The BTB/POZ Domain of the Regulatory Proteins Bric

Btb

Developmental Biology 237, 368–380 (2001)doi:10.1006/dbio.2001.0358, available online at http://www.idealibrary.com on

The BTB/POZ Domain of the Regulatory ProteinsBric a brac 1 (BAB1) and Bric a brac 2 (BAB2)Interacts with the Novel DrosophilaTAFII Factor BIP2/dTAFII155

Jean-Christophe Pointud,* Jan Larsson,† Bernard Dastugue,*and Jean-Louis Couderc* ,1

*Institut National de la Sante et de la Recherche Medicale U384, Laboratoire de Biochimie,UFR Medecine, 28, place Henri Dunant, 63001 Clermont-Ferrand, France; and†Department of Microbiology, Umeå University, SE-901 87 Umeå, Sweden

The BTB/POZ domain is an evolutionarily conserved protein–protein interaction domain present in the N-terminal regionof numerous transcription factors involved in development, chromatin remodeling, and human cancers. This domain isinvolved in homomeric and heteromeric associations with other BTB/POZ domains. The Drosophila BTB/POZ proteinsBric a brac 1 (BAB1) and Bric a brac 2 (BAB2) are developmentally regulated transcription factors which are involved inpattern formation along the proximo-distal axis of the leg and antenna, in the morphogenesis of the adult ovaries, and in thecontrol of sexually dimorphic characters. We have identified partners of the BAB1 protein by using the two-hybrid system.The characterization of one of these proteins, called BIP2 for BAB Interacting Protein 2, is presented. BIP2 is a novelDrosophila TATA-box Protein Associated Factor (TAFII), also named dTAFII155. We show that the BTB/POZ domains of

AB1 and BAB2 are sufficient to mediate a direct interaction with BIP2/dTAFII155. This provides a direct link betweenhese BTB/POZ transcription factors and the basal transcriptional machinery. We discuss the implications of the interactionetween a BTB/POZ domain and a TAFII for the molecular mechanisms of transcriptional control mediated by BTB/POZ

transcription factors. © 2001 Academic Press

Key Words: BTB/POZ domain; TAFII; Drosophila; transcriptional regulation.

INTRODUCTION

Many conserved protein domains involved in the devel-opmental regulation of gene expression have been identi-fied. The BTB/POZ domain (for Broad-complex, Tramtrackand Bric-a-brac/Poxvirus and Zinc fingers; Di Bello et al.,1991; Harrison and Travers, 1990; Godt et al., 1993; Kooninet al., 1991) is an evolutionarily conserved protein–proteininteraction domain that is found generally at theN-terminal end of numerous developmentally regulatedtranscription factors (Zollman et al., 1994; Bardwell andTreisman, 1994; Albagli et al., 1995). This domain isinvolved in homophilic as well as heterophilic protein–protein interactions (Bardwell and Treisman, 1994; Dhor-

1 To whom correspondence should be addressed. Fax: 334 73 27
61 32. E-mail: [email protected].
368

dain et al., 1995; Hoatlin et al., 1999; Kobayashi et al.,2000). This domain was first identified in Drosophila andpoxviruses and many examples of BTB/POZ domains arenow described from yeast to man (Albagli et al., 1995). Theycorrespond essentially to two major classes of proteins. Thefirst class comprises actin-binding proteins bearing theKelch motif (Xue and Cooley, 1993). The second classcomprises developmentally regulated transcription factorswhich bind to DNA, most of them via the presence of C2-H2

type zinc fingers. Classical transcription factors bind asdimers to dimeric DNA sites (McKnight and Yamamoto,1992). The BTB/POZ domain is a homomerization domain(Bardwell and Treisman, 1994; Dhordain et al., 1995) butthere are also examples of heteromerization between BTB/POZ proteins (Bardwell and Treisman, 1994; Hoatlin et al.,1999; Kobayashi et al., 2000). Li et al. (1997b) and Ahmad et
al. (1998) have shown that the BTB/POZ domain of the
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All rights of reproduction in any form reserved.

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369BTB Domain Interaction with Drosophila BIP2/dTAFII155

promyelocytic leukemia zinc finger (PLZF) protein forms atightly intertwined dimer. A surface-exposed groove linedwith conserved charged amino acids was identified at theBTB/POZ dimer interface of PLZF (Ahmad et al., 1998).This groove is highly suggestive of a peptide-binding sitethat may represent a site of interaction of the BTB/POZdomain with other non-BTB/POZ domain proteins. A mu-tational analysis of the BTB/POZ domain of PLZF demon-strated that dimerization of the BTB/POZ domain, togetherwith the conservation of charged amino acids in the grooveof the BTB/POZ domain, are critical for the transcriptionalactivity of PLZF (Melnick et al., 2000).

PLZF and LAZ3/BCL6 (Lymphoma-associated zinc finger3/B cell lymphomas 6) are transcriptional repressors impli-cated in human cancers that mediate their activity byrecruiting histone deacetylase complexes via their BTB/POZ domain (Deweindt et al., 1995; Dong et al., 1996;

hordain et al., 1998; David et al., 1998). Their BTB/POZdomain interacts directly with the nuclear co-repressorsN-CoR (nuclear receptor co-repressor) and SMRT (silencingmediator for retinoid and thyroid hormone receptor) (Dhor-dain et al., 1997; Hong et al., 1997; Guidez et al., 1998; Linet al., 1998). Recruitment of histone deacetylase complexesdoes not seem, however, to be a general mechanism forBTB/POZ transcriptional repressors (Deltour et al., 1999).Other BTB/POZ proteins are transcriptional activators thatprobably have to interact with the basal transcriptionalmachinery to activate gene expression. The BTB/POZ do-main of ZF5 is required for the transcriptional activation ofthe HIV-1 LTR (Kaplan and Calame, 1997). The BTB/POZprotein MAZR is able to activate the c-myc promoter(Kobayashi et al., 2000).

