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INTRODUCTIONThe Drosophila zinc-finger (Zn-finger) transcription factorSenseless (Sens) and its homologues have been shown to beinvolved in a variety of cell biological and developmental processes,including cell fate specification, differentiation, neurodegeneration,proliferation, apoptosis and stem cell self-renewal (Cameron et al.,2002; Chandrasekaran and Beckendorf, 2003; Frankfort et al.,2001; Gilks et al., 1993; Grimes et al., 1996; Hock et al., 2004;Hock et al., 2003; Jia et al., 1996; Karsunky et al., 2002; Nolo et al.,2000; Saleque et al., 2002; Shroyer et al., 2005; Tong et al., 1998;Tsuda et al., 2005; Zeng et al., 2004). Although the functions ofsens and its C. elegans homologue pattern of gene expression-3(pag-3) have been mainly studied in the context of the nervoussystem, most of the studies of the vertebrate homologues growthfactor independence 1 (Gfi1) and Gfi1b have focused onhematopoietic development. However, recent work has providedstrong evidence that Gfi1 is required in the nervous system(Dufourcq et al., 2004; Tsuda et al., 2005; Wallis et al., 2003).Indeed, the Gfi1 loss-of-function phenotype in mouse inner ear haircells is quite similar to the loss-of-function phenotype of sens inembryonic sensory organs (Nolo et al., 2000; Wallis et al., 2003).In addition, expression of sens and Gfi1 in fly and mouse sensoryorgan precursors depends on the activity of fly basic helix-loop-helix (bHLH)-type proneural proteins and their mouse orthologues,respectively (Bertrand et al., 2002; Brown et al., 2001; Hassan andBellen, 2000; Jafar-Nejad et al., 2003; Nolo et al., 2000; Wallis etal., 2003). Moreover, sens and proneural genes function in the same

pathway during sensory organ development, and there is evidencefor Gfi1/bHLH cooperation in vertebrate tissues (Brown et al.,2001; Frankfort et al., 2001; Frankfort et al., 2004; Jafar-Nejad etal., 2003; Kazanjian et al., 2004; Nolo et al., 2000; Shroyer et al.,2005; Wallis et al., 2003). Altogether, these observations stronglysuggest that some mechanisms involved in the expression andfunction of Gfi1/1b, PAG-3 and Sens (GPS) proteins areevolutionarily conserved.

GPS proteins are C2H2-type Zn-finger nuclear proteins. There aresix Zn fingers in Gfi1 and Gfi1b, five in PAG-3 and four in Sens.There is ~88-89% sequence identity between Sens Zn fingers andthe four C-terminal-most Zn fingers of Gfi1/1b and PAG-3 (Jafar-Nejad and Bellen, 2004). Both Gfi1 and Gfi1b have been shown tobind to the same consensus DNA element [taAATCac(a/t)gca; withthe core sequence in uppercase] using their Zn-finger domains.There is ample evidence that both proteins function as DNA-bindingtranscriptional repressors (Doan et al., 2004; Grimes et al., 1996;Jegalian and Wu, 2002; Tong et al., 1998; Vassen et al., 2005;Zweidler-Mckay et al., 1996), although it has been suggestedthat they can also act as transcriptional activators (Osawa et al.,2002; Sharina et al., 2003). As predicted from the evolutionaryconservation of their Zn fingers, PAG-3 and Sens strongly bind tothe Gfi1/1b consensus binding site (Aamodt et al., 2000; Jafar-Nejadet al., 2003). Indeed, the sequence identity between the Zn fingers ofthe GPS proteins goes beyond what is necessary to bind the sameDNA sequence, suggesting that GPS Zn fingers might playconserved functional roles other than DNA binding (Jia et al., 1997).

sens was isolated in a genetic screen designed to identify novelgenes involved in the development of the embryonic peripheralnervous system (PNS) in Drosophila (Salzberg et al., 1994). sens isexpressed in sensory organ precursors and their progeny in bothembryonic and adult PNS. In sens mutant embryos, sensory organprecursors (SOPs) form and divide but fail to differentiate properly,and instead undergo apoptosis (Nolo et al., 2000; Salzberg et al.,1994). Adult PNS development is also impaired in sens mutantclones, indicating that sens is required for the development of mostor all sensory organs in flies. Moreover, ectopic expression of sens

Senseless physically interacts with proneural proteins andfunctions as a transcriptional co-activatorMelih Acar1,*, Hamed Jafar-Nejad2,3,*, Nikolaos Giagtzoglou3, Sasidhar Yallampalli2, Gabriela David2,Yuchun He3, Christos Delidakis4 and Hugo J. Bellen1,2,3,†

The zinc-finger transcription factor Senseless is co-expressed with basic helix-loop-helix (bHLH) proneural proteins in Drosophilasensory organ precursors and is required for their normal development. High levels of Senseless synergize with bHLH proteins andupregulate target gene expression, whereas low levels of Senseless act as a repressor in vivo. However, the molecular mechanismfor this dual role is unknown. Here, we show that Senseless binds bHLH proneural proteins via its core zinc fingers and is recruitedby proneural proteins to their target enhancers to function as a co-activator. Some point mutations in the Senseless zinc-fingerregion abolish its DNA-binding ability but partially spare the ability of Senseless to synergize with proneural proteins and to inducesensory organ formation in vivo. Therefore, we propose that the structural basis for the switch between the repressor and co-activator functions of Senseless is the ability of its core zinc fingers to interact physically with both DNA and bHLH proneuralproteins. As Senseless zinc fingers are ~90% identical to the corresponding zinc fingers of its vertebrate homologue Gfi1, which isthought to cooperate with bHLH proteins in several contexts, the Senseless/bHLH interaction might be evolutionarily conserved.

KEY WORDS: Drosophila, Senseless

Development 133, 1979-1989 (2006) doi:10.1242/dev.02372

1Program in Developmental Biology, Baylor College of Medicine, Houston,TX 77030, USA. 2Department of Molecular and Human Genetics, Baylor College ofMedicine, Houston, TX 77030, USA. 3Howard Hughes Medical Institute, BaylorCollege of Medicine, Houston, TX 77030, USA. 4Institute of Molecular Biology andBiotechnology, FORTH and Department of Biology, University of Crete, Heraklion,GR-71110, Greece.

