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RESEARCH ARTICLE

E(y)1/TAF9 mediates the transcriptional output of Notch signalingin Drosophila

Gengqiang Xie1, Zhongsheng Yu2, Dongyu Jia1, Renjie Jiao1,* and Wu-Min Deng1,*

ABSTRACT

Transcriptional activation of Notch signaling targets requires the

formation of a ternary complex that involves the intracellular domain

of the Notch receptor (NICD), DNA-binding protein Suppressor of

Hairless [Su(H), RPBJ in mammals] and coactivator Mastermind

(Mam). Here, we report that E(y)1/TAF9, a component of the

transcription factor TFIID complex, interacts specifically with the

NICD–Su(H)–Mam complex to facilitate the transcriptional output of

Notch signaling. We identified E(y)1/TAF9 in a large-scale in vivo

RNA interference (RNAi) screen for genes that are involved in a

Notch-dependent mitotic-to-endocycle transition in Drosophila

follicle cells. Knockdown of e(y)1/TAF9 displayed Notch-mutant-

like phenotypes and defects in target gene and activity reporter

expression in both the follicle cells and wing imaginal discs.

Epistatic analyses in these two tissues indicated that E(y)1/TAF9

functions downstream of Notch cleavage. Biochemical studies in S2

cells demonstrated that E(y)1/TAF9 physically interacts with the

transcriptional effectors of Notch signaling Su(H) and NICD. Taken

together, our data suggest that the association of the NICD–Su(H)–

Mastermind complex with E(y)1/TAF9 in response to Notch

activation recruits the transcription initiation complex to induce

Notch target genes, coupling Notch signaling with the transcription

machinery.

KEY WORDS: E(y)1, TAF9, Notch pathway, Drosophila, TFIID

complex, Transcriptional regulation

INTRODUCTIONThe eukaryotic transcription factor IID (TFIID) complex,

comprising the TATA box-binding protein (TBP) and 13 or 14

TBP-associated factors (TAFs), plays an essential role in the

transcriptional regulation of gene expression, and all components

of this complex were generally thought to be required for RNA-

polymerase-II-initiated transcription in all eukaryotic cells

(Goodrich and Tjian, 2010; Thomas and Chiang, 2006).

However, an increasing number of studies suggest that some

TAFs found in different species are vital in specific events, such

as apoptosis, spermatogenesis and adipogenesis (Goodrich and

Tjian, 2010). For example, mouse TAF7L is specifically required

for male germ-cell differentiation (Cheng et al., 2007; Pointud

et al., 2003), and, in both zebrafish and mouse embryonic stem

cells, interaction between TRF3 and TAF3 is essential for

hematopoiesis (Bartfai et al., 2004; Hart et al., 2007; Hart et al.,

2009). Kalogeropoulou et al. have demonstrated that TAF4b is

specifically associated with c-Jun and other AP-1 family

members to regulate the expression of Integrin a6 in the

context of cancer progression (Kalogeropoulou et al., 2010).

Although a recent report has shown that TAF4 interacts with

Pygopus, a transcriptional activator of Wingless (Wg) signaling,

to induce transcription of naked cuticle in Drosophila (Wright

and Tjian, 2009), how other signaling pathways, such as Notch,

are regulated by specific TAFs is poorly understood.

Notch signaling is an evolutionally conserved pathway across

species that plays a pivotal role in many different developmental

events, including cell fate determination, control of cell

proliferation and apoptosis, and the maintenance of stem cells

(Artavanis-Tsakonas and Muskavitch, 2010; Guruharsha et al.,

2012; Tien et al., 2009). Dysregulation of Notch is implicated in a

number of diseases, including cancer (Louvi and Artavanis-

Tsakonas, 2012; Ntziachristos et al., 2014). Notch signaling

activation is mediated by a direct interaction between the Notch

receptor in one cell and its ligand on the neighboring cell (Diaz-

Benjumea and Cohen, 1995; Doherty et al., 1996). Such an

interaction induces two consecutive proteolytic processes that

result in the release of the Notch intracellular domain (NICD)

(Struhl and Adachi, 1998), which is then translocated to the

nucleus and activates transcription of its target genes by

interacting with the DNA-binding protein Suppressor of

Hairless [Su(H)] and the coactivator Mastermind (Mam) to

form a functional Su(H)–NICD–Mam ternary complex (Bailey

and Posakony, 1995; Lecourtois and Schweisguth, 1995).

Although numerous Notch target genes have been identified in

different tissues or organs during different developmental stages,

how the Su(H)–NICD–Mam ternary complex regulates their

expression is largely unclear. Recent findings in different systems

have suggested that chromatin-associated epigenetically-

regulatory mechanisms are very important for proper expression

of Notch target genes (Bray et al., 2005; Domanitskaya and

Schupbach, 2012; Endo et al., 2011; Kugler and Nagel, 2007;

Mulligan et al., 2011; Yu et al., 2013; Zeng et al., 2013). In flies,

histone chaperones Asf1 and Nap1 are differentially associated

with LID-associated factor (LAF) and RPD3-LID-associated

factor (RLAF) silencing complexes to mediate epigenetic

silencing at the Notch target Enhancer of Split [E(spl)] cluster

(Moshkin et al., 2009). A SIRT1–LSD1 co-repressor complex

regulates Notch target gene expression in both mammalian and

Drosophila cultured cells (Mulligan et al., 2011). Our very recent

study has demonstrated that the histone chaperone CAF-1

complex epigenetically and positively regulates Notch target

gene expression in Drosophila (Yu et al., 2013). But the most

basic questions of how the Su(H)–NICD–Mam complex activates

1Department of Biological Science, Florida State University, Tallahassee, FL32304-4295, USA. 2State Key Laboratory of Brain and Cognitive Science, Instituteof Biophysics, the Chinese Academy of Sciences, Datun Road 15, Beijing100101, China.

