore, we show that Hox-2.3 can specifi-

cally recognize previously known homeodomain-binding sites in vitro. Using Southwestern analysis we found no Hox-2.3 binding sites in the 5' part of the Hox-2 locus containing three genes including Hox-2.3.

RESULTS AND DISCUSSION

(a) High level production of Hox-2.3 protein in HeLa cells co-infected with Hox.2.3 recombinant vaccinia virus (reVV) To produce full-length Hox-2.3 protein, a reVV was constructed containing Hox-2.3 eDNA (VVH2.3( + )) under control of a phage T7 promoter and transcription terminator. A reconstituted eDNA (W. de Graaff, F.M. and J. Deschamps, unpublished data) was cloned in the insertion vector pT7-5 which was then used to produce a recombinant virus by homologous recombination (Mackett et al., 1985). A negative-control virus, VVH2.3( - ), was made by integration of the Hox-2.3 eDNA in reverse orientation. Expression of the Hox-2.3 gone was achieved by coinfecting cells with VVH2.3( + ) and VVTF7-3, a reVV expressing the phage T7 RNA polymerase gone from the VV promoter P7.5 (Fuerst et al., 1986). In Table I the reVVs used in the present study are listed. In co-infected HeLa cells, accumulation of Hox-2.3 protein migrating as a single 29-kDa band was observed 24 h after infection. This band was detected neither in uninfected HeLa cells, in cells infected with wt W , in cells co-infected with VVTF7-3 and VVH2.3( - ) (Fig. IA), nor in cells infected with VVH2.3( + ) or VVTF7-3 (not shown). The 29-kDa band was identified as Hox-2.3 protein in two further ways: (l) it co-migrated with Hox-2.3 protein obtained by translation in vitro (not shown) and (2) it was TABLE ! List of VV and reVVs

VV"

Insertsb

wt VV VVTF7-3 VVH2.3(+ ) VVH2.3(- )

wt Phage T7-RNA polymerasegone Hox-2,3 eDNA H,x.2.3 eDNA in reversedorientation

" The wt VV used was strain WR (American Type Culture Collection). The reVVs, VVH2.3(+ ) and VVH2.3(- ), were generated as follows: reconstituted HoXo2.3eDNA was treated with Exoill which [eft a 49-bp 5' non-translated region and a 21-bp 3' non-coding sequence. Insertion plasmids TT-H(+ ) and T7.H( - ) were constructed by ligatingthis Hox2.3 fragmentprovided with BamHI linkers in sense and reversed orientatk,n, respectivel), in pT7-5. This insertion vector contains a T7 prometer and transcription terminator, flanked by W TK-encoding sequences (Fu~rst ct al., 1987). Standard recombinant-DNAtechniques were used (Maniatis ct al., 1982}.Recombinant viruses were generated from x~I-VVfollowing standard methods (Mackett ¢t ai.. 1985).VVTFT3 was constructed ~ Fuerst ¢t al. (1986). t, Hclerologoussequences integrated in reVV.

197

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14Fig. 1. Gel.electrophoresis pattern and immunoblot of nuclear proteins from uninfected or (co-)infected HeLa cells. (Panel A) Fractionation in 0.1% SDS-15% polyacrylamide gel of nuclear proteins from uninfected (lane I), wt W (strain WR)-infected HeLa cells (lane 2) and from HeLa cells co-infected with VVTF7-3 and reWH2.3( - ), the negative-control virus (lane 3), or WH2.3( + ), the Hox-2.3 expression construct (lane 4). Co.infected cells were harvested 24 h p.i. Nuclei from approx, l0 s cells were loaded per lane. The sizes of marker polypeptides are indicated in kDa. Arrow point~ at Hox-2.3 protein. Proteins were stained with Coomassie Blue. (Panel B) Western blot of crude nuclear extracts from co-infected HeLa cells expressing or lacking Hox-2.3 protein. Lane 1: Co-infected HeLa cells expressing Hox-2.3, treated with pre-immune serum. Lanes 2 and 3: Co-infected HeLa cells, expressing or lacking Hoe.2.3, respectively, incubated with rabbit polyclonal serum containing specific anti Hox-2.3 antibodies. Methods. HeLa cells, grown in Dulbccco-modified Eagle's medium containing 7.5% FCS to 75°.o confluence, were inoculated with W according to Fuerst et al. (1987) at a multiplicity of 10 pfu/celi per recombinant. Crude nuclear extracts from co-infected cells 24 h p.i. were isolated by iysis with 20 mM Tris.HCI pH 7.2/10 mM EDTA/100 mM NaCI/i% Triton X.100. Nuclear proteins were separated by 0.1% SDS-15% PAGE and transferred to nitrocellulose (Towbin et al., 1979). All subsequent incubations were performed at 37°C. The Western blot was blocked i h in PBST (PBS/0.05% Twecn20)/10% NCS followed by several washes in PBST. Affinity-purified primary antibodies were diluted in PBST/10% NCS and applied for 1 h. After several PBST washes, the blot was further processed using the Protoblot system (Promega, Madison, WI). The antibodies were raised against an oligopeptide corresponding to aa ! 3--42 of the predicted Hox2.3 protein (Meijlink et al, 1987) synthesized on a Milligen Biosearch type SAM-2 using the Merrifield solid-phase coupling technique (Barany and Merrifield, 1980). Virgin New Zealand white rabbits were immunized subcutaneously gith peptide conjugated to keyhole-limpet hemocyanin with I-ethyl-3.(3-dimethylanfinopropyl)carbodiimidefollowing standard procedures (Harlow and Lane, 1988). Approx. 500 pg conjugate was injected and booster immunizations followed after 4, 8, 12 weeks. Antibody affinity-purification was done ~ith Reactigei (Pierce) according to the manufacturer's specifications.

