RESEARCH ARTICLE

Collaborative repressive action of the antagonistic ETStranscription factors Pointed and Yan fine-tunes geneexpression to confer robustness in DrosophilaJemma L. Webber, Jie Zhang, Alex Massey, Nicelio Sanchez-Luege and Ilaria Rebay*

ABSTRACTThe acquisition of cellular identity during development dependson precise spatiotemporal regulation of gene expression, withcombinatorial interactions between transcription factors, accessoryproteins and the basal transcription machinery together translatingcomplex signaling inputs into appropriate gene expression outputs.The opposing repressive and activating inputs of the Drosophila ETSfamily transcription factors Yan and Pointed orchestrate numerous cellfate transitions downstream of receptor tyrosine kinase signaling,providing one of the premier systems for studying this process. Currentmodels describe the differentiative transition as a switch from Yan-mediated repression to Pointed-mediated activation of common targetgenes. We describe here a new layer of regulation whereby Yan andPointed co-occupy regulatoryelements to repress geneexpression in acoordinated manner, with Pointed being unexpectedly required for thegenome-wide occupancy of both Yan and the co-repressor Groucho.Using even skipped as a test-case, synergistic genetic interactionsbetween Pointed, Groucho, Yan and components of the RNApolymerase II pausing machinery suggest that Pointed integratesmultiple scales of repressive regulation to confer robustness. Wespeculate that this mechanism may be used broadly to fine-tune theexpression of many genes crucial for development.

KEY WORDS: Gene regulatory network, Cell fate specification,Chromatin occupancy, Mesoderm development, RNA pol II pausing

INTRODUCTIONGenetic and epigenetic mechanisms together produce thespatiotemporal gene expression dynamics that drive accurate androbust developmental transitions. At the genetic level, combinatorialcodes of competing and collaborating transcriptional activators andrepressors are recruited to individual cis-regulatory enhancers todetermine precise gene expression outputs (Ma, 2005; Bauer et al.,2010). Analogously, at the epigenetic level, activating and repressivemarks facilitate open or closed chromatin states that respectivelypromote or preclude expression, and more nuanced regulation can beachieved by the simultaneous presence of activating and repressivemarks (Reynolds et al., 2013; Lagha et al., 2012). For example, atmany developmentally important genes, specific combinations ofinherently conflicting histone modifications permit RNA polymerase(pol) II to initiate transcription but then stall, keeping gene expression

off yet poised for rapid activation (Schwartz et al., 2010; Gaertneret al., 2012). Although it is known that chromatin looping canphysically coordinate the transcriptional complexes assembled atenhancers across a locus with the promoter-proximal complexes thatorchestrate RNA pol II pause and release, the mechanisms by whichthese two layers of regulation are actually integrated to fine-tune geneexpression dynamics during development are just beginning to beelucidated (reviewed by Gaertner and Zeitlinger, 2014; Liu et al.,2015; Meng and Bartholomew, 2017).

The Drosophila ETS transcriptional repressor Yan [also knownas Anterior open (Aop); Nüsslein-Volhard et al., 1984; Rogge et al.,1995] and activator Pointed (Pnt) provide a useful model system forexploring how activator and repressor inputs are balanced to controldevelopmental gene expression. Genetic and biochemical analysisof several enhancer elements, including the muscle heart enhancer(MHE) that drives the segmental pattern of even skipped (eve)expression in the cardiogenic mesoderm, has showcasedcompetition between Yan and Pnt for access to consensus ETSmotifs as a mechanism for directing rapid off-on gene expressiontransitions in response to upstream signals. Thus, prior to signaling,Yan outcompetes Pnt to repress target gene expression, therebystabilizing the uncommitted precursor state. Following pathwayactivation, Yan is targeted for rapid degradation, allowing Pnt accessto sites previously occupied by Yan. This turns on formerlyrepressed gene expression programs to promote a differentiativetransition (Brunner et al., 1994; Klaes et al., 1994; O’Neill et al.,1994; Rebay and Rubin, 1995; Hsu and Schulz, 2000).

The results of several recent studies motivated us to reconsider theuniversality of this regulatory mechanism with respect to all Yantarget genes and to ask whether more complicated Yan-Pntinteractions might also contribute to regulation of well-studiedtargets such as eve. First, chromatin immunoprecipitation-sequencing (ChIP-seq) studies have shown that Yan occupieschromatin in broad stretches of clustered peaks, bindingpreferentially to enhancers associated with developmentallyimportant genes and signaling pathway effectors (Webber et al.,2013a). Simple binary off-on regulation of all of these putativetargets seems unlikely. Second, a comparative survey of Yan andPnt protein expression throughout development revealed extensiveco-expression, particularly in tissues in which receptor tyrosinekinase (RTK) signaling levels are presumed low (BoisclairLachance et al., 2014). This raises the possibility of morecomplicated interactions than might be needed if their expressionwere always mutually exclusive as it is in the embryonic midline.Indeed, two recent studies focused on eve highlight the importanceof properly balanced Yan and Pnt repressive and activating inputs atthe MHE before, during and after a cell fate transition, andemphasize the use of long-range interactions between the MHE andother Yan-bound elements as a mechanism for ensuring robustReceived 26 March 2018; Accepted 17 May 2018

Ben May Department for Cancer Research, University of Chicago, Chicago,IL 60637, USA.

