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Nat Cell Biol. Author manuscript; available in PMC 2012 March 1.
Published in final edited form as:
PMCID: PMC3173870

The tumor suppressor L(3)mbt inhibits neuroepithelial proliferation and acts on insulator elements


In Drosophila, defects in asymmetric cell division often result in the formation of stem cell derived tumors. Here, we show that very similar terminal brain tumor phenotypes arise through a fundamentally different mechanism. We demonstrate that brain tumors in l(3)mbt mutants originate from overproliferation of neuroepithelial cells in the optic lobes caused by de-repression of target genes in the Salvador-Warts-Hippo (SWH) pathway. We use ChIP-seq to identify L(3)mbt binding sites and show that L(3)mbt binds to chromatin insulator elements. Mutating l(3)mbt or inhibiting expression of the insulator protein gene mod(mdg4) results in upregulation of SWH-pathway reporters. As l(3)mbt tumors are rescued by mutations in bantam or yorkie or by overexpression of expanded the deregulation of SWH pathway target genes is an essential step in brain tumor formation. Therefore, very different primary defects result in the formation of brain tumors, which behave quite similarly in their advanced stages.

Development of the Drosophila nervous system recapitulates many steps in mammalian neurogenesis. Neurons in the adult fly brain arise from stem cells called neuroblasts which undergo repeated rounds of asymmetric cell division during larval stages 1,2. After division, one daughter cell remains a neuroblast while the other is called the ganglion mother cell (GMC) and divides just once more into two differentiating neurons. Most larval neuroblasts are inherited from the embryo 3 but the so-called optic lobe neuroblasts (NB) located laterally on each brain lobe (Fig. 1d) pass through a neuroepithelial (NE) stage (Fig. 1e) and are therefore a particularly suitable model for mammalian neurogenesis 4,5. During early larval stages, the NE cells of the optic lobes (OL) proliferate and separate into the inner (IOA) and outer (OOA) optic anlagen (Fig. 1c, control, cross section) 6,7. During late larval stages, NE cells switch to a neurogenic mode. On the medial side, they generate optic lobe neuroblasts (OL NBs), which generate the neurons of the medulla, the second optic ganglion 4,8. OL neurogenesis is controlled by a wave of lethal of scute (l(1)sc) expression passing through the neuroepithelium from medial to lateral 5. The activity of the Jak/STAT pathway inhibits neural wave progression and thereby controls neuroblast number. Differentiation of neuroepithelial cells also involves the Notch, Epidermal Growth Factor (EGF) and Salvador-Warts-Hippo (SWH) pathways 9-13.

Figure 1
l(3)mbt is necessary to prevent tumorous over proliferation of the larval CNS

Characterization of Drosophila genes identified in brain tumor suppressor screens has demonstrated that defects in neuroblast asymmetric cell division result in the formation of stem cell derived tumors that metastasize and become aneuploid upon transplantation 14,15. These screens also identified lethal (3) malignant brain tumor (l(3)mbt) 16,17, a conserved transcriptional regulator 18 that is also required for germ-cell formation in Drosophila 19. L(3)mbt binds to the cell cycle regulators E2F 20 and Rb 21 but the relevance of these interactions is unclear. We show that in Drosophila, L(3)mbt regulates target genes of the Salvador-Warts-Hippo (SWH) pathway (Fig. 2i) that are important in proliferation and organ size control 22-24. The SWH-pathway is regulated by the membrane proteins Expanded (Ex) and Fat, which activate a protein complex containing the kinases Hippo and Warts to phosphorylate the transcriptional co-activator Yorkie 25-27. Yorkie acts together with the transcription factors Scalloped 28-31 and Homothorax 32 to activate proliferative genes like Cyclin E and the microRNA bantam (ban) 33 and Drosophila inhibitor of apoptosis 1 (diap1, thread in Flybase). Upon phosphorylation, Yorkie is retained in the cytoplasm and its target genes are not activated. In Drosophila the main role of the SWH-pathway is to limit proliferation in imaginal discs and its absence leads to tumorous overgrowth 34. In vertebrates, many homologs of key pathway members are tumor suppressors indicating that this function is conserved 34.