*These authors contributed equally to this work†Author for correspondence (e-mail: hbellen@bcm.tmc.edu)

Accepted 20 March 2006

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induces ectopic sensory organ formation, suggesting that Sens canplay an instructive role in this process (Jafar-Nejad et al., 2003; Noloet al., 2000). In Drosophila, the bHLH proneural proteins areinvolved in the selection of SOPs and implementation of neuronalfate in sensory lineages (Bertrand et al., 2002; Culi and Modolell,1998; Goulding et al., 2000; Huang et al., 2000; Jarman et al., 1993;Modolell, 1997; Villares and Cabrera, 1987). As DNA-bindingtranscriptional activators, proneural proteins bind E-box elementsand drive gene expression (Cabrera and Alonso, 1991; Murre et al.,1989; Van Doren et al., 1991). Given the evolutionary conservationof the role of bHLH proteins in neurogenesis, understanding howbHLH proteins regulate the expression of their target genes is ofinterest (Bertrand et al., 2002; Hassan and Bellen, 2000). We havepreviously presented in vivo and in vitro evidence that on a proneuraltarget gene enhancer with multiple E-boxes and a Sens-binding site(S-box), Sens can function as a repressor or activator, depending onthe level of Sens relative to proneural proteins (Jafar-Nejad et al.,2003). Although our findings suggested that binding of Sens to DNAmight oppose its transcriptional synergism with proneural proteins,the structural basis for this synergism and the mechanism for theswitch between the repressor and activator functions of Sens remainunknown.

Here, we dissect the mechanism of the transcriptional activationand repression mediated by the Sens protein on bHLH targetenhancers. We present evidence that besides binding DNA, the coreZn fingers of Sens physically interact with proneural proteins, andtherefore are responsible for both repression and co-activation. TheDNA-binding-dependent repression and the bHLH-binding-dependent co-activator functions of Senseless are separable both intranscription assays and in vivo. We propose that differential affinityof the Sens Zn fingers for their DNA recognition site versusproneural proteins allows dual function in the transcriptionalregulation of target genes.

MATERIALS AND METHODSFly strains, genetics and immunohistochemistryThe following fly lines were used in this study: y w, Canton S, UAS-sens (C6)(Nolo et al., 2000); C684-Gal4 (Manseau et al., 1997); y w; UAS-FLP;C684-Gal4 y+ FRT80B, UAS-sens-1CC, UAS-sens-2CC, UAS-sens-3CC,UAS-sens-4CC, y w; sens-g; sensE2 red e/TM3, Kr-GFP, y w; sens-1CCg;sensE2 red e/TM3, Kr-GFP, y w; sens-3CCg; sensE2 red e/TM3, Kr-GFP(this study); and Eq-Gal4/TM6B (Pi et al., 2001). To rescue the adult mitoticclones of sensE2 with sens genomic transgenes (sens-g, sens-1CCg or sens-3CCg), L+ Tb+ progeny of the following cross were inspected for regions ofbristle loss or yellow– bristles: y w; UAS-FLP; C684-Gal4 y+ FRT80B �y w; sens-Xg/L; sensE2 FRT80B/TM6, Tb. Quantification of the post-orbitalbristles was performed in y w eyeless-FLP; sensE2 FRT80B/w+ M(3)67CFRT80B (sens clones with no rescue) and y w eyeless-FLP; sens-Xg/+;sensE2 FRT80B/w+ M(3)67C FRT80B flies, in which sens-Xg stands forsens-g, sens-1CCg, sens-2CCg, sens-3CCg or sens-4CCg. Embryocollection, fixation and staining were performed using standard procedures(Bellen et al., 1992). Primary antibodies used in this study are rat �-Elav1:500 (7E8A10; DSHB) (O’Neill et al., 1994), guinea pig anti-Sens 1:1000(Nolo et al., 2000) and mouse mAb 22C10 1:100 (DSHB) (Zipursky et al.,1984). Secondary antibodies were from Molecular Probes (Alexa488-conjugated) and from Jackson ImmunoResearch Laboratories (Cy3 and Cy5conjugated), and were used at 1:500.

Preparation of the mutant sens genomic rescue and UASconstructssens open reading frame cloned in pBluescript was mutated using theQuikchange site-directed mutagenesis kit (Stratagene) to generate Zn-fingermutant versions of sens, which were then transferred to the pUAST vector.A 1769 bp fragment that includes the coding region for the Zn-fingerdomains of sens in the genomic construct of sens was obtained by digesting

the sens genomic rescue fragment with RsrII and AatII restriction enzymes.This 1769 bp RsrII-AatII fragment was cloned into a modified version ofpBluescript vector using AatII and RsrII sites. The Quikchange site directedmutagenesis kit was used to change the cysteines in Zn-finger domains intoalanines. The mutant 1769 bp AatII-RsrII fragment was then excised fromthe pBluescript vector and cloned into the 21 kb pCaSpeR4-sens genomicrescue construct, which was fully digested with RsrII and partially digestedwith AatII. Colonies were screened by PCR and positive colonies weresequenced to determine the correct insertions.

EMSA, S2 cell transfection and luciferase assaysEMSA was performed as described previously (Ou et al., 2000). Theproteins were in vitro translated using TNT Quick CoupledTranscription/Translation System kit (Promega). The R21 optimalGfi1/Sens-binding sequence (Jafar-Nejad et al., 2003; Zweidler-Mckay etal., 1996) and the Sens-binding site on the ac promoter were used as theprobes. Fifty times unlabeled probe was used as cold competitor.Transfection and luciferase assays were performed as described by Jafar-Nejad et al. (Jafar-Nejad et al., 2003). E-box and S-box mutagenesis wereperformed using the Quikchange site-directed mutagenesis kit. E-boxeswere mutated from CANNTG to AANNTT, and the S-box core was mutatedfrom AATC to GGTC. All constructs were verified by sequencing. Theprimer sequences are available upon request.

