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206 nature genetics • volume 32 • september 2002

Distinct in vivo requirements for establishment versusmaintenance of transcriptional repression

John C. Wheeler1,3*, Christine VanderZwan1,2*, Xiaoti Xu1, Deborah Swantek1, W. Daniel Tracey1,2

& J. Peter Gergen1

*These authors contributed equally to this work.

1Department of Biochemistry and Cell Biology and the Center for Developmental Genetics and 2Graduate Program in Genetics, Stony Brook University,Stony Brook, New York 11794-5140, USA. 3Present address: The Rothberg Institute for Childhood Diseases, Guilford, Connecticut, USA. Correspondenceshould be addressed to J.P.G. (e-mail: jgergen@notes.cc.sunysb.edu).

Low-level ectopic expression of the Runt transcription factorblocks activation of the Drosophila melanogaster segmenta-tion gene engrailed (en) in odd-numbered parasegments and isassociated with a lethal phenotype. Here we show, by using agenetic screen for maternal factors that contribute in a dose-dependent fashion to Runt-mediated repression, that there aretwo distinct steps in the repression of en by Runt. The initialestablishment of repression is sensitive to the dosage of thezinc-finger transcription factor Tramtrack. By contrast, the co-repressor proteins Groucho and dCtBP, and the histone deacety-lase Rpd3, do not affect establishment but instead maintainrepression after the blastoderm stage. The distinction betweenestablishment and maintenance is confirmed by experimentswith Runt derivatives that are impaired specifically for eitherco-repressor interaction or DNA binding. Other transcriptionfactors can also establish repression in Rpd3-deficient embryos,which indicates that the distinction between establishmentand maintenance may be a general feature of eukaryotic tran-scriptional repression.The odd-numbered stripes of the segment-polarity gene en areextremely sensitive to repression by Runt, the founding mem-ber of the Runx family of transcription factors. Embryos with

an increased gene dosage of runt (run) showed a delay in enactivation in cells located between the Runt pair-rule stripes(data not shown). This suggested that the reduction in Runtassociated with the resolution of the pair-rule pattern from anearlier broad band of expression was a prerequisite for activa-tion of en in these interstripe cells (Fig. 1a,b). Indeed, low-levelexpression of a GAL4-regulated UAS–run transgene (Fig. 1c)blocked activation of the en stripes (Fig. 1d,e). We identified thethreshold amount of Runt required for repression of en by con-trolling the level of GAL4-driven expression1. Expression ofother pair-rule and segment-polarity genes was normal at thisthreshold level of Runt (Fig. 1f–h and data not shown). Theseobservations confirm and extend previous results obtainedwith a heat-inducible hs-run transgene2 that indicate that Runtis a potent repressor of en.

The threshold amount of Runt required for en repression coin-cides with the amount that is associated with embryonic lethality.Experiments with other GAL4 and UAS–run lines indicate thatthere is a substantial increase in viability at half this amount ofectopic Runt1. We took advantage of this dose sensitivity to con-duct a genetic screen for factors that potentiate Runt activity. Weused a collection of chromosomal deficiencies to identify genomic

Fig. 1 Runt is a potent repressor of en. a, run mRNA is expressed in a broad bandthroughout the presegmental region of an embryo at the syncitial blastodermstage (nuclear division cycle 13). Expression extends from roughly 70% of theegg length (where 100% is the anterior pole) to nearly the posterior end, withhighest expression in the center. b, The fully resolved pair-rule pattern of runmRNA expression in a wildtype cellular blastoderm stage embryo comprisesseven stripes, each about four cells wide, separated by interstripe regions, alsoabout four cells wide. Note the lack of run mRNA accumulation in the regionseparating the third and fourth stripes (diamond). c, Accumulation of runmRNA in a cellular blastoderm embryo in which expression of the UAS–run232