Figure 2
Candidate screen reveals function for SWH-pathway in optic lobe proliferation

L(3)mbt contains three MBT domains which bind mono- or dimethylated histone tails 21,35. Biochemical experiments in vertebrates have suggested a role in chromatin compaction 21 but whether this role is conserved is not known. Results published while this paper was under review have shown that germline genes are upregulated in l(3)mbt mutant brains and are necessary for tumor formation 36. Our data indicate that L(3)mbt is bound to insulator sequences, which affect promoter-enhancer interactions and influence transcription 37,38. In Drosophila, the proteins CTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4) and GAF are found at insulator sequences but how these factors act is unknown 38.

Our data show that tumor formation in l(3)mbt mutants is initiated by the uncontrolled overproliferation of neuroepithelial cells in the optic lobes due to the upregulation of proliferation control genes normally repressed by the SWH-pathway. L(3)mbt is located at DNA sequences bound by chromatin insulators and we propose that the function of L(3)mbt as a chromatin insulator is essential for repressing SWH target genes and preventing brain tumor formation.


l(3)mbt tumors originate in the optic lobes

l(3)mbt mutants - like brat or lgl - showed a strong increase in neural stem cells positive for the neuroblast marker Deadpan (Fig. 1a and Supplementary Fig. S1a) resulting in an abnormal enlargement of the brain (Fig. 1a). However, in contrast to brat and lgl mutants 39-43, the asymmetric segregation of determinants was unaffected (Fig. 1b, Supplementary Fig. S1b and data not shown). Instead, we observed abnormal enlargement of the optic lobes. In wild type brains (Fig. 1c, control) the epithelial monolayer of the OOA forms OL neuroblasts at of its medial edge (Fig. 1d, e). In l(3)mbt mutants, the neuroepithelia of both IOA and OOA were massively expanded (Fig. 1c, middle and right panels). Initially, this led to a delay in OL neuroblast formation (Fig. 1c, top view) but later, OL neuroblasts were formed and their number was significantly increased (Fig. 1a, c). Unlike in wild type, neuroblast formation was also seen in the center of the OL epithelium (Fig. 1c, close up). To quantify the epithelial phenotype, we reconstructed optic lobe neuroepithelia (of IOA and OOA) in 3D (see methods). While OL epithelia were on average 1.2×105μm3 in wild type third instar larvae, their average size was significantly increased to 3×105μm3 in l(3)mbt76 mutants and 8.25×105μm3 in l(3)mbt76/Df(3R)D605 larvae (Fig. 1f). During first larval instar (L1), cell number and mitotic pattern within the OL of l(3)mbt mutants were normal and during early second instar (L2 early), the IOA and OOA separated normally (Fig. 1g). However, starting in late second instar (L2 late) the mutant epithelia expanded and started folding. Since tumors did not form in l(3)mbt clones and available RNAi lines did not cause OL phenotypes, we generated a small hairpin micro RNA 44 to test cell autonomy of the overproliferation defect. Expression of l(3)mbtshmiR in central brain (CB) neuroblasts resulted in depletion of L(3)mbt protein (Supplementary Fig. S1d) however, this did not cause overproliferation (Supplementary Fig. S1c). In contrast, when expressed in neuroepithelia of the optic lobes l(3)mbtshmiR caused a strong overproliferation in the IOA and OOA (Fig. 1h). Epithelial expansion was also seen in l(3)mbt mutant wing imaginal discs (Fig. 3b, c, data not shown and 16). In most adult wings, this resulted in a significant size increase (Supplementary Fig. S1e, f) but occasionally, very small deformed wings could also be observed. Taken together, these data suggest that overproliferation of neuroepithelial cells during late L2 stages initiates tumor formation in l(3)mbt mutants.