*Authors for correspondence (rjiao@sun5.ibp.ac.cn; wumin@bio.fsu.edu)

Received 4 April 2014; Accepted 23 June 2014

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 3830–3839 doi:10.1242/jcs.154583

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expression of Notch target genes and which cofactor(s) bring it tothe general transcription machinery are unanswered.

The Drosophila egg chamber is an ideal system to studythe regulation of Notch signaling and its developmentalconsequences (Bastock and St Johnston, 2008; Klusza and Deng,2011). During mid-oogenesis (stages 7–10A), Notch signaling is

activated in entire follicular epithelia by the germline-expressedligand, Delta (Dl) (Deng et al., 2001; Lopez-Schier and StJohnston, 2001). This activation induces the follicle cells from the

mitotic cycle into endoreplication, which is mediated by thesuppression of Cut and activation of Hindsight (Hnt; also known asPebbled) through Notch signaling (Sun and Deng, 2007; Sun and

Deng, 2005). As in other processes, this Notch-dependent cellcycle transition also requires Notch protein cleavage, which isachieved by c-secretase components Presenilin and Nicastrin.

In addition, the nuclear effector of Notch signaling Su(H) isneeded for the switch. The precise timing of Notch signalingduring mid-oogenesis is regulated through multiple mechanisms(Domanitskaya and Schupbach, 2012; Heck et al., 2012; Poulton

et al., 2011). Our recent finding demonstrated that the microRNApathway regulates the temporal pattern of Notch signaling byrepressing Delta-mediated inhibition of Notch in Drosophila

follicle cells (Poulton et al., 2011). Interestingly, transcriptionalcofactors are also involved in precise control of Notch signaling(Domanitskaya and Schupbach, 2012; Heck et al., 2012) – the

transcriptional co-repressor SMRTER inhibits Notch activity in atemporally restricted manner in follicular epithelium (Heck et al.,2012), whereas the transcriptional cofactor Corepressor for

element-1-silencing transcription factor (CoREST) promotesNotch signaling in a spatially restricted manner by affectinghistone H3 K27 tri-methylation and histone H4 K16 acetylation inthe follicle cells (Domanitskaya and Schupbach, 2012). In a genetic

RNA interference (RNAi) screen for regulators of Notch signalingin follicle cells, we identified e(y)1 as being required for properNotch signaling in mediating the mitotic-to-endoreplicating cycle

transition of follicle cells. Further studies showed that e(y)1 is alsorequired for the transcriptional regulation of Notch target genesduring wing development and in cultured S2 Drosophila cells.

Epistatic analyses in both follicle cells and adult wings suggestedthat E(y)1 functions downstream of the Notch cleavage step.Biochemical studies in S2 cells demonstrated that E(y)1 physicallyinteracts with both transcriptional effectors of Notch signaling,

Su(H) and NICD. Taken together, our data reveal that theassociation of the NICD–Su(H)–Mastermind complex with E(y)1in response to Notch activation recruits the transcription initiation

complex to induce Notch target genes, coupling Notch signalingwith the transcription machinery.

RESULTSe(y)1 is required for Notch-signaling-mediated follicle celltransition from mitotic cycle to endocycle duringDrosophila oogenesisTo identify genes that modulate Notch signaling, we performed alarge-scale in vivo RNAi screen for defects in the expression ofNotch target genes in Drosophila melanogaster follicle cells. The

follicle cell epithelium shows a temporal Notch activation patternduring stages 7–10A of oogenesis, with both negative andpositive targets identified (Sun and Deng, 2007; Sun and Deng,

2005). We tested around two thousand Transgenic RNAi Project(TRiP) RNAi lines (Ni et al., 2009; Ni et al., 2008; Ni et al.,2011), which contain either long double-strand (ds) hairpin (Ni

et al., 2009; Ni et al., 2008) or short hairpin RNAs (Ni et al.,

2011) (both referred to as RNAi) under the control of theupstream activating site (UAS). These lines were crossed to the

flip-out Gal4 driver (Ito et al., 1997; Pignoni and Zipursky, 1997)to generate random follicle-cell RNAi clones that were marked bythe expression of green and red fluorescent proteins (GFP andRFP, respectively). The follicle-cell RNAi clones were then

screened for defects in the expression pattern of the negativeNotch target Cut, a homeodomain-containing transcription factor(Sun and Deng, 2005). Cut is normally expressed in early

oogenesis (stages 1–6) and then downregulated at stage 7 uponNotch activation in follicle cells (Sun and Deng, 2005). Failure todownregulate Cut at stage 7 suggests a defect in Notch signaling.

From this screen, we found that knockdown of the gene CG6474

[enhancer of yellow 1 (e(y)1)], which encodes a homolog ofTAF9, a core component of the TFIID transcription initiation

complex (Soldatov et al., 1999; Thomas and Chiang, 2006),resulted in prolonged Cut expression in 74% (n572) of folliclecell RNAi clones at stages 7–8 (Fig. 1A), suggesting a Notchdefect in e(y)1-depleted follicle cells.

To confirm that E(y)1 is required for Notch signaling in folliclecells, we examined the expression of zinc-finger protein Hnt, apositive follicle-cell-specific Notch target, which is normally

expressed in the entire follicular epithelium from stages 7–10A(Sun and Deng, 2007). Downregulation of Hnt in mid-oogenesis

Fig. 1. E(y)1 is required for the Notch signaling pathway in Drosophila

follicle cells. (A,A9) Cut is upregulated in the e(y)1-RNAi follicle cells(expressing RFP) of a stage-7 egg chamber. (B,B9) Expression of Hnt issuppressed in the e(y)1-knockdown follicle cells (expressing RFP) of an eggchamber at stage 7. (C–D9) The expression of two direct reporters of Notchsignaling, E(spl)mb-CD2 and E(spl)m7-lacZ, labeled by the expression oflacZ in red (C) and of GFP in red (D), respectively, is markedly decreased ine(y)1-depleted follicle cells in mid-stage egg chambers. Nuclei were labeledwith DAPI (blue).