recognized in Western blot analysis by a rabbit anti-Hox2.3 polyclonal serum (Fig. 1B). This antiserum was raised against a synthetic peptide corresponding to a lessconserved domain of the Hox-2.3 protein. Pre-immune serum contained no antibodies against Hox-2.3. No other nuclem proteins from control co-infected cells are recognized. Specific binding of the anti-Hox-2.3 antibodies is blocked by an excess of synthetic peptide (data not shown). This is the first report on the use of this hybrid expression system to produce a transcription factor. Other studies reported its use to produce E. coli fl-galactosidase, viral glycoprotein antigens (Fuerst etal., 1987) and the Tlymphocyte membrane protein CDC4 (Berger et al., 1988). Conventional reWs have been used to express several trans-acting factor-encoding genes (De Th6 et al., 1990; Jackson et al., 1990; Schmid et al., 1989; Verrijzer et al., 1990). We estimated the amount of produced Hox-2,3 at at least 0.5 pg per 105 co-infected HeLa cells, as judged by Coomassie Brilliant Blue staining and comparison with marker proteins. In the studies mentioned above, similar or lower levels of expression were reported. RNA expression levels are reported to be extremely high in this hybrid expression system (approx. 30% of the total RNA; Fuerst and Moss, 1989) but at the protein level, expression is only several-fold higher than in conventional r e W systems (Fuerst et al., 1987). This is probably due to the low efficiency with which T7 transcripts are capped by W enzymes, resulting in peor translation (Paerst and Moss, 1989) Unfortunatel,,:, nuclear extraction (see Fig. 2 legend) resulted in ¢olisid~rable losses of Hox-2.3 protein, probably due to poor solubility. (b) Lack of evidence for modification The use of a eukaryotic system, rather than prokaryotic systems, often allows the direct detection and study of protein modification. For instance, DNA-binding studies of Hox-l.3 produced by a recombinant baculovirus, demonstrated that phosphorylated species formed more stable DNA-protein complexes in vitro and hzve a higher affinity for nuclear binding sites than unphosphorylated Hox-l.3 (Odenwald et al., 1989). Differences in phosphorylation of Hox-l.3 and many other proteins including for instance the Drosophila homeodomain protein fushi tarazu (Krause and Gehring, 1989), can be detected by normal SDS-PAGE. Some transcription factors synthesized by r e W have been shown to be post-translationally glycosylated and/or phosphorylated (Jackson et al., 1990; Menneguzzi et al., 1989). in the present study, we did not obtain evidence for post-translational modification of Hox-2.3 since it was always detected as a sharp single band on SDS-polyacrylamide gels. We cannot rule out post-translational modifica-