The functionally related proteins Bric a brac 1 (BAB1) andBric a brac 2 (BAB2) are nuclear BTB/POZ proteins whichare required during Drosophila development for patternformation along the proximo-distal axis of the leg andantenna, for the morphogenesis of the adult ovaries and thecontrol of sexually dimorphic characters (Godt et al., 1993;Godt and Laski, 1995; Sahut-Barnola et al., 1995; Kopp etal., 2000). In a two-hybrid screen with the BAB1 protein, wehave identified a protein, BIP2 (BAB Interacting Protein 2),which is a novel Drosophila TAFII, also named dTAFII155(Gangloff et al., 2001b). In this paper, we present themolecular characterization and the expression pattern ofBIP2, and we show that BIP2 interacts with the BTB/POZdomain of the two BAB proteins, providing a direct linkbetween BTB/POZ transcription factors and the basal tran-scriptional machinery.

MATERIALS AND METHODS

Plasmids and Constructs

All yeast and bacterial expression vectors were constructed byPCR using primers with the appropriate restriction sites andconstructs were verified by automated DNA sequencing. Details of
constructions are available on request. Fusions with the DNA-
Copyright © 2001 by Academic Press. All right

binding domain of GAL4 (GAL4DBD) were constructed in themulticopy vector pGBT9 containing the TRP1 marker. Fusionswith the activation domain of GAL4 (GAL4AD) were constructed inthe multicopy vector pACT2 containing the LEU2 marker. GSTfusions were constructed in the pGEX vectors. For the in vitrotranscription and translation experiments, the BIP2G77 cDNA wasubcloned in the KTRAB plasmid in frame with the human

b-globin ATG codon.

Yeast Two-Hybrid System

The bait used for the screen corresponds to a region of the BAB1protein (aa 65–256) containing the BTB/POZ domain and subclonedin frame in the pGBT9 vector. This construct was used to screen aDrosophila third larval instar cDNA library constructed in thepACT vector (gift from Dr. Elledge). The two-hybrid screen wasperformed essentially as described (Fields, 1993) in Y190 straincells (MATa, leu2, trp1, his3, gal4D, gal80D, URA3::GAL1-lacZ,LYS2::GAL-HIS3). Approximately 2 3 106 transformants werescreened for growth on a 2Leu 2Tryp 2His 1 25 mM 3-Amino

riazol (3AT) selective medium. Of 121 clones isolated on theelective medium, 65 were also positive for b-galactosidase activ-

ity. The GAL4DBD and GAL4AD plasmids of these positive clonesere segregated in yeast cells and GAL4AD plasmid DNA was

xtracted and transformed in Escherichia coli C600. The GAL4AD

plasmids were retransformed in yeast cells with the bait pGBT9-BAB165-256 or with the pGBT9-GAL4DBD vector alone. Of the 65positive clones, 53 clones interacted with pGBT9-BAB165-256 and did

ot transactivate the reporter genes with the pGBT9 vector alone.o quantify the different protein–protein interactions, the

b-galactosidase activity was measured as described (Miller, 1972)after the co-transformation of the Y190 strain cells with theappropriate pair of plasmids.

Northern Blot Analysis

For Northern blot analysis, poly(A)1 RNA was isolated by usingDynabeads Oligo(dT)25 (DYNAL). Adult flies, pupae, larvae, andembryos were frozen at 270°C and homogenized in 0.1 M Tris–HCl (pH 8.0); 0.5 M LiCl; 10 mM EDTA; 1% SDS; 5 mM DTT.After this step, the instructions from the manufacturer, DYNAL,were followed. The samples were separated on a 1.0%formaldehyde-agarose gel and blotted onto a GeneScreenPlus filter(DuPont-NEN) by using VacuGene Vacuum Blotting System. Pre-hybridization and hybridization of the filter were performed withUltrahyb solution (Ambion) at 42°C. A bip2 cDNA clone and aRpS3 cDNA 32P-labeled by random priming and purified with

harmacia S-200 HR column were used as probes. The membraneas washed 2 3 5 min in 23 SSPE at room temperature, 3 3 15 min

n 23 SSPE, 2% SDS at 65°C, and once in 0.13 SSPE at roomemperature for 15 min.

Production of Polyclonal anti-BIP2 Antibody

A region of the BIP2 protein corresponding to the amino acidresidues 719 to 909 was subcloned into the pGEX-4T1 expressionvector. The resulting glutathione S-Transferase (GST) fusion pro-tein was produced in E. coli and purified as described (Smith andJohnson, 1988) on glutathione agarose beads. The purified fusionprotein was used to immunize two rabbits (EUROGENTEC Bel
S.A.).
s of reproduction in any form reserved.

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370 Pointud et al.

Western Blot Analysis

Ovaries from 3- to 4-day-old females and third instar larvae weredissected in ice-cold 13 PBS and briefly sonicated in a dissociatingmedium (125 mM Tris–HCl (pH 6.8), 8 M urea, 4% SDS, 10 mMDTT, 1 mM PMSF). Embryos (0–2 h or 2–24 h) were dechorionatedand briefly sonicated in the same dissociating medium. Extractswere boiled 2 min, spun at 20,000g for 3 min. The supernatantswere electrophoresed on a 6% polyacrylamide and transferred ontonitrocellulose membranes (Bolt and Mahoney, 1997). The anti-BIP2polyclonal antibodies were diluted 1/20,000. Peroxidase-conjugated goat anti-rabbit IgG (heavy plus light chain) specificantibodies (Vector Laboratories Inc.) were used as secondary anti-bodies. Detection by an ECL 1 kit (Amersham) was performed by

sing standard methods.

Immunostaining on Larval Tissues

Late third instar larvae were dissected and antibody staining wasperformed as described by Sahut-Barnola et al. (1995). The anti-BIP2 polyclonal antibody was diluted 1/3000 and the peroxidase-conjugated goat anti-rabbit IgG (heavy plus light chain) specificantibodies were diluted 1/1000.

