Drosophila homologs of transcriptional mediator complex subunits

Drosophila homologs of transcriptionalmediator complex subunits are requiredfor adult cell and segment identityspecification

Muriel Boube,1 Christian Faucher, Laurent Joulia, David L. Cribbs,2 and Henri-Marc Bourbon

Centre de Biologie du Developpement-CNRS, 31062 Toulouse CEDEX 04, France

The origins of specificity in gene expression are a central concern in understanding developmental control.Mediator protein complexes regulate transcriptional initiation, acting as modular adaptors linking specifictranscription factors to core RNA polymerase II. Here, we identified the Drosophila homologs of 23 humanmediator genes and mutations of two, dTRAP240 and of dTRAP80 (the putative fly homolog of yeast SRB4).Clonal analysis indicates a general role for dTRAP80 necessary for cell viability. The dTRAP240 gene is alsoessential, but cells lacking its function are viable and proliferate normally. Clones reveal localizeddevelopmental activities including a sex comb cell identity function. This contrasts with the ubiquitousnuclear accumulation of dTRAP240 protein in imaginal discs. Synergistic genetic interactions support shareddevelopmental cell and segment identity functions of dTRAP240 and dTRAP80, potentially within a commoncomplex. Further, they identify the homeotic Sex combs reduced product, required for the same cell/tissueidentities, as a functional partner of these mediator proteins.

[Key Words: Mediator; SRB; transcription; cell identity; segment identity; homeotic; development]

Received April 26, 2000; revised version accepted September 28, 2000.

Transcription initiation is a central and highly regu-lated step in eukaryotic gene expression that unitescore RNA polymerase II (Pol II) with numerous otherfactors. These include Pol II–associated general tran-scription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, andTFIIH) and sequence-specific regulators bound to geneenhancers (for review, see Hampsey and Reinberg 1999;Malik and Roeder 2000). Work of the last 10 years hasbrought to light a new class of transcription factor com-plex, the mediator (MED). The first known mediatorcomponents, encoded by the yeast SRB/MED genes,were identified by dominant mutations suppressing theconditional lethality caused by a C-terminal domain(CTD) mutation of the Pol II large subunit (Thompson etal. 1993). The biochemically purified Srb proteins inter-act physically with core RNA Pol II in the form of largeprotein complexes (Thompson et al. 1993; Kornberg1999).

More recently, mediator complexes have likewisebeen identified in mammalian cells where, as in budding

yeast, they associate with the core Pol II to form a giantholoenzyme (Asturias et al. 1999). The mammalianMED complexes capable of stimulating basal transcrip-tion initiation in vitro contain ∼20 subunits, including atleast five proteins homologous to yeast Srb/MED pro-teins (Boyer et al. 1999; Gu et al. 1999; Ito et al. 1999;Naar et al. 1999; Rachez et al. 1999; Ryu et al. 1999).Several related complex forms that mediate transcrip-tion in vitro have been isolated through their physicalcontact with a spectrum of mammalian transcriptionfactors. These include nuclear receptors for thyroid hor-mone (TRAP complex; Ito et al. 1999) and vitamin D3(DRIP complex; Rachez et al. 1999), VP16, the p65 sub-unit of NF-�B, SREBP-1a (ARC; Naar et al. 1998, 1999),Sp1 (CRSP; Ryu et al. 1999), E1A (Boyer et al. 1999), andp53 (Ito et al. 1999). Alternative protocols have yieldedrelated mammalian complexes (human or mouse media-tor; Chao et al. 1996; Jiang et al. 1998; SMCC, Ito et al.1999) or subcomplexes (negative regulator of activatedtranscription, NAT, associated with human Srb10/Cdk8;Sun et al. 1998). These related complexes are viewed asversatile interfaces that link specific transcription fac-tors and the general Pol II machinery in a complex equi-librium (Lee et al. 1998). The MED complexes are mostoften considered transcriptional coactivators, and thisproperty has been used as the basis of their biochemical

1Present address: Department of Molecular and Cellular Biology, CID-CSIC, c/ Jordi Girona 18–26, Barcelona 08034, Spain.2Corresponding author.E-MAIL [email protected]; FAX 33-561-55-65-07.Article and publication are at www.genesdev.org/cgi/doi/10.1101/gad.179800.

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purification. Importantly, however, these complexes arenot dedicated activators, and some forms have also beendescribed as corepressors (Song and Carlson 1998; Sun etal. 1998).

A systematic examination of gene expression in yeastsrb mutants has revealed distinct families of target genesof varying size and composition for different Srb sub-units (Holstege et al. 1998). This functional diversity islikely to reflect a corresponding diversity of physicallyinteracting regulatory transcription factors. MED com-plexes appear to integrate regulatory information frommultiple transcription factors and relay that informationto the core Pol II (for reviews, see Parvin and Young 1998;Hampsey and Reinberg 1999; Kornberg 1999; Malik andRoeder 2000). In support of this view, recent work dem-onstrates that human ARC complex can interact withtwo transcription factors and parlay this input into asynergistic transcriptional response (Naar et al. 1998,1999). In metazoans, the dynamic developmental processrequires fine control of the gene expression program,presumably involving their MED complexes. However,for the moment little is yet known of their in vivo func-tions in development. The first known mutation of ametazoan MED subunit, in the sur2 locus of the nema-tode Caenorhabditis elegans, was isolated as a sup-pressor of activated Ras in vulval development (Singhand Han 1995). A MED complex has been identifiedin C. elegans and suggested to participate in regulation ofdevelopmental target genes (Kwon et al. 1999). Recently,gene inactivations have been described for two mousesubunits, Srb7 (Tudor et al. 1999) and TRAP220 (Ito et al.2000). Murine Srb7 corresponds to a core MED sub-unit required for yeast cell viability and is apparentlyrequired for cell viability in the mouse embryo as well.The inactivation of TRAP220, a subunit implicated inligand-dependent binding to thyroid hormone receptor,reveals diverse developmental defects in a variety of tis-sues.