transgene is driven by one copy of the NGT40 maternal GAL4 driver. Althoughexpression from the endogenous gene predominates, the accumulation of api-cally localized run mRNA transcripts in both cells in the interstripe regions (dia-mond) and cells outside the presegmental region of the embryo provides anindication of the amount of ectopic expression. d, Expression of en mRNA in awildtype embryo at the gastrula stage. Expression in all 14 parasegments isapparent at this stage, although expression in the odd-numbered stripes isweaker than in the even-numbered stripes. The diamond marks the seventhstripe, which arises in cells located between the third and fourth Runt stripes. e, Expression of the odd-numbered en stripes at the gastrula stage is blocked byectopic Runt expression. The diamond indicates the region where the seventhen stripe would be expressed normally. Repression of en at this stage isobtained in 90% of the embryos when one copy of the NGT40 driver is used todrive expression of the UAS–run232 transgene. f–h, This amount of ectopic Runtdoes not alter the mRNA expression patterns of eve (f), prd (g) or slp1 (h), threeother regulators of en.

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intervals that dominantly suppress the lethality associated withGAL4-driven expression of Runt (Methods). To identify singleinteracting genes, we tested P-element insertion lines and otherlines with mutations in selected deficiency intervals. We also usedthis strategy to test mutations in selected candidate genes thatwere not represented in the deficiency collection. These experi-ments identified four genes that have specific dose-dependentmaternal effects on the lethality produced by ectopic expression ofRunt (Table 1). Three of these, dCtBP, Groucho (Gro) and Rpd3,encode proteins that do not bind DNA but have been character-ized as co-repressors on the basis of their interactions with otherDNA-binding transcription factors2–8. The fourth gene, tram-track (ttk), encodes two different zinc-finger proteins that alsofunction as repressors in the D. melanogaster embryo9,10.

We examined the effect of each of these four genes on Runt-dependent repression of en using a stronger GAL4 driver toincrease the penetrance of repression. Reduced maternal activity

of all four genes restored en expression in thepresence of ectopic Runt, but with differences inthe timing of recovery. In embryos with reducedTtk activity, the odd-numbered en stripes wererestored in the blastoderm stage, with nearlywildtype expression of en (Fig. 2a,c,d). But withreduced maternal dosage of Rpd3, Gro ordCtBP, en expression was not restored untilgermband extension (Fig. 2b, e–h and data notshown). These results indicate that there may bea functional distinction between the establish-ment and maintenance of en repression in theearly D. melanogaster embryo.

The above experiments were based on dose-dependent interactions at threshold amounts ofectopic Runt. To further investigate the roles ofthese different factors, we examined the effectsof eliminating the maternal contribution togene expression by generating germlineclones11. Eliminating maternal Gro disrupts theexpression of several segmentation genes5,which makes it difficult to characterize the spe-cific role of Groucho in en regulation by thismethod. This approach was also not informa-tive for ttk, because germ cells that werehomozygous for any one of several ttk muta-

tions that we tested failed to complete oogenesis.By contrast, early gastrula-stage embryos derived from female

germ cells homozygous for the Rpd304556 mutation showed nor-mal expression of several pair-rule and segment-polarity genes,including the odd-numbered en stripes7. The Rpd304556 allelecontains a P-transposon insertion that reduced the maternaltranscripts to undetectable levels (data not shown). Ectopic Runtexpression in these Rpd3-deficient embryos repressed the odd-numbered en stripes (Fig. 3a,b). Although Runt repressed en inembryos that had little or no Rpd3, maintenance of this repres-sion was sensitive to a 50% decrease in Rpd3 activity, which pro-vides compelling genetic evidence for a distinction between theestablishment and maintenance of Runt-dependent repression.