Immunostaining on Polytene Chromosomes

Squashes of polytene chromosomes and antibody stainings wereperformed as described by Zink and Paro (1989). The anti-BIP2primary antibody was diluted 1/200. The anti-BAB2 primary anti-body (Laski and Godt, personal communication) was diluted 1/500.The Cy3-conjugated anti-rabbit and the FITC-conjugated anti-rat

FIG. 1. Two-hybrid screen with the BTB/POZ domain of BAB1. (two-hybrid screen (BAB1

65-256) is indicated with the double arrow. Notroups of overlapping cDNAs corresponding to BIP2 were recoverednd 15 clones of 0.7 kb (G75). (C) Growth on selective mediumonstructs. All the co-transformed yeasts grow on a -L-W medium (lnd either with the Gal4AD-BIP2G62 fusion, the Gal4AD-BIP2G77 fusio

medium (lane 4). The positive control is the co-transformation ofgrowth was observed when the GAL4AD-BIP2 fusions were transfordata not shown). No growth was observed when the GAL4DBD-BA

secondary antibodies were diluted 1/500. i


GST Pulldown Experiment

The BIP2G77 cDNA was subcloned in frame with the humanb-globin ATG codon in the KTRAB vector and the corresponding35S-methionine-labeled polypeptide was produced in vitro by usingthe TNT coupled reticulocyte lysate system from Promega. Pro-duction of the GST-BAB1 fusions in E. coli, purification, and GSTpulldown experiments were performed as described by Melcher andJohnston (1995).

BIP2/dTAFII155 Accession NumberThe BIP2/dTAFII155 gene sequence has been deposited in the

enBank/EMBL database under Accession No. AJ251945 andDNA sequence under Accession No. Y14999. The GadFly numberorresponding to the bip2 gene is CG2009.

RESULTS

Identification of BAB-Interacting Proteins

To identify potential partners for the BTB/POZ domain ofthe BAB proteins, we have used the two-hybrid system toscreen a third larval instar cDNA library with a region ofthe BAB1 protein containing the BTB/POZ domain (Fig.1A). Of 121 clones selected for Histidine autotrophy, 65clones were also positive for the activation of the secondreporter gene, lacZ. Out of these 65 clones, 53 interactedspecifically with the bait (GAL4DBD-BAB1

65-256) upon retrans-ormation in yeast. From these 53 positive clones, 26

hematic representation of the BAB1 protein. The bait used in thet the bait is larger than the BTB/POZ domain (aa 98–215). (B) Threethe screen: a single clone of 1.6 kb (G62), 10 clones of 1.0 kb (G77),

he yeasts co-transformed with the BIP2 constructs and the BAB1 and 2). The yeasts transformed with the GAL4DBD-BAB1

65-256 fusionwith the Gal4AD-BIP2G75 fusion are able to grow on a -H selectiveteracting proteins SNF1 and SNF4 (lane 3; Yang et al., 1994). No

with the GAL4DBD-SNF1 fusion (lane 3) or with the GAL4DBD alonefusion was transformed with the GAL4AD-SNF4 fusion (lane 4).

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(Fig. 1B), with 15 identical cDNAs of 0.7 kb (G75), 10identical cDNAs of 1.0 kb (G77), and a single cDNA of 1.6kb (G62). The yeast cells transformed with the baitGAL4DBD-BAB1

65-256 and with one of these three GAL4AD

fusions grow on a selective medium (Fig. 1C, lane 4). Thisgrowth is due to a specific interaction with BAB1 becauseno growth is detected with a co-transformation with theprotein kinase SNF1 (Fig. 1C, lane 3) or with other unre-lated proteins like Laminin or RAF (data not shown).Unexpectedly, these partial cDNAs did not contain a BTB/POZ domain and correspond to a new gene that we havecalled bip2 for Bab Interacting Protein 2. The 27 otherositive clones from the two-hybrid screen correspond tonother new gene that we have called bip1 for Bab Interact-

ing Protein 1. The function of the BIP1 protein will bediscussed elsewhere.

To get the full-length sequence for the bip2 gene, wescreened a l genomic library (Maniatis et al., 1978) using as

probe the G62 cDNA clone isolated from the two-hybridcreen. A phage containing the entire coding sequence forhe bip2 gene was isolated and sequenced. A complete bip2DNA was recovered by RT-PCR using as a templateoly(A)1 mRNAs extracted from third instar larvae. By

comparing the genomic sequence and the cDNA sequence,we determined that the bip2 gene is 6.5 kb in length, andcontains 6 introns and 7 exons (Fig. 2A). Using as a probe

FIG. 2. Structure of the bip2/dtafII155 gene and protein. (A) The b6 introns in a 6.5-kb genomic fragment. Solid boxes indicate the co59 untranslated region was determined by comparing the genomicextends 118 bp upstream the ATG codon. The 39UTR region wasclone LP05832. The 39UTR region extends 139 bp downstream theof the introns are indicated in base pairs. The origin of the genomprotein contains several domains involved in chromatin mediateddomain, an A/T hook motif, and a PHD-finger. The relative positiothe two-hybrid screen are shown below. G62 corresponds to aminoaminoacids 859 to 1091.