*Author for correspondence (irebay@uchicago.edu)

A.M., 0000-0003-0283-1142; I.R., 0000-0002-2444-3864

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regulation (Webber et al., 2013b; Boisclair Lachance et al., 2018).How Yan/Pnt-mediated regulatory mechanisms might becoordinated with epigenetic mechanisms that influence geneexpression is not known.In this study, we report the discovery of a completely unexpected

role for Pnt in recruiting or stabilizing Yan occupancy and repressionat regulatory elements across the genome. In wild-type embryos, wefind that Yan and Pnt have virtually identical genome-wideoccupancy patterns and that the two actually co-occupy individualenhancers. Whereas the majority of Pnt occupancy is Yanindependent, the majority of Yan occupancy is Pnt dependent, afinding that positions Pnt as an anchor with respect to establishingYan occupancy and repression. Further challenging the model ofexclusive Yan-Pnt regulatory antagonism, gene expression analysespredict that in addition to the classic opposing Pnt and Yan inputs atselect targets, Yan and Pnt together negatively regulate many targetgenes. Pnt also facilitates chromatin binding of the TLE co-repressorprotein Groucho, raising the possibility of context-specific roles forPnt as a repressor. Focusing on the target gene eve, synergisticinteractions between Pnt, Yan, Groucho and factors associated withpausing of RNA pol II fine-tune Eve expression to ensure robust cellfate specification. We propose that the collaborative action of anopposing activator-repressor pair establishes repressive complexesthat collaborate with the pol II pausing machinery to create a locus-wide poised state that both prevents spurious gene activation andensures timely induction of expression following signaling cues.

RESULTSYan and Pnt co-occupy regulatory regionsTo investigate how regulatory inputs from Yan and Pnt areintegrated across their target gene loci, we used ChIP-seq togenerate a genome-wide map of Pnt-bound regions in stage 11Drosophila embryos and then compared it with that of Yan. The twooccupancy profiles were strikingly similar, including at loci of theknown Yan/Pointed targets argos (aos), even skipped (eve) andmae(also known as edl) (Fig. 1A, Fig. S1A,B). Using high-confidencebound regions identified with the model-based analysis of ChIP-seq(MACS) peak-calling tool (Zhang et al., 2008), we calculated that82% of Yan-bound peaks overlapped with Pnt-bound peaks. In theinstances when a peak was called only in the Pnt dataset, visualinspection of the tag density pileups often revealed a subthresholdaccumulation of reads in the Yan sample (Fig. S1A,B). Consistentwith the similar binding landscapes, central motif enrichmentanalysis showed that the consensus sequences recognized byMothers against dpp (Mad) and ETS transcription factors were thetwo most enriched motifs in Pnt-bound peaks (Fig. S1C), exactly ashas been shown for Yan-bound peaks (Webber et al., 2013a).Assigning Pnt-bound regions to the nearest gene produced a list ofgenes with significant overlap with a similarly generated Yan targetlist, and thus near identical enrichment of gene ontology (GO) terms(Table S1, Fig. S1D).Although Yan and Pnt are co-expressed extensively in stage 11

embryos (Boisclair Lachance et al., 2014), we expected theoverlapping occupancy profiles to reflect mutually exclusive Yanor Pnt binding to specific enhancers, consistent with currentunderstanding of their antagonistic relationship. To assess this, weselected a subset of bound regions for which an ability to respondappropriately to Pnt and Yan activating and repressive inputs hadbeen previously demonstrated in S2 cell transcriptional reporterassays (Webber et al., 2013a). To our surprise, sequential ChIP(ChIP-reChIP) followed by qPCR revealed Yan-Pnt co-occupancyat six of the seven regions tested (Fig. 1B). This suggested that the

mechanisms that organize Yan and Pnt chromatin occupancy andcontributions to gene expression regulation are more complicatedthan previously assumed.

Pnt facilitates Yan recruitment across the genomeYan-Pnt co-occupancy of an enhancer could result from eitherinterdependent or independent recruitment. Based on the acceptedmodel of Yan and Pnt function in which Yan-mediated repressionmaintains cells in an uncommitted progenitor-like state, we predictedthat Yan is more likely to be the initiator, perhaps recruiting Pnt topoise bound target regions for subsequent activation in response tosignaling. We therefore first asked whether binding of Pnt to its targetregions depends uponYan by examining Pnt chromatin occupancy inyan null mutant embryos. In contrast to our predictions, the bindinglandscape of Pnt was broadly conserved in the absence of Yan(Fig. 1C,D, Table S2), suggesting that Pnt recruitment occursprimarily independently of Yan.

To determine whether Yan recruitment was similarly independentof Pnt, we profiled Yan chromatin occupancy in pnt null mutantembryos. In contrast to expectations, comparison of ChIP-seq signalprofiles revealed a global reduction in Yan occupancy (Fig. 1E,F).This findingwas validated independently byChIP-qPCR at all targetstested (Fig. 1G). Indirect immunofluorescence analysis confirmedcomparable Yan protein levels in wild-type and pnt mutant embryos(Fig. S2), ruling out the most trivial explanation for globally reducedoccupancy. In further support for a direct role for Pnt in facilitatingYan recruitment to chromatin, Yan occupancy was preferentiallyreduced at regions identified as bound by both Yan and Pnt versusregions identified as bound byYan alone (Fig. 1H).We conclude thatPnt plays a crucial and unexpected role in the recruitment and/orstabilization of Yan binding across the genome.

Pnt collaborates with Yan to mediate repressive functionYan’s unanticipated dependency upon Pnt for proper occupancymotivated us to consider a non-canonical role for Pnt as a repressor,and a collaborative rather than antagonistic relationship with Yan inthis capacity. The central prediction was that the expression of genessubject to such Pnt-Yan cooperative repression should increaseupon loss of either Pnt or Yan. To test this, we utilized anunpublished analysis of mRNA expression changes in pnt or yanmutant embryos that we had performed with a custom Agilentmicroarray made with probes from Yan-bound genes identified byChIP (for ChIP targets, seeWebber et al., 2013a). We first identifiedprobes for which expression was significantly changed (P<0.05) inpnt mutants versus wild type (Fig. 2A) and then selected a handfulof up- or downregulated targets for qPCR validation. Comparison ofarray and qPCR results revealed broad agreement between the twodatasets, confirming the overall quality of the array data (Fig. 2B).As a second point of validation, we determined whether mRNAlevels of the known Yan/Pnt targets aos, mae and eve exhibited theexpected opposite response to loss of Pnt or Yan. Consistent withexpectation, expression of aos and mae was reduced in pnt mutantsand increased in yan mutants. In contrast, although eve levels wereelevated in the absence of Yan, they were not significantly changedin pnt mutants, a finding perhaps in keeping with the stochastic andrather modest loss of Eve expression that has been described in pntmutant embryos (Halfon et al., 2000).