Runt interacts directly with Groucho through a conserved car-boxy-terminal Val-Trp-Arg-Pro-Tyr (VWRPY) motif2. Thisobservation suggested an alternative approach for investigating therole of Groucho in Runt-dependent repression of en. We found

Fig. 2 Dose-dependent maternal effects on establishment and maintenance ofen repression. Expression of en mRNA as visualized by in situ hybridization inembryos at the gastrula (a,c,e,g) and germband extended (b,d,f,h) stage. Theembryos shown are heterozygous for the UAS–run232 transgene and derivedfrom females that are heterozygous for the NGT40 and NGTA maternal GAL4drivers. This specific combination produces about 50% more activity thanobtained with NGT40 alone. a–d, In genetic backgrounds that are otherwisenormal, the increase in ectopic Runt expression produced by this combinationblocks expression of the odd-numbered en stripes in all gastrula stage embryos(a) and this repression is maintained through germband extension (b). Runt-dependent repression of en is potentiated by the maternal dosage of ttk.Expression of en mRNA in odd parasegments is nearly wild type in both gas-trula (c) and germband extended (d) stage UAS–run expressing embryos thatare derived from females heterozygous for ttk1. Similar suppression of Runt-mediated repression is obtained with the ttk1e11 and ttk02667 mutations (datanot shown). e, Ectopic Runt represses the odd-numbered en stripes in all gas-trula stage embryos derived from females that are heterozygous for theE(spl)E48 mutation. But this repression in not maintained during germbandextension. f, Intermediate restoration of en mRNA expression is observed inmore than 50% of the embryos from E(spl)E48 heterozygous females; about25% of germband extended embryos in this cross have wildtype en expression.Similar results were obtained with E(spl)BX22. g,h, Runt-dependent repressionof en is observed in all gastrula stage embryos from females that are heterozy-gous for the Rpd304556 mutation (g), but is not maintained during germbandextension (h). Similar results were obtained with CtBP (not shown).

Table 1 • Genes with a dose-dependent maternal effect on Runt-mediatedlethality

Gene Mutationa UAS–run viability (n)b UAS–lacZ activityc

Control ss e ro 3% (421) 100%dCtBP 03463 15% (551) 92%Gro E(spl)E48 30% (188) 84%Rpd3 04556 31% (692) 81%Ttk 1e11 21% (383) 96%

aResults for representative specific mutant alleles of the indicated genes. An isogenized chromo-some carrying mutations in ss, e and ro was used as a control for outcrossing the NGT40 stock. Inter-actions with dCtBP, Rpd3 and Ttk were identified initially from the suppression of Runt-mediatedlethality by the deficiency chromosomes Df(3R)ry615 (29% viability, 203 sibs), Df(3L)GN50 (36% viabil-ity, 102 sibs) and Df(3R)faf-BP (40 % viability, 250 sibs), respectively. Gro was examined as a candidategene on the basis of previous studies. The mesA allele of dCtBP also suppressed Runt-mediatedlethality (32% viability, 139 sibs). The E(spl)BX22 mutation did not show a significant interaction in theUAS–run viability assay (3% viability, 153 sibs). Several different Ttk mutations were tested with thefollowing viabilities: Ttk1, 19% (270 sibs); Ttk5346, 58% (129 sibs); TtkB330, 33% (157 sibs); Ttk10556,27% (181 sibs); TtkRM730, 2% (176 sibs); TtkD2-50, 8% (199 sibs); Ttkosn, 0% (323 sibs). bFemales carry-ing one copy of the NGT40 driver were generated by crosses between homozygous NGT40 femalesand males carrying the mutations to be tested. Virgin females from these crosses that were het-erozygous for a mutation and one copy of NGT40 were subsequently mated with males heterozy-gous for the UAS–run232 transgene, located on the second chromosome, and for the CyO balancerchromosome. The numbers report the percentage viability of straight winged UAS–run progeny rel-ative to their curly winged, CyO siblings. The number of siblings recovered is given in parentheses.cAs a control for the nonspecific maternal effects of different mutant chromosomes on GAL4-drivengene expression, heterozygous females were mated to males homozygous for a UAS–lacZ transgene.β-galactosidase activity was measured for 3–5 embryos in 2–3 experiments for each maternal geno-type. Embryos from control ss e ro heterozygous females were tested as a standard. The individualaverages relative to the ss e ro average for each experiment are as follows: dCtBP03463 (82 and 103),E(spl)E48 (62, 82 and108), Rpd304556 (69, 69 and 106) and Ttk1e11 (64 and 120).