198 A

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Fig. 2. Binding of Hox-2.3 protein to common homeodomain recognition sites in mobility.shift assays. (Panel A) Cytoplasmic and nuclear extracts from co-infected cells were incubated with an 32P-end-labeled NPI or NP2T oligos (see Table II). Of (NH4)2SO4-precipitated nuclear extract (N) and c:,tosolic extract (C) 2 #g and 10/~g was incubated with 1 ng probe and 3 #g poly(dl-dC).(dl-dC):Arrows (in panels A and B) indicate a band that was specifically shifted in the presence of Hox-2.3. (Panel B) Gel shift of labeled NP6, (TAA)s and (TAA)2o by cytoplasmic extracts containing or lacking Hox2.3. ( + ) and ( - ) indicate the presence or absence of Hox-2.3 protein in the incubation mixture. Methods. Nuclear extracts were prepared by a modification of the procedure described by Dignam et ai. (1983) using buffer A (10 mM Hepes.KOH pH 8.2/1.5 mM MgCI2/10 mM KCI/0. ! % NP-40) for cell disruption, and buffer C (20 mM Hepes.KOH pH 8.2/1.5 mM MgCI,/0.2 mM EDTA/0.6 M NaCi/25% glycerol) and buffer D (20 mM Hepes.KOH pH 8.2/0.2 mM EDTA/0.5 M NaCI/20% glycerol) for nuclear protein extraction and dialysis, respectively. The buffers were supplemented with ! mM PMSF and 1 mM DTT. Cytoplasmic extracts were mixed with one vol of 2 mM EDTA/2 mM D'i'r/40% glycerol/0.036% NP-40/20% sucrose. (NH4)2SO4-precipitation of nuclear extract was carried out at 25?/0 saturation during 30 min. The precipitate was resuspended in buffer C and dialysed against buffer D. The band shift assays were exactly as described by Zwartkruis et ah (1991).

tions that escape detection by SDS-PAGE or overloading of the modification system due to the vast quantities of Hox-2.3 protein produced.

(c) Hox-2.3 binds to common homeodomain recognition sequences To test whether functional Hox-2.3 protein is synthesized, its DNA-binding ability was examined in various DNA-binding assays. In previous studies it was found that a number of homeodomain proteins bind specific elements in the engrailed and ultrabithorax promoter regions (Beachy et al., 1988; Desplan et al., 1988; Hoey and Levine, 1988; Hoey et al., 1988; Hayashi and Scott, 1990). On the basis of these observations, the Antp-related Hox-2,3 homeodomain is expected to bind these sites. Oligos containing one or several copies of the consensus binding site, 5'TCAATTAAAT (Desplan et ai., 1988; Hoey and Levine 1988), referred to as the NP binding site, and repeats of the trinucleotide TAA (Bcachy et ah, 1988) were used in mobility-shift assays (kindly supplied by C. Desplan~ se¢ Table !!). In the presence of Hox-2.3 an extra bandshift occurred when a nuclear extract from co-infected cells expressing HoxQ.3 was incubated with NPl or NP2T (Fig. 2A). A second band, visible in lane N P I / + , was in

control experiments (not shown) found to be non-specific and not consistently reproducible. When a cytoplasmic extract containing Hox-2.3 was used, more NP2T prob e was shifted (Fig. 2A, NP2T, lanes C vs. lanes N). NP6 was also specifically bound by cytoplasmic Hox-2.3. A difference in binding activity was observed between (TAA)s and (TAA)2o. (TAA)2o is specifically shifted by cytoplasmic extract containing Hox-2.3 but (TAA)5 is not (Fig. 2B). Although in these experiments the DNA-binding activity of TABLE II Oligos used in DNA-binding assays Oligo"

Sequence"

NPI NP2T NP6 (TAA)~ (TAA),.

TCAATTAAATga TCAA'I-rAAATgaTCAATTAAATga (tcATTTAATTGA)4(TCAATI'AAATga), TAATAATAATAATAA (TAA)I~(TfA)~

' Oligos used in various in vitro DNA-binding assays. O]igos NPI, NP2T and NP6 are described by Desplan et al. (19~8). Oligos containing TAA repeats are described by Beach, ctal. (1988). " Nuclcotide sequences of oligos. Loger-casc Icttc~ represent nonessential spacer sequences.

199 Hox-2.3 was determined qualitatively, the results suggest a higher affinity for a NP site than for TAA repeats. In the case of (TAA)5 a specific complex was never observed, whereas with NP1 a slightly retarded band was always observed. Surprisingly, cytoplasmic extract had higher specific DNA-binding activity than nuclear extract although it contained smaller amounts of Hox-2.3, as judged from S D S PAGE. A likely explanation would be that a large portion of the protein present in the nuclear fraction is aggregated and/or not soluble, whereas by definition the cytoplasmic fraction contains only soluble protein. Cytoplasmic extracts from metabolically labeled co-infected cells incubated with [35S]L-methionine for 30 rain contained approx. 10% of the total labeled Hox-2.3 due to rapid accumulation of Hox-2.3 in the nuclear fraction (data not shown). In DNase I footprinting analysis protection of the multiple NP sites from nuclease degradation was observed when binding reactions contained Hox-2.3 protein (Fig. 3). In the case of NP6 a clear protection pattern appeared with nuclear extract containing Hox-2.3. Sites of enhanced cleavage were visible between the NP sites. DNase I protection of NP2T was not complete when DNA was incubated with an amount of extract giving the full protection of NP6. However, the intensity of the bands corresponding t~ m in the NP sites was reduced and hypersensitive sites dk!, occur. The amount of Hox-2.3 protein in cytoplasmic extracts was not sufficient to give detectable protection in DNase I footprinting reactions. Obviously, this technique is less sensitive than a band-shift assay, since it requires a much higher occupancy of the specific DNA sequence by the binding protein. Furthermore, protection of TAA repeats in (TAA)2o by Hox-2.3 has been observed (data not shown) and Hox-2.3 recognized an Ant/, site (5'-ANNNNCATTA; M011er et at., 1988) in the Xenopus Wnt-I promoter and protected this site from DNase I degradation (X. Gad, G.A. Kuiken, W. Baarends, J.G. Schiithuis, R.d.J., J.G. Koster and O.HJ. Destr~e, submitted).