GST pull-down experimentsGST fusion proteins were expressed using pGEX-4T1 vector (AmershamBiosciences) in BL-21 pLys(S) cells (Novagen). sens and different sensfragments were cloned into pGBKT7 vector (Clontech) in-frame with the Nterminal c-myc tag and in vitro translated using TNT Quick CoupledTranscription/Translation System kit (Promega). The same protocol asdescribed by Giagtzoglou et al. (Giagtzoglou et al., 2003) was used toperform GST pull-down experiments. Detection was performed by westernblot using anti-c-myc antibody (9E10, DSHB).

Co-immunoprecipitationssens open reading frame was cloned into pCMV-HA vector (Clontech) in-frame with the N-terminal HA tag. achaete open reading frame was clonedinto p3XFLAG-CMV-10 expression vector (Sigma) in-frame with N-terminal 3�FLAG tag. Lipofectamine 2000 (Invitrogen) was used totransfect COS-7 cells according to the manufacturer’s protocol. A total of24 �g of DNA was transfected per 100 mm culture dish. Thirty-six hoursafter transfection cells in each culture dish were washed twice with cold PBSand were then harvested in 700 �l IP buffer [50 mM Tris-HCl (pH 8.0), 150mM NaCl, 0.5% NP40, 5 mM EDTA, 0.1 mM PMSF and Complete proteaseinhibitor (Roche)] for 45 minutes to obtain whole cell lysate. Uponcentrifugation at 16,000 g, the supernatant was used for the IP reaction. Anti-HA monoclonal agarose conjugate (Sigma) was used for Co-IP reactionsbased on the product user manual. For washing steps the above-mentionedIP buffer was used.

Statistical analysis of the post-orbital bristle numbersOne-way ANOVA with Scheffe error protection was used to determine thestatistical significance of the differences between the number of post-orbitalbristles in the genotypes under study. The P values are determined using ‘t-test for independent samples’.

RESULTSZn finger 1, 2 and 3 are required for Sens DNAbindingWe have previously shown in an S2 cell transcription assay that theSens protein can act as a transcriptional repressor and activator,depending on its relative abundance to the proneural proteins (Jafar-Nejad et al., 2003). The reporter construct used in that study consistsof the ac proximal enhancer/promoter region upstream of the fireflyluciferase coding sequence (ac-luc; Fig. 1A). This ac enhancercontains a Sens-binding site (S-box) and three E-boxes, knownbinding sites for proneural proteins. Proneural proteinsheterodimerize with Daughterless (Da) via their bHLH domains and

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bind to the E-boxes on ac-luc to upregulate transcription (Cabreraand Alonso, 1991; Caudy et al., 1988; Van Doren et al., 1991; VanDoren et al., 1992). Depending on the amount of ac and daexpression constructs transfected, the luciferase expression from thisreporter can be increased 10 to 1000 times the basal level. To obtainthe optimal sensitivity in the transcription assays, we use low levelsof proneural expression constructs (1-2 ng) to assess thetranscriptional activation potential of Sens (activation assay), andhigher levels of proneurals (10 ng) to assess the transcriptionalrepression potential of Sens (repression assay). In the absence of Acand Da, Sens does not activate or repress ac transcription.

Based on evolutionary conservation with its vertebratehomologues, Sens can be divided into two domains: an N-terminaldomain of 414 amino acids, which shows little homology with other

GPS proteins, and a C terminal domain of 127 amino acids, whichexhibits strong homology with other GPS proteins and contains fourhighly conserved C2H2-type Zn fingers. We aligned Sens to itsclosest homologue from the mosquito Anopheles gambiae, which isthought to have diverged from Drosophila about 180 million yearsago (Devenport et al., 2000), and found nine conserved stretches of6-10 amino acids in the Sens N-terminal domain. Mutationalanalysis of the conserved stretches followed by transcription assaysindicate that the individual conserved motifs in the N-terminaldomain are not important for the activation and repression mediatedon ac by Sens (data not shown).

The four C2H2-type Zn-finger domains of the GPS proteins,which mediate DNA binding, are shown in Fig. 1B (Nolo et al.,2000; Zweidler-Mckay et al., 1996). Deletion analysis of Gfi1 Zn

1981RESEARCH ARTICLEMechanisms of Senseless synergism

Fig. 1. Zn-finger domains of Sens mediate DNA binding, repression and activation. (A) Schematic of the ac-luc reporter used in the S2 celltranscription assay. E1, E2 and E3 represent the E-boxes, and S represents the S-box in the 470 bp ac proximal enhancer. (B) Alignment of the fly (D.m) Sens Zn-finger (ZF) domains with the corresponding Zn-finger domains of Homo sapiens (H.s) Gfi1 and Caenorhabditis elegans (C.e) PAG-3shows that Zn-finger domains of GPS proteins and the linker regions that connect them are highly conserved. Stars represent the cysteines in theC2H2 structure. Squares represent the amino acids that are predicted to contact DNA in C2H2-type Zn-finger domains. Circles represent the aminoacids in linkers that have the potential to be phosphorylated. Blue boxes denote divergent amino acids. (C) EMSA assay using a previouslycharacterized probe (R21, see Materials and methods) and Sens with different types of Zn-finger mutations. Sens loses its ability to bind to DNA ifthe cysteines in Zn finger 1, 2 or 3 are mutated. The amino acids that were predicted to contact DNA in Zn fingers 2 and 3 but not in Zn finger 1seem to be crucial for DNA binding. Zn finger 4 seems to be dispensable for DNA binding. (D) Activation assays in S2 cells show that all Zn fingersare involved in the synergism with proneural proteins to upregulate the expression of the ac-luc reporter. Sens fails to synergize with proneuralproteins when either Zn finger 2 or 3 is mutated, but exhibits some synergism upon mutating Zn finger 1 or 4. (E,F) Repression assays on the ac-lucreporter using wild-type and Zn-finger mutant Sens expression constructs. Sens loses its ability to repress the ac-luc reporter at low levels if Zn finger1, 2 or 3, but not 4, is mutated.