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(stages 7–10A) is an indication of disrupted Notch signaling infollicle cells (Sun and Deng, 2007). Indeed, Hnt downregulation

was detected in 68% (n551) of e(y)1-knockdown follicle cells(Fig. 1B). Both Cut upregulation and Hnt downregulation weredetected in an independent e(y)1-RNAi line (no. 6474R-1) from theNational Institute of Genetics (NIG) that was used to knock down

e(y)1 expression in mid-staged egg chambers (supplementarymaterial Fig. S1A; data not shown).

To further exclude potential off-target effects on Notch

signaling caused by e(y)1-RNAi, we set out to analyze thephenotypes of e(y)1 mutants. An allele named e(y)1190 wasgenerated through imprecise excision of a P-element that had

been inserted into the 59-UTR of the e(y)1 locus (Fig. 2A; seeMaterials and Methods for details). This allele was bothhemizygous and homozygous lethal during early larval stages

and could be rescued to adulthood by a duplication[Dp(1;3)DC335 or Dp(1;3)DC336] harboring the e(y)1 genomicsequence (data not shown). Flippase-flippase recognition target(FLP-FRT)-induced follicle cell mitotic clones of e(y)1190

showed upregulated levels of Cut and downregulated levels ofHnt during stages 7–8 when compared with neighboring wild-type cells (Fig. 2B–C0), supporting our findings in e(y)1

knockdown cells.Notch-induced transcription activation can be assessed in a

more direct manner by using transgenic reporters. Both E(spl)mb-

CD2 (de Celis et al., 1998; Furriols and Bray, 2001) andE(spl)m7-lacZ (Assa-Kunik et al., 2007; Pines et al., 2010), which

contain the Su(H) binding sites, are expressed in follicle cellsduring mid-oogenesis upon Notch activation. Indeed, the

expression of these two reporters was substantially reduced ine(y)1-RNAi follicle cells (Fig. 1C,D). In addition, the expressionof another follicle-cell-specific Notch reporter broad-early-

enhancer-lacZ (brE-lacZ), recently identified by our laboratory

(Jia et al., 2014), was also decreased in e(y)1-depleted folliclecells (supplementary material Fig. S1B). These results suggestthat e(y)1 is required for proper transcriptional regulation of the

Notch target genes in follicle cells.A developmental consequence of Notch activation in a follicle

cell during stage 7 is the induction of a switch from the mitotic

divisions of early oogenesis to endoreplication cycles (endocycles)(the M/E transition) during mid-oogenesis (Deng et al., 2001;Lopez-Schier and St Johnston, 2001). To determine whether e(y)1

is required for this Notch-dependent event, we examined nucleisize and expression of mitotic markers in mosaic egg chamberscontaining follicle-cell clones that expressed RNAi against e(y)1.Knockdown of e(y)1 resulted in smaller and more densely

distributed nuclei than those of the neighboring wild-type folliclecells, suggesting a failure in the M/E switch (supplementarymaterial Fig. S2A9). To determine whether e(y)1-knockdown

follicle cells remained in the mitotic cycle, we monitored twomitotic markers – phosphorylated histone H3 and Cyclin B (CycB)– by staining the mosaic egg chambers. In wild-type egg chambers,

phosphorylated histone H3 and CycB oscillate in early mitoticcycles up to stage 6 and are absent during endoreplication stages

Fig. 2. Generation of an e(y)1 null mutantallele, e(y)1190, and characterization of itsNotch defects in follicle cells and adultwings. (A) Genomic organization of the e(y)1

locus, with the p{GT}-BG00948 P-elementinsertion site (white triangle) and the deletionin e(y)1190 (black double arrows). The singleblack arrow denotes the transcription start siteof e(y)1 and its direction. Black bars indicatethe coding regions of e(y)1; white barsindicate 59- and 39-UTRs. Scale bar: 200 bps.(B–C0) Stage-7 egg chambers bearinge(y)1190 homozygous mutant follicle-cellclones labeled by the absence of RFP andoutlined in white (B,C). Egg chambers werestained for Cut (B9) and Hnt (C9), both shownin green. Yellow arrows (B9,B0,C,C0) point topolar cells, in which Cut is normallyexpressed when Hnt is suppressed. Cellnuclei were stained with DAPI (blue)(B0,C0). (B0,C0) Merged images. (D,E) Ane(y)1190 mosaic wing that was induced atlarval stages has a defective wing edge(D), which can be fully restored to a normalwing margin by a duplication line carrying thee(y)1 genomic region (E).

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(stages 7–10A) (Deng et al., 2001; Shcherbata et al., 2004),whereas in e(y)1-knockdown follicle cells, both markers showed

random expression after stage 6 (supplementary material Fig.S2B,C). Taken together, these data indicate that e(y)1 is potentiallyrequired for the Notch-dependent M/E switch in Drosophila

follicle cells.

Disruption of e(y)1 affects the expression of Notch targetgenes in the wing imaginal disc and cultured S2 cellsTo assess whether e(y)1 also modulates Notch signaling in othertissues, we examined the expression of two well-documentedNotch target genes, wg and cut, in the wing imaginal disc. During

wing disc development, the Notch signaling pathway is activatedat the dorsal–ventral boundary by an interaction between theNotch receptor on one cell and its ligand (Delta or Serrate) on the

neighboring cell (Diaz-Benjumea and Cohen, 1995; Dohertyet al., 1996). Because e(y)1 mutant cells in the wing discgenerated by FLP-FRT mosaic analysis displayed very poorviability (supplementary material Fig. S3B,B9), we employed

the RNAi line to study the role of e(y)1 in Notch signalingduring wing development. To circumvent early larval lethalityof engrailed-Gal4 (en-Gal4)-induced e(y)1-RNAi (data not

shown), we used the temporally controlled Gal4-Gal80ts

system (McGuire et al., 2003) to express UAS-e(y)1-RNAi inthe posterior compartment of a wing disc marked by GFP

expression. The control discs did not show detectable changes inWg (Fig. 3I) or Cut expression (Fig. 3J) (compare the GFP-positive posterior region with the GFP-negative anterior region).