(d) Southwestern analysis of DNA.binding activity Hox-2.3 A Hox-2.3-containing extract fractionated by SDSPAGE was subjected to Southwestern analysis, a method aimed at characterizing specific DNA recognition by immobilized renatured protein (Miskimins et at., 1985; Vinson et al., 1988). In situ DNA-binding specificity of Hox2.3 was determined by incubating Western b;ot strips containing or lacking Hox-2.3 protein with labeled probes (Fig. 4 and Table II). After incubation with oligos NP2T, NP6 or (TAA)2o, a 29-kDa band was observed after autoradiography, indicating binding by Hox-2.3. Although the (TAA)2o probe gives a high background ofother DNA-

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Fig. 3. DNase ! protection of homeodomain-binding sites in NP2T and NP6 by Hox-2.3 protein. DNase I footprint analysis of Hox-2.3-binding activity was performed with NP2T and NP6 (see Table). Probes were end-labeled by filling-in the appropriate restriction site with Pollk. (Panel A) Labeled NP6 was incubated with increasing amounts (0.33 ~g, i #g, 3 #g and 9/~g; lanes 3-6, respectively) of nuclear extract containing Hox-2.3 protein. Protected NP sites are marked by vertical bars, hypersensitive sites are indicated with a horizontal arrow. Lane C corresponds to the reaction mixture incubated ~ ~,h 9 ~g control extract. Symbol no ext denotes the absence of extract in footprint reaction. G/A indicates marker sequencing ladder identifying G and A bases. (Panel B) DNase i protection of the anti-sense strand of NP6. The amounts of extract used in this experiment were 0.5/Ag, 2 og and 8 ttg; lanes 2-4, respectively. (Panel C) Footprinting of NP2T. NP2T probe was incubated with 8/~g nuclear Hox-2.3 or control extract (H and C, respectively). Methods. DNase l footprinting reactions were carried out essentially as described by Pruijn et al. (1987). Strand-specific labeling of fragments was performed by filling in the appropriate restriction site with Pollk and [ ~.32pldATP. Fragments were isolated by preparative TBE-5% PAGE after digestion with another endonuclease. Various amounts ofnuclear extract in a 50 ~l DNA-binding reaction containing 20mM Hepes.KOH pH 7.5/I mM MgCI2/! mM DTT/0.02% NP-40/125 mM NaCI/I ng labeled probe/150 ng poly(dldC).(dl-dC)/5% glycerol were incubated for 30 rain at room temperature. To this binding mixture, 4 pl 50 mM MgCI2 with 67-400 ng DNase I (Boehringer, grade ll) freshly diluted from 10 mg/ml stock was added. DNase I digestion was terminated after I rain by adding 150 t~l of stop mix consisting of 4 mM EDTA-NaOH ptl 8.0/0.04°0 SDS/5 pg/ml salmon-sperm DNA. After phenol:chloroform ( I:1 ) extraction the DNA was precipitated and rinsed ~ith 70°0 ethanol before loading on a 8 M urea- 10% polyacrylamide gel.