Although a handful of studies have uncovered roles for Pnt innegative regulation of gene expression, including hid in the embryo,yan in the eye disc and asense in the larval brain (Kurada andWhite,1998; Rohrbaugh et al., 2002; Zhu et al., 2011), Pnt has beencharacterized exclusively as a transcriptional activator (Klämbt,

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1993; Scholz et al., 1993; Brunner et al., 1994; O’Neill et al., 1994;Gabay et al., 1996; Schwartz et al., 2010). We were thereforeintrigued by the set of genes upregulated in the pnt mutant.Although some of these expression increases could reflect indirectregulation, because the genes used in the analysis were selectedbased on chromatin occupancy, the approach should enrich forchanges resulting from loss of direct Pnt-mediated regulation.Focusing on genes with upregulated expression, which wouldreflect loss of Pnt repressive inputs, we assessed their response toloss of Yan. If the ability of Pnt to repress transcription depends onits ability to recruit Yan, then a similar set of genes should beupregulated in both mutants; indeed, a strong positive correlationwas observed (R2=0.7, P<0.0001; Fig. 2C).To gain insight into the developmental processes that might be

regulated by coordinated Pnt-Yan repression, we identifiedthe ontologies of the upregulated genes using the PANTHERclassification system (Mi et al., 2013). Upregulated genes were

enriched for categories associated with muscle cell fate commitmentand cardioblast differentiation. These GO terms were absent fromontology analyses performed with downregulated genes (Table S3).Considering these differences in light of the Yan and Pnt expressionpatterns in the stage 11 embryo suggests that in the mesoderm,where Yan and Pnt are co-expressed and RTK signaling levels arelow (Gabay et al., 1997; Boisclair Lachance et al., 2014), the twocollaborate as repressors to stabilize the unspecified state.

Groucho is recruited to Yan and Pnt co-occupied regionsA second prediction of a model in which Pnt contributes repressivefunction to gene regulation is that it should recruit co-repressorproteins, either directly or through its interaction with Yan. Toidentify likely candidates, we examined the modENCODE database(Contrino et al., 2012; www.modencode.org) to compare availableco-repressor genome-wide occupancy patterns with those of Yanand Pnt. The binding landscape of the co-repressor Groucho (Gro)

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Fig. 1. Pnt recruits Yan to chromatin. (A)Comparison of ChIP-seq read density forPnt-GFP (Pnt) and Yan across argos (aos),with RefGene gene track shown belowprofiles. Asterisk marks the region assessedby ChIP-qPCR. (B) Sequential Yan-PntChIP-qPCR analysis plotted as fold increaserelative to mock-treated control, normalizedto a negative control region (NC1; Webberet al., 2013a), using mean±s.e.m. from two(cv-2 and lace) or more separateexperiments. (C,E) ChIP-seq read densityprofiles for Pnt-GFP and Yan from wild-type(WT) and yan or pntmutant embryos at neur.Red shading highlights an example ofstatistically significant reduction in Yanoccupancy. (D,F) Pie charts showing theproportion of Pnt or Yan peaks gained or lostin the reciprocal mutant background. (G)ChIP-qPCR analysis of Yan occupancy atcandidate target regions from either control(wild-type) or pntmutant embryos. Data fromat least three separate experiments areplotted as mean±s.e.m. normalized to anegative control region. (H) Comparison ofratios of Yan read density in pnt mutantsrelative to the wild-type control. Yan-boundregions that do not intersect with a Pnt-bound peak were less affected by loss of pntthan peaks that intersect Pnt. **P<0.01(Student’s t-test).

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immediately stood out. Because the published datasets were notappropriately stage-matched to our work, we performed ChIP-seqanalysis of Gro in stage 11 wild-type embryos. The resultsconfirmed the similarity of the Gro binding landscape to that ofYan and Pnt. Intersecting high confidence peaks of Gro with theYan and Pnt datasets revealed a 54% and 37% overlap, respectively,and heat-map analysis of Yan/Pnt co-bound regions suggested evengreater overlap (Fig. 3A).To determine whether proper Gro occupancy requires Pnt, we

performed ChIP-seq analysis of Gro in a pnt mutant background.Western blot analysis revealed no significant change in Gro proteinlevels in pntmutant versus wild-type embryos (Fig. S3A,B) and Grooccupancy was only moderately affected at regions of the genomewithout nearby Pnt binding (Fig. 3B,C). In contrast, analogous toour finding of reduced Yan occupancy in pnt null animals, Grobinding was reduced in regions of the genome where Gro and Pntprofiles normally overlap (Fig. 3C). Comparison of the ChIP-seqpeaks suggested that loss of Gro occurred at regions that alsodisplayed reduced Yan occupancy in pnt null embryos. Forexample, Gro occupancy was lost across the neuralized locus(Fig. 3D), in patterns similar to those observed for Yan loss, but wasbarely reduced at the turtle locus, which does not bind Yan(Fig. 3E). Plotting the ratio of Gro occupancy at bound regions inpnt mutants relative to the wild-type control confirmed that thereduction of Gro in the absence of pnt is more severe at Yan andGro co-occupied sites, than at sites that are not bound by Yan(Fig. 3F). Taken together, these data indicate that Pnt recruits bothGro and Yan to common regulatory elements, raising the possibilityof coordinated Yan-Pnt-Gro occupancy and repression of theassociated target gene.We tested this prediction by correlating gene expression changes

in pnt mutant embryos with the changes in Yan and Gro occupancydescribed above. Of the 320 genes associated with Yan andGro occupancy loss in pnt mutants, 129 were represented in thecustom microarray. Of these, 107 were differentially expressed inthe absence in of pnt, with 72% displaying upregulated expression(Fig. 3G). Using the converse approach, upregulated genesidentified in the microarray had reduced Yan and Gro signalintensity in pnt mutants relative to wild type; this list included thevalidated Gro target E(spl)mbeta-HLH (Fig. S3C, Fig. S4). We

conclude that the loss of Yan and Gro occupancy that occurs in pntmutant embryos reflects a novel mechanism by which Pnt recruitsand collaborates with these two repressive factors to negativelyregulate expression at a significant subset of target genes.