Figure 3
SWH target genes are misregulated in l(3)mbt mutants

Signaling pathways controlling optic lobe proliferation

As staining of l(3)mbt mutants for aPKC and Actin did not reveal any change in apical basal polarity (Fig. 2a, b) we used the GAL4C855a driver line to express dominant active, dominant negative and RNAi constructs for the major signaling pathways, epigenetic complexes and epithelial polarity genes (Supplementary Fig. S2e). The GAL4C855a driver is expressed in the IOA and OOA from first to third larval instar and in imaginal disc epithelia (Supplementary Fig. S2a, b). We analyzed epithelial tissue size in the IOA, the OOA and the imaginal discs and estimated the number of OL neuroblasts (Supplementary Fig. S2c, d, e and see methods).

Activation of the epidermal growth factor (EGF) pathway promoted epithelial growth mainly in the IOA (Fig. (Fig.2c,2c, S2e). Activation of the Jak/STAT pathway increased epithelial size (Fig. (Fig.2d,2d, S2e) 5 whereas deregulation of Dpp or over activation of FGF pathways did not cause any visible phenotypes (Fig. 2c, d).

In contrast, inhibition of the SWH-pathway (Fig. 2i) resulted in a phenotype similar to l(3)mbt mutants (Fig. 2f, h, Supplementary Fig. S2e; see also 11). expanded (ex) RNAi in the optic lobes (Supplementary Fig. S2c) caused epithelial overproliferation similar to what has been described for ex mutants 11. Overexpression of Hippo together with the apoptotic inhibitor P35, strongly reduced the size of optic lobe epithelia (Fig. 2e). Upon expression of non-phosphorylatable Yorkie 26,27, the size of neuroepithelia in the IOA and OOA was five to ten fold increased (Fig 2f, g) a phenotype also observed by Reddy et al. (2010) 11. A similar albeit milder phenotype was observed upon expression of the Yorkie target ban (Fig. (Fig.2h,2h, S2e). Thus, inhibition of the SWH-pathway or overexpression of its target genes can recapitulate the increased OL proliferation seen in l(3)mbt mutants.

L(3)mbt tumor formation involves the SWH-pathway

The SWH-pathway inhibits expression of diap1, ban, and – as part of a negative feedback loop – ex (Fig. 2i) 45. To investigate SWH-pathway activity we used a diap1-GFP4.3 reporter, in which the second transcriptional start site of diap1 (TSS2) controls GFP expression (see below for a map of the diap1 locus and Reference 30). In wild type brains diap1-GFP4.3 is expressed in the neuroepithelium of the optic lobes during second and early third larval instars but becomes restricted to three small stripes during mid third instar (Fig. 3a). However, in l(3)mbt mutants, diap1-GFP4.3 remained expressed throughout the overgrowing neuroepithelium (Fig. 3b, note that the high GFP expression in the control is in lamina cells). Moreover, in wing imaginal discs, diap1-GFP4.3 was upregulated in the entire wing pouch in l(3)mbt mutants (Fig. 3b). The ex-lacZ reporter was only moderately expressed in wild type epithelia of the OOA but was upregulated in l(3)mbt mutants (Fig. 3c). A similar upregulation was seen in the wing imaginal disc (Fig. 3c). To test ban-miRNA activity we used a negative GFP-sensor carrying multiple ban binding sites 46. When ban is active this GFP-sensor is downregulated. In control optic lobes the ban-GFP-sensor was not detectable indicating strong ban expression. The limited dynamic range of the ban-GFP-sensor did not allow us to detect further ban upregulation in the optic lobes (data not shown). In l(3)mbt mutant wing discs, however, GFP was almost completely lost indicating a strong increase in ban activity (Fig. 3d). Thus, l(3)mbt inhibits the expression of SWH target genes. These effects were cell autonomous and not a consequence of tumor formation since expression of l(3)mbtshmiR in the posterior compartment of the wing disc using GAL4en (engrailed-GAL4) increased expression of ban (Fig. 3f), diap1-GFP4.3 (Fig. 3e) and ex-lacZ (Fig. 3g) only in this compartment.