genomic DNA that contains the entire bip2 gene for in situ


hybridization to polytene chromosomes from salivaryglands, we have located the bip2 gene on the fourth chro-mosome at the map position 102B (data not shown). TheCanadian chromosome four genome project has localizedthe bip2 gene in its cosmid contigs close to the zaro-4 geneLocke et al., 2000) at the position 102B7-8 (R. Hodgetts,personal communication). The bip2 gene is annotated inhe Drosophila genome project under the GadFly Accessiono. CG2009 (Adams et al., 2000).The bip2 gene encodes a putative protein of 1406 amino

cid residues with a predicted molecular weight of 155 kDand a basic isoelectric point of 9.35. By comparing themino acid sequence of BIP2 with the databases and bysing a search program that identifies protein motifs

http://www.isrec.isb-sib.ch/software/PFSCAN_form.html),e determined that BIP2 contains several known motifs

mplicated in chromatin mediated transcriptional regula-ion (Fig. 2B). The BIP2 protein contains in its N-terminalegion a Histone-Fold domain [amino acids (aa) 1–75, Fig.B]. This motif was first defined as a structural commoneature of the four core histones (Arents et al., 1991).equence comparisons showed that several TAFIIs con-

served in yeast, Drosophila, and vertebrates contain aHistone-Fold domain that has similarities to the histonesH2A, H3, or H4 (Kokubo et al., 1994; Hoffmann et al., 1996;Xie et al., 1996). The Histone-Fold domain is involved in

ene is located on the fourth chromosome and contains 7 exons andsequences and open boxes indicate the untranslated regions. Theence of the bip2 gene with the EST clone LD15321. The 59UTRmined by comparing the genomic sequence of bip2 with the ESTcodon and contains an AATAAA polyadenylation signal. The sizesder corresponds to the ATG codon of the bip2 gene. (B) The BIP2scriptional regulation. The BIP2 protein contains a Histone-Foldthe entire protein of the three groups of BIP2 cDNAs identified in

s 554 to 1062 of BIP2, G77 to aminoacids 757 to 1091, and G75 to

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372 Pointud et al.

histone-like pairs (Nakatani et al., 1996; Xie et al., 1996;Birck et al., 1998; Gangloff et al., 2000, 2001a). The pres-ence of a Histone-Fold domain at the N terminus of BIP2suggests that BIP2 could be a new Drosophila TAFII. It wasdemonstrated that BIP2 is the Drosophila homologue of theyeast yTAFII47 and that it forms a histone-like pair selec-ively with dTAFII24 (Gangloff et al., 2001b). BIP2 wasenamed BIP2/dTAFII155.

The BIP2/dTAFII155 protein contains also a PHD fingerPlant Homeo Domain-finger) at its C-terminus (aa 1342–392, Fig. 2B). The PHD-finger or LAP domain (Leukemia-ssociated-Protein domain) is an atypical class of zincngers with a C4-H-C3 structure (Aasland et al., 1995; Saha

et al., 1995). These atypical zinc fingers have been sug-ested to be involved in DNA-binding and/or protein–rotein interaction (Linder et al., 2000). BIP2/dTAFII155

contains also a putative A/T hook motif (aa 574–586)(Aravind and Landsman, 1998).

The BTB/POZ Domains of BAB1 and BAB2 AreSufficient for the Interaction with BIP2/dTAFII155

The BTB/POZ domain of several proteins, such asTramtrack, ZID (zinc finger protein with interaction do-main), LAZ3/BCL6, and PLZF, has been shown to mediatehomophilic protein-protein interactions (Bardwell andTreisman, 1994; Dhordain et al., 1995; Dong et al., 1996).

he proteins BAB1 and BAB2 contain in their N-terminalegion a BTB/POZ domain (BAB1

BTB and BAB2BTB). Here, we

show that the BAB BTB/POZ domains are able to bothhomomerize (Fig. 3A, lanes 1 and 2) and heteromerize (Fig.3A, lane 3) in a yeast two-hybrid system. Given thisobservation, we would have expected to isolate cDNAs thatencode BTB/POZ domains from our yeast two-hybridscreen. However, the identified BAB interacting factor BIP2does not contain a BTB/POZ domain. This prompted us toanalyze the interaction between BIP2 and BAB1 in moredepth. The bait BAB1

65-256 used in the screen is larger than theBTB/POZ domain alone (aa 95–215). Therefore, we testedwhether the BTB/POZ domain of BAB1 is sufficient tomediate an interaction with BIP2. In addition, we wereinterested to determine whether BIP2 can also interact withthe BTB/POZ domain of BAB2. Yeast cells were trans-formed with the construct GAL4AD-BIP2G75 (Fig. 2B) thatallowed the expression of the portion (aa 859–1091) of theBIP2 protein that had previously been shown to mediate theinteraction with BAB1, and with a second construct thatexpressed either the BTB/POZ domain of BAB1 (GAL4DBD-BAB1

BTB) or BAB2 (GAL4DBD-BAB2BTB) or the BAB165-256 peptide

(GAL4DBD-BAB165-256). These cells were able to grow on the

elective medium (Fig. 3B, lanes 2, 4 and 6). The activity ofhe reporter protein b-galactosidase was measured in these

yeast cells (Fig. 3C). The interactions detected in yeastbetween the BTB/POZ domain of BAB1 and the three BIP2peptides that had been isolated in the original two-hybridscreen (BIP2G75, BIP2G77, and BIP2G62, Fig. 3C, lanes 5–7) are
ery similar to the interactions detected between the bait i

AB165-256 and the different BIP2 peptides (Fig. 3C, lanes 1–3).

The BTB/POZ domain of BAB2 is also able to mediate aninteraction with BIP2 (Fig. 3C, lanes 9–11). To define moreprecisely the domain of BIP2 that interacts with the BTB/POZ domain of the BAB proteins, we generated deletionclones of BIP2G75, the smallest bip2 cDNA that had beensolated due to its interactions with BAB1. None of theselones was able to interact with the BTB/POZ domain ofhe BAB proteins in the yeast two-hybrid system (data nothown). These experiments show that the BTB/POZ do-ains of BAB1 and BAB2 are sufficient for the interactionith BIP2/dTAFII155 and that BIP2G75 is the minimal region

equired for this interaction.