Pnt mediates repressive inputs at eveHaving defined a novel role for Pnt in recruitment of Yan and Gro,we next investigated how these interactions influence expression ata specific locus. The heart identity gene eve provided an idealvantage point to do this because of the already deep mechanisticunderstanding of how Yan repressive and Pnt activating inputs areorganized at specific enhancers (Halfon et al., 2000; Webber et al.,2013b; Boisclair Lachance et al., 2018). In stage 11 embryos, Eve isexpressed in segmentally arrayed clusters of cells in the developingcardiogenic mesoderm. Yan and Pnt exert antagonistic inputs at thelevel of a pattern-driving MHE, such that in yan mutant embryosextra Eve+ cells are specified, whereas in pnt mutant embryos, thenumber of Eve+ cells specified is reduced (Halfon et al., 2000).Additional repressive input is provided by D1, a Yan-responsiveelement, deletion of which results in elevated and more variable Eveexpression (Webber et al., 2013b).

Matching the pattern of Yan occupancy (Webber et al., 2013a), tagdensity profiles of Pnt at the eve locus revealed enrichment at both theD1 and MHE regulatory regions (Fig. 4A); Pnt occupancy at the D1region, which genetically appears to be dedicated to dampening Eveexpression (Webber et al., 2013b), further supports the suggestion ofa role for Pnt in repressive regulation. Analysis of the Yan genome-wide ChIP dataset in pnt mutant embryos suggested that Pnt isrequired for proper Yan occupancy at both the MHE and D1; ChIP-qPCR confirmed this dependency (Fig. 4B). To test whether Yan andPnt are co-bound, we performed sequential ChIP. Although we wereunable to detect co-occupancy at the eve MHE, simultaneousoccupancy was detected at the eve D1 region (Fig. 4C). Oneexplanation for the negative results at the MHE is that we are simplybelow the detection threshold. Indeed, both the genome-wide ChIPdata sets and the ChIP-qPCR confirmation experiments always showlow enrichment at the MHE, perhaps indicating that Yan/Pntoccupancy/co-occupancy of this pattern-driving enhancer occurs inonly the small subset of mesodermal cells from which Eve+

pericardial cells are specified. Alternatively, Yan and Pnt might

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co-occupy the D1 but not the MHE, with 3D interactions enablingD1-bound Pnt to recruit/stabilize Yan occupancy at both the D1 andthe MHE. Although further testing will be required to distinguishbetween these possibilities, in support of the latter, long-rangeinteractions between the D1 andMHE stabilize Yan occupancy at thetwo elements (Webber et al., 2013b; Boiclair Lachance et al., 2018).Because complete loss of pnt results in reduced Eve expression,

we devised an alternative genetic strategy to assess repressivefunction. We reasoned that embryos heterozygous for Yan mightprovide a suitably sensitized background to reveal a role for Pnt-mediated repressive regulation. We first assessed Eve expressionlevels in animals heterozygous for either yan or pnt and comparedthese with Eve levels in double heterozygotes. The yan and pnt loss-of-function alleles were fully recessive, with no significant changein Eve expression detected relative to wild-type control (Fig. 4D). Incontrast, in yan/+;pnt/+ embryos, Eve levels were significantlyelevated and extra Eve+ cells were specified (Fig. 4D,E). Werepeated the experiment using a functional Eve-YFP BAC transgene(Webber et al., 2013b) and again measured elevated Eve levels indoubly heterozygous animals compared with single heterozygotes(Fig. S5). Together, these data suggest a cooperative function forYan and Pnt in negative regulation of eve.

We extended the dose-sensitive genetic interaction analysis toassess involvement of the co-repressor Gro, occupancy of which atboth the eveMHE and D1 is reduced in the absence of pnt (Fig. 4A).Consistent with previous studies of Gro’s repressive input at eve(Helman et al., 2011), we observed increased Eve levels and extraEve+ cells in gro/+ embryos; both phenotypes were enhanced inpnt/gro doubly heterozygous animals (Fig. 4D,E). We conclude thatYan, Pnt and Gro work collaboratively to negatively regulate Evelevels and Eve+ cell fate specification in the cardiogenic mesoderm.

Pnt integrates with pausing machinery to maintain a poisedstateThe inherent conflict of recruiting both active and repressive histonemarks to a given bound region is characteristic of a balancedchromatin state, whereby genes are held silent, but poised fortranscriptional activation (Schwartz et al., 2010; Gaertner et al.,2012). Analogously, at the transcription factor level co-occupancyby the activator-repressor pair Pnt/Yan could both preventinappropriate activation of eve under sub-threshold signalingconditions and prime the locus for rapid transcriptional activationfollowing the onset of upstream signaling. Meta-analysis of publiclyavailable ChIP-seq data for three different chromatin modifications

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Fig. 3. Pnt recruits the co-repressor Gro to Yan-boundregions. (A) Heat-map analysis ofYan, Pnt-GFP (Pnt) and Grooccupancy at the top 250 Yan/Pnt-bound regions. Each row representsan individual peak that spans 2 kb,inversely sorted by ChI signal andcentered around each peak midpoint.(B) Ratios of Gro occupancy in pntmutants relative to the wild-typecontrol show that Gro bound regionsthat do not intersect with a Pnt-boundpeak were relatively unaffected byloss of pnt, whereas Gro binding wasreduced at regions normally bound byPnt. ****P<0.0001 (Student’s t-test).(C) Average signal intensity plotsshow reduced Gro occupancy occurspredominantly at peaks with wild-typePnt binding. (D,E) Read densityprofiles for Pnt-GFP, Gro and Yanfrom wild-type or mutant embryos atthe neur and turtle (tutl) loci. Redshaded regions contrast thecoordinate loss of Yan and Grooccupancy at neur in the absence ofpnt with the lack of change in Gro attutl where Yan is not normally bound.(F) Gro peaks not bound by Yan arenot significantly reduced in pntmutants whereas Gro peaks thatoverlap Yan in wild-type conditionsare significantly reduced.****P<0.0001 (Student’s t-test).(G) The set of genes associated withboth Yan and Gro peak loss in the pntmutant is enriched for genes withsignificantly elevated expression inthe microarray.