A better understanding of the in vivo physiologicalroles of the MED complexes in metazoan developmentrequires the continued genetic analysis of mutations oftheir subunits in model organisms. Toward this end, wehave identified 23 Drosophila homologs of human MEDsubunits in the recently completed genomic sequence.We have cloned two of these and identified loss-of-func-tion mutations for each. Both mutations are recessivelethal, revealing the functions of the genes as essential.However, the two show marked differences, as one isrequired for cell viability and the other is required for theintegrity of the organism. These mutations have allowedus to identify discrete roles for MED protein functions inthe control of adult segment and cell identities. Syner-gistic genetic interactions between the two putativeMED subunits indicate a shared developmental func-tion, potentially within a Drosophila complex. Theirsynergistic interactions with the homeotic Sex combsreduced (Scr) locus further implicate the SCR homeo-domain protein as a functional partner whose activityis modulated by MED protein activity during develop-ment.

Results

Identification of 23 Drosophila homologs of humanmediator proteins and cloning of pap/dTRAP240

A new Drosophila gene, poils aux pattes (pap; cytologi-cal locus 78A1-3), was identified in a P-elementscreen for dominant genetic modifiers of cell identityfunctions of the homeotic loci Scr (Hox-A5/B5) andproboscipedia (pb; Hox-A2/B2; Pattatucci et al. 1991;Cribbs et al. 1995). The Scr selector gene confers protho-racic identity, while pb alone induces maxillary iden-tity. Together, the Scr and pb selectors show a combina-torial behavior leading to specification of the adult labialpalps (mouthparts). Low-level ectopic expression of PBprotein from an hsp70–pb mini-gene, the HSPB ele-ment, induces several dose-sensitive cell-identityphenotypes (Cribbs et al. 1995; Boube et al. 1997) thatwere used to screen for second-site dominant modi-fier mutations. Of 5000 new autosomal P insertionstested, only one, in the pap locus, showed dose-sensitiveenhancement of the distal sex comb (described below)induced by the HSPB element. Starting from the P-ele-ment molecular tag, a 50-kb pair interval encompassingthe pap gene was cloned. Analysis of genomic andcomplementary DNA (cDNA) sequences indicates atranscription unit spanning at least 22 kb and generatinga ∼10-kb mRNA (Fig. 1a). The exonic P insertion residesupstream of the first in-frame ATG of an open readingframe (ORF) of 2618 amino acids (see Fig. 1a). Full rever-sion of lethality by mobilizing the P element, and therescue of lethality by a ubiquitin–cDNA construct (notshown), confirmed that this ORF corresponds to pap.The ORF encodes the unique Drosophila counterpart ofTRAP240/ARC250, recently identified as a subunit ofthe human thyroid hormone receptor–associated protein(TRAP) or activator-recruited cofactor (ARC) proteincomplexes (Ito et al. 1999; Naar et al. 1999). The Dro-sophila PAP protein shows 27% overall identity (40%similarity) with human TRAP240 and 27% identity(39% similarity) with its C. elegans counterpart. Thisconservation extends across the proteins but is highestin the N- and C-terminal regions (Fig. 1b). We thereforeconsider pap as the presumptive fly homolog ofTRAP240.

The recent availability of the Drosophila genomic se-quence (Adams et al. 2000) and the collection of corre-sponding cDNAs through the Berkeley Drosophila Ge-nome Project (BDGP; Rubin et al. 2000) allowed us toidentify a single putative Drosophila homolog for each ofthe 23 known human TRAP/ARC subunits (Fig. 2a; Ma-terials and Methods). For simplicity, we will refer tothese proteins and their genes in the text as TRAPs whenmore than one name exists for the same entity (Malikand Roeder 2000). While the existence of a biochemicalentity remains to be demonstrated, the observed struc-tural conservation of such a large number of MED genesprovides clear circumstantial evidence for the existenceof a fly mediator complex (dMED) similar to the purifiedcomplexes from worms (Kwon et al. 1999) and mammals(above).

Fly mediator genes in cell and segment identity

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Figure 1. pap encodes the fly homolog of the human transcriptional mediator subunit TRAP240. (a) Molecular organization of thepap/dTRAP240 region (cytological position 78A1-3, chromosome 3L). The position of the recessive lethal P-lacW element insertionallele pap1 is shown with the map of this region. (E) EcoRI; (B) BamHI; (S) SalI. The pap53 allele deletes the entire coding portion ofexon 1 (amino acids 1–23). The transcription unit is shown with boxes representing exons; ATG is the presumptive initiator codon,filled boxes indicate protein coding region, and broken lines indicate introns (the precise site of transcription initiation is not known).(b) Protein sequence comparisons of TRAP240 homologs from Drosophila (d), human (h), and Caenorhabditis elegans (c). Because ofthe length of the proteins, only the most conserved regions (the N and C termini) are shown. Identical amino acids are shaded in blackand conservative changes in gray. The Genbank accession numbers for the fly and human proteins are AF227214 and AF117754,respectively. The putative C. elegans homolog is derived from EMBL accession number Q93442, with aligned sequence beginning ata conserved methionine.