However, in en-Gal4, UAS-GFP; tub-Gal80ts.e(y)1-RNAidiscs, levels of Wg or Cut in the GFP-expressing posteriorcompartment were decreased as compared with those in theanterior compartment (Fig. 3K,L). These results demonstrate that

e(y)1 is involved in the expression of Notch target genes in thewing disc.

To determine whether E(y)1 is necessary for the transcription

of Notch target genes when Notch signaling is artificiallyinduced, we employed a well-documented transient Notchactivation system in cultured S2 cells by transfecting a Notch-

expressing plasmid, pMT-Notch (Krejcı and Bray, 2007; Yuet al., 2013). In this assay, the overexpressed full-length Notchcan be cleaved to produce a constitutively active form of NICD(denoted by NICD) upon treatment with EDTA. NICD translocates

into the nucleus and physically interacts with Su(H), whichactivates the transcription of Notch target genes. In this transientNotch activation system, assuming that E(y)1 is required for

regulating Notch target gene expression, the induced expressionof Notch target genes by pMT-Notch should be compromisedby a perturbation of e(y)1. As expected, the introduction of

interfering dsRNAs against e(y)1 [dse(y)1], but not against GFP(dsGFP), into the Notch-activating S2 cells led to a significantreduction in expression of the primary endogenous Notch target

genes E(spl)m7 and E(spl)m8 (also known as E(spl)m7-HLH andE(spl)m8-HLH, respectively) (Fig. 5D,E). These results suggestthat E(y)1 is also required for artificially induced Notch signalingin cultured S2 cells.

e(y)1 genetically interacts with components of theNotch pathwayTo determine whether e(y)1 is functionally involved in Notchsignaling in the wing imaginal disc, we knocked down e(y)1

through use of a tissue-specific Gal4 driver, C96-Gal4, which is

expressed in the cells of the dorsal–ventral boundary of the wing

Fig. 3. Genetic interaction between e(y)1 and Notch in wingdevelopment. (A) A wild-type adult wing with a normal wing margin.(B) Knockdown of e(y)1 under the C96-Gal4 driver caused partial wingmargin loss, and Notch heterozygous flies (N264-39/+) showed very mild wingmargin notching in the distal region (C). Combination of [C96-Gal4.e(y)1-RNAi+N264-39/+], however, resulted in nearly complete wing margin defects(D). An e(y)1 mutant allele [e(y)1190] was generated; see Materials andMethods for details. Trans-heterozygotes of N264/39/e(y)1BG00948 (E) showeda similar phenotype to Notch heterozygotes (C), with the same penetrance(64 out of 132 in E versus 49 out of 101 in C). e(y)1190 heterozygous fliesshowed no visible wing defects (data not shown), whereas introducing onecopy of e(y)1190 into N264-39/+ flies substantially enhanced the wing margindefects of N264-39/+ (F). (G,H) Synergistic enhancement of notching of thewing margin between N1/+ and e(y)1190/+. N1 is a hypomorphic Notch allele,with 12 out of 81 wings showing mild notching when heterozygous(G, compared with 49 out of 101 of N264/39). When a copy of e(y)1190 wasincorporated, the notched wing margin phenotype was visibly enhanced, andpenetrance was remarkably increased to 50 out of 82 (H). (I–L) Knockdownof e(y)1 in wing discs compromised the expression of Notch target genes.Anterior and posterior compartments of wing discs are separated by whitelines; anterior is to the left and posterior to the right. (I,J) Wing discs fromwild-type control flies showed normal expression patterns of Wg (red, I) andof Cut (red, J) in the third-instar larvae. (K,L) Wg level was obviously reducedin the e(y)1-depleted posterior compartment marked by the expression ofGFP (green) (K, compared with internal control level in the anteriorcompartment). The level of Cut was also dramatically decreased in theposterior e(y)1-RNAi area (L). Superscript Ri after a gene denotes RNAiagainst that gene.

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disc, the precursor of the adult wing margin (Gustafson andBoulianne, 1996; Saj et al., 2010). Defective Notch signaling in

the dorsal–ventral boundary leads to loss of wing margins (deCelis et al., 1996). Depletion of e(y)1 under the C96-Gal4 driveralso led to partial wing margin loss in 96.9% (n564) of wings(Fig. 3B), similar to the Notch loss-of-function phenotype. This

notched wing margin phenotype was also observed in mosaicadult flies in which e(y)1190 random clones were induced at larvalstages by heat-shock-induced Flp (hsFLP) (Fig. 2D). Notch

heterozygous animals (N264-39/+) showed a mild wing marginnotching in the distal region in 48.5% (n5101) of wings (Fig. 3C).Combination of C96-Gal4.e(y)1-RNAi and N264-39/+ resulted in a

synergistically enhanced notched wing phenotype in terms of bothpenetrance and expressivity (100%, n532; Fig. 3D). Furthermore,although e(y)1 heterozygous flies [e(y)1190/+] did not show any

visible wing defects (data not shown), incorporation of a copy ofthe e(y)1190 mutation into Notch heterozygotes greatly enhancedthe wing margin notching phenotype in both expressivity (Fig. 3Fcompared with Fig. 3E) and penetrance [increasing from 48.5%

(n5132; Fig. 3E) to 90% (n560; Fig. 3F)]. Similarly, e(y)1190 alsodisplayed a synergistic interaction with another Notch allele, N1

(Fig. 3G,H). In addition to dorsal–ventral boundary formation in

the wing disc, Notch signaling also plays an important role in cellfate determination through lateral inhibition (Artavanis-Tsakonasand Muskavitch, 2010). Indeed, we observed lateral specification

defects in bristle and wing vein development when e(y)1 wasdepleted (supplementary material Fig. S3C–H). These geneticexperiments suggest that e(y)1 functionally interacts with the

Notch pathway during Drosophila wing development.