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from 4-8 h embryos, which includes the stage 11 time point used inour ChIP experiments, provided circumstantial evidence that evemay be poised. Specifically, the combination of negligibleH3K27ac, which exclusively marks active enhancers, prominentH3K4me1, a mark of both poised and active enhancers, andprominent H3K27me3, a mark that in the absence of H3K27acindicates a poised state, suggests that the eve locus may be poised(Rada-Iglesias et al., 2011; Bonn et al., 2012; Koenecke et al., 2017;Fig. 5A).Poised genes commonly employ an RNA pol pausing strategy

whereby RNA pol II is recruited and initiates transcription, but thenpauses downstream of the transcription start site (TSS) until it receivesthe appropriate signaling cues for pause release (reviewed by Gaertnerand Zeitlinger, 2014). Recent work inDrosophila cell lines implicatesGro in RNA pol II pausing (Kaul et al., 2014), an intriguingassociation given our discovery of a role for Pnt in recruiting Gro,including to the eve MHE and D1 enhancers (Fig. 3D,E, Fig. 4A).Furthermore, meta-analysis of genome-wide ChIP datasets revealedfrequent overlap between Yan and Pnt occupancy patterns with thoseof components of the pausing machinery, including Trithorax-like(Trl, also known as GAGA factor) and Bric à brac 1 (Bab1) (Fig. 5B;Contrino et al., 2012; Tsai et al., 2016). Focusing on eve, the locusbears hallmarks of pausing with high pol II occupancy near the TSS,together with low incidence of H3K4me3 and overlap with thepausing factor Trl (Fig. 5C; Contrino et al., 2012; Lee et al., 2008;Fuda et al., 2015; Gaertner et al., 2012; Tsai et al., 2016).Using eve as our model, we assessed genetic interactions between

Pnt and members of the pausing machinery. We first tested whetherheterozygosity for either Trl or bab1 could influence Eve expressionand observed no significant change in Eve levels (Fig. 6A). Incontrast, embryos doubly heterozygous for either pnt and Trl or pntand bab1 displayed significantly increased Eve levels with acorresponding increase in the number of Eve+ cells specified(Fig. 6A,B). Similar changes in Eve expression and number of Eve+

cells were observed in embryos doubly heterozygous for either Trland bab1 or yan/gro and Trl (Fig. 6A,B, Fig. S6). A trend towardsincreased Eve expression was also observed in pnt/Nelf-E double

heterozygotes, although the relative increase was not statisticallydifferent from control, perhaps because of the maternal contributionof Nelf-E and/or the multi-subunit nature of the NELF complex(Wang et al., 2010; Wu et al., 2005). Survival to adulthood ofanimals doubly heterozygous for either pnt and Trl or pnt and bab1was half that of any of the three single heterozygotes (Fig. 6C),suggesting that interactions between Pnt and the pausing machinerymay play a broader role in development beyond eve.

DISCUSSIONThe precision with which multipotent cells commit to specializedfates relies on regulated de-repression of gene transcription. Thus, instem cells and in early embryos, concomitant with the initial openingof chromatin domains by pioneer factors, conflicting epigeneticmarks deposited at the promoters ofmany developmentally importantgenes recruit yet stall RNA pol II, thereby maintaining repression andmultipotency. How such epigenetic-based repressive poising iscoordinated with the transcription factors that respond to inductivecues to direct specific cell fate transitions as development proceeds isnot well understood. Our study positions the ETS1 homolog andtranscriptional activator Pointed (Pnt) as a key integration pointbetween the transcriptional repressive complexes that assemble atregulatory elements across a locus and the molecular complexes thatestablish, maintain and release RNA pol II pausing. The results notonly re-define the classic Yan-Pnt cell fate switch paradigm inDrosophila, but more broadly uncover a novel strategy by whichgenetic and epigenetic regulation is coordinated to confer robustnessto developmental cell fate transitions.

The accepted model for Yan and Pnt function predicts mutuallyexclusive occupancy at enhancers, with RTK signaling triggeringthe transition from an initial Yan-bound repressed state inuncommitted progenitors to a subsequent Pnt-bound activatedstate that drives cell fate acquisition. Our study paints a differentpicture in which Pnt plays a role in establishing and stabilizing thatinitial Yan-bound repressed state, and in fact co-occupies manyregulatory elements with Yan. It is important to note that becausethe sequential ChIP analysis was not a genome-wide study and

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Fig. 4. Yan, Pnt and Gro collaborate to fine-tuneeve expression. (A) Read density profiles forPnt-GFP, Gro and Yan from wild-type or mutantembryos at the eve locus. Red shading shows thatPnt-GFP occupancy is broadly maintained at theMHE and D1 in the absence of Yan, whereas Yanand Gro occupancy is reduced at both elements inthe absence of Pnt. (B) ChIP-qPCR analysis of Yanoccupancy at the D1 and MHE in stage 11 wild-typeor pnt mutant embryos. Data from at least fiveseparate experiments are plotted as mean±s.e.m.normalized to a negative control region.(C) Sequential ChIP detects Yan-Pnt co-occupancyat the D1 but not at the MHE. Fold increase relativeto mock-treated control and normalized to anegative control region is plotted. Bars representmean±s.e.m. of at least six independentexperiments. (D) Quantification of average Evelevels per cluster in different genetic backgrounds.Box plots depict measurements from at least 70clusters. ***P<0.001; ****P<0.0001 (ANOVA withTukey’s multiple comparison test). (E) Bar chartsdepicting the frequency of clusters with differentnumbers of Eve+ cells from at least seven embryos.

