RESEARCH ARTICLE Open Access Modifiers of notch ... · RESEARCH ARTICLE Open Access Modifiers of notch transcriptional activity identified by genome-wide RNAi Philippos Mourikis2†,

RESEARCH ARTICLE Open Access

Modifiers of notch transcriptional activityidentified by genome-wide RNAiPhilippos Mourikis2dagger Robert J Lake3dagger Christopher B Firnhaber1 Brian S DeDecker1

Abstract

Background The Notch signaling pathway regulates a diverse array of developmental processes and aberrantNotch signaling can lead to diseases including cancer To obtain a more comprehensive understanding of thegenetic network that integrates into Notch signaling we performed a genome-wide RNAi screen in Drosophila cellculture to identify genes that modify Notch-dependent transcription

Results Employing complementary data analyses we found 399 putative modifiers 189 promoting and 210antagonizing Notch activated transcription These modifiers included several known Notch interactors validatingthe robustness of the assay Many novel modifiers were also identified covering a range of cellular localizationsfrom the extracellular matrix to the nucleus as well as a large number of proteins with unknown functionChromatin-modifying proteins represent a major class of genes identified including histone deacetylase anddemethylase complex components and other chromatin modifying remodeling and replacement factors Aprotein-protein interaction map of the Notch-dependent transcription modifiers revealed that a large number ofthe identified proteins interact physically with these core chromatin components

Conclusions The genome-wide RNAi screen identified many genes that can modulate Notch transcriptionaloutput A protein interaction map of the identified genes highlighted a network of chromatin-modifying enzymesand remodelers that regulate Notch transcription Our results open new avenues to explore the mechanisms ofNotch signal regulation and the integration of this pathway into diverse cellular processes

BackgroundThe Notch (N) cell-surface receptor is the central ele-ment of one of the handful of signaling pathways thatare evolutionary conserved throughout metazoansNotch signaling directs the development of multicellularorganisms through membrane-anchored interactionsbetween adjacent cells The response to Notch signalsvaries greatly and can result in diverse biological conse-quences such as cell proliferation differentiation orapoptosis Notch signaling is initiated by the binding ofthe transmembrane Notch receptor with one of itsligands Delta or Serrate expressed on the surface of aneighboring cell [1] The receptor-ligand interactioninduces a series of proteolytic events that releases theNotch intracellular domain (Nicd) which then

translocates to the nucleus and complexes with tran-scription factors and co-activators to regulate targetgene expression Nicd together with Suppressor of Hair-less [Su(H)] a DNA binding protein in the CSL (CBF1Su(H)Lag2) family and mastermind (mam) proteinsform the core transcriptional complex of the signalingpathway with the Enhancer of Split [E(spl)] locus beingthe most thoroughly characterized downstream tran-scriptional target The Notch signaling pathway ismodulated at many levels Notch protein abundancetrafficking and post-translational processing as well asthe regulated formation of repressive and promotingcomplexes on the DNA The final cell fate outcome is acomplex interplay between all the cellular factors thatregulate Notch activityWe designed a genome-wide RNA interference (RNAi)

screen using a Drosophila cell culture-based systemaimed to identify novel proteins that directly influencethe signaling capacity of the core Notch pathway Thisgenome-wide RNAi screen was performed on

Correspondence dedeckercoloradoedudagger Contributed equally1Department of Molecular Cellular and Developmental Biology University ofColorado Boulder CO 80309 USAFull list of author information is available at the end of the article

Mourikis et al BMC Developmental Biology 2010 10107httpwwwbiomedcentralcom1471-213X10107

copy 2010 Mourikis et al licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (httpcreativecommonsorglicensesby20) which permits unrestricted use distribution and reproduction inany medium provided the original work is properly cited

Drosophila Kc167 cell cultures that were transfectedwith a construct that expresses a constitutively activemembrane-tethered form of the Notch receptor NΔecn[2] Notch pathway activity was monitored by measuringthe transcriptional response of a luciferase-reporter genecassette (m3-luc) containing the native promoter ele-ment of the E(spl)m3 gene [3] the most Notch respon-sive E(spl) target in cell culture [4]The results of our study reveal the identity of proteins

that influence the signaling output of the core Notchpathway Employing complementary data analyses wefound 399 putative modifiers - 189 promoting and 210antagonizing Notch signaling These included severalknown Notch interactors validating the robustness of theassay and our experimental approach Molecules residingin the extracellular matrix (2) the plasma membrane(3) the cytosol (16) and the nucleus (26) as well asa large number of proteins with unknown function andlocalization (53) were also recovered (Table 1)To further analyze and categorize our dataset the

Notch signaling modifiers identified in the study werecombined with physical interaction data from publicdatabases The interaction map that was generatedrevealed classes of interacting Notch modifiers such asmRNA processing and ribosomal proteins The networkanalysis also highlighted a central core of chromatin reg-ulating genes including chromatin modifying enzymesand remodelers that interact directly with the Su(H)DNA binding complex