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Figure 2. (Legend on following page)






pap and dTRAP80 encode putative mediator subunitsrequired for development

Among the new dMED genes identified was dTRAP80 atcytological position 90F1-2 on chromosome 3R (Fig. 2b).It encodes a predicted dTRAP80 protein of 642 aminoacids exhibiting 40% identity (59% similarity) to its hu-man counterpart. The majority of putative DrosophilaMED genes, including pap, appear to lack a homolog inthe complete Saccharomyces cerevisiae genome se-quence. S. cerevisiae SRB4 encodes a core component ofthe Srb mediator complex required for the expression ofvirtually all yeast genes (Holstege et al. 1998). The genewas identified by dominant mutations that directly sup-press a Pol II CTD mutation (Thompson et al. 1993). Forthe dTRAP80 protein, low but potentially significantoverall structural conservation (16% identity, 48% simi-larity) was detected with Srb4 proteins from the MEDcomplexes of the yeast S. cerevisiae and Schizosaccha-romyces pombe (Thompson et al. 1993; Spahr et al.2000). The overall identity between these two yeast Srb4proteins is only 25% (60% similarity), with conservationmost pronounced in a region with predicted �-helicalcharacter between amino acids 214–313 of S. cerevisiaeSrb4 (40% identity, 72% similarity; see Fig. 2c). Bothprimary sequence and predicted helical character areconserved within this interval in metazoan TRAP80moieties, attaining 34% identity between humanTRAP80 and S. pombe SRB4. The corresponding se-quences appear unique in the budding yeast and Dro-sophila genomes, arguing against a novel reiterated do-main. Thus, despite the low level of overall identity,these observations are good evidence for homology of themetazoan TRAP80 genes with yeast SRB4.

One lethal P-insertion mutation from the BDGP col-lection (Spradling et al. 1999) is situated within thedTRAP80 coding sequence. This insertion, dTRAP801, islocated downstream of the apparent initiator ATGwithin the same exon (Fig. 2b). The cloning and the iden-tification of mutations in these two putative DrosophilaMED genes allowed us to initiate an in vivo assessmentof their physiological roles in normal development.dTRAP801 mutants die as second-instar larvae with noobvious cuticular defect. The initial pap1 P-element in-sertion and most derived imprecise excisions (includingthe molecular null allele pap53; see Fig. 1a) are recessiveembryonic lethals. pap− embryos appear normal apart

from discrete cuticular defects of the embryonic mouth-parts. Thus, both functions are essential for viability,and pap is detectably required for normal embryonic de-velopment. Ubiquitous accumulation of pap anddTRAP80 mRNA was observed by in situ hybridizationin embryos of all stages and in larval imaginal discs (notshown). The presence of mRNA in early embryos furthersuggests that a maternal contribution partially compen-sates for the absence of zygotic expression for both papand dTRAP80.

dTRAP80 loss of function is cell lethal

The prototypical Srb4 protein is required for transcrip-tion of nearly all Pol II–dependent promoters in yeast(Holstege et al. 1998). If dTRAP80 encodes the functionalhomolog of Srb4, it is predicted to participate in all as-pects of mediator function, and the dTRAP80− conditionshould be cell lethal. The survival of dTRAP80− embryosto second-instar larvae (above) suggested that mater-nally contributed dTRAP80 mRNA suffices for embry-onic survival. To test the consequences of removingdTRAP80 while minimizing the complication of mater-nal contribution, we employed mitotic recombination.From heterozygous mother cells, twin clones of daughtercells homozygous for each of the two chromosome armswere induced, one carrying dTRAP801 (or dTRAP80+)and the other its wild-type homolog (plus the associatedcuticular markers Stubble [Sb] and ebony [e]). ThedTRAP80+/+ or dTRAP80−/− cells of interest were iden-tified by their bristle shape (Sb+). Where dTRAP80+

yielded 150 clones, none were observed withdTRAP80−/− for an equivalent sample size. We concludethat dTRAP80 is required for cell viability in the adultepidermis. This result provides independent supportfor a general cellular role of dTRAP80 consistent withthe sequence-based interpretation that it encodes a flySrb4.

pap encodes a ubiquitous nuclear proteinwith localized developmental functions

To examine the distribution and cellular localization ofPAP protein, a polyclonal antiserum was generatedagainst its N-terminal region, then enriched by affinitychromatography with the same PAP protein fragment

Figure 2. Identification and genomic localization of Drosophila melanogaster homologs to known human MED genes. These includedTRAP80, an apparent metazoan homolog of yeast SRB4. (a) Cytological positions of 21 predicted dMED loci are shown. These geneshave been annotated by the Genome Annotation Database of Drosophila (GadFly; http//www.fruitfly.org/annot/) and are listed inMaterials and Methods. Homology searches identified dTRAP80 as the apparent metazoan homolog of yeast SRB4 (see c). (b) Genomicorganization of the dTRAP80 locus (cytological interval 90F1-2 of chromosome 3R) is shown. The dTRAP80 region map, (E) EcoRI; (N)NotI; (K) KpnI; (Bg) BglII, indicates the position of the recessive lethal dTRAP801 P-element insertion, previously referred to asl(3)s2956 (Spradling et al. 1999). The exon–intron organization is shown beneath with filled boxes for protein coding region and brokenlines indicating introns. (c) Aligned protein sequences of TRAP80/Srb4 homologs from D. melanogaster (d), human (h), Schizosac-charomyces pombe (sp), and Saccharomyces cerevisiae (sc). Genbank accession numbers are AF244916, AF117657, CAB10081, andL12026, respectively. A Caenorhabditis elegans TRAP80 homolog was also detected (not shown; Genbank accession numberCAB76740). Identical amino acids are shaded in black, and conservative changes (Taylor 1986) in gray. Positions of aligned amino acidspredicted to adopt helical (H) or extended (E) secondary conformations (http//www.ibcp.fr/predict.html) are indicated above thealignment.