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because whole embryos rather than single cells were profiled, it ispossible that Yan-Pnt co-occupancy only occurs at a small subset oftargets and that the broad similarity in overall occupancy patternsprimarily reflects a mix of exclusively Yan or exclusively Pntbinding at the same enhancers in different cells. Arguing againstthis, the genome-wide loss of Yan occupancy in pnt mutants andnormal Pointed occupancy in yan mutants positions Pointed as thecritical determinant and/or stabilizer of Yan binding and repression,with co-occupancy likely relevant to the mechanism.We therefore speculate that Pnt is required first to set up Yan

occupancy and repression and second to respond to RTK signalingby activating target gene expression; its activating function is thusepistatic to its repressive role, explaining why predominantly loss-of-function phenotypes have been described for pntmutants. Use ofthe same transcription factor to dictate both the repressive regulation

that maintains the initial multipotent state and the subsequentactivation that changes it, enables a level of temporal coordinationof gene expression dynamics that may be crucial to the robustness ofdifferentiative transitions. We also note that although Yan/Pntfunction has been studied primarily in the context of RTK signaling,the two are co-expressed in many tissues across development,including those presumed to have low RTK signaling input (Gabayet al., 1997; Boisclair Lachance et al., 2014), and co-occupyregulatory elements across a broad swath of signaling pathwaygenes and crucial developmental regulators that are unlikely allto be regulated downstream of RTK signaling. Thus, Pnt-Yan-Groenhancer co-occupancy may provide a modular repressivemechanism that can be adapted to a variety of regulatory situations.

How Pnt-Yan co-occupancy is organized/facilitated by the DNAsequence of each enhancer will be interesting to explore. One

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Fig. 5. Chromatin marks and TF occupancy associatedwith the eve locus predict a poised chromatin state.(A) The eve locus is associated with the poised chromatinsignature comprising H3K27me3 and H3K4me1 anddepletion of H3K27ac. ChIP-seq profiles of modEncodedatasets (H3K27me3: stage 4-8 h embryos, modEncode811; H3K4me3: stage 4-8 h embryos, modEncode 778;H3K27ac: stage 4-8 h embryos, modEncode 835) werevisualized using IGB. Yan and Pnt ChIP datasets are shownfor reference. The RefGene gene track is shown below theprofiles. Blue boxes depict peaks called using IGB and a97% threshold. (B) Trl and Bab1 are associated with Yan-,Pnt- andGro-bound regions. Aggregate binding profiles of Trl(GAF: stage 8-12 embryos, modEncode 3397) and Bab1(Bab1: 0-12 h embryos, modEncode 628) were generated forregions of the genome bound by Yan, Pnt and Gro.(C) ChIP-seq profiles of Trl, H3K4me3 and RNA pol II (GAF:stage 8-12 embryos; H3K4me3: stage 4-8 h embryos,modEncode 790; RNA pol II: stage 4-8 h embryos,modEncode 846) at the eve locus. Blue boxes depict peakscalled using a 97% threshold with IGB.

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possibility is that Pnt initially interacts with all ETS binding sites toopen up a regulatory element, but then gets displaced at a subset ofsites upon recruitment of Yan, perhaps remaining bound only atsites required for subsequent activation. Alternatively, distinctsequence preferences rather than affinity differences might result inPnt occupancy of only a specific subset of ETS binding sites,leaving others free for Yan to bind. This model also supports avariation in mechanism by which Pnt and Yan are recruited jointly,rather than sequentially, to establish the initial repressed state, withco-occupancy essential to stabilize Yan binding. Our recent workexploring how the cis-regulatory organization of the eve MHEorganizes Yan and Pnt inputs supports the idea that distinctsequence preferences enable simultaneous occupancy and hencecomplex integration of repressive and activating inputs (BoisclairLachance et al., 2018).The mechanism by which Yan occupancy depends on Pnt also

remains to be elucidated. One possibility is that Pnt recruits Yandirectly. However, to date our efforts to detect Yan-Pnt protein-protein interactions, either in vitro, in two-hybrid screens, orin standard co-immunoprecipitation experiments, have yieldednegative results. Indirect protein-level interaction mechanisms,such as bridging the complex with Gro or with another transcriptionfactor, may thus be more likely. The strong overlap between Madand ETS binding sites noted in Yan/Pnt-bound regions genome-wide (Webber et al., 2013a) makes the Dpp effector Mad anintriguing candidate. Alternatively, rather than nucleating specificprotein complexes, Pnt may establish or interpret a local chromatinstate that permits Yan binding. Analogous pioneer-like activity hasbeen described for a few other ETS factors, including PU.1 (alsoknown as Spi1) and ETV2 (reviewed by Iwafuchi-Doi and Zaret,2014; Kanki et al., 2017). As pnt encodes two alternatively splicedproducts, Pnt-P1 and Pnt-P2, that contain the same DNA-bindingdomain but different amino-terminal activation domains and exhibitdifferent patterns of expression and signal responsiveness (Klämbt,1993; Scholz et al., 1993; O’Neill et al., 1994; Brunner et al., 1994;Gabay et al., 1996; Shwartz et al., 2013), it will be important tore-evaluate the role of each isoform during cell fate transitions withrespect to the establishment of Yan/Gro binding, target generepression and the subsequent switch to activation.Regardless of the precise mechanism, Yan’s reliance on Pnt for its

stable recruitment provides a plausible explanation for a previous

unexpected finding that doubling Yan dose does not lead to increasedor ectopic DNA occupancy (Webber et al., 2013a). More broadly, thestrategy of the activator recruiting the repressor could provide anoccupancy feedback circuit that buffers against fluctuations inactivator or repressor concentrations. For example, the standardcompetition model predicts that a multipotent cell with lower thannormal Pnt will over-recruit Yan; we speculate that such Yan-dominated repression would be sluggish in response to inductivecues. In contrast, a system in which Yan occupancy depends on Pntmight be buffered against such variation, because the consequence oflower Pnt levels would be less efficient Yan recruitment, whichshould maintain the appropriate Yan-Pnt balance.