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fingers has shown that Zn fingers 3-5 of Gfi1, which correspond toZn fingers 1-3 of Sens, are required for DNA binding (Zweidler-Mckay et al., 1996). To begin to assess the precise role of individualZn fingers in the repressor and activator functions of Sens, wemutated each Zn finger in Sens and examined the ability of themutant Sens proteins to bind DNA in electromobility shift assays(EMSA). We generated two types of mutants for each Zn finger. Inthe first group (Sens-1CC, Sens-2CC, Sens-3CC and Sens-4CC), wemutated the two cysteines in the C2H2 structure to alanines (Fig. 1B,stars). These mutations probably disrupt the structure of theindividual Zn fingers. In the second group (Sens-1RTT, Sens-2QDK,Sens-3QNT and Sens-4RDR), we altered the amino acids that havebeen predicted to directly contact DNA to alanines (Fig. 1B, boxes)(Pavletich and Pabo, 1991; Zweidler-Mckay et al., 1996). Sincethese amino acids are not crucial for the Zn-finger structure(Blancafort et al., 2004), these mutations should abolish directcontact with specific DNA targets but at least partially preserve theoverall Zn-finger structure.

To determine protein-DNA interactions and relative bindingaffinities of the mutant Sens proteins for DNA, we used twodifferent probes in EMSA assays. To detect weak protein-DNAinteractions, we used as a probe a previously characterized Gfi1-binding site called R21, to which the wild-type Sens is able to bindstrongly (Fig. 1C, lane 2). As shown in Fig. 1C, Sens-1CC, Sens-2CC and Sens-3CC proteins lose their ability to bind the R21 probe,suggesting that Zn fingers 1, 2 and 3 are required for DNA binding(Fig. 1C, lanes 4, 6, 8). However, in agreement with Gfi1 data(Zweidler-Mckay et al., 1996), Sens-4CC can bind DNA,suggesting that Zn finger 4 is not essential for DNA binding (Fig.1C, lane 10). As shown in Fig. 1C, the second group of Sens Zn-finger mutants behave somewhat differently in the EMSA. Sens-2QDK, Sens-3QNT and Sens-4RDR behave similarly to their CCcounterparts, indicating that the amino acids predicted to directlycontact the R21 probe in Zn fingers 2 and 3 are crucially importantfor DNA binding. However, unlike Sens-1CC, Sens-1RTT is stillable to bind the R21 probe, albeit weaker than wild-type Sens andZn-finger 4 mutants (Fig. 1C, lane 12). This difference suggests thatalthough Zn finger 1 is required for DNA binding, its role in DNAbinding is more complex than a direct contact between the RTTamino acids and DNA.

We also used the S-box in the ac promoter as a probe in theEMSA assay to determine the binding affinities of mutant Sensproteins for the endogenous Sens-binding site. Wild-type Sens andSens-4CC but not Sens-1CC, Sens-2CC nor Sens-3CC are able tobind to the S-box probe (data not shown). Moreover, in line with theR21 data, the Sens-1RTT binds much weaker than wild-type Sensand Sens-4CC. Note that the Sens-4CC binding affinity for the S-box is weaker than wild-type Sens, suggesting that although Znfinger 4 is not essential for DNA binding, it may increase thestrength of Sens-DNA interaction.

Zn fingers play different roles in the activationand repression conferred by SensTo determine the importance of each Zn-finger domain for theactivation and repression mediated by Sens, we tested the mutantsin the S2 cell transcription assay. In our activation assay (ac-da,2ng), wild-type Sens can synergize with Ac-Da and increase thetranscription induced by Ac-Da about 18 times (Fig. 1D). Sens-2CCand Sens-3CC failed to synergize with Ac-Da (Fig. 1D). Sens-4CCand especially Sens-1CC exhibited significantly less synergism thanwild-type Sens. Western blot analysis shows that wild-type Sens andall mutant Sens constructs are expressed at similar levels, indicating

that the difference between the activator potential of mutant versionsof Sens is not simply due to their level of expression or their stability(data not shown). Similar results were obtained for Sens-1RTT,Sens2-QDK, Sens-3QNT and Sens-4RDR (data not shown). Thesedata indicate that all Zn fingers cooperate in the Sens/bHLHsynergism. However, Zn fingers 2 and 3 are indispensable for thisprocess.

To test the ability of the Zn-finger mutants to repress actranscription, we used our ‘repression assay’. Low levels of wild-type Sens repress transcription in this assay and as the Sens toproneural ratio increases, the Sens activity switches from a repressorto an activator (Fig. 1E,F) (Jafar-Nejad et al., 2003). Sens-4CC andSens-4RDR behave essentially as wild-type proteins in this assay(Fig. 1E,F). By contrast, mutations in Zn fingers 1, 2 or 3 abolish therepression function of Sens, corroborating the correlation betweenSens DNA binding and repression. Interestingly, Sens-1CC andSens-1RTT display transcriptional activation at a lower Sens toproneural ratio compared with the wild-type Sens (Fig. 1E,F),providing further evidence for the negative contribution of SensDNA-binding to its ability to synergize with proneural proteins.Similar to the data obtained from the ‘activation assay’ (Fig. 1D),Sens proteins with mutations in Zn finger 2 or 3 do not show anypremature synergism with Ac-Da (Fig. 1E,F), highlighting the roleof these core Zn fingers in both synergism and repression. Together,these data indicate specific roles for the Zn fingers in repression andactivation (Table 1).

Zn fingers 2 and 3 are required for bristlespecificationTo explore the role of DNA-binding and repressor versus activatorfunction of Sens in its ability to induce extra SOPs in vivo, weemployed the Gal4/UAS system to express Zn-finger mutantversions of Sens in flies (Brand and Perrimon, 1993). Wild-type sensand the CC mutants were ectopically expressed using the thorax-specific driver Eq-Gal4 (Pi et al., 2001; Tang and Sun, 2002).Expression of wild-type Sens using this driver induces numerousextra bristles on the thorax (Fig. 2B) (Pi et al., 2004). Sens-1CC andSens-4CC are both able to induce many extra bristles (Fig. 2C,F).However, although immunohistochemical staining with anti-Senseless antibody indicates comparable expression levels from alltransgenes (data not shown), Sens-2CC and 3CC are unable toinduce extra sensory organs (Fig. 2D,E). As Sens-1CC is unable tobind DNA, the data suggest that DNA binding is not essential forSOP induction upon Sens overexpression. Moreover, the inability ofSens-2CC and Sens-3CC to generate extra sensory organs correlateswell with their inability to synergize with Ac-Da in transcriptionassays (Table 1) and suggests a link between Sens transcriptionalsynergism and SOP induction.