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(Materials and Methods). The specificity of the antiserawas confirmed by immunostaining of embryos and ofimaginal discs overexpressing PAP in the posterior com-partment of each segment (engrailed–GAL4/UAS–pap) orof discs harboring mitotic clones of pap53 cells (Materialsand Methods). In overexpression experiments, strongstaining was limited to posterior cells, whereas in clonesof pap53 cells, the signal was no longer detected (notshown). Immunostaining experiments with this specificanti-PAP serum show that pap mRNA is translatedthroughout the imaginal tissues, as predicted by themRNA distributions. PAP protein accumulation is pre-dominantly nuclear (Fig.3a,b,d,e), in agreement with arole in the general transcription machinery.

To examine functional requirements for the essentialpap gene in adult development, we generated mitoticclones of mutant cells. In marked contrast to dTRAP80(above), clones were obtained showing that normal papfunction is not required for cell viability. Clones inducedduring larval development (Fig. 3) led to distinct conse-quences in different tissues. First, adult Drosophila me-lanogaster males normally align a single row of special-ized bristles, the sex comb teeth, on the first tarsal seg-ment of the prothoracic (T1) leg. These sex comb teeth

are not found elsewhere. In contrast, clones of pap mu-tant cells situated in the distal second tarsal segmentdifferentiated as ectopic sex comb teeth (Fig. 3c). Normalpap function thus opposes sex comb cell fate in this po-sition. This role appears cell autonomous, as all observedectopic sex comb teeth were mutant for pap (see Fig. 3c).In contrast, clones within the normal sex comb or bor-dering it did not affect the number of cells adopting thisfate (Fig. 3c). Second, clones localized elsewhere in theT1 leg, or at any position in the T2 and T3 legs, werewithout effect (not shown). Third, clones in the maxil-lary palps are associated with malformations. Finally,large clones in the wing blade, the notum, or the anten-nae led to apparently normal pattern. Therefore in con-trast with ubiquitous accumulation of PAP in epidermalcells, pap function is required for normal development inonly a subset of those cells. Taken together, these datastrongly suggest that developmental pap activity may beregulated according to the tissue and cell, being requiredfor some identities but dispensable for others.

Previous biochemical characterization identified hu-man TRAP240 as a component of the ubiquitous MEDcomplexes and, hence, of the general transcription ma-chinery. As such, pap/dTRAP240 mutations might have

Figure 3. Expression and activity of the pap/dTRAP240 locus. (a,b,d,e) Localization of PAP in imaginal discs from third-instar larvaeusing a monospecific rabbit polyclonal serum. Shown are a prothoracic leg imaginal disc (a) with enlargement (b), and a wing imaginaldisc (d) with enlargement (e). PAP protein is expressed in most or all imaginal cells (a,d) and accumulates preferentially in the nucleus(b,e). Mutant clones at the distal extremity of the second tarsal segment in the T1 legs (c) exhibited an ectopic distal sex comb (dsc).Ectopic sex comb teeth (small circles) are yellow (pap−). A maximum of three ectopic teeth were observed in any pap− clone. Withinthe normally placed sex comb (sc) at the distal end of the first tarsal segment of this sample, three mutant cells (marked with circles)are interspersed with pap+ cells. This sex comb is of normal size. Hence, the pap− condition affected neither sex comb cell differen-tiation nor rotation for alignment. (f) Twin spot clonal analysis of pap function in imaginal disc development. Adjacent (twin) clonesof pap−/− (no GFP; black arrow) and pap+/+ cells (two copies of ubiquitin–GFP; white arrow) are identified by expression of thecell-autonomous GFP marker in a third-instar larval wing imaginal disc. Note the similar sizes and distributions of the wild-type andmutant cells.






adverse consequences for cell physiology and growth.Consequently, the discrete changes in cell identity ob-served could reflect a spurious fragilization of the cellrather than a concrete developmental role for pap in cellspecification. To address this concern, clonal analysiswas performed (Materials and Methods and above) usinga green fluorescent protein (GFP) cell marker to examinethe proliferation and distribution of mutant cells relativeto wild type. Clones of cells homozygous for pap53 andthe reciprocal wild-type product were induced in first- orsecond-instar larvae, then compared in imaginal discsof late third-instar larvae. An example is shown in Fig-ure 3f, where twin clones are observed in a wing imagi-nal disc. Adjacent clones of mutant cells (black) are aslarge as their wild-type counterparts (white) and simi-larly distributed relative to the axes of the imaginaldisc. Similar results were obtained in other imaginal tis-sues, including the labial and T1 leg discs (examples inFig. 4 below). These results reinforce the conclusion thatthe distal sex comb cell identity transformation observedin pap mutant clones reflects a normal, localized func-tion of this gene in this position and not an indirectconsequence of an impaired general transcription ma-chinery function.

pap and dTRAP80 interact synergisticallywith the homeotic Sex combs reduced locusin cell and segment identity specification