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that GAL4-driven expression of Runt∆8, a Runt derivative withoutthe VWRPY motif (Fig. 4a), repressed en at the blastoderm stage(Fig. 4b), but this repression was not maintained and the full pat-tern of 14 stripes emerged during germband extension (Fig. 4c).These observations corroborate results obtained with a heat-shockexpression system, which indicate that Runt-dependent repressionof en at the blastoderm stage is independent of Groucho2. Ourresults using GAL4-driven expression of Runt show, however, thatthe Runt–Groucho interaction is important for maintainingrepression at later stages.

The distinction between establishment and maintenance wassubstantiated further by experiments with RuntCK, a derivativecontaining two mutations in the Runt domain that specificallydisrupt DNA binding12 (Fig. 4d). Unexpectedly, RuntCK was aseffective as wild-type Runt in establishing repression during theblastoderm stage when expressed using the GAL4 system (Fig. 4e).But, as found for Runt∆8, the repression by RuntCK was notmaintained (Fig. 4f). On the basis of these results, we concludethat DNA binding by Runt and the Runt–Groucho interaction arenot required for the initial repression of en at the blastodermstage, but that both interactions are essential for maintaining thisrepression at later stages.

These results suggest a two-step model for Runt-dependentrepression of en (Fig. 5). Initial repression at the blastoderm stageis sensitive to ttk dosage. The two proteins encoded by ttk, Ttk-p69 and Ttk-p88, are expressed maternally and degraded in earlyembryogenesis, becoming nearly undetectable by the onset ofgastrulation10. Genetic tests of the ability of different ttk alleles tosuppress the effects of ectopic Runt indicated that Ttk-p88 mightbe more relevant to the repression of en than is Ttk-p69. The Ttk1

mutant, which disrupts Ttk-p88 (ref. 13), suppressed Runt-mediated lethality, whereas TtkD2-50 and Ttkosn, which disruptTtk-p69 (ref. 14), had little effect (Table 1). The Ttk-p88 andTtk-p69 isoforms have different zinc-finger domains and distinct

DNA-binding specificities9,15. The initial repression of en doesnot require high-affinity DNA binding by Runt. We propose thatthe DNA binding by Ttk-p88 targets Runt to the en cis-regulatoryregion and that Runt and Tramtrack cooperate to prevent tran-scription of en, perhaps by interacting directly with componentsof the basal transcriptional machinery. The interaction betweenRunt and Tramtrack is not necessarily direct and might be medi-ated by other proteins. Indeed, our genetic screen identifiedanother interval whose deficiency suppressed the establishmentof en repression by Runt.

Maintenance of repression after the blastoderm stage is a seconddistinct phase of en regulation. Stable repression requires bindingof both DNA and Groucho by Runt and involves genetic interac-tions with dCtBP, Groucho and Rpd3. Groucho and Rpd3 are pre-sent as a complex in the D. melanogaster embryo16. We proposethat stable repression involves recruitment of the Groucho–Rpd3complex by a DNA-bound form of Runt (Fig. 5). The histonedeacetylase activity of Rpd3 will then prevent subsequent activa-tion by reducing accessibility to transcriptional activators, thetranscriptional machinery or both.

Is the initial establishment of en repression by Runt and Tram-track essential for the maintenance phase of repression, or are thesetwo mechanistically independent pathways of Runt-mediatedrepression? If the second phase were independent of the initialestablishment phase, then the expectation would be that reducingmaternal Tramtrack would cause derepression at the blastodermstage, but this repression would soon be re-established. This wasnot observed, however, which suggests that Runt- and Tramtrack-dependent establishment is a prerequisite for the subsequent Grou-cho- and Rpd3-dependent maintenance of en repression.