We speculate that this precisely poised Pnt-Yan-mediatedrepressive state is achieved through close coordination with theRNA pol II pausingmachinery (summarized in Fig. 7). Several piecesof evidence support this idea. For example, not only is Pnt essentialfor proper Yan occupancy, but it also recruits the co-repressor Gro tothe same set of regulatory elements. Prior genome-wide analyses ofGro occupancy and function in embryos and cultured cells haveshown that Gro-regulated genes are enriched for epigeneticmarks andpromoter proximal transcripts commonly associated with pausedRNA pol II (Kaul et al., 2014; Chambers et al., 2017). Ourdemonstration of eve de-repression in embryos doubly heterozygousfor gro and Trl provides the first genetic evidence of a possible directmechanistic link between Gro repressive complexes and the RNApol II pausing machinery. Our study also emphasizes the likelyimportance of Pnt to the Gro-paused RNA pol II connection. Forexample, included among the set of genes showing coordinatelydisrupted Yan-Gro occupancy and de-repression in pnt mutantembryos is E(spl)mbeta-HLH, a target previously shown to beregulated by Gro-dependent RNA pol II pausing in cultured cells(Kaul et al., 2014 and Fig. S4). The web of synergistic geneticinteractions between pnt, yan, gro andmutants in RNA pol II pausingfactors, such as Trl, Bab1 and NELF, further supports such a model.

RNA pol II pausing establishment and release is also closelylinked to Polycomb (PcG) repressive complexes in both Drosophilaandmammals (Schwartz et al., 2010; Gaertner et al., 2012; Bernsteinet al., 2006; Ferrai et al., 2017) and PcG repression has in turn beenconnected to Gro (Abraham et al., 2015). For example, a recent studydescribes how recruitment of the Hox family transcriptionalactivators AbdA and Ubx reduces PcG binding at RNA pol II

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cells from at least three embryos. (C) Adult survivalrates of indicated genotypes, plotted as mean±s.e.m.of at least three independent experiments.

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paused genes to promote release and transcriptional activation(Zouaz et al., 2017). Similarly, ETS1, the mammalian ortholog ofPnt, promotes release of paused RNA pol II to activate angiogenicgene expression, although connections to PcG complexes were notinvestigated (Chen et al., 2017). Trl, which our study connectsgenetically to Pnt-Gro-Yan repressive mechanisms, helps direct PcGproteins to Polycomb response elements, or PREs, and thuscontributes to PcG repressive activity (Mahmoudi et al., 2003;Mishra et al., 2003; Mulholland et al., 2003). Given that eve is a PcGtarget gene, with a validated PRE (Dura and Ingham, 1988; Fujiokaet al., 2008; Kim et al., 2011), it may provide an ideal context forelucidating the molecular levels of integration between Yan-Pnt-Groand PcG repressive complexes in relation to RNA pol II pausing.In conclusion, we propose that, analogous to the use of conflicting

epigenetic marks to poise RNA pol II, the inherent conflict of co-occupancy by an activator-repressor pair such as Pnt-Yan establishesan exquisitely sensitive and dynamic repressive mechanism thatconfers robustness to developmental gene expression regulation.Because loss or misexpression of ETS transcription factorscontributes to many cancers and because oncogenic transformationrelies on dysregulated use of normal developmental pathways,exploration of these ideas in mammalian systems may provide newinsight into human disease.

MATERIALS AND METHODSChromatin immunoprecipitationAll chromatin immunoprecipitation (ChIP) was from stage 11 embryos(approximately 5-7 h after egg lay) processed as previously described(Webber et al., 2013a) and summarized in supplementary Materials and

Methods. Sequential ChIP of Yan and Pnt was performed by firstimmunoprecipitating Yan (guinea pig anti-Yan; Webber et al., 2013a).The protein-DNA complex was eluted in reduced volume and diluted 1:10in ChIP lysis buffer before performing the second round of ChIP with arabbit GFP antibody (rabbit anti-GFP, A6455, Invitrogen, Lot# 1603336). Ano antibody control (mock-treated) processed identically to experimentalsamples was included. For ChIP-seq, two biological replicates and an inputsample were sequenced on an Illumina Hi-seq instrument according to theIllumina protocols. The raw sequence data were aligned to the April 2006D.melanogaster genome using BWA (Li and Durbin, 2009). Followingstandard practice in the field (Robertson et al., 2007; Rozowsky et al., 2009;Zhong et al., 2010), having first confirmed consistency between replicates,the two IP reads were combined and peak detection performed using MACSsoftware with an mfold of 3.40 and otherwise default parameters. MACS-defined bound regions were assigned to the nearest gene using the UCSCgenome browser and gene ontology enrichment analyses were performed(see supplementary Material and Methods for further details). Top-scoringbound regions were also subjected to Centrimo analysis to identify centrallyenriched binding motifs in the Pnt ChIP-seq dataset (see supplementaryMaterials andMethods for more details). SPPwas used to calculate genome-wide tag density profiles. Default parameters were used, with the exceptionthat the scale.by.dataset.size=T option was used to normalize tag density bythe total dataset size to make it comparable across samples (Kharchenkoet al., 2008). ChIP-qPCR was performed as previously described (Webberet al., 2013a) and is summarized in supplementary Materials and Methods.

Microarray analysisA custom expression array was designed on the Agilent GE 8×15 Kplatform. The microarray included 7080 probes for putative Yan targetgenes identified by Webber et al. (2013a) (designed using the Earraysoftware by Agilent), 1894 probes for random genes and 536 control probes.Total RNA was extracted from stage 11 wild-type, yan null or pnt nullembryos with TRIzol Reagent (Invitrogen) following the manufacturer’sprotocol, and purified using the RNeasy Mini Kit (Qiagen). Total RNAwaslabeled and hybridized to the microarray using the Quick Amp One-ColorLabeling Kit (Agilent) as described by the manufacturer. Triplicateexperiments for each genotype were performed. Expression array datawere first analyzed by the Feature Extraction Software (Agilent) usingdefault parameter settings, and the processed signals for the probes werethen used for downstream analysis. Linear models were generated for eacharray using only the probes in the random gene set, and then signals werenormalized across all arrays. After normalization, the average signal for eachprobe across triplicate experiments of each genotype was calculated, andsignal fold changes between wild type and the pnt or yan mutants werecomputed. t-tests were performed at the probe level, and probes with aP-value less than 0.05 were selected as significant (Table S4). Formicroarray validation, RNA was isolated, reverse transcribed and theresultant cDNA subjected to Real-Time PCR as described in thesupplementary Materials and Methods.