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Table 1. Comparison of the in vitro and in vivo roles of SensZn fingers

Wild type 1CC 2CC 3CC 4CC

DNA binding in EMSA + – – – +Repression in S2 cells + – – – +Synergism in S2 cells High Low – – MediumEctopic bristle upon OE + + – – +Rescue lethality + – ND – NDRescue embryonic PNS Full Partial ND – NDRescue adult PNS Full Partial – – Full

Summary of the data obtained from EMSA, transcription assay, overexpression (OE)and rescue experiments performed using wild-type and Zn-finger mutant versions ofSens. ND, not determined.

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DNA binding is not essential for some of the Sensfunctions in vivoSo far, our data suggest that Sens DNA binding is dispensable foradult bristle formation by Sens. To better assess the in vivo role ofDNA-binding in Sens function, we performed in vivo rescueexperiments. We generated four sens genomic rescue constructs,each mutated in one of the Zn fingers (sens-1CCg, sens-2CCg, sens-3CCg, sens-4CCg), and established several independent transgeniclines for each construct. We first determined the ability of sens-g,Sens-1CCg and Sens-3CCg mutant genomic transgenes to rescue theexternal sensory organs in adult sensE2 clones, which lose almost allmechanosensory bristles (Fig. 3A). As shown in Fig. 3B, sens-g isable to rescue all the bristles in the sensE2 clones. sens-1CCgtransgene is able to rescue many bristles in a sensE2 clone, asevidenced by the presence of yellow– bristles (Fig. 3C). However,sens-3CCg fails to rescue the bristle loss phenotype in mutant clones(Fig. 3D). To compare the in vivo function of the wild-type andmutant sens genomic transgenes in a quantitative manner, wegenerated large adult sens mutant clones in the head using theeyeless-Flp system (Newsome et al., 2000), and tested each sensgenomic transgene for its ability to rescue the loss of post-orbitalbristles in sens clones. In animals with sens mutant clones butwithout a rescue transgene, the number of post-orbital bristles is lessthan a third of the wild-type number (Fig. 3E and data not shown).A wild-type genomic transgene as well as sens-4CCg fully rescuesthe bristle loss (Fig. 3E an data not shown), and sens-1CC causes apartial restoration to about two-thirds of the wild type. By contrast,sens-2CC or sens-3CC do not rescue (Fig. 3E and data not shown).In addition, the rescue data for bristles perfectly correlate with therescue data of eye size and morphology (data not shown), whereSens has been shown to be required for R8 photoreceptorspecification (Frankfort et al., 2001). As 1CC, 2CC and 3CC all

disrupt the DNA binding and 1CC partially preserves the synergism,these data corroborate our cell culture and in vivo overexpressionobservations and indicate that the sens requirement for adult bristledevelopment corresponds to the activator function of Sens.

We also tested sens-1CCg and sens-3CCg transgenes for theirability to rescue the lethality and the PNS development in sensmutant embryos. Unlike the wild-type sens rescue transgene (sens-g), sens-1CCg and sens-3CCg are not able to rescue the lethality of

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Fig. 2. Zn fingers 2 and 3 are indispensable for the bristle-inducing ability of Sens. (A) Thorax of a wild-type (Canton S) fly.(B) Ectopic expression of wild-type sens with Eq-Gal4 driver induces theformation of many extra bristles. Ectopic expression of Sens with amutant Zn finger 1 or 4 with Eq-Gal4 driver can also induce extra bristleformation (C,F). Sens loses its ability to induce ectopic bristle formationif either Zn finger 2 or 3 is mutated (D,E). Midline bristle loss isobserved in flies expressing Sens, Sens-1CC and Sens-4CC, which isdue to a closure defect during thorax development when Sens retainsactivity. Sens-2CC and Sens-3CC do not display the dorsal closurephenotype.

Fig. 3. A sens genomic transgene can partially rescue the adultPNS phenotypes of sens mutant flies when Zn finger 1 ismutated. (A) Bristles fail to develop in sensE2 mitotic clones generatedby the C684-Gal4 driver (see Materials and methods). (B) sens-g is ableto rescue all bristles in sensE2 mitotic clones (bristles in clones aremarked with yellow). (C) sens-1CCg is able to rescue some of thebristles in sensE2 mitotic clones. (D) sens-3CCg fails to rescue the bristleloss in sens mutant clones. Arrows in C,D indicate lost macrochaetae;the arrowhead in C indicates a rescued macrochaetae. Mutant areas inA-D are marked with white lines. (E) Quantification of number of post-orbital bristles formed in flies that have large sens mutant clonesgenerated by the eyeless-FLP system. Error bars represent the standarderror of the mean. y w was used as the wild-type (WT) strain. For eachgenotype, post-orbital bristles of 7-24 flies were counted. Thedifferences among ‘no transgene’, sens-2CCg and sens-3CCg are notstatistically significant, neither are the differences among wild type,sens-g and sens-4CCg. However, the bristle number for sens-1CCg issignificantly different from all other genotypes and again shows partialrescue (P<0.0001).

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embryos that are homozygous for a null allele of sens (Table 1).Staining for neuronal markers reveals a significant rescue of PNSdevelopment with the sens-1CCg transgene (data not shown).However, the rescue is not complete, as some of the neurons are lost.In addition, the neurons exhibit differentiation, axon guidance andfasciculation defects (data not shown), indicating that Sens isrequired for neuronal differentiation in the embryonic PNS. Bycontrast, sens-3CCg does not rescue the PNS defects associated withsensE2 (data not shown, Table 1), although staining with anti-Sensantibody shows that both transgenes are able to produce similarlevels of Sens protein (data not shown). As Sens-1CC and Sens-3CChave no DNA-binding activity in our assay conditions, the dataindicate that some key aspects of Sens function in embryonicsensory organ development are not dependent on DNA binding.