Fig. 3 Rpd3-independent repression by Runt, Sloppy-paired 2 or Even-skipped.Wildtype expression of mRNAs from en (a) and odd (d) in gastrula stageembryos. Expression of the odd-numbered en stripes is blocked by ectopicexpression of Runt (b) or Sloppy-paired 2 (c) in embryos that lack maternalRpd3 activity. Similarly, ectopic expression of Even-skipped leads to repressionof odd in the even-numbered parasegments of embryos from both wildtypefemales (e) and Rpd3 germline clone females (f). Embryos were generated bymating females that produce eggs from germ cells homozygous for theRpd304556 mutations with males homozygous for a hs-run (b), hs-slp2 (c) or hs-eve transgene (f), or by mating a wildtype female and an hs-eve male (e).Embryos were heat shocked (37 °C) for 5 min (b) or 10 min (c,e,f) and allowedto recover for 20 min (25 °C) before processing for in situ hybridization.

Fig. 4 Groucho recruitment and DNA binding are required for maintenanceof en repression. a, The Runt∆8 derivative. The Runt domain (shadedsquare) mediates binding to DNA (horizontal bar). The Runt∆8 protein lacksthe 25 carboxy-terminal amino acids, including the VWRPY motif that medi-ates interaction with Groucho. b, Females homozygous for the NGT40 driverwere mated with males homozygous for UAS–run[∆8]4-3. Expression of Runtat this level is lethal and associated with fully penetrant repression of en atthe blastoderm stage. c, Repression is not maintained, and 98% of theembryos show expression of en mRNA in odd-numbered parasegments dur-ing germband extension. d, The RuntCK derivative in which the Runtdomain (filled square) contains two specific amino-acid substitutions thatspecifically disrupt DNA binding12. e, Females homozygous for the NGT40driver were mated with males homozygous for UAS–run[CK]77. Expressionof Runt at this level is lethal and associated with fully penetrant repressionof en at the blastoderm stage. f, Repression is not maintained duringgermband extension. As for wildtype Runt, en is the most sensitive target ofboth the Runt∆8 and RuntCK proteins, and other segmentation genes areexpressed normally in embryos that express threshold levels of these Runtderivatives (not shown).

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Rpd3-deficient embryos have a pair-rule segmentation pheno-type7. But the expression of several segmentation genes, includ-ing the initial expression of en, is normal in Rpd3-deficientembryos. Defects in en are first apparent during germbandextension7, the stage at which Rpd3 acts to maintain Runt-dependent en repression. These observations indicate that thecentral function of Rpd3 in segmentation may be to maintainrepression that is initially established by Runt and other pair-ruletranscription factors. The defects in en expression in Rpd3-defi-cient embryos have been interpreted as being caused indirectlyby the failure of Even-skipped (encoded by eve) to repress odd-skipped (odd)7. But Even-skipped retains the ability to repressodd in Rpd3-deficient embryos (Fig. 3d–f). Thus, as was foundfor Runt, Rpd3 is not required for Even skipped–dependentrepression. Similar results are obtained with Sloppy-paired 2, aforkhead-domain transcription factor (encoded by slp2) that alsorepresses the odd-numbered en stripes (Fig. 3c). These resultsindicate that Rpd3 may be important for maintaining repressiondependent on Runt, Even-skipped and Sloppy-paired 2, which isestablished initially through other molecular mechanisms.

Rpd3 participates in repression by several transcription factors inorganisms ranging from yeast to humans17. Our ability to separatethe role of Rpd3 in maintenance from a possible role in establish-ment was facilitated greatly by the temporally dynamic yet repro-ducible program of transcriptional regulation that controls cell fatespecification in the early D. melanogaster embryo. The powerfulgenetic advantages offered by this system will be invaluable for dis-secting further the in vivo mechanisms of activation and repressionby Runt and other eukaryotic transcription factors.