E(y)1 mediates the transcriptional output of Notch signalingthrough association with NICD and Su(H)The Notch signaling pathway is delicately regulated at multiplelevels, including ligand and receptor post-translationalmodification, endocytosis and vesicle trafficking, and epigeneticregulation of target gene expression (Fischer et al., 2006; Fortini

and Bilder, 2009; Tien et al., 2009; Yu et al., 2013; Zeng et al.,2013). Interaction between Notch ligand and receptor induces twoconsecutive proteolytic cleavages of the Notch receptor, S2 and

S3, which results in the release of NICD (Guruharsha et al.,2012). Translocation of the cleaved NICD to the nucleus activatestranscription of the target genes through formation of a ternary

complex with the DNA-binding protein Su(H) and the coactivatorMam (Guruharsha et al., 2012). e(y)1 putatively encodes a corecomponent of the TFIID transcription initiation complex, which

is required for polymerase-II-mediated gene expression (Dynlachtet al., 1991). Thus, two possibilities for its role in Notch signalingexist – first, E(y)1 is required for the transcriptional expression ofa core component or a regulator of the Notch pathway; second,

E(y)1 is directly engaged in the transcriptional control of Notchtarget genes that are mediated by the core NICD–Su(H)–Mamcomplex. To distinguish between these two possibilities, we

conducted epistatic analyses in follicle cells by expressingtransgenes that encode components acting in different steps ofthe Notch pathway. These transgenic constructs include a

membrane-tethered active form of Notch, NEXT; a constitutivelyactive form of Notch, NICD (Rebay et al., 1993); and a fusionprotein Su(H)–VP16 comprising the DNA-binding domain of

Su(H) and the strong activation domain of viral transcription-factor

Fig. 4. Epistatic analyses of E(y)1 in the Notchpathway in endocycling follicle cells and adultwings. (A–D9) E(y)1 regulates Notch signalingdownstream of Notch proteolytic cleavage in mid-stagefollicle cells. Cell nuclei were stained with DAPI (blue;A,B,C,D). Flip-out clones were marked by theexpression of RFP (red; A,B,C,D) and outlined bywhite lines. Mid-stage egg chambers were stained forCut in green (A–D9). Because a stau:GFP transgenewas recombined in the hsFLP-carrying Xchromosome, punctate green signals were detectedwithin the germ cells. (A,B,C,D) Merged pictures ofthree channels; (A9,B9,C9,D9) Single channel of Cutstaining. (A,A9) Follicle cell clones of e(y)1-RNAi in amid-stage egg chamber showed Cut upregulation.(B–C9) Expression of NEXT (B,B9) or NICD (C,C9) failedto stop Cut upregulation caused by e(y)1 depletion.(D,D9) Induction of the strong Su(H)–VP16 activatorprevented Cut expression in mid-stage e(y)1-RNAifollicle cells. (E–H) Ectopic expression of Su(H)–VP16suppressed the notched wing margin defect caused byC96-Gal4-driven e(y)1 RNAi. (E) C96-Gal4 controladult flies had normal wing margins. (F) A typical lossof wing margin in C96-Gal4.e(y)1-RNAi flies. (G) Co-expression of NICD under the same C96-Gal4 driverdid not suppress wing margin defects caused by e(y)1

RNAi. (H) Co-expression of Su(H)–VP16 substantiallysuppressed the wing margin loss resulting from C96-

Gal4.e(y)1-RNAi. All wings were from male flies.Superscript Ri after a gene denotes RNAi againstthat gene.

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protein VP16 (Kidd et al., 1998; Shyu et al., 2009). NEXT isproduced after S2 cleavage and NICD after S3 cleavage. Because

the majority of Notch regulators and core components functionupstream of either S2 cleavage or S3 cleavage, if E(y)1 is requiredfor the expression of one of the necessary proteins, expression ofNEXT and/or NICD would rescue the defects caused by e(y)1

depletion. However, if E(y)1 is directly involved in the expressionof Notch target genes, NEXT and/or NICD would have no effect, butSu(H)–VP16 might activate Notch target gene expression in the

absence of e(y)1, because Su(H)–VP16 is a more potenttranscription activator than Su(H) and can activate a Notch targetindependently of Notch (Kidd et al., 1998). Because, in addition,

the VP16 activation domain can directly interact with multiplecomponents of different general transcription factor complexes(Hall and Struhl, 2002), we speculate that expression of certain

Notch targets by Su(H)–VP16 might bypass E(y)1. Expression ofthese constructs can therefore distinguish whether the involvementof E(y)1 is at the level of target gene expression or componentexpression. Cut upregulation in e(y)1-knockdown follicle cells was

employed as a readout in this epistatic analysis. During stages 7–8of oogenesis, 76% (n566) of e(y)1-RNAi follicle cell clonesshowed Cut upregulation (Fig. 4A). Similar percentages of Cut

upregulation were detected when NEXT (79%, n546; Fig. 4B) orNICD (70%, n582; Fig. 4C) was co-expressed with e(y)1-RNAi.By contrast, Cut upregulation resulting from e(y)1-RNAi was

dramatically reduced by co-expression of Su(H)–VP16 (24%,n557; Fig. 4D). These results suggest that the involvement ofE(y)1 in Notch signaling is probably not related to the expression

of Notch regulators or core components upstream of the release ofNICD. We employed similar epistatic analysis for the function of

E(y)1 in the adult wing. As seen in Fig. 3B, C96-Gal4.e(y)1-RNAi adults had notched wing margins (Fig. 4F). Although co-

expression of NICD did not alleviate this defect (Fig. 4G), C96-

Gal4.e(y)1-RNAi animals expressing Su(H)–VP16 showedgreatly alleviated wing margin defects (Fig. 4H). C96-

Gal4.Su(H)–VP16 alone displayed typical Notch gain-of-

function phenotypes during Drosophila wing development(supplementary material Fig. S4). Taken together, these epistaticresults in both follicle cells and developing wings suggest that

E(y)1 acts downstream of NICD and is engaged in transcriptionalactivation of Notch target genes.