The BIP2/dTAFII155 Protein Interacts in Vitroith the BAB1 Protein

To confirm in vitro the interactions detected in vivo inyeast, we performed a glutathione S-Transferase (GST)pulldown assay between the proteins BAB1 and BIP2. Dif-ferent parts of the BAB1 protein were produced in E. coli asfusion proteins with the GST, and purified on agarose beadscoated with glutathione (Fig. 4A). The construct GST-BAB1

BTB corresponds to a fusion between the GST and theTB/POZ domain of BAB1. The construct GST-BAB1

65-256

contains the region of BAB1 used as bait in the two-hy-brid screen fused to the GST. The largest construct GST-BAB1

65-497 contains the N-terminal half of the BAB1 proteinfused to the GST. The BIP2G77 cDNA was subcloned inrame with the human b-globin ATG codon in the KTRAB

vector and the corresponding protein was produced in vitroin a rabbit reticulocyte lysate in the presence of 35S-

ethionine. An interaction is detected between BIP2 andhe BTB/POZ domain of BAB1 (Fig. 4B, lane 3). Highermounts of BIP2 are retained with the construct GST-AB1

65-256 (Fig. 4B, lane 4) and almost all the BIP2 input isretained with the construct GST-BAB1

65-497 (Fig. 4B, lane 5).he fact that larger fragments of the BAB1 protein mediatestronger interaction than the BTB/POZ domain alone

uggests that the regions adjacent to the BTB/POZ domainacilitate the interaction in vitro. Altogether, these datandicate that the BTB/POZ domain of BAB1 is sufficient initro for an interaction with BIP2/TAFII155 and that thenteraction is probably direct.

bip2/dtafII155 mRNA and Protein Are Presenthroughout Drosophila Development

A high stringency Northern blot analysis was performedby using the BIP2G77 cDNA (Fig. 2B) as probe. A singleranscript of 4.5 kb is detected throughout Drosophilaevelopment (Fig. 5A). The estimated size of 4.5 kb isonsistent with the predicted size of the bip2 mRNA. Highevels of the bip2 transcript are detected in the early embryoFig. 5A, lane 1). This transcript might correspond to ma-ernally deposited transcript as a ubiquitous maternal RNA
s detected in newly laid eggs by tissue in situ hybridization

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(data not shown). The bip2 transcript is also detected inorthern blot experiments in 2- to 24-h-old embryos, third

nstar larvae, prepupae, pupae, and adult males and femalesFig. 5A, lanes 2–7).

Polyclonal antisera were collected from two rabbits im-unized with a fusion protein containing 190 amino acid

esidues (aa 719–909) from the BIP2 protein fused to theST. These antisera were used to check the presence of the

FIG. 3. Interaction of the BTB/POZ domains of BAB1 and BAB2 inctivity in two-hybrid assays and growth on selective medium ofonstructs. The different combinations between hybrids with the G

BTB/POZ domains of BAB1 and BAB2 mediate BTB-BTB interactioBAB2

BTB corresponds to the BTB/POZ domain of BAB2 (aa 195–305).and 2). BAB1

BTB heteromerize with BAB2BTB (lane 3). As controls, the

or the Gal4DBD-BAB2BTB fusion (lanes 4 and 5). (B) The yeast cel

Gal4DBD-BAB1BTB fusion, the Gal4DBD-BAB2

BTB fusion or with the Gal4and 6). No growth was observed when the GAL4AD-BIP2G75 fusioobserved when the Gal4DBD-BAB1

BTB, the Gal4DBD-BAB2BTB, or the Gal4

and 5). (C) The BTB/POZ domains of BAB1 and BAB2 are sufficien the two-hybrid screen (see Fig. 1). BAB1

65-256 contains the BTB/POZecovered from the two-hybrid screen with BAB1

65-256 (see Figs. 1 adomain of BAB1 is sufficient to interact with the clones of BIP2 (lthe clones of BIP2 (lanes 9–11). As controls, the Gal4AD alone wasfusion or the Gal4DBD-BAB2

BTB fusion (lanes 4, 8, and 12). Each b-galith three independent clones.

IP2 protein at different stages of development. A single (


and of approximately 160 kDa is detected throughoutrosophila development with both antisera (Fig. 5B). The

ize of this protein is consistent with a predicted size of 155Da for BIP2, and this protein was not detected using thereimmune sera (data not shown). We therefore assumehat this 160-kDa protein corresponds to BIP2. BIP2 isetected in protein extracts from 0- to 2-h-old embryos, 2-o 24-h-old embryos, third instar larvae, and adult ovaries

yeast two-hybrid system. Quantification of b-galactosidase (b-gal)yeast cells co-transformed with the BIP2 constructs and the BABBD and hybrids with the Gal4AD are shown below each lane. (A) TheAB1

BTB corresponds to the BTB/POZ domain of BAB1 (aa 98–215).1BTB and BAB2

BTB homomerize in the yeast two-hybrid system (lanesAD alone was co-transformed either with the Gal4DBD-BAB1

BTB fusionnsformed with the GAL4AD-BIP2G75 fusion and either with theAB1

65-256 fusion are able to grow on a selective medium (lanes 2, 4,s transformed with the GAL4DBD alone (lane 7). No growth wasAB1

65-256 fusions were transformed with the GAL4AD alone (lanes 1,r the interaction with BIP2. BAB1

65-256 corresponds to the bait usedain of BAB1. BIP2G75, BIP2G77, and BIP2G62 correspond to the clones

). These clones interact with BAB165-256 (lanes 1–3). The BTB/POZ

5–7). The BTB/POZ domain of BAB2 is sufficient to interact withansformed either with the Gal4DBD-BAB1

65-256 or the Gal4DBD-BAB1BTB

idase quantification is the result of two independent experiments

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374 Pointud et al.