200

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specific competitor

SRV

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14.4Fig. 4, Characterization of Hox-2.3-binding activity by Southwestern analysis. Nuclear extracts from co-infected cells were separated by 0.1% SDS-15% PAGE and transferred to nitrocellulose. The Western blot was divided in strips after renaturing the immobilized nuclear pr6tcins and incubated with different s2P-labeled probes. NP2T, NP6 and (TAA),o are described in Table Il. The lanes denoted with CON were strips incubated with polylinker DNA from pGEM3Z plasmid. SRV is a 150-bp fragment containing genomic (upstream Hox.2.3) sequences harboring two Hox-i.3-biading sites. A 100-fold molar excess of unlabeled fragment was added to the binding reaction in the lane with '100 ×' at the top. ( + ) and ( - ) refer to presence or absence of Hox-2.3 protein on nitrocellulose strip. Sizes of standard proteins are indicated in kDa. Methods. A protocol originally described by Vinson et al. (1988) was used, with some modifications. Western blots of (NH4),SO4-precipitated nuclear extracts were washed three times with binding buffer consisting of 25 mM Tds.HCI pH 7.5/50 mM NaCi/2 mM DTT/2 mM EDTA/0.3% BSA/0.025% NP-40 for a period of 30 min. Incubation in denaturing buffer containing 7 M guanidine hydrochloride/50 mM Tris.HCI pH 8.0/2 mM DTT/2 mM EDTA/0.3% BSA for 45 min was followed by 4 x 5 min washes of renaturing buffer containing 2 x binding buffer except for BSA (0.3%) and NP-40 (0. !%). After an overnight incubation in renaturing buffer the blot was washed 3 x 5 min in binding buffer. The blot was then incubated 3 h with labeled probe (10s-5 x i0 s cpm) in a small volume of binding buffer complemented with 6 pg poly(dl-dC).(dl-dC). Washes of the blot in large volumes of binding buffer containing 0. i M NaC! were prolonged until radioactivity was no longer detected in the washing buffer. The blot was autoradiographed ! day at -80°C with the aid of an intensifying screen. All handling was performed at room temperature.

binding proteins including histones, a strong band corresponding to Hox-2.3/DNA complex is also visible. In contrast, labeled unrelated polylinker sequences from the plasmid pGEM-3Z were not bound, confirming that the binding is selective. Addition of unlabeled NP6 competitor to the labeled NP6 double-stranded DNA probe reduces the intensity of the band indicating that binding is specific. These results show that denaturation and subsequent renaturation did not affect the DNA-binding specificity of Hox2.3. in Drosophila, homeodomain recognition elements were observed to be involved in auto- and cross-regulatory mechanisms for the transcriptional control of several homeobox genes (reviewed in Hayashi and Scott, 1990). A fragment located upstream from the Hox-2.3 gene was reported to contain two Hox-i.3 binding sites (Odenwald et al., 1989). namely 5"-ATTCATI'AT]" and 5'-CCACATTACT at nt positions - ! 13 and -161 relative to the tsp, respectively (Meijlink ¢t al., 1989; Verrijzer et al., 1988).

These sequence elements are therefore candidates to play a role in cross-regulation of Hox-2.3 by Hox-l.3. To see whether they are involved in autoregulation of Hox-2.3, we investigated their binding to Hox-2.3 protein. However, the SRV fragment (see Fig. 4), which contains both sites, failed to bind to Hox-2.3 protein. Protection of these Hox-l.3 sites could not be detected, either, in a DNase ! footprinting analysis using maximum amount of Hox-2.3 containing nuclear extract (data not shown). These results, therefore, do not support involvement of the Hox-1.3 binding sites in auto-regulation of Hox-2.3. (e) Enrichment of potential targets by Southwestern blots

To search for possible natured !-!ox-2.3 target sites in the Hox-2 cluster we used the Southwestern-blot mapping technique for selection of fragments from a pool of labeled fragments (LeLong et al., 1989; Miskimins et al., 1985). Different genomic ~. clones were used: clones L2, L3 (Meijlink et al., 1987) and L5 (T.Hoeijmakers and F.M., un-

201 published), together containing the Hox-2.3, Hox-2.4 and Hox-2.5 genes and their flanking sequences. The 2 clones were digested with endonuclease Taql and, after labeling, incubated with Western blots containing reVV-produced Hox-2.3. Bound fragments were eluted from the immobilized proteins and analysed by agarose gel-electrophoresis. In Fig. 5, the results obtained with clone L5 are shown. From the pool of labeled fragments incubated with the renatured Hox-2.3 several fragmentg are bound preferen-