Previous studies have shown that specific transcription factors

can directly interact with specific TAFs to recruit the transcriptionalmachinery to their target genes (Kalogeropoulou et al., 2010;Wright and Tjian, 2009). Su(H) is the executive core transcription

factor of Notch signaling. Genetic analyses encouraged us tohypothesize that Su(H) physically interacts with E(y)1 to recruit thetranscriptional machinery to initiate Notch target gene expression.To test this hypothesis, we turned to the Drosophila S2 cell system

again. We co-transfected two constructs to express Flag-taggedE(y)1 and Myc-tagged Su(H), respectively, both under the controlof the Actin5C promoter in S2 cells. As expected, Flag-tagged

E(y)1 was able to immunoprecipitate Myc-tagged Su(H) (Fig. 5A).The NICD–Su(H)–Mam ternary complex has been reported toregulate Notch target gene activation in many organisms, and we

therefore asked whether E(y)1 is also physically associated withNICD to promote Notch activation. Indeed, the association betweenE(y)1 and NICD was detected in a co-immunoprecipitation assay

(Fig. 5B). Thus, E(y)1 mediates the transcriptional output of Notchsignaling by interacting with NICD and Su(H) biochemically.

Fig. 5. E(y)1 is physically associated with NICD andSu(H) and required for Notch target gene expressionin S2 cells. (A) Flag-tagged E(y)1 interacts with Myc-tagged Su(H) in Drosophila S2 cells. (B) Myc-taggedNICD is associated with Flag-tagged E(y)1 in S2 cells.Flag-tagged E(y)1 and Myc-tagged Su(H) (A), and Myc-tagged NICD and Flag-tagged E(y)1 (B), were co-transfected into cultured S2 cells. Total cellular extractswere prepared for the co-immunoprecipitation assaywith IgG (control) or the indicated antibodies. Antibodiesused for immunoprecipitation (IP) in A,B are indicated atthe top, and the proteins detected by western blottingafter immunoprecipitation are indicated to the left. Inputlanes represent 5% of the S2 cell extracts that wereused for immunoprecipitation. (C) E(y)1 protein levelsare greatly reduced by treatment with dse(y)1 but notdsGFP in S2 cells, whereas NICD and Tubulin proteinlevels were unchanged by treatment with dse(y)1.(D,E) Expression of two Notch target genes, E(spl)m7

(D) and E(spl)m8 (E), strongly induced by transfectedMyc-tagged NICD in S2 cells, was significantlysuppressed by e(y)1 knockdown. The basal expressionof E(spl)m7 and E(spl)m8 is very low, indicated bytransfection of the pAc5.1 empty vector, shown in theleft side of each chart, and e(y)1-RNAi manipulation hadlittle effect on their expression. E(spl)m7 and E(spl)m8

levels were increased 39-fold (D) and fivefold (E),respectively, when Notch signaling is induced bytransfection of pAc5.1-Myc-NICD in normal S2 cells.Knockdown of e(y)1 significantly suppressed thisactivation induced by the expression of NICD.***P,0.001.

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Requirement of other TAFs in Notch signalingThe results described thus far indicate that E(y)1 is required for theNICD–Su(H)–Mam-complex-mediated transcriptional program of

Notch signaling. E(y)1, the Drosophila homolog of human TAF9,was initially identified as one of the core components of the TFIIDcomplex (Dynlacht et al., 1991; Goodrich et al., 1993). To determinewhether E(y)1 is a specific factor of the TFIID complex for Notch

signaling and/or whether all components of the TFIID complex arerequired for Notch target gene activation, we examined Notchactivity during Drosophila wing development after depleting other

components of the TFIID complex through RNAi obtained from theTRiP. Knockdown of TAF12 by the en-Gal4 driver did not obviouslyaffect Notch activity, as revealed by the expression of Wg (Fig. 6A),

and consistently, the adult wings of C96- Gal4.TAF12-RNAianimals had normal wing margins (Fig. 6D). However, ablation ofTAF1 under the en-Gal4 driver visibly decreased the level of Wg in

the posterior compartment of the wing disc (Fig. 6B), andexpectedly, overexpression of TAF1-RNAi induced by C96-Gal4

caused notched wing margins in adult flies (Fig. 6E). Furthermore,we tested several RNAi lines corresponding to specific components

of the TFIID complex (TAF2, TAF5 and TAF6) available at theVienna Drosophila RNAi Center under the same C96-Gal4 driver,and all of them showed notched wing margin defects with different

expressivity (data not shown). These results suggest that allcomponents of the TFIID complex we tested, except TAF12, arerequired for Notch signaling, at least during Drosophila wing

development. The e(y)1, e(y)2 and e(y)3 genes were geneticallyidentified as the respective mutations enhance the phenotype of they2 mutation (Georgiev and Gerasimova, 1989; Georgiev et al.,1990), but only E(y)1 is believed to be a component of the TFIID

complex. To investigate whether e(y)2 or e(y)3 plays a similar role inNotch signaling as that of e(y)1, we compromised e(y)2 function byexpression of e(y)2-RNAi and found that it was not required for

Notch activity during wing development (Fig. 6C,F), implying thatunlike e(y)2, e(y)1 is specifically required for Notch signaling.

DISCUSSIONThe Notch signaling pathway has been well documented in avariety of tissues and species, ranging from Drosophila to

humans (Artavanis-Tsakonas and Muskavitch, 2010; Fortini and

Bilder, 2009; Tien et al., 2009). The regulation of this pathwayoccurs at different levels and is especially well-characterized forthe stages before nuclear entry of NICD (Andersson et al., 2011;

Guruharsha et al., 2012; Tien et al., 2009). Recent findings fromour laboratory and others have shown that Notch signaling can beregulated epigenetically by the CAF1 chromatin chaperonecomplex or by the SWI/SNF chromatin remodeling complex in

different tissues (Yu et al., 2013; Zeng et al., 2013). Our studiesaddress how the Su(H)–NICD–Mam transcriptional complex ofthe Notch pathway interacts with the general transcription factor

complexes to initiate expression of Notch target genes. Theresults suggest that E(y)1/TAF9, a component of the TAFIIDtranscription initiation complex, directly interacts with Su(H)–

NICD–Mam complex and recruits the TFIID complex to mediatethe transcriptional activation of Notch signaling in Drosophila.