Results and DiscussionDevelopment of a robust assay to measure changes inNotch transcriptional activityA reporter assay was developed to measure Notch activ-ity in a high-throughput Drosophila cell-based approachThe assay consists of three components 1) a Notchactivity reporter construct with two tandem copies ofthe E(spl)m3 promoter positioned upstream of the fireflyluciferase gene (m3-luc) [3] 2) the constitutively activemembrane-tethered form of the Notch receptor with theextracellular domain removed (NΔecn) driven by theviral OpIE2 promoter 3) a control construct that

constitutively expresses firefly luciferase also driven bythe viral OpIE2 promoter (con-luc) Con-luc was used tonormalize signal intensity relative to transfection effi-ciency cell density and viability and general effects onOpIE2-mediated transcriptionTo test the sensitivity and specificity of the Notch

activity assay a series of experiments were performed incells treated with interfering RNA targeting known com-ponents of the Notch signaling pathway Cells wereincubated with dsRNA against mastermind (mam)Hairless (H) and the major downstream co-transcriptionfactor Suppressor of Hairless (Su(H)) and then split andtransfected for three assays N-induced (NΔecn gtm3-luc) luciferase expression levels were measured relativeto either con-luc (Figure 1A) or uninduced E(spl)m3promoter (m3-luc) (Figure 1B) Uninduced promoterlevels were also tested by normalizing m3-luc measure-ments with corresponding con-luc signals (Figure 1C)As predicted we found that targeting Su(H) and mam

with RNAi in cells expressing activated Notch resultedin a sharp reduction of the reporter luciferase activity(Figure 1A and 1B) Conversely knock-down of Su(H)increased the basal activity of the m3-luc reporter in theabsence of NΔecn (Figure 1C) These opposing effectsof Su(H) RNAi on E(spl)m3 expression are consistentwith the dual roles of Su(H) as a transcriptional repres-sor in the absence of Notch activation as well as a tran-scriptional activator when complexed with processedNicd in the nucleus [4] In contrast RNAi against Hair-less resulted in a marked decrease in the ratio ofinduceduninduced signal of m3-luc (Figure 1B) Thisdecrease is expected due to the specific de-repression ofthe uninduced Notch target promoter when H isknocked down (Figure 1C) and shows that there isrobust Su(H)H complex repressor activity in the unin-duced Kc167 cells The different ratios for H RNAitreatment obtained by the two different normalizationmethods (Figures 1A and 1B) highlights the additionalmechanistic information that can be deduced when nor-malizing by the uninduced E(spl)m3 promoter activityHairless acts as a repressor in the uninduced cells buthas no apparent role in Notch activated cells

Table 1 Cellular distribution of Notch modifiers selected by the complimentary analysis methods

Analysis Method Total Extracellular Membrane Cytosolic Nuclear Unknown

NΔecn gt m3-luccon-luc (Activators) 153 8 (52) 2 (26) 28 (183) 40 (261) 73 (477)

NΔecn gt m3-luccon-luc (Repressors) 130 0 5 (38) 17 (131) 27 (208) 81 (623)

NΔecn gt m3-lucm3-luc (Activators) 75 0 1 (13) 12 (160) 28 (373) 34 (453)

NΔecn gt m3-lucm3-luc (Repressors) 74 0 2 (27) 13 (176) 5 (203) 44 (595)

Total (discounting duplicates) 399 2 3 16 26 53

The majority of the proteins are of unknown cellular localization andor function Of the annotated proteins most are predicted to reside in the nucleus followedby cytosolic proteins with a small percentage found in the plasma membrane and extracellular matrix

Splitting the cells into three different assays alsoallows the uninduced Notch target promoter measure-ment to be used as an alternative and specific controlfor Notch induced activity This additional control flagsdsRNA treatments that may specifically affect transcrip-tion of the viral OpIE2 promoter RNAi treatment maymodulate either the signal of interest andor the controlsignal and the resulting ratios may be altered indistin-guishably between these possibilities Whereas this sec-ond control will sort a subset of these dsRNAs asdefinitively altering Notch target transcription (positiveby both normalization methods)The Notch activity assay responded in a predictable

and specific manner to RNAi of known Notch signalingcomponents and these data establish our experimentalset-up (robotic cell transfer normalization methodsincubation time) as optimal for detecting changes inNotch transcriptional activities

Genome-wide RNAi screen and data analysisThe RNAi screen was performed using a dsRNA libraryfrom the Drosophila RNAi Screening Center (DRSC)containing a total of 23560 dsRNAs targeting knownand predicted gene products After four days of RNAitreatment cells were uniformly dispensed by roboticliquid handling into microplates containing the differenttransfection mixes (con-luc m3-luc and NΔecn gtm3-luc) Each assay was performed in duplicate and fireflyluciferase activity was measured 24 h after transfectionFor data analysis we eliminated all wells containing

dsRNA with more than one off-target as predicted bythe Drosophila RNAi Screening Center (DRSC) Of thedsRNA in the final hit lists 12 contained a single pos-sible predicted off-target and are noted in the datatables Data from the screen were analyzed by the twocomplementary methods described above (see Figures1A and 1B) Prospective hits were selected as dsRNAs

Figure 1 Validation of the Notch activity reporter and application for high throughput RNAi Validation of the m3-luc reporter by RNAitargeting of known Notch pathway components Notch induced E(spl)m3 signal normalized against A the control viral promoter orB uninduced E(spl)m3 promoter transcription C Uninduced E(spl)m3 promoter expression normalized by control promoter For each dsRNA 32independent wells were measured in a 384-well plate format Each box encloses 50 of the data with the median value displayed The errorbars mark the full range excluding the shown outliers D Schematic of the automated high throughput screen in 384-well plates DrosophilaKc167 cells were incubated with a unique dsRNA per well After a four day incubation the cells were split into three different transfection mixesin duplicate Firefly luciferase signals were read 24 h after transfection

that significantly perturbed the Notch induced signal(NΔecn gtm3-luc) normalized by the control promoter(con-luc) resulting in 153 hits with significantly low and130 with significantly high signals respectively (Figure2A and Additional file 1) A complementary set of hitswere selected with signals from Notch induced reporter(NΔecn gtm3-luc) normalized by the uninduced promo-ter (m3-luc) resulting in 74 hits with significantly lowand 75 hits with significantly high signals (Figure 2Band Additional file 2) Analyzing the data by these twomethods provided a full spectrum of Notch signalingeffectors that could be further categorized by theirrespective activities Hits that scored in both normaliza-tion methods represent the subset of genes that eitheraffect Notch induced transcription specifically or have