The Protein BIP2/dTAFII155 Is Present in All themaginal Structures of the Third Larval Instar

As the partners of the BIP2 protein, BAB1 and BAB2, areexpressed and required in third instar larvae, we used theanti-BIP2 antibodies to determine the expression pattern ofthe BIP2 protein during the third larval instar. The BIP2protein is ubiquitously expressed in the major imaginaldiscs, i.e., the leg imaginal discs, the eye-antennal disc, thehaltere disc, the wing disc, and the genital disc (Figs. 6A, 6B,6C, 6D, 6E, respectively). The BIP2 protein is nuclear in allthese imaginal discs. Along the larval intestinal tract, theBIP2 protein is detected mainly in the imaginal structures,like the salivary gland imaginal ring, the foregut imaginalring of the proventriculus, the hindgut imaginal ring be-tween the midgut and the hindgut, and the midgut imaginalhistoblasts (Figs. 6F, 6G, 6H, 6I, respectively). BIP2 is alsoexpressed in some of the larval tissues of the digestive tract,such as posterior region of the proventriculus (Fig. 6G), thesalivary glands (Fig. 6J), and the Malpighian tubules (Fig.6K). Furthermore, the BIP2 protein is expressed and accu-mulates in the nuclei of the pericardial cells (Fig. 6L), thering glands (Fig. 6M) and the fat body (Fig. 6N). Finally, BIP2is expressed in the larval ovary (Fig. 6O) and in the testis(Fig. 6P). In the ovary, the protein BIP2 is present at higherlevels in the terminal filament cells (Fig. 6O, TFC) than in

FIG. 4. The BIP2/dTAFII155 protein interacts in vitro with the BAroteins were produced in E. coli, purified and analyzed by SDS-PAG

2) corresponds to a fusion of the GST with the BTB/POZ domain ofGST with the amino acids 65 to 256 of BAB1 (corresponding to theto a fusion of the GST with the amino acids 65 to 497 of BAB1. Tproteins are indicated in kDa on the right. The molecular masses oBAB1 interact in vitro. Agarose beads coated with the GST proteinin vitro transcribed and translated peptide corresponding to the BIGST alone is not able to retain the BIP2 peptide (lane 2) whereas alvitro translated and 35S-labeled BIP2 peptide is shown in lane 1. Tautoradiography.

other cells of the ovary.


In the third instar larva, BIP2 is expressed and accumu-ates in the nuclei in a wide range of tissues. All themaginal structures of the third instar larva express highevels of BIP2. In addition, the BIP2 protein is expressed inome of the larval tissues but not in all of them. Tissues,uch as the anterior region of the proventriculus, theosterior region of the ovary (Fig. 6O), the gut (Figs. 6G, 6H,I), the central nervous system, and the lymph gland (dataot shown), appear not to express BIP2 or to express onlyow levels of BIP2.

The Protein BIP2/dTAFII155 Binds to Many Sitesn Polytene Chromosomes

The BAB2 protein is expressed in the salivary glands atthird larval instar, and it binds to many sites on thepolytene chromosomes of the salivary gland cells (Fig. 7A;Godt and Laski, personal communication). We have shownthat the BIP2 protein binds on polytene chromosomes tomany sites representing mainly decondensed transcription-ally active chromatin (Gangloff et al., 2001b). To analyzewhether BIP2 binding sites colocalize with BAB2 bindingsites on chromosomes, we performed immunostainings onpolytene chromosomes using the anti-BIP2 antibody and ananti-BAB2 antibody. The BIP2 protein binds to many more

rotein. (A) Glutathione S-Transferase (GST) and GST-BAB1 fusionectrophoresis, and stained with Coomassie blue. GST-BAB1

BTB (lane1 (aa 98–215). GST-BAB1

65-256 (lane 3) corresponds to a fusion of theused in the two-hybrid screen). GST-BAB1

65-497 (lane 4) correspondsolecular masses of the GST protein and of the GST-BAB1 fusionein standards (lane 1) are indicated in kDa on the left. (B) BIP2 andth the different GST-BAB1 fusion proteins were incubated with ancDNA clone. This peptide was labeled with 35S-methionine. TheGST-BAB1 fusion proteins retain BIP2 (lanes 3–5). The input of inroteins were analyzed by SDS-PAGE electrophoresis followed by

B1 pE elBABbaithe m

f protor wiP2G77

l thehe p

sites than the BAB2 protein on polytene chromosomes


p

hmcmh

s1nadt(OtKdpd

iT

tbBRd


(Figs. 7A–7C) and a subset of the BIP2 binding sites colo-calizes with BAB2 binding sites (Fig. 7B). A strong band ofBAB2 protein is detected at the bab locus itself (Fig. 7C,arrowhead). BIP2/dTAFII155 also accumulates at this spe-cific site suggesting that the BAB2 and BIP2/dTAFII155roteins can colocalize on DNA in vivo.

DISCUSSION

The evolutionary conserved motif known as the BTB/POZ domain is present in many proteins that play a crucialrole in development in Drosophila, many of them byregulating transcription. In humans, this domain has been

FIG. 5. The bip2/dtafII155 mRNAs and proteins are presenthroughout Drosophila development. (A) Developmental Northernlot analysis of poly(A)1 mRNAs. The blot was hybridised with aIP2 cDNA probe. The blot was reprobed with RpS3 to controlNA loading. A single transcript of the predicted size of 4.5 kb isetected in all stages of development with the BIP2G77 probe. (B)

Western blot analysis of proteins extracted either from 0- to 2-h or2- to 24-h embryos, from total third instar larvae (L3) or from adultovaries (OV). The blot was probed with an anti-BIP2 polyclonalantibody. A band of approximately the predicted size of BIP2 (155kDa) was detected in all the protein extracts.

involved in leukemogenesis (Chen et al., 1993; Chaplin et c


al., 1995). From a yeast two-hybrid screen with the BTB/POZ transcription factor BAB1 we have identified andcharacterized a BAB Interacting Protein that is a novelDrosophila TAFII. BIP2/dTAFII155 contains, like its human

omologue hTAFII140, an N-terminal Histone-Fold do-ain, and a C-terminal PHD finger. Sequence comparisons,

o-immunoprecipitation, and yeast two-hybrid experi-ents, have shown that BIP2/dTAFII155 is the Drosophila

omologue of yTAFII47, and that it forms a histone-like pairselectively with dTAFII24 (Gangloff et al., 2001b).