To test whether the SWH-pathway is important for tumor formation in l(3)mbt mutants, we investigated genetic interactions. In l(3)mbt and ban double mutants, neuroepithelial size was significantly reduced and tumors did not form (Fig. 4a, compare Supplementary Fig. S4a). Neuroepithelial size was reduced from 4.5×105μm3 in ban1/+; l(3)mbt76 to less than 1×105μm3 in ban1; l(3)mbt76 larvae at the same developmental stage (Fig. 4b). Overexpression of Expanded (Ex) rescued the overproliferation in l(3)mbt76 mutants (Fig. 4c, d; OL neuroepithelial size 3.3×105μm3 in l(3)mbt76, 1.8×105μm3 in GAL4C855a>UAS-Ex, l(3)mbt/+ or in GAL4C855a>UAS-Ex, l(3)mbt76). Finally, tumor size in l(3)mbt mutants was reduced by removing one copy of yorkie (Fig. 4e and Supplementary Fig. S4b, c, d; OL neuroepithelial size 3×105μm3 in l(3)mbt76/E2 and 2.2×105μm3 in l(3)mbt76/l(3)mbtE2; yki/+). Thus the SWH-pathway and its target genes are important for tumor formation in l(3)mbt mutants.

Figure 4
SWH-pathway shows genetic interaction with l(3)mbt

L(3)mbt is a nuclear protein

The SWH-pathway regulates gene expression by excluding Yorkie from the nucleus 26,27. Surprisingly, neither the subcellular localization of Yorkie (Supplementary Fig. S3b and data not shown) nor the expression of the associated transcription factor Scalloped 28-31 (Supplementary Fig. S3a) were changed in OL epithelia or wing discs of l(3)mbt mutants, indicating that L(3)mbt does not influence SWH signaling activity.

To analyze the subcellular localization of L(3)mbt we generated a specific antibody (Fig. 5a). In Drosophila embryos and in larvae, L(3)mbt was nuclear in interphase and dispersed in the cytoplasm during mitosis (Fig. 5a and data not shown). This staining was specific since it was lost from l(3)mbt mutant larvae (data not shown and Western Blot analysis in Supplementary Fig. S5c) and upon l(3)mbtshmiR expression (Supplementary Fig. S1d and Fig. 3e, f, g). The localization was confirmed in flies expressing functional RFP-L(3)mbt or GFP-L(3)mbt fusions (see also Supplementary Fig. S5a, b). Live imaging of larval neuroblasts expressing RFP-L(3)mbt revealed that the protein accumulates in nuclear dots in interphase cells (Fig. 5b). When expressed in salivary glands, L(3)mbt localized to multiple bands on polytene chromosomes that were often characterized by reduced DAPI staining (Fig. 5c). Thus, L(3)mbt is a nuclear protein that most likely exerts its function by associating with chromatin.

Figure 5
L(3)mbt localizes to the nucleus

L(3)mbt binds to SWH-pathway target loci

To test chromatin binding we performed chromatin immunoprecipitation (ChIP) followed by quantitative PCR analysis from third instar larval brains and imaginal discs (see methods). diap1 can be transcribed from three promoters, TSS1, TSS2 and TSS3 28,30. L(3)mbt bound moderately to TSS2 and strongly to TSS1 (Fig. 6a). Consistent with this, diap1-GFP5.1 (contains TSS1) was expressed in OL neuroblasts and neurons and strongly upregulated in l(3)mbt mutants (Fig. 6b). This is surprising because previous reports concluded that TSS1 is not expressed in wing or eye imaginal discs 28,30. For TSS3, we detected weak binding of L(3)mbt, however, the relevance of this interaction is unclear (Fig. 6a). L(3)mbt did not bind to the bxd Polycomb response element (PRE) in the Ubx locus (Fig. 6a) 35 but bound to other SWH target genes like Cyclin E (Fig. 6a) 47.