opposing effects between induced and uninduced tran-scription such as Su(H) (Figure 2C area a) Hits thatscored only for Notch induced signal (NΔecn gtm3-luc)normalized by the viral promoter (con-luc) primarilyselected for genes that affect the Notch induced anduninduced transcription by the same percentage (Figure2C area b) The histone deacetylase Rpd3 and theBrahma complex subunit Bap55 fell into this category(Additional file 1A) Hits that scored only for Notchinduced signal (NΔecn gtm3-luc) normalized by theuninduced E(spl)m3 promoter (m3-luc) represent genesthat primarily affect uninduced reporter transcriptionsuch as the repressor complex component Hairless andthe Brahma complex chromatin remodeling factormoira (Figure 2C area c)

Figure 2 RNAi data analysis overview Histograms of targeted genes binned by standard deviations from the mean (z-scores) A Histogram ofz-scores for Notch-induced E(spl)m3 reporter (NΔecn gtm3-luc) normalized by the signal from the control reporter (con-luc) (Additional file 1)B Signals of Notch-induced E(spl)m3 reporter (NΔecn gtm3-luc) normalized by the signal from the non-induced E(spl)m3 reporter (Additional file2) Cutoffs for genes selected are highlighted in grey for both A and B C Plot of the z-scores from histogram 2A on the x-axis and histogram 2Bon the y-axis Regions outside the red box are listed as potential hits and the overlaps between the two normalization methods are shaded inblue (area a) Area a represents the subset of genes that either affect Notch induced transcription specifically or have opposing effects oninduced and non-induced reporter transcription (eg Su(H)) RNAi for this overlapping set was redesigned and retested (Additional file 5) Area brepresents genes that affect both Notch induced and non-induced transcription by similar percent amounts (eg heph and Bap55) Area crepresents genes that primarily affect non-induced reporter transcription specifically (eg H RpL19 and Bap170) Red andor boxed genes haveknown genetic interactions with Notch Blue are chromatin components Yellow are mRNA processing factors and Green are ribosomalcomponents (Minute class)

Classification of the identified proteinsClassification of modifiers identified in the screen wasbased upon gene ontologies (GO terms) as reported byFlybase [5] These classes are shown as a percentage ofgenes with that GO term and median z-scores of thatclass (Additional file 3) Certain classes showed particu-larly significant z-scores For instance activators ofNotch induced transcription as normalized by the con-trol reporter (con-luc) contained 10 chromatin-associated factors 65 of the hits and 16 transcriptionfactors representing 105 Both these classes have amedian z-score of -29 placing these groups in the top02 of the calculated genome-wide distribution (Addi-tional file 3C) Of the identified genes 90 have predictedand known human orthologs associated with humangenetic disorders (Additional file 4) [6]

Known Notch pathway interactors found by the RNAiscreening methodThirteen genes that have been described to geneticallyinteract with Notch were identified (Figure 2C boxedgene names) Among these the core Notch pathwaytranscription factor Su(H) and the repressor Hairlessfurther validated the screening method We also recov-ered the known negative regulator of Notch signalingSuppressor of deltex [Su(dx)] encoding a cytoplasmicprotein that functions as an E3 ubiquitin ligase that ubi-quitinates membrane-anchored Notch [7] and prickle(pk) encoding a transcription factor known to play arole in E(spl)mδ gene expression [8] Nine other genes(heph al Sos toe Vg Lobe lid Nipped-A and RpL19)were identified that have been shown to geneticallyinteract with Notch signaling but whose mechanisticlevel of integration into the Notch pathway are under-stood to varying degrees [9-17]An in vivo RNAi screen for Notch activity has recently

been published that is based on bristle and wing mor-phology and as a different approach to this transcrip-tional based study the overlap was minimal [18] Of the14 genes listed in the previous study that have knowngenetic interactions with Notch only tramtrack (ttk) iscommon to both screens The direct transcription basedmethod of our study would be expected to be better sui-ted to identify transcription and chromatin factors asindicated by the strong scores of repressor componentsand core chromatin components identified (Additionalfile 3) In contrast the phenotype-based study was moresensitive to membrane trafficking machinery makingthe two studies complimentary

Protein interaction network of Notch transcriptionmodifiersAn interaction network was generated to map physicalinteractions between the Notch transcriptional activity

modifiers identified in the screen and core componentsof the Notch signaling pathway (Figure 3) This interac-tion map was generated by combining physical interac-tion data (eg two-hybrid and Co-IP data) from theDroID [19] and BioGRID [20] databases with theNotch-dependent transcription modifiers identified inthis genome-wide study This map does not include theknown genetic interactions identified between the candi-date genes and caution should be noted as to thepresence of possible false positives in the protein inter-action dataThe importance of chromatin in Notch regulation has

recently become apparent and this transcription-basedscreen was suited to uncover this class of regulators Onaverage chromatin-modifying genes scored relativelyhigh in the data analysis (Additional file 3) The interac-tion map reveals a central core of chromatin modifyingcomponents that have multiple physical connections tothe nuclear elements of the Notch pathway such as Su(H) and H (Figure 3B) Many of these chromatin com-ponents are known to interact genetically and physicallywith the Notch pathway [1421-23]The protein interaction network also shows a number of

protein classes that have no known mechanistic link toNotch transcriptional regulation For these classes of mole-cules (mRNA processing proteins and ribosomal factorsdiscussed later) the network suggests that they may beaffecting Notch signaling through direct interactions withthese core chromatin components (Figure 3D and 3E)