The ectopic distal sex comb induced by pap clones is areadily visible cell identity marker that reflects normalpap function. Ectopic distal sex comb teeth are inducedin appropriately positioned cells lacking any pap func-tion (above). This phenotype is also observed at low fre-quency with certain Scr and pb gain-of-function alleles(ScrScxP and hsp70–pb [HSPB] mutations; Pattatucci et al.1991; Cribbs et al. 1995). This effect of the ScrScxP alleleis enhanced in pap heterozygotes (Table 1). Functions ofthe homeodomain transcription factor SCR specify pro-thoracic identity, including sex comb cell fate. The in-

duction of distal sex combs by HSPB also depends on Scractivity, as it is no longer detected in Scr heterozygotes(see Table 1). Ectopic sex comb differentiation is en-hanced in HSPB/pap53 heterozygotes, but this effect isabolished in Scr heterozygotes (Table 1). These observa-tions of dose-sensitive interactions indicate a synergisticfunctional link between Scr and pap in this cell identityspecification. pap and dTRAP80 both encode the soledetected fly homologs to human proteins identified bytheir presence in the MED complex. If the enhancementof the distal sex comb phenotype in pap heterozygotes iscaused by limiting mediator function, double heterozy-gotes with dTRAP80 should aggravate this condition.We therefore tested whether dTRAP80 acts togetherwith pap in this cell identity decision. The loss-of-func-tion allele dTRAP801 is fully recessive, as for pap53. Incontrast, 10% of pap53 +/+ dTRAP801 males possess anectopic distal sex comb tooth, revealing a cooperativefunction in these cells (Table 1). Synergistic enhance-ment of the ectopic sex comb caused by ScrScxP is like-wise observed in double heterozygotes (Table 1). Thesefunctional data indicate a shared function of PAP anddTRAP80, again suggesting the existence of a Drosophilamediator complex. They further suggest that at least onecommon function of PAP and dTRAP80 acts to antago-nize Scr activity in distal sex comb differentiation.

Apart from the prothorax, normal Scr activity is alsorequired for development of the adult labial palps, whereit acts in a combinatorial fashion with the homeotic pbgene (Cribbs et al. 1995; Percival-Smith et al. 1997). Thisled us to examine the effects of pap and dTRAP80 mu-tations on adult mouthparts formation. The wild-typelabium is typified by the presence of pseudotrachealrows used for drinking and the absence of a segmentalappendage. The hypomorphic pb4/pb5 genotype leads toa transformation of distal labium to antennal arista (Fig.4a), with a concomitant reduction of the pseudotracheae.In this sensitized context, changes in relative Scr activitycan be readily detected. Reduced pap or dTRAP80 activ-ity in heterozygotes enhances the labial-to-leg transfor-mation, as seen by the appearance of leg-specific celltypes: sex comb teeth in males, bracted bristles, and ter-minal claws (Table 2). In pap dTRAP80, double hetero-zygotes leg structures often entirely replace labial pseu-dotracheae (Fig. 4c). As in the leg, this effect of pap anddTRAP80 mutants on labial development is synergistic(see Table 2). These data provide further support for ashared role of PAP and dTRAP80 proteins opposed, inthis case, to the leg-forming activity of Scr in the labialtissue.

pap and the homeotic selector locus Scr act in parallel

The observed effects of pap and dTRAP80 mutations inthe T1 legs and labium may most simply be rationalizedas consequences of increased Scr activity. This could re-sult from augmented regulatory activity of SCR proteintoward Scr target genes. Alternatively, it might reflecthigher Scr gene expression with greater quantities ofSCR protein. To distinguish between these two possibili-

Table 1. Scr and dMED in distal sex comb (DSC) cellidentity

Genotype

Percentagelegs with

DSC

MeanDSCteeth

per leg

Percentageof legs with

three or moreDSC teeth

pap53/+ 0 (n = 200) 0 0ScrScxP 3 (n = 182) 0.04 0ScrScxP/pap53 15 (n = 102) 0.15 0HSPB 6 (n = 344) 0.06 0HSPB/Scr4 0 (n = 252) 0 0HSPB/pap53 54 (n = 206) 0.54 1HSPB/pap53Scr4 0 (n = 171) 0 0dTRAP801/+ 0 (n = 200) 0 0pap53 dTRAP801/++ 10 (n = 372) 0.1 0ScrScxP/dTRAP801 9 (n = 140) 0.1 0ScrScxP/pap53 dTRAP801 34 (n = 150) 0.4 10

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ties, we examined SCR homeoprotein accumulation byindirect immunofluorescence in pb4/pb5 labial imaginaldiscs that give rise to mixed labial/antennal or pb4/pap1

pb5 dTRAP801, yielding T1 leg identities. Nuclear SCRprotein does not detectably increase with the transitionto T1 leg identity (Fig. 4, cf. b and d). These data indicatethat PAP and dTRAP80, acting in parallel or downstreamof Scr, negatively modulate SCR protein function in la-bial tissue.

The above experiments were performed in heterozy-gotes for pap and dTRAP80. In the sensitized geneticcontext employed, slight but functionally importantchanges in SCR accumulation could potentially pass un-detected in this test. We therefore tested the molecularepistatic relations between pap and Scr in homozygousmutant cells to determine whether Scr gene expressiondepends on normal pap function and vice versa. Both Scrand pap are normally expressed throughout the labialand T1 leg imaginal discs, and both confer detectablephenotypes there as described above. Mitotic clones ofhomozygous pap−/− or Scr−/− cells were induced in first-

and second-instar larvae and identified in mature third-instar imaginal discs by the cell autonomous GFPmarker and by the accumulation of SCR or PAP proteinsexamined in these cells of known genotype. Representa-tive results for SCR protein accumulation are shown forpap− clones in the labial (Fig. 4e,f) and T1 leg (Fig. 4g,h)imaginal discs. No change in SCR accumulation was de-tected in pap−/− cells compared with neighboring wild-

Table 2. dMED mutations and adult labial segmentdevelopment

GenotypeSex combteeth (%)

Claws(%)

pb4/pb5 6 0pb4/pb5 pap1 18 41pb4/pb5 dTRAP801 1 1pb4/pb5 pap1 dTRAP801 53 62

Note. Counts (n > 100 in all cases) refer to half labia (derivedfrom a single imaginal disc).