To further support the notion that Sens DNA-binding isdispensable for adult bristle formation, we generatedphosphomimetic mutations in each of the three linker motifs in SensZn-finger domain by changing the first serine or threonine residues(Fig. 1B, circles) to glutamic acid, and tested them in vitro and invivo. It has been shown that phosphorylation of the first threonine orserine residue in the conserved linker motifs that connect adjacentC2H2-type Zn fingers results in a severe decrease in the ability ofthe corresponding Zn-finger protein to bind DNA, regardless of their

specific recognition site (Dovat et al., 2002; Jantz and Berg, 2004).EMSA assays indicated that phosphomimetic mutation in linker 3did not cause any detectable effect on Sens-DNA interactions andphosphomimetic mutation in linker 1 caused a slight decrease in theaffinity of Sens for DNA (Fig. 4A). However, we did not detectDNA-binding for Sens with a phosphomimetic mutation in linker 2(Sens-L2-T-E, Fig. 4A). We next performed ‘activation assays’ inS2 cells and found that, similar to Sens-1CC, Sens-L2-T-E canpartially synergize with Ac-Da (data not shown). Finally, wegenerated transgenic flies that express Sens-L2-T-E and ectopicallyexpressed mutant Sens using the Eq-Gal4 driver to test if it caninduce extra SOP formation. We found that although weaker thanwild-type Sens, misexpression of Sens-L2-T-E can induce numerousextra bristles (Fig. 4B,C). These data support the notion that SensDNA-binding is not essential for the generation of extra bristles invivo and further establish a correlation between the Sens-proneuralsynergism in cell culture and bristle induction by Sens in vivo.

Proneural proteins are able to recruit Sens to theac regulatory regionThe proximal ac enhancer used in the ac-luc reporter has three E-boxes as well as one S-box (Fig. 1A). Removal of the three E-boxesshows that they are important for transcriptional activation by Ac-Da or Sc-Da in S2 cell co-transfection experiments and for properexpression of an ac-lacZ transgene in vivo (Martinez et al., 1993;Van Doren et al., 1992). As our data indicate that binding of Sens tothe S-box in the ac enhancer is not necessary for Sens-proneuralsynergism, we assessed the role of each E-box in this process. To this

RESEARCH ARTICLE Development 133 (10)

Fig. 4. Sens with a phosphomimetic mutation in linker 2 is ableto induce bristle formation but does not bind the R21 probe.(A) EMSA assay using wild-type Sens and mutant Sens versions withphosphomimetic mutations in the linker regions that connect the Znfingers. R21 oligonucleotide, to which Sens binds strongly, was used asprobe. A phosphomimetic mutation in linker 1 (Sens-L1-S-E) reducesthe Sens-DNA interaction. However, a phosphomimetic mutation inlinker 2 (Sens-L2-T-E) does not show any DNA-binding activity underthese conditions. The gel on the right shows 20% of the input for themutant proteins and 20% of the wild-type Sens input is shown in Fig.6E. (B,C) Ectopic expression of sens-L2-T-E using Eq-Gal4 driver inducesformation of extra bristles at both 25°C and 28°C, as shown in B andC, respectively.

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Fig. 5. Sens is recruited to the ac regulatory region via protein-protein interactions. (A) An S2 cell transcription assay using the ac-luc reporter with mutations in various combinations of E-boxes. wt,wild type; E1-3, mutant E-boxes 1-3 (see Fig. 1A). Blue bars representthe presence, and red bars represent the absence, of the S-box on theac-luc reporter. The absence of the S-box does not affect the expressionmediated by Ac and Da when no Sens protein is present.(B) Amplification of the expression induced by Ac and Da upon additionof Sens. The absence of the S-box significantly increases the ability ofSens to synergize with proneural proteins on the ac-luc reporter.

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end, we generated ac-luc reporter constructs with all combinationsof single, double and triple mutant E-boxes in the presence andabsence of the S-box.

We first established the contribution of each E-box to thetranscriptional activity conferred by the Ac and Da in the absence ofSens. As shown in Fig. 5A, luciferase expression is highest when allthree E-boxes are present and lowest when all three E-boxes aremutated, in agreement with previous observations (Van Doren et al.,1992). Although each individual E-box seems to contribute to thewild-type level of transcription, E1 is more important than E2 andE3. Similar results were obtained when low levels of ac-da (2 ngeach) were used in transfection assays (data not shown). These dataalso serve as a control to show that when the sens expression vectoris not co-transfected with ac-da, presence or absence of the S-boxdoes not affect the luciferase expression mediated by Ac-Da.

We next co-transfected sens together with proneurals andcalculated the ratio between luciferase expression conferred by Ac-Da in the presence and absence of Sens as an amplification ratio(Fig. 5B). Absence of the S-box in an otherwise wild-type constructresults in a significant enhancement of luciferase expression in thepresence of Sens (Fig. 5B). The amplification ratio becomes evenhigher in E1, E2 and E3 single mutants. Moreover, when the S-box

is mutated, even the presence of E1 or E2 site alone is enough tomediate Sens-proneural synergism. However, when Ac and Da arenot able to bind DNA (triple E1, E2, E3 mutant), Sens is unable tosynergize with proneural proteins. Together, these data stronglysuggest that Sens can be recruited to the ac promoter by the Ac-Daprotein complex to potently activate transcription.

Sens interacts with proneural proteins via its Zn-finger domainsAs the activator function of Sens fully depends on the presence ofproneural proteins and at least one intact E-box in the enhancer, wehypothesize that Sens is recruited to the ac enhancer not only viaDNA interaction but also through interaction with proneuralproteins. To test this hypothesis, we first performed a co-IPexperiment by expressing HA-tagged Sens and Flag-tagged Ac inCos-7 cells. Indeed, antibodies against HA-Sens permittedprecipitation of the Flag-Ac protein, suggesting that Ac and Sensinteract in vivo (Fig. 6A). To test if Sens and other proneural proteinsalso interact, we examined if GST-tagged Ac, Scute (Sc) and Atonal(Ato) can pull down tagged Sens. We find that Sens physicallyinteracts with all of the bHLH-type proneural proteins tested here(Fig. 6B) and also with the ubiquitously expressed bHLH-type