Epistatic analysis of candidate genesThe subset of candidate Notch modifiers that over-lapped between the two normalization methods (28genes) was retested with redesigned dsRNAs (Additionalfile 5) Luciferase reporter activity was assessed in cellsin which Notch had been activated by either the mem-brane tethered NΔecn or the downstream intracellularNicd aiming to discriminate between factors that regu-late Notch processing at the plasma membrane versusfactors that affect Notch signaling downstream in thenucleus (Figures 4 and 5) Of the re-designed dsRNA79 retested by either normalization method 67 re-tested the m3-luc normalized signal and 64 the con-lucnormalized signal Three genes were identified thatexclusively promote the activity of the membrane boundNotch and may function to inhibit the intramembraneproteolysis of the receptor This class includes Patj andtwo genes of unknown function CG7099 and CG17189(Figure 4B Class IV and Figure 5C Class IV) The solu-ble protein Patj has not been shown to modulate Notchactivity directly but is known to associate with thetransmembrane protein Crumbs that in turn is knownto repress Notch activity [24] Crumbs is a central regu-lator of epithelial apical-basal polarity in Drosophila and

has been shown to down regulate g-secretase activityand the membrane proteolysis of Notch [24] Our obser-vation in Kc167 cell culture a non-polarized cell linesuggests that Patj may be modifying Notch signaling notvia influencing the localization of the receptor butinstead by acting in the Crumbs-based complex to downregulate membrane proteolysis of Notch In contrastRNAi against nuclear factors such as Su(H) His33A ampB

Nipped-A ttk and Sin3A (Figures 4 and 5) had similareffects on NΔecn and Nicd induced transcription indi-cating interactions with Notch downstream of the pro-teolytic processing events These results demonstratethat the screening method identified components ofNotch signaling that modulate activities that take placeon the plasma membrane as well as nuclear and chro-matin-based regulation

Figure 3 Protein-protein interaction map of Notch transcription modifiers The Notch interaction network was generated by connectingthe Notch transcription modifiers identified in the genome-wide study with protein-protein interaction links (eg two-hybrid and Co-IP datafrom the DroID database [19]) This resulting network included 126 genes (nodes) with 237 physical interactions (edges) Genetic interactionswere not used for the network and the resulting map was drawn using Cytoscape [51] A These physical links are shown in relation tocomponents of the activated Notch pathway (N and Su(H)) and the Notch repressor complex (Su(H) H CtBP and gro) shown in redB Expanded view of the chromatin factors identified in this study that form the central core of the interaction network (blue) C Ttk is a knowndownstream target of Notch signaling The transcriptional and physical interaction data suggests that this factor may have a positive feedbackrole in Notch induced transcription D Factors with roles in mRNA processing (yellow) The interaction network suggests that these proteins maybe working though the chromatin machinery to modulate Notch transcription E The interaction network suggests the possibility of a similarchromatin based mechanism for the class of ribosomal proteins known as Minute The network file is included with the supplemental data(Additional file 6) and can be viewed in detail using the open source Cytoscape viewer httpwwwcytoscapeorg

Factors involved in chromatin modificationThe transcription-based screening method using anendogenous E(spl)m3 promoter sequence was particu-larly useful for identifying chromatin components Weidentified several chromatin factors previously shown toaffect Notch-dependent transcription A component ofthe SAGA histone acetyltransferase complex Nipped-Awas identified Nipped-A the Drosophila homologue ofyeast Tra1 and mammalian TRAP proteins is a key fac-tor of the SAGA complex It has been shown previouslythat reduced Nipped-A dosage enhances the wingnotching phenotype of both mastermind and Notchmutants [1425] The RNAi treated cell culture datademonstrates that Nipped-A promotes transcription atthe E(spl)m3 promoter both in the presence and absenceof activated Notch (Figures 4 and 5) This shows thatthe result of Nipped-A function is independent ofwhether active Nicd is localized on the target promoterWe also identified several homologues of components

of the Rpd3 histone deacetylase co-repressor complexincluding Sin3a Sds3 (CG14220) a putative ortholog ofSAP130 (Sin3A Associated Protein 130 CG11006) andRpd3 itself (Figures 4 5 and Additional file 1B) Whenthese factors were targeted by RNAi there was anincrease in Notch-induced reporter transcription consis-tent with the role of the Rpd3 complex and histone

deacetylation as a transcriptional inhibitor [26] Conver-sely knocking down Sin3a had the opposite effect onthe uninduced baseline activity of the E(spl)m3 promo-ter (Figure 5C) Thus unlike the histone acetylationcomplex (SAGA) the activity of the deacetylation com-plex (Sin3A) on the E(spl)m3 promoter is dependent onthe presence of activated NotchThe screen identified several components of the chro-

matin remodeling complex Brahma Brm AssociatedProtein 55 (Bap55) Brm Associated Protein 170(Bap170) polybromo and moira (mor) A previous Dro-sophila phenotype based screen has found a geneticinteraction between the Notch ligand Delta and anothercomponent of the Brahma complex brahma (brm) [21]Loss of function brm alleles were found to enhanceDelta mutant phenotypes in eye and bristle development[21] The various Brahma components identified in thisstudy show a complex array of effects on the transcrip-tion of the E(spl)m3 promoter some consistent withpreviously described loss of function brm alleles whileothers opposing RNAi directed against Bap55 and poly-bromo demonstrated a specific reduction in Notchinduced transcription (Figure 2C area b) that is consis-tent with the previously observed role of brm in Notchsignaling during Drosophila development [21] Unex-pected are the Brahma subunits identified that modulate