MethodsStrains and transgenes. The NGT40 germline transformant strain,which uses the nanos promoter to drive maternal expression of a GAL4mRNA that also includes the 3′ UTR of an α-tubulin gene, has beendescribed1. We generated a related strain, NGTA, by mobilizing the sametransposon onto the third chromosome. The NGTA driver has 50% ofthe activity of NGT40 on the basis of measurements of β-galactosidaseactivity in extracts from embryos with a UAS–lacZ transgene. Thus, thecombined NGT40;NGTA strain is 1.5 times more active than thehomozygous NGT40 strain, and about 3 times more active than NGT40heterozygotes. The UAS–run232, UAS–en4-1, UAS–dpp42B.4 andUAS–lacZ4-1-2 transgenes have been described1.

The UAS–run[CK]77 line contains a second chromosome insertion ofthe P-transposon P{UAS–run[CK].L}. We generated this P-transposon byreplacing a BglII fragment of p[UAS–run.T] with the analogous fragmentfrom the plasmid pB:runt[CK], which contains the Cys127Ser andLys199Ala mutations12, and established germline transformants by stan-dard protocols. RNase protection assays indicated that the UAS–run[CK]77

transgene was expressed comparably to the UAS–run232 transgene. Expres-sion of the UAS–run[CK]77 transgene was lethal at the levels obtained in

embryos from homozygous NGT40 females, whereas 26% of theUAS–run[CK]77 progeny survived at the lower expression level obtained inembryos from heterozygous NGT40 females.

The UAS–run[∆8]4-3 line contains a second chromosome insertion ofthe P-transposon P{UAS–run[∆8].S}. The protein expressed by this con-struct carries a Flag epitope tag sequence Asp-Tyr-Lys-Asp-Asp-Asp-Lysinserted between amino acids 455 and 456 and lacks amino acids 485–509of the normal Runt protein. We generated this construct using the ExSitePCR-based site-directed mutagenesis kit and the run cDNA plasmidpB:ED[Bam-8, ∆KS]18 as described12. The primer sequences and cloningstrategy used to generate this construct are available from the authors onrequest. We created plasmid p[UAS:runt[∆8]] by subcloning a BamHIfragment from the pB:runt[Flag∆8] construct into BglII linearized pUAS:Tvector19. The P{UAS–run[∆8].S}4-3 transformant line was recovered bystandard P-element-mediated germline transformation. Expression of thistransgene was lethal in embryos from homozygous NGT40 females,whereas 55% of the UAS–run[∆8]4-3 progeny survived at the lower expres-sion level obtained in embryos from heterozygous NGT40 females.

The strain carrying the heat-inducible hs-run transgene has beendescribed20. We obtained similar heat-inducible hs-eve19B (ref. 21) and hs-slp24A2-3 (ref. 22) strains from G. Struhl and M. Leptin, respectively.Strains representing the ‘deficiency kits’ and a few P-element insertion andlethal mutations in selected intervals, the ttk mutations ttk1ell, ttk1 andttk02667, and the Rpd3 mutation Rpd304556 were provided by the Blooming-ton Drosophila Stock Center. We obtained stocks carrying the ttkD2-50,ttkB330, ttk10556, ttk5346, ttkrM730 and ttkosn mutations from C. Klämbt(Univ. of Munster) and the Gro mutations E(spl)E48 and E(spl)BX22 from U.Banerjee (Univ. of California, Los Angeles).

In situ hybridization. In situ hybridization was carried out as described23.We synthesized digoxigenin-labeled (Boehringer Mannheim) RNA ribo-probes to detect the en, eve and run mRNA transcripts as described1,20. Theprobe for sloppy-paired 1 (slp1) was synthesized with T7 RNA polymeraseusing an EcoRI-linearized plasmid containing a PCR fragment of 600 bp ofslp1 subcloned into the BamHI site of pBS(KS+). The probe for odd wassynthesized with T7 RNA polymerase using a NotI-linearized, odd cDNAfragment of 2 kb cloned into the EcoRI site of pBS(KS+). M. Weir (Wes-leyan Univ.) provided the slp1 and odd plasmids. The probe for paired(prd) was synthesized with T3 RNA polymerase using a NotI-linearizedtemplate that contained a cDNA insert with an NdeI site engineered at thefirst AUG codon subcloned into the NdeI site of pAR3039. This plasmidconstruct was constructed by J. Treisman and provided by S. DiNardo(Univ. of Pennsylvania).