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--O

--u

Fig. 5. Selective binding by Hox-2.3 in Southwestern analysis. A mouse genomic clone containing the Hox-2.5 gene and surrounding sequences was digested with Taql and end-labeled with Pollk. (Panel A) Autoradiography of nitrocellulose strips with immobilized nuclear proteins from co.infected HeLa cells producing Hox-2.3 incubated with these fragments. Lane I and 2 show strips washed either with binding buffer containing 0. I M NaCI or 0.15 M NaCI. U is an aspecific DNA.binding histone species serving as internal control. HOX indicates Hox-2.3 protein. (Panel B) Gel-electrophoretic analysis of labeled genomic clone fragments that eluted from DNA-binding proteins on the Southwestern blot shown in panel A. Restriction fragments were fractionated in an agarose gel. Lane 0 shows the input of labeled fragments. The denotations on top of each lane correlate gith DNA-binding proteins indicated m panel A. Molecular weights of protein markers and size of DNA markers are as indicated. Metlmds. Southwestern.blot mapping was performt~l as described in Fig. 4. except that blots were pre-incubated 30 rain with 30 gg pol)~dldC).(dl-dC) per ml binding buffer prior to the binding reaction. Fragments were eluted in 20 mM Tris-HCi pH 8/0.1 mM EDTA/I M NaCI/O.i°. SDS at 60=C for 45 min. Precipitation was carried oul ~Jth addition of 5 pg salmon-sperm DNA. After rinsing ~ith 70% ethanol and drying, the fragments ~erc fractionated b)- 1.25°0 agarose gel electrophore~is in TBE.

tially, but with different affinities. Some fragments remained bound after washing with buffer containing 0.15 M NaCI, indicating the presence of homeodomain-binding sites in these fragments. A fragment of about 1.2 kb, present in all clones, that was bound most strongly, was identified as a 2 fragment. The 2 genome contains many putative homeodomain-binding sites and 2 sequences were shown to be bound specifically by engrailed homeodomain (Desplan et al., 1985). No fragments from the mouse genomic inserts were strongly bound. In conclusion, we have not obtained evidence for ability of Hox-2.3 protein to bind in the vicinity of its own genomic region in spite of its ability to recognize certain artificial binding sites. In concert with this, we observed earlier that reporter-gene constructs containing regulatory genomic Hox.2.3 sequences co-transfected with a Hox.2.3 expression construct did not significantly alter reporter-gene transcription indicating absence of Hox-2.3 target sites (W. de Graaff, J. Deschamps and F.M., unpublished data). The Southwestern technique may be used to isolate sequences containing specific Hox-2.3 binding sites from randomized oligos and to identify natural target sequences in genomic DNA. (f) Conclusions

(I) The hybrid reW/T7-RNA polymerase expression system provides a simple and rapid method to synthesize large amounts of functional Hox-2.3 protein in mammalian cells. (2) In mobility-shift and DNase I footprinting assays, the Hox-2.3 protein binds to DNA sequences containing one or several 5'-TCAATTAAAT elements, a common homeodomain-recognition site. (TAA)a0 was bound but (TAA)5 not at all. Renatured Hox-2.3 protein binds in Southwestern analysis to the same recognition sites. Remarkably, in this assay recognition sites of Hox-l.3, an Antp-type homeodomain protein differing slightly from Hox-2.3, are not recognized. (3) Using Southwestern-blot mapping, an in vitro DNAbinding assay allowing detection of specific binding sites in DNA fragments, we found no target sites of Hox-2.3 in the 5' part of murine HOX-2 locus containing Hox-2.3 and two other genes.

ACKNOWLEDGEM ENTS

We thank Leo Heijnen for technical assistance concerning W virus work. VVT7-3 and pT7-5 were a kind gift of Dr. B. Moss. Dr. C. Desplan kindly provided M I3 vectors containing the oligos listed in Table li. We are grateful to Prof. Siegfried W. de Laat for helpful discussions. We owe special thanks to Prof. Willy J.M. Spaan for his support

202

during the work involving reW and for reading the manuscript.