Figure 4 Analysis of retested genes A Retested genes (from selected set Figure 2C area a) that show significantly reduced signaling whendown regulated by RNAi Signals are shown as (+-) percent deviation from the control RNAi signal Three general classes are shown All threeclasses down regulate both soluble (Nicd) and membrane bound (NΔecn) Notch-induced signal yet have different effects on the E(spl)m3promoter in the absence of active Notch Class I genes have positive Class II neutral and Class III negative effect on the uninduced signalB Selected set of retested genes that show significantly enhanced signaling when down regulated by RNAi Two classes of hits are noted ClassIV is only effective on the membrane bound form of Notch (NΔecn) while class V is effective on both membrane bound and soluble forms(Nicd) All deviations are calculated to be significant by two-tailed t-test with p-values lt 005 from control RNAi treatment (Additional file 5 forfull statistics)

Figure 5 Modulation of Notch transcription for subset of retested genes A The two constitutively active Notch constructs used todetermine epistatic relationships in the pathway NΔecn and Nicd NΔecn is a truncated form of N missing the extracellular domain that isinitially membrane bound NΔecn undergoes constitutive cleavage to form the soluble Nicd that is transported to the nucleus to activatetranscription Su(H) is the canonical Notch pathway transcription factor that represses transcription in the absence of Nicd and is essential for theNicd activated transcription of targets such as E(spl)m3 B Transcriptional response to RNAi treatment of selected retested genes that promoteNotch signaling The E(spl)m3 reporter was induced with either NΔecn or Nicd or left in the uninduced repressed state All three classes downregulate both soluble (Nicd) and membrane bound (NΔecn) Notch-induced signal yet have different effects on the E(spl)m3 promoter in theabsence of active Notch Class I genes have positive Class II neutral and Class III negative effect on the repressed signal C Transcriptionalresponse to RNAi treatment of selected retested genes that repress Notch signaling Two classes of hits are noted Class IV is only effective onthe membrane bound form of Notch (NΔecn) while class V is effective on both membrane bound and soluble forms (Nicd) Error bars representthe standard error of the mean (SEM) Significant deviation from control RNAi treatment calculated by two-tailed t-test with a p-value lt 005(Additional file 5 for full statistics)

transcription from the uninduced E(spl)m3 promoterBap170 and mor The screen reveals that both of thesecomponents specifically mediate transcription from theuninduced E(spl)m3 promoter while Bap170 activatesand mor represses (Figure 2C area c)In addition to chromatin modifying complexes a new

interaction between the histone variant H33 and Notchsignaling is seen RNAi treatment of either genomic copyof the H33 histone variant (H33A and H33B) shows adramatic decrease in Notch activated transcription(Figures 4 and 5) The histone variant H33 has beenshown to be incorporated into the promoters of activelytranscribed genes in a replication independent process tomaintain transcription and its influence on Notch tar-geted transcription remains to be explored [27]A major question that arises from these data is how

specific can the identified chromatin factors be to regu-lating Notch transcription It has recently been notedthat chromatin components are more selective in func-tion than was previously thought Surprisingly there arenow a handful of examples where modulating theexpression of a single target gene can rescue the pheno-type associated with a null mutation in a chromatinremodeling complex component [28] By immunopreci-pitation and mass-spec analysis it has recently beenshown that the Notch repressor complex contains ahost of chromatin modifying components [22] Theseidentified components include Sin3A Rpd3 lid Bap55and moira factors that were also uncovered in thisscreen as modifiers of Notch target transcription Thisrepressor complex has been shown to be recruited toNotch target promoters by Su(H) and this interactionmay provide a mechanism for targeting the activity ofthese chromatin factors to Notch signaling [2223] Thisis consistent with the observation that the genetic inter-actions demonstrated between this repressor complexand Notch were not seen when tested against a host ofother signaling pathways [2223] Control reporter tran-scription levels in this study indicated that targetingthese chromatin genes by dsRNA did not significantlyreduce cell viability and growth over the course of thefive-day RNAi incubation in culture The screen datashows that Notch signaling may be particularly sensitiveto the levels of these chromatin components in the cellwhile the protein interaction network confirms thatmany of these chromatin factors physically interact withSu(H) and Hairless suggesting a mechanism to explainthis observationRegulation of histone position and modification are

known factors that determine the ldquocontext dependentrdquonature of Notch signaling during development Thesefactors differentially interpret the signals received fromthe cell surface by recording an epigenetic history onthe target promoter This transcription-based screen

revealed new chromatin factors that can be further stu-died for their role in Notch-mediated development

mRNA processing factorsThe genome-wide transcription assay revealed two otherclasses of proteins not conventionally associated with tran-scriptional regulation A number of ribosomal componentsand proteins associated with mRNA processing werefound to regulate transcription of the activated Notch tar-get gene (Figures 2 and 3) What is unexpected aboutthese interactions is their relative specificity as was for thechromatin components Again any RNAi treatments inthe genome screen that significantly effected cell viabilityor general transcription were excluded from the analysisIn addition all Notch induced target transcription signalswere subsequently normalized to either the control signalor the uninduced Notch target promoter This analysisdemonstrated that knocking down these components ofthe ribosome and splicing machinery did not significantlyaffect general cell viability and had a relatively specificeffect on Notch target transcriptionA number of mRNA splicing and processing compo-