Tag density analysis and generation of heat mapsSorted bed files were produced using the BEDOPS wig2bed script (Nephet al., 2012). To manage the large number of calculations, a Visual BasicApplication (VBA) for Microsoft Excel was used to generate matrices ofread density data for groups of given bound regions ±5 kb from the midpointof each individual region. The matrices were used for (1) calculating ratiosof transcription factor (TF) occupancy in mutant versus wild type, (2)producing aggregate read density profiles by averaging read density acrossall peaks and (3) generating TF binding heat maps. For further details, seesupplementary Materials and Methods.

Differential binding analysisBed files of MACS-defined bound regions and sequence aligned reads forwild-type and mutant datasets for each factor were generated. The differentialbinding analysis software, MAnorm (Shao et al., 2012), was used to generatea merged set of bound regions for each factor with quantitative values ofdifferential binding and associated P-values for each peak. These datasetswere used to describe patterns of peak gain or peak loss, where peak

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loss corresponds to normalized M values of >0.5 and peak gain of <−0.5(Fig. 1D,F, Tables S2 and S5).

Eve quantificationFor quantification of Eve levels and numbers of Eve+ cells, embryos werestained as described byWebber et al. (2013a) with either 1:10mouse anti-Eve[3C10, Developmental Studies Hybridoma Bank (DSHB)] or 1:1000 rabbitanti-GFP (A6455, Invitrogen, 1603336). Using a Zeiss 880 confocalmicroscope, serial 0.8 µm z-sections were taken through the Eve-positivemesodermal cells and maximum projections generated. Expression intensitywas calculated as the mean pixel intensity for each Eve+ cluster minus themean background pixel intensity, normalized to the average cluster intensityof the control imaged in the same session. For cell counts, Eve+ cells werecounted by going through z-stack projections of the relevant slices. For rescueexperiments, stage 11 embryos of each genotype were hand-selected,transferred to vials and incubated at 25°C until adults emerged.

Yan and Gro quantificationFor Yan expression analysis, wild-type or pntΔ88 embryos were stained asdescribed by Boisclair-Lachance et al. (2014) with 1:10,000 guinea pig anti-Yan (Webber et al., 2013a). For western blot analysis described in Fig. S3,stage 11 wild-type or pntΔ88 embryos were dechorionated in 50% bleachand homogenized in 50 µl of SDS sample buffer [250 mM Tris-Cl (pH 8),10% SDS, 50% glycerol, 50% β-mercaptoethanol, 0.04% bromophenolblue]. Samples were passed through a 27G needle 10 times and boiled for10 min prior to running on an 8% SDS-PAGE gel. After transfer toPVDF, blots were probed with 1:100 mouse anti-Gro (DSHB) antibody and1:2000 mouse anti-tubulin (T9026, Sigma) antibody, which served as aloading control.

Drosophila strains and geneticsThe following stocks were obtained from the Bloomington Drosophila StockCenter: w1118, pntΔ88/TM3,Sb1, w1;Trl13C/TM6B,Sb1,Tb1, Df(3L)babAR07,bab1AR07,bab2AR07/TM6B,Tb1 and y1,w67c23; P{w+mC]y+mDint2=EPgy2}EY07065/TM3, Sb1,Ser1. Additional stocks used include: groMB36

(Jennings et al., 2008), pntAF397 (Rebay et al., 2000), yanER443 andyanE833 (Karim et al., 1996), Pnt-GFP (Boisclair Lachance et al., 2014), andEve-YFP (Webber et al., 2013b). To allow genotyping of stage 11 embryos,stocks were rebalanced over twist-Gal4>UAS-GFPmarked second and thirdchromosome balancers.

Statistical analysisData are presented as mean±s.e.m. except where otherwise described andminimum sample sizes are reported in each figure. Data were plotted andanalyzed for statistical significance using Graphpad Prism software.Statistical significance was determined either by a two-tailed t-test whereappropriate, or alternatively with a one-way ANOVA in combination withTukey’s multiple comparison tests to compare two or more groups. P-valuesless than 0.05 were considered to be statistically significant.

AcknowledgementsWe thank Pieter Faber, MikaykaMarchuk and Abhilasha Cheruku in the University ofChicago Genomics Facility for help with ChIP-seq and microarray. Jean-FrancoisBoisclair Lachance, Kohta Ikegami, Rebecca Spokony and Matthew Slatteryprovided many helpful discussions and comments on the manuscript. Weacknowledge the Bloomington Drosophila Stock Center (NIH P40OD018537) andthe Developmental Studies Hybridoma Bank (created by the NICHD of the NIH)for reagents.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: J.L.W., J.Z., N.S.-L., I.R.; Methodology: J.L.W., N.S.-L.;Software: J.L.W., A.M., N.S.-L.; Validation: J.L.W.; Formal analysis: J.L.W., A.M.;Investigation: J.L.W., J.Z., A.M.; Resources: A.M.; Data curation: J.L.W.; Writing -original draft: J.L.W.; Writing - review & editing: J.L.W., I.R.; Visualization: J.L.W.,A.M.; Supervision: J.L.W., I.R.; Project administration: J.L.W., I.R.; Fundingacquisition: J.L.W., I.R.

FundingThis work was supported by grants from the American Heart Association(12POST12040225/Jemma Webber/2012-2014 and 15POST22660028/JemmaWebber/2015 to J.L.W.) and the National Institute of General Medical Sciences (R01GM080372 to I.R.), and by the Genomics Core Facility through a University ofChicago Cancer Center Support Grant (P30 CA014599). N.S.-L. was supported inpart by the National Institute of General Medical Sciences (T32 GM007281) and theNational Eye Institute (R01 EY12549 to I.R.). Deposited in PMC for release after12 months.

Data availabilityChIP-seq and microarray data from this study have been deposited at GeneExpression Omnibus with accession numbers GSE114092 and GSE114209,respectively.

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/doi/10.1242/dev.165985.supplemental

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