REFERENCES Akam, M.: Hox and HeM: homologous gene clusters in insects and vertebrates. Cell 57 (1989) 347-349. Barany, G. and Merrifield, R.B.: Solid-phase peptide synthesis. In: Gross, E. and Meienhofer, J. (Eds.), The Peptides, Analysis, Synthesis, Biology, Vol. 2, Academic Press, New York, 1980, pp. 1-18L Beachy, P.A., Krasnow, M.A., Gavis, E.R. and Hogness, D.S.: An Uitrabithorax protein binds sequences near its own and the Antem~apedia promoter. Cell 55 (1988) 1069-1081. Bergcr, E.A., Fuerst, T.R. and Moss, B.: A soluble recombinant polypeptide comprising the amino-terminal half of the extracellular region of the CD4 molecule contains an active binding site for human immunodeficiency. Prec. Natl. Acad. Sci. USA 85 (1988) 2357-2361. Cho, K.W.Y., Goetz, J., Wright, C.V.E., Fritz, A., Hardwicke, J. and DeRobertis, E.M.: Differential utilization of the same reading frame in a Xenopus horace box gone encodes two related proteins sharing the same DNA-binding specificity. EMBO J. 7 (1988) 2139-~149. De Th6, H., Del Mar Vivanco.Ruiz, M., Tiolais, P., Stunner, berg, H. and Dejean, A.: Identification of a retinoic acid responsive element in the rctinoic acid receptor fl gene. Nature 343 (1990) 177-180. Deschamps, J. and Meijlink, F.: Mammalian homcobox genes in normal development and ncoplasia. CRC Crit. Rev. Oncogen. 3 (1992) 117173. Desplan, C., Thies, J. and O'Farreil, P.H.: The Drosophila developmental gene, engrailed, encodes a sequence-specific DNA binding activity. Nature 3 ! 8 (1985) 630-635. Desplan, C., Thies, J. and O'Farrell, P.H.: The sequence specificity of homeodomain~DN A interaction. Cell 54 (1988) 108 !- 1090. Dignam, J.D., LeBowitz, R.M. and Roedcr, R.G.: Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Rcs. I1 (1983) 1475-1489. Driever, W. and Ntlsslein.Volhard, C.: The bicoid protein is a positive regulator of htmchback transcription in the early Drosophila embryo. Nature 337 (1989) 138-143. Duboule, D. and Doll6, P.: The structural and tunctional organization of the murine HOX gene family resembles that of Drosophila homeotic genes. EMBO J. 8 (1989) 1497-1505. Fuerst, T.R., Niles, E,G., Studier, F.W. and Moss, B.: Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Prec. Natl. Acad. Sci. USA 83 (1986) 8812-8126. Fuerst, T,R., Earl, P.L. and Moss, B.: Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes. Mol. Ceil. Biol, 7 (1987) 2538-2544. Fuerst, T.R, and Moss, B.: Structure and stability of mRNA synthesized by vaccinia virus-encoded bacteriophage T7 RNA polymerase in mammalian cells, J. Mol. Biol. 206 (1989) 333-348. Gehring, W.J.: Horace boxes in the study of development. Science 236 (1987) 1245-1252. Graham, A., Papalopulu, N. and Krumlauf, R.: The murine and Drosophila homeobox gene complexes have common features of organisation and expression. Cell 57 (1989) 367-378. Harlow, E. and Lane, D.: Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988. Hayashi, S. and Scott, M.P.: What determines the specificity of action of Drosophila homeodomain proteins. Cell 63 (1990) 883-894. Herr, W. and Sturm, R .... TZ'c POU domain: a large c~nserved region A

.

II

in the mammalian pit-l, oct-l, ocl-2, and the Caerorhabditis elegans unc-86 gene products. Genes Dev. 2 (1988) 1513-1516. Hoey, T. and Levine, M.: Divergent horace box proteins recognize similar DNA sequences in Drosophila. Nature 332 (1988) 858-861. Hoey, T., Warrior, R., Manak, J. and Levine, M.: DNA-binding activities of the Drosophila melanogaster even-skipped protein are mediated by its homeo domain and influenced by protein context. Mol. Cell. Biol. 8 (1988) 4598-4607. Ingham, P.W.: The molecular genetics of embryonic pattern formation in Drosophila. Nature 335 (1988)25-34. Jackson, S.P., MacDonald, J.J., Lees-Miller, S. and Tjian, R.: GC box binding induces phosphorylation of SP1 by a DNA-dependent protein kinase. Cell 63 (1990) 155-165. Kissinger, C.R., Lui, B., Martin-Blanco, E., Kornberg, T.B. and Pabo, C.O.: Crystal structure of an engrailed homeodomain-DNA complex at 2.8 A; resolution: a framework for understanding homeodomainDNA interactions. Cell 63 (1990) 579-590. Krause, H.M. and Gehring, W.J.: Stage-specific phosphorylation of the fushi tarazu protein during Drosophila development. EMBO J. 8, 1197-1204. Laughon, A. and Scott, M.P.: Sequence of a Drosophila segmentation gene: protein structure i~omologywith DNA-binding proteins. Nature 310 (1984) 25-31. Lelong, J., Prevost, G., Lee, K. and Crepin, M.: South western blot mapping: a procedure for simultaneous characterization of DNA binding proteins and their specific genomic DNA target sites. Analyt. Biochem. 179 (1989) 299-303. Mackett, M., Smith, G.L. and Moss, B.: The construciiun and characterization of vaccinia virus recombinants expressing foreign genes. In: Rickwood, D. and Haines, B.D. (Eds.), DNA Cloning. A Practical Approach, Vol. 2., IRL Press, Washington, DC, 1985, pp. 191211. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. McGinnis, W., Levine, M.S., Hafen, E., Kuroiwa, A. and Gehring, W.J.: A conserved DNA sequence in homeotic genes of Drosophila Antennapedia and Bithorax complexes. Nature 308 (1984)428-433. Meijlink, F., De Laaf, R., Verrijzer, P., Destr~e, O., Kroezen, V., Hilkens, J. and Deschamps, J.: A mouse homeobox containing gene on chro. mosome !1: sequence and tissue-specific expression. Nucleic Acids Rex. 15 (1987) 6773-6786. Meijlink, F., De I.aaf, R., Verrijzer, P., De Graaff, W. and Deschamps, J.: Regulation of expression of the Hox2.3 gene. In: De Laat, S.W., Bluemink, J.G. and Mummery, C.L. (Eds.), Cell )o Cell Signals in Mammalian Development. NATO ASI Series, Vol. H26, SpringerVerlag, Berlin, 1989, pp. 23-41. Menneguzzi, G., Lathe, R., Kieny, M.P. and Veer, N.: The E2 trans. activating protein of bovine papillomavirus type 1 (BPVI) is serinephosphorylated in vivo. Oncogene 4 (1989) 1285-1290. Miskimins, W.K., Roberts, M.P., McCleUand, A. and Ruddle, F.H.: Use of a protein-blotting procedure and a specific DNA probe to identify nuclear proteins that recognize the promoter region of the transferrin receptor o;ene. Prec. Natl. Acad. Sci. USA 82 (1985) 6741-6744. MUller, M., Affolter, M., Leupin, W., erring, G., Wathrich, K. and Gehring, WJ.: Isolation and sequence-specific DNA binding of the Antemzapedia homeodomain. EMBO J. 9 (1988) 4299-4304. Nelson, H.B. and Laughon, A.: The DNA-binding specificity of the Drosophila fushi tarazu protein: a possible role for DNA bending in homeodomain recognition. New Biol. 2 (1990) 171-178. Odenwald, W.F., Garbern, J., Arnheiter, H., Tournier-Lasserve, E. and Lazzarini, R.A.: The Hox-l.3 homeo box protein is a sequence-specific DNA-binding phosphoprotein. Genes Dev. 3 (1989) 158-172.