1985RESEARCH ARTICLEMechanisms of Senseless synergism

Fig. 6. Sens binds proneural proteins throughits Zn-finger domains. (A) An IP assay showsthat Sens interacts with Ac in COS-7 cells. (B) Invitro translated c-myc-tagged Sens interacts withbacterially expressed GST-tagged proneuralproteins in a GST pull-down assay. Input laneshows 20% of the input used in the GST pull-down assay. (C) Sens binds to Sc through its Zn-finger domains. Bacterially expressed GST-taggedSc protein can interact with in vitro translated full-length Sens (a), and Sens Zn-finger domains (c),but not with Sens N terminus (b), which lacks theZn-finger domains. (D) Sens can still bind GST-tagged Sc if any of its Zn-finger domains aredeleted (b,c,d,e). However, Sens loses its ability tobind Sc if Zn finger2, linker2 and Zn finger3 aredeleted together (f). The bottom gel showswestern blot analysis using anti c-myc antibody todetect 20% of the input. (E) Sens-L2-T-E binds Scin a GST pull-down assay. The binding is weakerthan wild-type Sens, in line with the weaker bristlephenotype (see Fig. 4B,C). Input lanes show 20%of the actual input.

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protein Daughterless (Jafar-Nejad et al., 2006), in agreement withthe observation that Sens can synergize with various proneuralproteins in vivo (Nolo et al., 2000; Quan et al., 2004).

To identify the Sens domain that binds to proneural proteins, weperformed GST pull-down experiments using GST-Scute andseveral fragments of the Sens protein that are tagged with c-myc. Asshown in Fig. 6C, the Zn-finger domains of Sens mediate theinteraction between Sens and Scute. In addition, the data also showthat the Zn-finger domains alone are sufficient to mediate theinteraction.

Our cell culture, ectopic expression and rescue experimentsindicate that Zn finger 2 and Zn finger 3 are indispensable for Sensfunction in cell culture and in vivo. To examine if Zn finger 2 andZn finger 3 are involved in the interaction between Sens andproneural proteins, we deleted each Sens Zn finger individually andtested mutant Sens proteins in GST pull-down assays for theirability to interact with Scute. We find that Sens is still able tointeract with Scute in the absence of any of the four Zn fingers (Fig.6D, lanes b-e), indicating that the Sens-bHLH interaction does notdepend on a single Zn finger. However, when both Zn finger 2 andZn finger 3, and the linker between them are removed, Sens is nolonger able to interact with Scute (Fig. 6D, lane f). These dataindicate that the core region of the Sens Zn-finger domain, whichcontains Zn finger 2 and Zn finger 3, mediates the interactionbetween Sens and Scute. To further support the notion that Sens-proneural interaction is required for bristle formation, we tested theSens-L2-T-E mutation, which fails to bind DNA but is able toinduce bristle formation upon overexpression, for its ability tointeract with Sc. As shown in Fig. 6E, Sens-L2-T-E can bind Sc,although weaker than wild-type Sens. Altogether, these datastrongly suggest that Sens-proneural interaction is necessary for therecruitment of Sens to proneural target enhancers and for its bristle-inducing ability. However, the data also indicate that the binding toproneural proteins is not sufficient and that these Zn fingers haveadditional functions.

Sens acts as a repressor when bound to DNA, andas an activator when bound to proneural proteinsAs the ac promoter may have other binding sites for unknownfactors that are also involved in the synergism between Sens andproneural proteins, we performed another set of transcription assayson an artificial reporter consisting of five UAS sites and a thymidinekinase promoter, UAS-tk-luc (Fig. 7A). Giagtzoglou et al. recentlyshowed that the Scute protein is able to induce luciferase expressionfrom the UAS-tk-luc reporter when fused with the Gal4-DNAbinding domain (Gal4DBD) (Giagtzoglou et al., 2005). If bindingof proneural proteins is enough to recruit Sens to an enhancer, weanticipate synergism in this assay when Scute or Achaete is fused tothe Gal4DBD. As shown in Fig. 7B, Sens is able to synergize withfull-length Scute and Achaete, strongly suggesting that Sens isbrought to the enhancer via proneural proteins.

E(spl) proteins are known to be negative regulators of proneuralprotein expression and function (Culi and Modolell, 1998; Delidakisand Artavanis-Tsakonas, 1992; Giagtzoglou et al., 2003; Jimenezand Ish-Horowicz, 1997; Knust et al., 1992). E(spl)m7 was shownto inhibit Scute-mediated transcriptional activation by binding to Scdirectly (Giagtzoglou et al., 2005). As shown in Fig. 7C, Sens is notable to synergize with the Scute protein in the presence of theE(spl)m7, providing further support to the hypothesis that Sens canonly synergize with proneural proteins when proneural proteins areable to induce transcription.

Our previous data indicate that repression mediated by low levelsof Sens requires DNA binding, and that Sens DNA binding has anegative role on the synergism between Sens and proneurals. To testif proneural proteins can synergize with Sens when it is tethered toDNA, we generated Sens-Gal4DBD fusion protein. This fusionprotein significantly represses the basal level of luciferase expressionfrom the UAS-tk-luc reporter (Fig. 7D). The repression is alsoobserved in the presence of high levels of proneural proteins. Theseobservations provide further evidence that Sens, when bound toDNA, indeed acts as a repressor.

RESEARCH ARTICLE Development 133 (10)

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Fig. 7. Sens can synergize with proneural proteins onthe UAS-tk-luc reporter in S2 cell transcriptionassays. (A) Schematics of the UAS-tk-luc reporterconstruct. (B) S2 cell transcription assays using UAS-tk-lucreporter. Sens expression alone does not have a significanteffect on the basal expression of the UAS-tk-luc reporter.However, Sens is able to synergize with Sc and Ac.(C) E(spl)m7 strongly inhibits the expression induced bythe Sc-Gal4DBD and the synergism between Sens and Sc-Gal4DBD. (D) When fused to Gal4DBD, Sens acts as astrong repressor on the UAS-tk-luc reporter. Ac and Sc failto synergize with DNA-bound Sens.