Many of the BTB/POZ transcription factors have beenhown to be transcriptional repressors (Deweindt et al.,995; Oyake et al., 1996; Li et al., 1997a). Some of them, butot all (Deltour et al., 1999), mediate their repressivectivity, via their BTB/POZ domain, by recruiting histoneeacetylase complexes to promoters of their respectivearget genes, thereby inducing a repressive chromatin stateDavid et al., 1998; Dhordain et al., 1998; Lin et al., 1998).ther BTB/POZ proteins function as transcriptional activa-

ors (Kaplan and Calame, 1997; Okada and Hirose, 1998;obayashi et al., 2000). Here, we show that the BTB/POZomains of BAB1 and BAB2 are sufficient to interact with aotential transcriptional co-activator, the TAFII factor BIP2/TAFII155. As BIP2/dTAFII155 is a component of TFIID

(Gangloff et al., 2001b), this interaction provides a directlink between the BTB/POZ transcription factors and thebasal transcriptional machinery and suggests a new mecha-nism for the transcriptional regulation by BTB/POZproteins. Interestingly, interactions between BTB/POZdomains and TAF proteins may mediate either atranscriptional activation or repression. Classical transcrip-tional activators act through a direct interaction betweentheir activation domain and the co-activators of the basaltranscriptional machinery (Pugh and Tjian, 1990; Dynlachtet al., 1991). A number of TAFs have been identified asdirect transcriptional targets of activators in vitro (Good-rich et al., 1993; Chen et al., 1994; Farmer et al., 1996).One interpretation of the interaction between a givenBTB/POZ domain and a TAFII is that this BTB/POZ proteins an activator that directly interacts with TFIID or otherBP-free TAFII-containing complexes (for review, see Bell

and Tora, 1999) to activate the transcription of its targetgenes. An alternative possibility is that the interactionbetween a BTB/POZ domain and a TAFII mediates repres-sion or anti-activation by interfering with the basal tran-scriptional machinery. It has been shown that the co-repressor N-CoR can directly interact with TFIIB, hTAFII32,and hTAFII70 to mediate signals from repressors to the basalmachinery (Muscat et al., 1998). BIP2/dTAFII155 and itshomologue hTAFII140 contain a PHD-finger at their Cterminus. While the function of the PHD-finger is un-known, it has been suggested that it is involved in protein–protein interactions. This domain is present in proteinsassociated with a repressor activity, like histone deacety-lases, and in interactions with heterochromatin (Aasland etal., 1995). The BIP2/dTAFII155 and hTAFII140 proteins
ould mediate, through their PHD fingers, interactions

376 Pointud et al.

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.

em

p

Bdtswc

bbh

mBtBow

atihigma

cwB(BMt(


between BTB/POZ proteins, and repressor activities. BTB/POZ domains could mediate repression of transcription byrecruiting histone deacetylase complexes to generate aclose conformation of the chromatin, and/or by directcontacts with members of the basal transcriptional machin-

FIG. 6. The BIP2/dTAFII155 protein is expressed in all the imaginand stained with an anti-BIP2 polyclonal antibody. BIP2 is expressehe haltere disc (C), the wing disc (D), and the genital disc (E). Thentestinal tract, such as the salivary gland imaginal ring (F, arrowindgut imaginal ring between the midgut and the hindgut (H, arros also detected in a posterior region corresponding to a domain of Blands (J), Malpighian tubules (K), pericardial cells (L), ring glands (Male gonads (P). In the larval ovary (O), the highest levels of BIP2 ar

FIG. 7. BIP2 colocalizes with BAB2 at specific sites on polytenehromosomes. DNA was stained with Hoechst 33258 (blue). BAB2as detected with secondary antibodies coupled to FITC (green).IP2 was detected with secondary antibodies coupled with Cy3

red). BIP2 binds to several sites at the tip of 3L chromosome (A).AB2 shows a prominent binding site at the bab locus in 61F (C).erging of both images (B) shows that BIP2 and BAB2 colocalize at

hree loci (white lines) and notably at the bab locus in 61Farrowhead).

re separated by dashed lines). Scale bars are 50 mm, except for (M) and


ry locking them into a nonfunctional complex or confor-ation.Very few non-BTB/POZ proteins have been identified as

artners of the BTB/POZ domains of Drosophila proteins.The interaction of the Tramtrack BTB/POZ domain withthe corepressor dCtBP (C-terminal binding protein) (Wen etal., 2000) and the BTB/POZ domain of GAGA with theheterologous proteins N-CoR and SMRT (Huynh and Bard-well, 1998) in in vitro experiments have been briefly re-ported. Since the BTB/POZ domains of the Drosophilanuclear proteins are strongly conserved in evolution (Zoll-man et al., 1994), it is probable that other Drosophila

TB/POZ proteins interact directly, via their BTB/POZomain, with one of the central components of the preini-iation complex, like BIP2/dTAFII155, to regulate the tran-cription of their target genes. As a given TAF can interactith more than one activator and a single activator can also

ontact multiple TAFs, we suggest that BIP2/dTAFII155could interact with other Drosophila BTB/POZ proteinsand/or that different BTB/POZ domains can contact differ-ent TAFs. The BTB/POZ transcriptional repressor HIC-1and its avian homologue gFBP-B do not seem to mediatetheir repressive effect by recruiting histone deacetylasecomplexes as they fail to interact with the co-repressorsSMRT, N-CoR, and mSIN3A and as their transcriptionalrepression is unaffected by Histone Deacetylase (HDAC)inhibitors (Deltour et al., 1999). The repressive function ofHIC-1 and gFBP-B could be mediated by a direct interactionetween their BTB/POZ domains and components of theasal transcriptional machinery such as the vertebrateomologue of BIP2/dTAFII155.Although the BTB/POZ domain of BAB1 is sufficient for

the interaction with BIP2/dTAFII155, our in vitro experi-ents show that the regions of BAB1 adjacent to the

TB/POZ domain facilitate this interaction. This observa-ion could reflect different folding properties of the differentAB1 fusion proteins or could imply a direct involvementf the BTB/POZ adjacent regions to stabilize the interactionith BIP2/dTAFII155. This could indicate that the regions

adjacent to the BTB/POZ domain could give another levelof specificity to the BTB/POZ proteins for an interactionwith a given protein like a TAF.