203 Otting, G., Qian, Y., Billeter, M., MOiler, M., Affolter, M., Gehring, W.J. and WOthrich, K.: Secondary structure determination for the Antennapedia homeodomain by nuclear magnetic resonance and evidence for a helix-turn-helix motif. EMBO J. 7 (1988) 4305-4309. Piccini, A., Perkus, M.E. and Paoletti, E.: Vaccinia as an expression vector. Methods Enzymoi. 153 (1987) 545-563. Pruijn, G.J.M., Van Driel, W., Van Miltenburg, R.T. and Van der Vliet, P.C.: Promoter and enhancer elements containing a conserved sequence motif are recognized by nuclear factor III, a protein stimulating adenovirus DNA replication. EMBO 3.6 (1987) 3771-3778. Sasaki, H., Yokoyama, E. and Kuroiwa, A.: Specific DNA binding of the two chicken Deformed family homeodomain proteins, Chox-l.4 and Chox-a. Nucleic Acids Res. 18 (1990) 1739-1747. Schmid, W., Strahle, U., Sch0tz, G., Schmitt, L and Stunnenberg, H.: Glucocorticoid receptor binds cooperatively to adjacent recognition sites. EMBO J. 8 (1989) 2257-2263. Scott, M.P, and Weiner, A.J.: Structural relationships among genes that control development: sequence homology between the Antennapedia, UItrabithorax and fushi tarazu loci of Drosophila. Proc. Natl. Acad. Sci. USA 81 (1984)4115-4118.

Scott, M.P., Tamkun, J.W. and Hartzell lIl, G.W.: The structure and function of the homeodomain. Biochim. Biophys. Acta 989 (1989) 25-48. Towbin, H., Staehelin, T. and Gordon, .L: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad Sci. USA 76 (1979) 43504354 Verrijzer, P., de Graaff, W., Deschamps, 3. and Meiilink, F.: Nucleotide sequence of the Hox2.3 gene region. Nucleic Acids Res. 16 (1988) 2729. Verrijzer, C.P., Kal, A.J. and Van der Vliet, P.C.: The DNA binding domain (POU domain) of transcription factor oct-I suffices for stimulation of DNA replication. EMBO 3.9 (1990) 1883-1888. Vinson, C.R., LaMarco, K.L., Johnson, P.F., Landschulz, W.H. and McKnight, S.L.: In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage. Genes Dev. 2 (1988) 801-806. Zwartkruis, F., Hoeijmakers, T., Deschamps, 3. and Meijlink, F.: Characterization of the Hoxo2.3 promoter: involvementof the transcription factor USF (MLTF). Mech. Dev. 33 (1991) 179-190.