Figure 4. pap and dTRAP80 functions contribute to segmental identity specification. Labial palp identity is determined by the jointaction of Scr and pb. (a,c) Loss-of-function mutations of pap/dTRAP240 and dTRAP80 affect labial palp development, favoring T1 legdevelopment. (a) The mutant combination pb4/pb5 (where pb5 is a protein-null allele and pb4 is a hypomorph leading to a C-terminallytruncated PB protein) transforms distal labial palps to antennal aristae (arista), whereas the proximal labium retains normal pseudo-tracheal rows (pst). (c) The pap1 and dTRAP801 mutations are recessive in this tissue but enhance the pb4/pb5 mutant phenotype.Shown are mouthparts of a pb4/pap1 pb5 dTRAP801 adult female. Morphology is altered compared to a; pseudotracheae are replacedby apparent leg structures, and claws replace the aristae. (b,d) Scr acts with pb in the labium to promote labial (or antennal/labial)identity but transforms the labium to distal T1 leg in the absence of pb. Accumulation of SCR protein was examined in imaginal labialdisc cells of pb4/pb5 mutants, giving rise to mixed antennal/labial identities (b) or pb4/pap1 pb5 dTRAP801 mutants that givepredominantly T1 leg tissue (d). Nuclear SCR protein was detected throughout the discs in both cases, as it was for wild type. SCRaccumulation is not increased and, indeed, may be somewhat reduced (cf. b and d), in the discs giving rise to T1 legs. (e–h) Screxpression examined in clones of pap53 homozygous cells. (e) Shown is a labial imaginal disc harboring a twin spot clone of +/+ (white,vertical arrow) or of pap53/pap53 cells (black, diagonal arrow). (f) Expression of the SCR homeodomain protein is seen in the same labialdisc. The clone is outlined and indicated by the diagonal arrow. SCR accumulation is unaltered in the mutant cells. (g) This T1 legimaginal disc harbors a prominent clone of pap53/pap53 cells (black, limit indicated by arrow). (h) SCR protein accumulation in thesame T1 disc. The clone is outlined and its border indicated by the arrow. SCR accumulation is unchanged in the pap− cells.






type cells in either tissue. These results obtained in ho-mozygous pap− cells confirm that Scr gene expression, asmeasured by accumulation of the nuclear homeodomainprotein, is not detectably affected by altered pap functionin these tissues. Conversely, PAP protein accumulationwas unchanged in Scr−/− cells (not shown), indicatingthat pap transcription is likewise independent of Scrfunction in these tissues. These reciprocal experiments,coupled with the results in heterozygotes describedabove, provide molecular evidence that the pap anddTRAP80 loci act in parallel with homeotic Scr functionin distal sex comb and labial identity specification.

Discussion

The MED complexes are believed to serve as adaptinginterfaces between regulatory proteins bound to specificDNA sequences and the RNA Pol II that executes thisinput. Overall complex architecture and physical inter-actions with RNA Pol II appear relatively unchanged be-tween yeast and mammals (Asturias et al. 1999). In thiswork, we found a single fly homolog for each of 23known human MED proteins. The presence of so manydetectable fly homologs conforms with the observedconservation of overall complex structure between yeastand mammals and clearly supports the existence of a flycomplex. The synergistic interactions detected in testsof double heterozygous mutant combinations for papand dTRAP80 loss-of-function mutations revealed ashared developmental function. The most direct inter-pretation incorporating these two complementary linesof evidence is that these proteins act in vivo within theshared confines of a fly mediator complex, similar tothose detected in worms and mammals.

Whether pap and dTRAP80 act within a mediatorcomplex remains unproven, and elucidating this pointmust await the biochemical demonstration of a fly com-plex containing both proteins. Still, the presence of somany recognizable homologs clearly suggests the exis-tence of a metazoan complex, leading us to consider herethe developmental and evolutionary implications of thisworking hypothesis.

The metazoan complex is likely to act as a motor forcefor evolving genetic programs: overall complex architec-ture might reflect constraints imposed by interactionwith RNA Pol II, while even limited modifications ofsubunit sequences could alter contacts with specifictranscription factors that amplify the modification. Spe-ciation and morphological divergence reflect changes ingene expression programs. Clarifying how these MEDprotein functions modulate transcription should shedlight on the constraints limiting or favoring evolutionarychange. One of the two Drosophila genes isolated here,dTRAP80, encodes the apparent fly homolog of the pro-totypical yeast complex member Srb4. In contrast, pap/dTRAP240 does not possess a detectable yeast equiva-lent. One intriguing possibility is that the pap gene mayreflect a metazoan-specific MED protein, conceivably re-placing a yeast protein of analogous function. The over-all number of protein components of yeast and mammals

appears similar, and some subunits of the yeast Srb com-plex are present in human MED as well. However, otherssuch as pap are detectable in nematodes, flies, and mam-mals but apparently absent in yeast. It is not yet clearwhether truly species-specific MED subunits exist. Still,the emergence or exchange of additional metazoan-spe-cific subunits, as well as mixing and matching amongvarious modular subcomplexes such as those detected inyeast (Lee and Kim 1998; Lee et al. 1999), may have beenan important element contributing to the diversificationof genetic programs in complex organisms.