Drosophila e(y)1 was initially phenotypically characterized by

enhancing the phenotype of the y2 allele when mutated (Georgievet al., 1990). A following study demonstrated that e(y)1 is highlyexpressed in follicle cells and oocytes during Drosophila oogenesisand that decreased e(y)1 transcription causes dramatic

underdevelopment of the ovaries and sterility of female flies(Soldatov et al., 1999). Consistent with this, our previous studiesindicate that the effect of e(y)1 loss in follicle cells is stronger than

that in the wing disc, suggesting that the intensity of e(y)1

involvement in Notch signaling is probably context dependent. Aprotein–protein interaction assay has revealed direct binding

between E(y)1/TAF9 and the activation domains of VP16 andp53 (Goodrich et al., 1993; Thut et al., 1995). The human homologof E(y)1 TAF9 has also been identified as a crucial protein that isrequired for p53- and VP16-dependent activation of transcription

(Uesugi et al., 1997). A recent report has shown that TAF4interacts with Pygopus, a transcriptional activator of Wg signaling,to induce transcription of naked cuticle (Wright and Tjian, 2009).

Our study suggests that TAF9 mediates the transcriptional outputof Notch signaling. It is very likely that, for a given signalingpathway, a specific TAF in the TFIID complex is required for

transcriptional output of this pathway, which is mediated throughdirect interaction between the TAF and a transcriptional effector.

Genome-wide transcriptome studies in different cell types from a

variety of species have revealed a considerable diversity in the

Fig. 6. Effects of depleting e(y)2 or other TAF-encoding genes on Notch signaling. (A–C) Confocalimages of wandering third-instar wing discs with en-Gal4

driving GFP expression plus the indicated constructs.Wing discs were stained for Wg (red). GFP (green) marksthe posterior compartment of wing discs in which en-Gal4

is expressed. (D–F) Pictures of adult wings from fliesexpressing the indicated constructs induced by the C96-

Gal4 driver. Superscript Ri after a gene denotes RNAiagainst that gene.

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expression of Notch-induced target genes (Aoyagi-Ikeda et al.,2011; Chadwick et al., 2009; Krejcı et al., 2009; Meier-Stiegen

et al., 2010; Weerkamp et al., 2006), which might explain thefunction of Notch signaling in so many different cellular contexts.However, the basis for this transcriptome diversity is only partiallyunderstood. The conventional view is that Su(H) is statically

associated with its target enhancers and that it repressestranscription when Notch is not activated (Andersson et al.,2011). Upon Notch activation, NICD, together with Mam, then

displaces co-repressors and brings co-activators to the Su(H)–NICD–Mam complex, which leads to transcriptional activation oftarget genes, but a systemic analysis of Su(H) binding at 11 E(spl)

Notch target genes has revealed that in Drosophila, Su(H)occupancy at target loci is largely different before and after Notchactivation (Krejcı and Bray, 2007). Prevailing models imply that the

general TFIID complex binds to core promoters even in the absenceof a specific transcription activator (Goodrich and Tjian, 2010). Ourbiochemical study shows that E(y)1/TAF9 can interact with bothNICD and Su(H). We propose that, after Notch-activation-induced

cleavage, the TFIID complex mediates the change of Su(H)occupancy on Notch target gene enhancers through the associationbetween E(y)1/TAF9 and the Su(H)–NICD–Mam ternary complex,

leading to transcriptional activation of Notch target genes.Many Notch target genes have been found in different tissues,

and their activation by Notch signaling is controlled in a

spatiotemporal manner (Borggrefe and Oswald, 2009). In ourstudies, although many TAFs of the TFIID complex that we testedwere required for Notch signaling during Drosophila wing

development, TAF12 seemed to be dispensable for Notchsignaling during wing margin formation, consistent with studiesshowing that some TAFs are not required for activation of certaingenes in specific tissues (Goodrich and Tjian, 2010). In addition, the

effect of e(y)1 loss in the wing disc appears to be weaker than that infollicle cells, suggesting that the intensity of e(y)1 involvement inNotch signaling is probably context dependent. Whether E(y)1 is

required for Notch target gene expression in all tissues remainsunclear. It is plausible that certain TAFs are involved in regulatingthe output and intensity of the expression of Notch target genes in a

spatial- and temporal-specific manner. Our study suggests that,upon Notch signaling activation, Su(H) is released from the Su(H)–Hairless complex to form a transcriptional activator complex withNICD and Mam and this Su(H)–NICD–Mam complex is then

recruited by E(y)1/TAF9 to the TFIID transcription initiationcomplex to activate expression of Notch target genes.

MATERIALS AND METHODSGeneration and characterization of the e(y)1190 alleleFly P element w1118 P{GT1}e(y)1BG00948 was inserted 78 base pairs (bps)

upstream of the start codon of the e(y)1 gene. Isogenized P-element flies

that are both homozygous and hemizygous viable and fertile were used

for generation of the e(y)1190 allele by a standard P-element-mediated

imprecise jump-out strategy. Excisions of P{GT1}e(y)1BG00948 that were

hemizygous lethal were sequenced by the use of a pair of primers (59-

CATAAGCTCACCGATTTC-39 and 59-CCTCCATCTTGAGATCTC-

39) that flanked the genomic region of the P{GT1}e(y)1BG00948

insertion site. The e(y)1190 mutation is a deletion of 339 bps including

part of the 59-UTR, exon 1, intron 1 and part of exon 2 of the e(y)1 locus

(see Fig. 2A).