nents were found to interact with Notch-activated tran-scription (Figure 2A and 2C) As expected theseproteins demonstrated extensive physical interactionswith each other (Figure 3D) Unexpectedly these mRNAmodifying proteins show physical interactions with thecore chromatin components identified in this transcrip-tion based screen (Figure 3D) The polypyrimidine tractbinding proteins Sex lethal (Sxl) and hephaestus (heph)were found to repress and activate Notch promoteractivity respectively in our cell culture assay Heph waspreviously found to interact genetically with Notch sig-naling during wing development [12] Other mRNA pro-cessing components such as the non-sense mediateddecay factors Upf1 Upf2 and Smg1 were found to mod-ulate Notch activated transcription in the analysis(Figures 2 and 3D) These mRNA components may beinteracting indirectly with Notch transcription throughtheir mRNA processing functions - for instance by spe-cifically controlling the mRNA processing of transcriptsfor an essential Notch signaling factor such as Su(H)The network suggests a possible alternate mechanism toexplain the interaction between the identified mRNAprocessing factors and Notch transcription one that ismediated though the chromatin machinery (Figure 3D)In plants components of the nuclear cap-binding

complex (including Cbp20) functionally interact withmicroRNA (miRNA) processing components such asArs2 giving these proteins dual roles in splicing andmiRNA processing [29] The role of Cbp20 in miRNAprocessing was also confirmed in Drosophila and mam-malian systems [3031] The nuclear cap-binding com-plex component Cbp20 was found to mediate Notch

transcription in this study (Figure 2) and demonstratesphysical interactions with the chromatin remodelingcomponent Ssrp (Figure 3D) The interaction networksuggests that the miRNA processing activity of Cbp20may be targeted to Notch signaling through interactionswith the chromatin remodeling machinery

Ribosomal factors and the classical Minute mutationsA complex of ribosomal proteins was identified thatmodulated Notch reporter transcription (Figures 2 and3E) This class of translation factors included the largeribosomal subunit RpL19 that belongs to the Minutegenetic class The Minutes are a class of ribosomal genemutations that are homozygous lethal delay cellulargrowth when heterozygous and have a rich history ofstudy [3233] Of interest RpL19 has been shown to bea modifier of Notch signaling [153435] In fact theMinute class of genes was first described in detail asmodifiers of Notch signaling in 1929 [36] Since thenribosomal components have been widely observed aseffectors of Notch [2537-39] The Notch transcriptionreporter measurements compliment these long-standingyet mechanistically unknown genetic interactions Onemechanism proposed to explain the relatively specificgenetic interactions between Minute mutations andNotch is the possibility of specific translational effectsFor instance the translation of long transcripts such asthe one encoding Notch itself may be sensitive to lowerlevels of specific ribosomal components In contrast analternative hypothesis has been presented that theseribosomal proteins may have post-translational effectson key components of Notch signaling [34] Minute pro-tein mutations are not found in the active site of theribosome as the peptide synthesis reaction is catalyzedexclusively by RNA in the core but rather on the sur-face of the ribosome Current structural and biochemicalstudies have demonstrated post-translational roles forthese surface coating ribosomal proteins [40] Thisincludes the folding of nascent peptide chains eitherdirectly on the surface of the ribosome or by the co-recruitment of protein chaperones The protein-proteininteraction map suggests that these types of post-trans-lational interactions may be directed towards the corechromatin components of the Notch network (Figure5E) Such a direct mechanism could explain the tran-scriptional effects described in this study as well as thelong-standing genetic observations between Notch andthe Minute class of mutations

Transcription factors that affect Notch-dependenttranscriptionAnalysis of the genes identified in the screen revealed anumber of transcription factors that affect Notch-depen-dent transcription Among these are cnc and maf-S that

are known to form a strong transcriptional activatorcomplex [41] RNAi targeting of either of these twogenes strongly suppressed both the Notch-induced aswell as non-induced E(spl)m3 reporter activity (Addi-tional file 1A) Also among the 15 transcription factorsthat promote Notch activity we found the DNA bindingprotein Deaf-1 Cnc maf-S and Deaf-1 are reported tointeract with the Hox protein Deformed (Dfd) to regu-late segmentation but their roles in other developmentalevents are not known [42] Our results provide a possi-ble role of these proteins in Drosophila development bypromoting Notch signalingAnother transcription factor that we found to play an

agonistic role in Notch signaling is the homeobox con-taining protein Aristaless (al) (Figure 5B) Al has beententatively linked to Notch signaling as it cell autono-mously represses the Notch ligand Delta in the pretarsusduring leg morphogenesis [10] It is possible that al isinvolved in a Notch-mediated lateral inhibition mechan-ism where al expressing cells remain undifferentiated byfavoring active Notch signaling whereas their neighbor-ing cells are free to express Delta and differentiate Ithas also been shown that Notch mutant clones in thedeveloping leg disk show diminished al levels suggestingthat al is a Notch target gene This would be the pre-dicted relationship in a lateral inhibition system wherea Notchal positive feedback loop would amplify theNotch activity differences between neighboring cellsTwo additional transcription factors that have been

previously shown to be involved in leg morphogenesiswere found to promote Notch signaling Bonus (bon) ahomologue of the vertebrate TIF1beta transcriptionalcofactor [43] and crooked legs (croI) a zinc finger pro-tein [44] Notch signaling is known to play an importantrole in Drosophila leg development and the recovery ofthese two transcription factors as modifiers of Notch-induced E(spl)m3 expression suggests that bon and croImay function to modulate Notch target gene output inthe developing leg [45]We also identified the Drosophila orthologues of two

mammalian proto-oncogenes kayak (c-fos) and c-Mybas positive regulators of Notch-signaling Although adirect functional link between these proteins and Notchsignaling has not been described kayak has been shownto interact genetically with Hairless [46] and c-Mybgenetically interacts with bon a novel Notch modifierdescribed above [47] In addition our data reveals asynergistic relationship between the positive regulator ofRas signaling 14-3-3ε and Notch Once again the pro-tein interaction network shows extensive contactsbetween 14-3-3ε and the chromatin machinery suggest-ing a mechanism for modulating Notch target transcrip-tion through Su(H) mediated chromatin modificationsInteractions between Notch and oncogenic pathways are