Ample evidence indicates that yeast Srb complex sub-units have diversified functions (Holstege et al. 1998).Some subunits such as Srb4 are required for transcriptionof nearly all yeast genes, whereas others are specific fordiscrete, largely nonoverlapping gene subsets. Informa-tion concerning the in vivo physiological roles of themetazoan MED complexes and their subunits remainssparse. A knock-out mutation of the ubiquitously ex-pressed mouse Srb7 gene suggests a requirement for cellviability (Tudor et al. 1999). Similarly, our clonal analy-sis indicates a requirement of dTRAP80 for cell viabilityin Drosophila, in agreement with a general role in tran-scription. In marked contrast, disruption of murineTRAP220 shows that this gene is essential for normaldevelopment, but lethality is associated with a complexvariety of defects in several tissues and is not cell lethal(Ito et al. 2000). Similarly, the analysis of pap activitysuggests a much more specific and restricted role for thisgene. No effect on larval cell proliferation was detected,and the tissue-specific consequences of pap mutationsfor adult cells range from no effect to cell or segmentidentity transformations.

The large size of known complexes, and the functionaldiversity observed for different yeast Srb subunits (Hol-stege et al. 1998), suggest their aptitude to receive andintegrate multiple cellular inputs. A previous study ofthe C. elegans sur-2 gene focused on its role as a geneticsuppressor of a Ras/MAP kinase pathway in vulval de-velopment, indicating a functional connection betweencell signaling and mediator function (Singh and Han1995). The human TRAP and DRIP complexes wereidentified by their biochemical interactions with thyroidand vitamin D3 nuclear hormone receptors, and a differ-ent subunit, human TRAP220, mediates ligand-specificcomplex binding to several nuclear receptors (Yuan et al.1998). The heightened affinity of this specific ligand-bound complex and its enhancement of in vitro tran-scription, in contrast to the nuclear receptor–TRAP220couple, seems likely to represent a recurrent theme forMED utilization in the dynamic expression of geneticprograms during development.

This diversity underlines the need for detailed in vivoelucidation of the roles of different mediator proteins.Are all MED protein functions effected within the frame-work of a single complex, or can these proteins also par-ticipate in other biochemical roles? The clonal analysisfor pap indicates a marked discordance between spatialgene expression and gene activity. These observationsmay represent evidence for positional information inter-

Boube et al.





preted through a mediator complex, which is in accordwith a previous proposal that MED complexes mayact as a sort of molecular control panel (Hampsey andReinberg 1999). It will be important to better under-stand the rules of in vivo mediator protein function, no-tably in transcriptional initiation where potential con-trol points include complex assembly, nuclear localiza-tion of subunits or complexes, and posttranslationalmodifications such as phosphorylation by protein ki-nases (Singh and Han 1995; Jiang et al. 1998; Boyer et al.1999) as elements permitting the rapid integration ofhighly specific spatial information from the cellular en-vironment.

Most descriptions ascribe a coactivator role to MEDfunction. Interestingly, though, observations in vitrohave also identified specific subcomplexes that serve astranscriptional corepressors (Song and Carlson 1998; Sunet al. 1998; Balciunas et al. 1999). Our genetic tests iden-tify pap as a functional antagonist of Scr, whereas testsof molecular epistasis indicate that these genes act inparallel. The observed antagonism with Scr could re-sult indirectly, reflecting the complex circuitry of thegenetic program. However, dose-sensitive synergisticfunctional interactions such as those detected heremay also indicate molecular interactions—which are di-rect or nearly so—through the intermediary of an as yetunidentified homeotic cofactor. Scr is expressed andrequired throughout the T1 and labial tissues. How-ever, this requirement is not uniform: SCR-expressingcells give rise to the diverse cell types composing anadult segment. The antagonistic effect of pap on Scrfunction described here is position-specific within theT1 leg, apparently limited to cells yielding the distal sexcomb. These apparently paradoxical observations mightreflect a crucial role of the MED complex in integratingdiverse inputs. In this manner, the localized effect thatappears as a negative regulation of Scr might reflectrather the de-repression of a target gene coregulated byScr with other specific factors. A MED function con-tributing to a combinatorial output that integrateshomeotic input with those of other specific transcriptionfactors would be a coherent role for segmental differen-tiation.

In a study of the roles of Srb subunits in the yeast geneexpression program, extensive functional diversitywas observed among the subunits (Holstege et al. 1998).In the present initial analysis of two MED genes inDrosophila, functional overlap but also clear differ-ences were noted between dTRAP80 and pap. The iden-tification of the dMED genes should now facilitatethe identification of their mutations toward the detailedin vivo characterization necessary for comprehend-ing how the diversity of individual subunit functionscan be integrated in a working machine with an im-portant developmental role. The convergence of datafrom studies of related complexes in yeast, mammals,C. elegans, and now Drosophila should soon afford aclearer picture of how mediators mediate transcrip-tional regulation throughout development and evolu-tion.

Materials and methods

Fly techniques

Standard Drosophila culture media and culture methods wereemployed.

Isolation of pap mutations

The HSPB transgenic element induces ectopic distal sex combsat low frequency. Among 5000 new autosomal insertions gen-erated by mobilization of an X-linked P-lacW element (http://flybase.bio.indiana.edu/), the pap1 allele (for poils aux pattes,“hairy legs”) enhanced distal sex comb frequency in combina-tion with the HSPB:4d line. Phenotypic revertants and impre-cise excisions were generated by remobilizing the pap1 insertionelement with �2-3 transposase from a stable �2-3 chromosomalinsertion (http://flybase.bio.indiana.edu/).