Maternal suppressor assay. We carried out the screen for dose-dependentmaternal suppressors of ectopic Runt lethality as follows. HomozygousNGT40 flies were crossed to individuals from balanced D. melanogasterstocks that carry different deficiency chromosomes. The chromosomalintervals spanned by the 160 different deficiency chromosomes tested rep-resent about 70% of the D. melanogaster genome. We collected virginfemales that were heterozygous for NGT40 and each particular deficiencychromosome from these crosses and mated these females withUAS–run232/CyO males. We determined the viability of the UAS–run prog-

Fig. 5 Genetics of establishmentand maintenance of repressionby Runt. a, Establishment of enrepression by Runt and Tram-track (Ttk). The idea that estab-lishment involves binding ofTramtrack to DNA (horizontalline) is supported by geneticinteractions indicating that theTtk-p88 isoform is involvedspecifically in en repression. Ttk-p88 and Ttk-p69 differ in theirzinc-finger DNA-binding domains and DNA-binding specificity9. Runt is shown as not bound to DNA and as not interacting with Groucho (Gro) on the basis ofthe efficient establishment of en repression by the RuntCK and Runt∆8 derivatives, respectively. The genetic interaction between Runt and Tramtrack, which maynot reflect a direct protein–protein interaction, is depicted as two sets of parallel lines. b, Factors involved in the maintenance of en repression by Runt. In thismodel, DNA-bound Runt recruits a Groucho–Rpd3 complex through the conserved C-terminal VWRPY motif. We propose that the histone deacetylase activity ofRpd3 modifies chromatin at the en locus, thereby stably maintaining repression at subsequent stages when Runt and Tramtrack are no longer present in the cell.

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eny relative to the number of CyO progeny. Previous experiments hadshowed that a single copy of the NGT40 driver reduced the viability ofUAS–run progeny to less than 10% in this assay. Forty-nine of the deficien-cies increased the viability of female UAS–run progeny to more than 30%of female viability or allowed the viability of several UAS–run males.

To determine whether the suppression was specific, we tested differentdeficiencies for their effects on the lethality associated with NGT-drivenexpression of UAS–dpp, UAS–en or both transgenes, as well as on theamount of β-galactosidase activity produced by NGT-driven expression ofa UAS–lacZ strain as described1. We classified the suppression of UAS–runlethality as nonspecific if the deficiency allowed more than 50% viability ofUAS–dpp progeny (from a background of 1%), allowed more than 5% via-bility of UAS–en progeny (from a background of 0%) or led to more than atwofold reduction in the levels of NGT-driven β-galactosidase activity. Weselected eight intervals for further analysis on the basis of these specificitytests. P-element insertion lines and other mutations that mapped in theseintervals were obtained from the Bloomington Stock Center and tested in asimilar fashion. We generated Rpd3 germline clones by recombining theRpd304556 line with FRT79D to generate a stock Rpd304556 FRT79D/TM3. Weused this stock in conjunction with hs:FLP; ovoD/Cx D/TM3 as described11

with the exception that heat shocks were carried out on three consecutivedays instead of two.

AcknowledgmentsWe thank the Drosophila Stock Center in Bloomington and our colleagueswho provided Drosophila stocks and plasmid DNAs; S. Kramer and L.-H. Lifor the p[UAS–run[CK]] construct and the initial UAS–run[CK]transformant line, respectively; X. Ning for help with generating theUAS–run[∆8] construct and transformant lines; T. Kalkan for help withcharacterizing the effects of deficiency chromosomes; and J. Landry andmembers of the Gergen laboratory for comments on the manuscript. Thiswork was supported by grants from the NIH and the NSF to J.P.G.

Competing interests statementThe authors declare that they have no competing financial interests.

Received 5 April; accepted 1 June 2002.

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