of particular interest as the involvement of Notch incancer biology and stem cell maintenance is becomingincreasingly apparentAn unexpected Notch target transcription modifier

identified in the screen is the Notch target gene Tram-track (ttk) We found that targeting of ttk with dsRNAresulted in reduced Notch activity (Figures 4 and 5) Incontrast ttk expression itself has been shown to increasein response to ectopic Notch activity [48] The RNAitreatment data suggest that ttk may function in a posi-tive feedback mechanism to promote Notch activatedtranscription and the network analysis suggests that thisinteraction may be mediated by a direct contact withNotch itself (Figure 3C)

ConclusionsA complementary genome-wide RNAi approach hasrevealed a subset of factors that modulate Notch targettranscription that may not have been found by tradi-tional genetic approaches For instance pleiotropiceffects combined with non-saturating mutagenesis mayhave obscured the detection of some components in tra-ditional genetic screens Several novel modifiers wereidentified in this RNAi transcription-based screen andthese factors will be further investigated for their preciseroles in the regulation of Notch signaling during devel-opment In addition the interaction network of thesefactors suggests that many may work through contactswith chromatin machinery components that are in turndirected to Notch target promoters by the transcriptionfactor Su(H)

MethodsDNA constructsConstitutively active Notch constructs were made withcDNA encoding either membrane-tethered DrosophilaNotch (NΔecn) constitutively activated by the removalof the extracellular domain or the soluble intracellulardomain (Nicd) [2] These truncated Notch constructswere cloned into the pIZ-V5His expression vector(Invitrogen) producing non-tagged proteins The controlexpression plasmid (con-luc) was constructed by cloningfirefly luciferase into the same pIZ-V5His expressionvector The luciferase reporter construct (m3-luc) con-tains a 14 kb tandem duplication of E(spl)m3 upstreamregulatory sequences cloned into a pGL2-Basic vector(Promega) as described [3]

Genome-wide RNAi methodA total of 23560 dsRNAs made available from the Dro-sophila RNAi Screening Center (DRSC) at HarvardMedical School were screened by the following methodKc167 cells were washed three times and resuspendedin serum-free Sangrsquos M3 medium (Sigma) at a

concentration of 5 times 105 cellsml Using a robotic liquidhandler 104 cells (20 ml) were uniformly dispensed intothe wells of 384-well polypropylene plates containingdsRNA and incubated for 45 min at room temperatureAn equal volume of M3 medium containing 10 fetalbovine serum was added and incubated for four daysOn day four the RNAi-treated cells were diluted with100 μl of medium mixed and 20 μl were dispensed intothe wells of six new 384-well plates pre-aliquoted with20 μl of transfection mix The six plates contained thethree different transfection mixes each in duplicateTransfection mixes were prepared with Effectene Trans-fection Reagent (Qiagen) following the manufacturerrsquosguidelines Luciferase activity was measured 24 h posttransfection using the Steady-Glo Luciferase Assay Sys-tem (Promega)This method requires only two plasmids to be

transfected at one time and gave acceptable signal tonoise ratios for high throughput screening in 384-well plate format (Figure 1) Whereas the conven-tional renilla dual-glo assay was not robust enough toscale to 384 well format with this Notch reporter sys-tem using an endogenous target In contrast to path-ways with soluble ligands the reporter andconstitutively active Notch constructs are required totransfect the same cell to activate transcription Withthe renilla dual-glo system adding the control con-struct required the co-transfection of three individualplasmids and this reduced the signal to noise ratio toinsufficient levels

Data analysisDuplicate measurements for each of the three signalswere averaged Notch specific E(spl)m3 promoter in thepresence of activated Notch (NΔecn gtm3-luc) the E(spl)m3 promoter alone (m3-luc) and the unrelated viralpromoter OplE2 (con-luc) The NΔecn gtm3-luc signalwas normalized two different ways by either the m3-lucor con-luc signals The z-scores of the log2 ratios werecalculated by using the standard deviation and mean ofthe measurements that corresponded to the 96-wells ofthe original dsRNA stock plates [49] To remove ratiosthat contained data that did not sufficiently replicate inthe original duplicate measurements a distribution wascalculated for the individual errors (estimated from theduplicate data sets) and ratios with an associated errorz-score above 3 were removed from further analysis(09 of the total data)To remove data associated with dsRNA that greatly

reduced general transcription or cell viability a distribu-tion of the signals from the control promoter (con-luc)was calculated and data with z-scores below -2 wereremoved (4 of the total wells) All calculations weredone by in house software written in JAVA

Hits were chosen as those log2 ratios with a z-scoreabove 2 or below -2 for NΔecn gtm3-luc normalized bythe viral promoter OplE2 (con-luc) For the data set nor-malized by the E(spl)m3 promoter alone (m3-luc) a z-score above 18 or below -18 was used The m3-lucnormalized distribution had more defined outliers indi-cating a better data set As a consequence m3-luc nor-malized data distribution had higher kurtosis as seen bya slightly sharper peak in Figure 2 This does not changethe rank order or relative differences in the hits of thatdata set but to make the cut-offs more equivalentbetween the two normalization methods the differentcut-off values were used

RNAi retest procedureGenes were chosen for retesting that were selected aspositive by both normalization methods (Figure 2c areaa) This second set of 28 dsRNAs were independentlyredesigned by the method of Arziman et al with no pre-dicted off targets and are listed in Additional file 5 [50]DNA templates for T7 reactions were generated by