In situ hybridization

Antisense probes were generated by in vitro transcription fromlinearized plasmids, using T3 or T7 RNA polymerases in thepresence of digoxygenin-UTP (Boehringer Mannheim). Probespecificity was verified by hybridization to embryos or larvaewith localized overexpression of the corresponding mRNA (en-grailed–GAL4/UAS–pap or UAS–TRAP80).

Identification of the dTRAP801 allele

This allele, previously referred to as l(3)s2956 (Spradling et al.1999), was identified by adjacent sequences located withindTRAP80 coding sequences.

Clonal analysis

Mitotic recombination experiments were performed using theFLP/FRT recombination system (Xu and Rubin 1993). To exam-ine pap function, we employed the strong or null pap53 allelethat deletes the putative initiator ATG. For adult effects, y whsFLP; mwh jv pap53 FRT-2A/TM3, Sb females were crossedwith y w; Dp(1 ;3)scS4 y+ M(3)i55 FRT-2A/TM3, Sb males. FLPrecombinase was induced by heat shock in first- or second-in-star larvae (1 h, 37°C). Sb+ adults were examined for pap−

Minute+ clones, using the associated cell-autonomous markersy, mwh, and jv. For twin spot analysis, pap− clones were in-duced and examined in imaginal discs using the cell autono-mous GFP marker expressed from the ubiquitin-63E (UB) pro-moter (Lee et al. 1988). For these experiments, y w hsFLP; mwhjv pap53 FRT-2A/TM6B, Hu Tb females were crossed with w;UB–GFP FRT-2A males. For analysis of Scr mutant clones, weemployed a chromosome carrying the FRT-82B insertion andthe null Scr1 allele (kindly provided by A. Percival-Smith) and achromosome carrying FRT-82B with UB–GFP (3R; obtainedfrom the Indiana University Drosophila Stock Center). Cloneswere induced by heat shock (1 h, 37°C) during first or secondinstar, and discs prepared from late L3 larvae. Fixation was for20 min, 22°C with 4% paraformaldehyde. GFP expression wasanalyzed by confocal microscopy.

DNA techniques

Standard techniques were employed for cloning, blotting, andsequence analyses.

Cloning and structure of pap

A 9.5-kb EcoRI genomic DNA fragment 3� to the pap1 insertionsite cloned by plasmid rescue was used to initiate the isolation






of 50 kb of genomic DNA encompassing pap and to probe cDNAlibraries. The intron–exon structure of pap/dTRAP240 was de-termined by comparison of genomic and cDNA sequences. Fortransgenic rescue, a pap minigene was constructed containingthe ubiquitin promoter and a fusion of pap cDNA (the 5� exons;see Fig. 1a) and genomic (3� exons) sequences, and transgeniclines were established following microinjection according tostandard methods (Cribbs et al. 1995). GenBank databasesearches used the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/). Protein sequence alignments were performed us-ing Clustal W software. Details are available on request.

Structure of dTRAP80

The intron–exon structure of dTRAP80 was determined bycomparison of genomic and cDNA (clone SD10038 from BDGP)sequences. Genbank data base searches and protein sequencealignments were as above.

Localization of dMED homologs

Listed below are the identified Drosophila genes correspondingto known human mediator proteins (see Fig. 2a), followed bytheir GadFly identifiers: dARC105 (CG4184); dTRAP26/TRFP (CG18267), dARC32 (CG13867); dTRAP95 (CG5465);dTRAP150�/SUR2 (CG3695); dTRAP170/RGR1 (CG12031);dTRAP36 (CG8609); dTRAP100 (CG7999); dTRAP56/CDK8(CG10572); dTRAP15/NUT2 (CG5057); dTRAP230 (CG8491);pap/dTRAP240 (CG9936); dTRAP220 (CG7162); dTRAP18/SOH1 (CG1057); dTRAP37 (CG1245); dTRAP32/MED6(CG9473); dTRAP34/MED7 (CG6529); dTRAP33/cycC/SRB11(CG7281); dTRAP80/SRB4 (CG7957); dARC42 (CG4703);dARC92 (CG12254). dARC70 (CG1793) is situated on the smallchromosome IV (not shown), and dTRAP19/SRB7 (CG17397)could not yet be mapped.

Generation of anti-PAP antiserum

Recombinant hexahistidine-Pap fusion protein including thefirst 366 amino acid residues of Pap protein was used to immu-nize rabbits (Eurogentec SA). Recombinant GST–Pap fusion pro-tein blotted to a nitrocellulose filter permitted affinity purifica-tion of anti-Pap antisera. Immunostainings employed commer-cially available reagents. Details are available on request.

Acknowledgments

This manuscript was much improved through the comments ofJ. Casanova, G. Gimenez, S. Gonzalez, C. Benassayag, M.Crozatier, B. Glise, and A. Vincent, whom we thank for theircritical reading. We thank the Indiana University DrosophilaStock Center and S. Kerridge, A. Percival-Smith, and N. Perri-mon for fly stocks; W. Gehring for the anti-Scr serum; P. Co-chard for help with the confocal microscope; and A. Monier andA. Lepage for expert technical help. We also acknowledge thecontributions of A. Roques, Se. Peyrefitte, and E. Vanrobays tothe structural characterization of pap. This work benefited fromthe ongoing support of the Centre National de Recherche Sci-entifique (CNRS) and grants from the Association pour la Re-cherche sur le Cancer (ARC) and from the MinistEre del’Education Superieure et la Recherche (MESR) of France. M.B.was financed by graduate fellowships from the MESR and theARC.

The publication costs of this article were defrayed in part bypayment of page charges. This article must therefore be hereby

marked “advertisement” in accordance with 18 USC section1734 solely to indicate this fact.

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Drosophila homologs of transcriptional mediator complex subunits