Fly stocks and geneticsFor clonal analyses, the mutation of e(y)1190 was recombined with

hsFLP122 and FRT19A on the X chromosome. Two independent UAS-

e(y)1-RNAi lines were used to knock down e(y)1 levels in this paper –

e(y)1TRiP#HMS00336 from the Bloomington Drosophila Stock Center and

e(y)16474R-1 from the National Institute of Genetics, Japan. To induce

random clones in follicle cells, the flip-out Gal4 driver [hsFLP;

act.CD2 (or y+).Gal4] was crossed to flies carrying an RNAi or an

overexpression construct under the control of the UAS promoter or

combination of interest. Their adult progeny were heat-shocked twice at

37 C for 30 minutes and then cultured in a wet-yeast-pasted vial at 29 C

for 2–3 days before dissection. w ubi-GFP M(1)osp FRT19A was a gift

from Richard S. Mann (Estella and Mann, 2010). Other fly stocks and

genotypes related to the figures listed are listed in supplementary material

Table S1.

ImmunocytochemistryAntibody staining was performed as previously described (Deng et al.,

2001). Antibodies from the Developmental Studies Hybridoma Bank

(University of Iowa, Iowa City, IA) were against Cut (1:20), CycB (1:10),

Wg (1:20) and Hnt (1:15). Other antibodies used were against

phosphorylated histone H3 (rabbit, 1:400; Upstate), CD2 (mouse, 1:50;

Serotec) and b-Galactosidase (rabbit, 1:2000; Sigma). Nuclei were co-

stained with DAPI (1:1000, Invitrogen) for 15 minutes at room

temperature. Images were captured on a Zeiss LSM-510 confocal

microscope in the Biological Science Imaging Resource Facility at

Florida State University. Figures were processed and arranged in Adobe

Illustrator.

S2 cell culture, transfection and RNAi assaysDrosophila S2 cells were cultured at room temperature in Hyclone

serum-free insect cell culture media (Roche). Transfection of S2 cells

was performed using FuGENE HD transfection kit reagents (Roche)

following the manufacturer’s instructions. The constructs used for

transfections are available upon request. The S2 cells were normally

harvested 48 hours after transfection for further experiments. Full-length

Notch expression was induced by using 500 mM CuSO4 for 24 hours

after transfection with pMT-Notch (Fehon et al., 1990). dsRNA was

prepared with the RiboMAX large-scale RNA production system-T7 kit

(Promega) as previously described (Huang et al., 2011). Primers used

were – e(y)1 forward 59-TTAATACGACTCACTATAGGGGAGA-

TCATGTCCATCCTGAAGGAG-39 and e(y)1 reverse 59-TTAATACGA-

CTCACTATAGGGGAGACGCTAGTTGGTCACAAACTC-39; control

GFP forward 59-TTAATACGACTCACTATAGGGGAGAATGGTG-

AGCAAGGGCGAGGAGCTG-39 and GFP reverse 59-TTAATACGAC-

TCACTATAGGGGAGACTTGTACAGCTCGTCCATGCCGAGAG-39

(bases in bold indicate T7 promoter sequences). For a 6-well plate, cells in

2 ml of medium in each well were treated with 15 mg dsRNA for 4 days

before plasmid transfection or induction with CuSO4.

Co-immunoprecipitation assayTotal protein extracts from S2 cells were prepared in immunoprecipitation

lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA

pH 7.4, 1% Triton X-100, 0.1% SDS) in the presence of protease inhibitors

[1 mM PMSF; protease inhibitor cocktail (Calbiochem)]. For co-

immunoprecipitation assays, extracts were incubated with specific

antibodies and protein A/G agarose beads (Abmart) at 4 C overnight

before washes and elution. Immunoprecipitates were boiled in 26SDS

loading buffer for elution from the beads. Rabbit anti-Flag (1:100, Sigma)

and rabbit anti-Myc (1:200, Sigma) antibodies were used for co-

immunoprecipitation experiments; mouse anti-E(y)1 (1:1000, Sigma),

rabbit anti-Flag (1:1000, Sigma), rabbit anti-Myc (1:2000, Sigma) and

mouse anti-Tubulin (1:2000, Sigma) antibodies were used for western blots.

AcknowledgementsWe thank Trudi Schupbach (Princeton University, Princeton, NJ), Richard S.Mann (Columbia University, New York, NY), the Bloomington Drosophila StockCenter, the Vienna Drosophila RNAi Center, the National Institute of Genetics andthe TRiP at Harvard Medical School for fly stocks; and the Developmental StudiesHybridoma Bank for antibodies. We also thank Thomas J. Fellers from theBiological Science Imaging Resource of Florida State University for technicalsupport; Jen Kennedy, William Palmer and Gabriel Calvin (Florida StateUniversity, Tallahassee, FL) for critical reading and helpful comments on themanuscript; Zhiqiang Shu (Florida State University, Tallahassee, FL) for assisting

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screen for e(y)1 mutant alleles; and members of the Deng and Jiao laboratoriesfor feedback and suggestions.

Competing interestsThe authors declare no competing interests.

Author contributionsW.-M.D., G.X. and R.J. conceived and designed the experiments. G.X., Z.Y. andD.J. performed the experiments. G.X., Z.Y., R.J. and W.-M.D. analyzed the data.G.X., W.-M.D. and R.J. prepared and wrote the paper with input from Z.Y. and D.J.

FundingThis work was supported by grants from the National Natural Science Foundationof China [grant number 31271573 to R.J. and 31228015 to W.-M.D.]; and a grantfrom the 973 program [grant number 2012CB825504 to R.J.]. W.-M.D. issupported by the National Science Foundation [grant number IOS-1052333]; andthe National Institutes of Health [grant number R01 GM072562]. Deposited inPMC for release after 12 months.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.154583/-/DC1

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RESEARCH ARTICLE Journal of Cell Science (2014) 127, 3830–3839 doi:10.1242/jcs.154583

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