PCR from Kc167 cell genomic DNA and dsRNA wasproduced using the MEGAscript RNAi kit (Ambion)Per well 25 ml of Kc167 cells at a concentration of 8 times105 cellsml were incubated with 125 μg of dsRNA (05mgml stock) for 1 h in serum-free M3 medium M3medium with 10 FBS (75 μl) was then added and incu-bated for 4 days On the fourth day 125 μl of mediumwas added and treated cells were split into 4 wells with50 μl per well each containing 50 μl of the followingtransfection mixes prepared as above a con-luc b m3-luc c m3-luc amp pIZ-NΔecn d m3-luc amp pIZ-NicdLuciferase levels were measured after 25 h as aboveRetests were done in quadruplicate for each dsRNA andthe results are given in Additional file 5 for the 22 posi-tive retests that have p-values lt 005 (compared withcontrol dsRNA treated cells)

Notch interaction network constructionThe Notch interaction network was generated by com-bining physical interaction data (eg two-hybrid data)from the DroID database [19] (that contained interac-tions from the BioGRID database [20]) with Notch tran-scription modifiers identified in the genome-wide studyGenetic interactions were not used for the networkmap The resulting network was drawn using Cytoscape[51] and the data can be found in additional file 6 Thenetwork file can be viewed in detail using the opensource Cytoscape viewer httpwwwcytoscapeorg

Orthology predictionAll orthology predictions for candidate genes were madeusing InParanoid [52]

Additional material

Additional file 1 A Excel table of screening data for Notch inducedtranscription normalized by the unrelated control promoter (NΔecngt m3-luccon-luc) Hits listed from initial screen with potential agonistsof Notch induced transcription listed in A and potential antagonistslisted in B

Additional file 2 Excel table of screening data for Notch inducedtranscription normalized by the uninduced E(spl)m3 promoter(NΔecn gt m3-lucm3-luc) Hits listed from initial screen with potentialagonists of Notch induced transcription or antagonists of uninduced E(spl)m3 transcription listed in A and vice versa in B

Additional file 3 Figure representing the distribution of Notchmodifiers by gene ontology classes Pie chart distributions forpercentage of genes represented in major gene ontology classes andcorresponding box plots of median z-scores for the various classes Boxplots are graphed as in Figure 1a A Distribution of genes that enhancethe Notch induced signal as normalized by the uninduced E(spl)m3promoter B Distribution of genes that suppress the Notch inducedsignal as normalized by the uninduced E(spl)m3 promoter C Distributionof genes that enhance the Notch induced signal as normalized by theunrelated control promoter (con-luc) D Distribution of genes thatsuppress the Notch induced signal as normalized by con-luc

Additional file 4 Excel table of orthology predictions Humanorthologs of the Drosophila genes identified in the screen werepredicted using InParanoid [52] Human diseases associated with thepredicted genetic counterparts are also listed

Additional file 5 Excel table of re-test data for the redesigneddsRNA Genes were chosen for retesting that were selected as positiveby both normalization methods (Figure 2c area a) This second set of 28dsRNAs were independently redesigned by the method of Arziman et alwith no predicted off targets and are listed in Additional file 5[50]Retests were done in quadruplicate for each dsRNA and the results aregiven for the 22 positive retests that have p-values lt 005 (comparedwith control dsRNA treated cells)

Additional file 6 Notch interaction network file A network file thatcan be viewed in detail using the open source Cytoscape viewer httpwwwcytoscapeorg[51] The Notch interaction network was generated byusing Notch transcription modifiers identified in the genome-wide studyas nodes and physical interactions (eg two-hybrid data) for edgesGenetic interactions were not used for the network map

AcknowledgementsWe thank S Artavanis-Tsakonas for generous support of this work Forproviding the RNAi library we thank N Perrimon and the Drosophila RNAiScreening Center (DRSC) Screening resources and expertise were providedby C Shamu and the ICCB - Longwood screening facilityThis work was supported by NIH grants HG003616 and NS26084 under SArtavanis-Tsakonas The Drosophila RNAi Screening Center (DRSC) issupported by grant 2R01GM067761-05 from the NIGMS division of NIH

Author details1Department of Molecular Cellular and Developmental Biology University ofColorado Boulder CO 80309 USA 2Stem Cells amp Development Departmentof Developmental Biology Pasteur Institute CNRS URA 2578 Paris France3Epigenetics and Progenitor Cells Keystone Program Fox Chase CancerCenter Philadelphia PA 19111 USA

Authorsrsquo contributionsPM RL and BD designed and performed experiments analyzed data andwrote the manuscript CF performed experiments and analyzed data Allauthors read and approved the final manuscript

Received 30 March 2010 Accepted 19 October 2010Published 19 October 2010

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doi1011861471-213X-10-107Cite this article as Mourikis et al Modifiers of notch transcriptionalactivity identified by genome-wide RNAi BMC Developmental Biology2010 10107

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Abstract

Background

Results

Conclusions

Background

Results and Discussion

Development of a robust assay to measure changes in Notch transcriptional activity

Genome-wide RNAi screen and data analysis

Classification of the identified proteins

Known Notch pathway interactors found by the RNAi screening method

Protein interaction network of Notch transcription modifiers

Epistatic analysis of candidate genes

Factors involved in chromatin modification

mRNA processing factors

Ribosomal factors and the classical Minute mutations

Transcription factors that affect Notch-dependent transcription

Conclusions

Methods

DNA constructs

Genome-wide RNAi method

Data analysis

RNAi retest procedure

Notch interaction network construction

Orthology prediction

Acknowledgements

Author details

Authors contributions

References