The BTB/POZ transcriptional activity may also dependon the BTB/POZ potential to form hetero-oligomers. The

ctures of third instar larvae. Late third instar larvae were dissectedhe major imaginal discs, the leg disc (A), the eye-antennal disc (B),protein is detected in all the imaginal structures along the larval

e foregut imaginal ring of the proventriculus (G, arrowhead), thend the midgut imaginal histoblasts (I). In the proventriculus, BIP2pression (G, arrow). A clear nuclear staining is detected in salivary

nd fat body (N). BIP2 is expressed in the female gonads (O) and theected in the terminal filament cells (TFC, some terminal filaments

l strud in tBIP2), thw), a

AB ex), a

e det
(O), 10 mm.

na(BtttudrmtBbec

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378 Pointud et al.

transcription factors Bach2 and MAZR are BTB/POZ pro-teins involved in hematopoiesis and in development ofmouse limbs (Oyake et al., 1996). The BTB/POZ domain ofMAZR mediates its homomerization but it can form het-eromers with the BTB/POZ domain of Bach2 (Kobayashi etal., 2000). The MAZR protein is able to activate the c-mycpromoter (Kobayashi et al., 2000). Interestingly, the combi-

ation of MAZR and Bach2 potentiates this transcriptionalctivation while Bach2 alone is a transcriptional repressorMuto et al., 1998; Kobayashi et al., 2000). The BAB1 andAB2 proteins can homomerize and heteromerize throughheir BTB/POZ domains. They can also heteromerize withhe BTB/POZ domain of other BTB/POZ transcription fac-ors, Broad-Complex and Tramtrack (Pointud and Couderc,npublished data). These results suggest that BTB/POZomain-mediated oligomerization may serve as combinato-ial codes for gene expression. A single BTB/POZ domainay mediate transcriptional activation as a homomer and

ranscriptional repression as a heteromer with anotherTB/POZ domain, or vice versa. The possible combinationsetween different BTB/POZ proteins would provide differ-nt interfaces of interaction with nuclear co-repressors oro-activators.The crystal structure of the BTB/POZ domain of PLZF

evealed a tightly intertwined dimer with a surface-exposedroove. This pocket is composed of some of the mostonserved residues of the BTB/POZ sequence and has a highharge density (Ahmad et al., 1998). The minimal domainf the BIP2/dTAFII155 protein required for the interactionith the BTB/POZ domains of the BAB proteins corre-

ponds to a highly charged region comprising multipleysine, glutamic acid and aspartic acid residues (aa 859–091). This charged region of BIP2/dTAFII155 is well con-

served in its homologues hTAFII140 and mTAFII140(Gangloff et al., 2001b). The charged-exposed groove of theBTB/POZ dimer may bind this charged region of BIP2/dTAFII155 or hTAFII140 through electrostatic interactions.A mutational analysis of the BTB/POZ domain of PLZFshow that this putative binding site is crucial for the properfolding of the protein and thus for the dimer formation, butit is especially critical for the transcriptional activity ofPLZF (Melnick et al., 2000). It is tempting to speculate thatmutations in this region, that do not impair dimer forma-tion but abolish the transcriptional activity of PLZF, act bypreventing the binding of transcriptional co-repressors orco-activators to the dimer of BTB/POZ domains. Theseconserved amino-acids are present in the BAB BTB/POZdomains, so it will be possible to test whether such muta-tions prevent the interaction between the dimer of BTB/POZ domains and non-BTB/POZ proteins like BIP2/dTAFII155.

The BAB proteins may regulate the expression of severalgenes at different steps of Drosophila development. TheBAB2 protein binds to several sites on polytene chromo-somes (this work; Godt and Laski, personal communica-tion). During leg morphogenesis, bab is required for seg-
mentation and for the specification of segment identity in

the tarsus (Godt et al., 1993). bab is also required for adultvary morphogenesis (Sahut-Barnola et al., 1995; Godt andaski, 1995). Finally, Kopp et al. (2000) have shown that theAB proteins are involved in the genetic control of theexually dimorphic abdominal pigmentation and the ab-ominal A5–A7 segment morphology in Drosophila mela-ogaster. Since BIP2/dTAFII155 is expressed in all the

tissues that express and require BAB, it is possible thatBIP2/dTAFII155 mediates all the BAB functions. The BABproteins could be transcriptional activators as well as tran-scriptional repressors, depending on the promoter consid-ered and it will be of great interest to identify BAB targetgenes and cis-regulatory sequences recognized by the BABroteins. However, the BAB proteins, unlike most of thether BTB/POZ proteins, do not contain a zinc finger DNAinding domain but a new type of DNA binding domainhat we are currently characterizing. Clearly, the identifi-ation of bab downstream genes is crucial to understandhether the interaction between BIP2/dTAFII155 and the

BTB/POZ domain of the BAB proteins is critical for BTB/POZ-mediated transcriptional activation or repression.

ACKNOWLEDGMENTS

We thank S. Lambel and V. Calco for excellent technical assis-tance. We thank O. Bardot, I. Davidson, D. Godt, F. Laski, D.Leprince, and G. Prive for critical readings of the manuscript,suggestions, and English corrections. The Western blot experi-ments were performed thanks to advice from G. Berson. We thankB. Viollet for the KTRAB vector and R. Hodgetts for communicat-ing results before publication. The two-hybrid third larval instarcDNA library was kindly provided by S. Elledge. J-C.P. wassupported by a fellowship from the MESR (Ministere del’Enseignement Superieur et de la Recherche). J.L. was supported bygrants from the Swedish Natural Science Research Council. J-L.C.was supported by Grant GREG#59/95.

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Received for publication March 21, 2001Revised May 21, 2001Accepted June 8, 2001

Published online August 15, 2001


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The BTB/POZ Domain of the Regulatory Proteins Bric