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DISCUSSIONSensory organ precursor development in flies requires the functionof proneural proteins and Sens (Frankfort et al., 2001; Frankfort etal., 2004; Jafar-Nejad et al., 2003; Koelzer and Klein, 2003; Nolo etal., 2000). Expression of sens is under the direct control of theproneural proteins (Jafar-Nejad et al., 2003) and SOPs co-expressproneural proteins and Sens (Jafar-Nejad et al., 2003; Nolo et al.,2000; zur Lage et al., 2003). We have previously shown that highlevels of Sens synergize with proneural proteins to upregulate theexpression of the ac gene via the ac proximal enhancer, a knowntarget of proneural proteins (Jafar-Nejad et al., 2003; Van Doren etal., 1992). However, low levels of Sens oppose the activator functionof proneural proteins on this enhancer. Intriguingly, our datasuggested that Sens DNA binding has a negative role in thesynergism between proneural proteins and Sens, but the molecularmechanism underlying this dual role in transcriptional regulationwas not elucidated.

Based on our current data, we propose the following model for therole of Sens in transcriptional regulation of proneural target genes insensory precursors. Early in the proneural cluster, proneural geneexpression is under the control of proneural and E(spl) proteins.At this stage, proneural genes start to engage in a positiveautoregulatory loop by binding to the E-boxes in their ownenhancers (Culi and Modolell, 1998; Van Doren et al., 1992).Initially, low levels of Sens bind DNA rather than the proneuralproteins via its Zn fingers because it has a higher affinity for DNA(Fig. 8A). When bound to DNA, Sens acts as a repressor. As Sens

interacts with several E(spl) proteins (Jafar-Nejad et al., 2003),recruitment of E(spl) through Sens might contribute to the negativeregulation of proneural target enhancers (Fig. 8A). As the level ofproneural proteins increases, proneural proteins induce more Sensexpression. This will lead to saturation of the S-boxes (Fig. 8B).Additional Sens will bind proneural proteins via its core Zn-fingerdomains and act as a co-activator to increase the transcriptioninduced by proneural proteins (Fig. 8C). We propose that the switchbetween the repressor and co-activator functions of Sens depends onthe conformational state of its Zn fingers. In this model, binding toproneural proteins will allow the Sens Zn fingers to adopt analternative conformation compared to the DNA-bound state. Thiswill enable Sens to cooperate with co-activators already recruited byproneural proteins, or to recruit new co-activators to further increasethe ability of proneural proteins to increase the expression of theirtarget genes in some contexts. This conformation-based hypothesisis supported by our observation that even point mutations in Sens Znfingers that are dispensable for proneural interaction still causesevere reduction in the synergism between Sens and proneuralproteins.

Multiple lines of evidence suggest that Sens acts as atranscriptional co-activator for bHLH proneural proteins. First, Sensis required for the upregulation and maintenance of proneural geneexpression in the wing margin chemosensory SOPs (Nolo etal., 2000). Second, Sens synergizes with proneural proteins toupregulate the expression of the ac proximal enhancer in S2 cellassays (Jafar-Nejad et al., 2003). Third, ectopic expression of Sensinduces ectopic proneural gene expression (Lai, 2003; Nolo et al.,2000). Fourth, Sens physically binds bHLH proteins via the coreregion of its Zn-finger domain. Fifth, Sens can not inducetranscription in the absence of proneural proteins. It should bementioned that our in vitro and in vivo observations indicate thatDNA binding is not essential for the ability of Sens to act as a co-activator and to induce SOP formation. Therefore, as SOPsaccumulate high levels of both proneural proteins and Sens, it islikely that proneural target enhancers that do not contain a Sens-binding site might also be a target for proneural-Sens transcriptionalsynergism.

Similar to its vertebrate homologues (Grimes et al., 1996; Tong etal., 1998), Sens can function as a transcriptional repressor whenbound to DNA (Fig. 7). Mutational analysis of Sens Zn fingers alsoindicates a link between DNA binding and the repressor function ofSens: those Sens mutants that do not bind DNA (1CC, 2CC and3CC) fail to repress ac transcription, whereas mutating Zn finger 4,which does not play a major role in DNA binding, does not affect therepressor function of Sens. Although the repressor function seemsto be less crucial than the co-activator function in vivo, our datasuggest that the repressor function of Sens also contributes to its rolein PNS development.

Sens physically interacts with proneural proteins via its Zn-finger domains, which are highly conserved between Sens and itsvertebrate homologues. In addition, Sens can synergize with themouse Ato homologue Math1 (Atoh1 – Mouse GenomeInformatics), when the two proteins are co-expressed in flies(Quan et al., 2004). Together, these observations suggest that theSens-bHLH interaction is evolutionarily conserved. In otherwords, vertebrate bHLH proteins such as Math1, Mash1 (AScl1– Mouse Genome Informatics) and Math5 (Atoh7 – MouseGenome Informatics), which are co-expressed with Gfi1 inmouse tissues (Kazanjian et al., 2004; Wallis et al., 2003; Yanget al., 2003), might be able to recruit Gfi1 to their targetenhancers.

1987RESEARCH ARTICLEMechanisms of Senseless synergism

Fig. 8. A model for the dual role of Sens Zn fingers in thetranscriptional regulation of proneural target genes. E representsE-box, S represents S-box. The four ovals in Sens depict Zn fingers. SeeDiscussion for details.

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In conclusion, our data suggest that Sens, a C2H2-type Zn-fingerprotein, binds to bHLH proneural proteins via its core Zn-fingerdomains and acts as a co-activator of the expression induced byproneural proteins. Sens can bind to various bHLH proteins andsynergize with fly proteins, as well as some of their vertebratehomologues in vivo. These data, together with other examples of Zn-finger/bHLH synergism (Bellefroid et al., 1996), suggest thatphysical and genetic interactions of this type might be a commonmechanism for Zn-finger/bHLH cooperation during development.

We thank H. Lacin for technical assistance; H. Tsuda for advice on mammaliancell culture experiments; A. Flora, G. Halder, S. K. Lee, A. Rajan, E. Seto, A.-C.Tien and K. Venken for critical reading of the manuscript; and theDevelopmental Studies Hybridoma Bank for antibodies. This work wassupported by grants from NASA and funds from HHMI. H.J.B. is an HHMIinvestigator. H.J.-N. is supported by HHMI and the NIH Medical GeneticsResearch Fellowship Program grant T32-GMO7526.

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1989RESEARCH ARTICLEMechanisms of Senseless synergism