Figure 6
L(3)mbt binds at the TSS and regulates SWH target genes and Jak/STAT pathway activity

To determine binding sites of L(3)mbt on a genome-wide level, we used Solexa-sequencing. Results from two highly correlated independent ChIP experiments (Pearson correlation 0.8835, Supplementary Fig. S6a) showed that L(3)mbt bound close to TSS (Fig. 6c and see methods). We assigned each bound region to the closest gene (see methods) and conducted a biological pathway (KEGG; Fig. 6d) and gene ontology (GO; Supplementary Fig. S6c) analyses of the predicted target genes 48. As the SWH-pathway is not yet annotated in these databases it was manually added to our analysis. SWH target genes are among the top 10 pathways overrepresented among L(3)mbt bound genes (data not shown). We identified seven out of eleven known and predicted SWH target genes (ban, CycA, CycB, CycE, E2f, diap1, fj, ex, Mer, wg, Ser) and found a significant enrichment (p-value 0.025) of these targets among genes bound by L(3)mbt (Fig. 6f, Supplementary Fig. S6e; 3% FDR). Importantly, the functional binding site at TSS1 of the diap1 locus (Fig. 6a) was confirmed (Fig. 6f). In addition, we found a low occupancy peak at a conserved site 23kb upstream of the ban transcription unit (Fig. 6f). Thus, L(3)mbt binds to multiple SWH target genes.

In our KEGG analysis we also found enrichment for Jak/STAT signaling genes (Fig. 6d and Supplementary Fig. S6e). Jak/STAT signaling previously has been shown to regulate optic lobe neuroepithelial growth 5. Indeed, the 10xSTAT92E-GFP sensor was significantly upregulated in both OL neuroepithelia and wing discs of l(3)mbt mutants 49 (Fig. 6e). Finally, we found “oocyte maturation” among the top 20 processes enriched in the KEGG and GO analysis (data not shown and Supplementary Fig. S6c). L(3)mbt bound close to the TSS of vasa (vas), bag of marbles (bam) and benign gonial cell neoplasm (bgcn) (Fig. 6h), which may explain the defects in germ cell formation in l(3)mbt mutants 19,50.

Microarray analysis of various Drosophila brain tumors has identified a set of genes that are specifically deregulated in l(3)mbt mutants and are termed L(3)mbt signature (MBTS) genes 36. 63% of these MBTS genes and 85% of the MBTS genes with a described germline function had L(3)mbt bound within the next +/−2kb (Fig. 6g, Supplementary Fig. S6d), suggesting that the identified L(3)mbt bound regions have in vivo relevance.

L(3)mbt binds to insulator elements

To analyze L(3)mbt binding specificity, we searched for DNA motifs enriched among L(3)mbt binding sites. Among seven DNA consensus motifs, four matched the consensus for the chromatin insulators CP190, BEAF-32, CTCF and Su(Hw) (Fig. 7a) 51-54 whereas, three did not match any known consensus motif (Supplementary Fig. S7a). Using published ChIP-chip data 51 we determined the overlap in binding sites (Fig. 7b). We found a strong overlap of L(3)mbt binding sites with class I chromatin insulators CP190, BEAF-32 and CTCF (Fig. 7b) whereas the overlap with the class II insulator protein Su(Hw) is smaller.

Figure 7
L(3)mbt binds at insulator-bound regulatory domains and influences Abd-B expression

The best-studied locus for insulator proteins is the Hox gene cluster of the Bithorax complex (BX-C) 37,55. Remarkably, L(3)mbt binding sites within this locus correlated strongly with CP190 and CTCF but less with Su(Hw) (Fig. 7c). To test whether L(3)mbt binding sites in the Hox-cluster are functional, we analyzed the expression of the homeotic gene Abdominal-B (Abd-B). CTCF mutants show a characteristic downregulation of Abd-B at the posterior end of the larval ventral nerve cord (VNC) 55. We found a significant reduction of Abd-B in l(3)mbt mutant larval CNS (Fig. 7d) that closely resembled the change seen in CTCF mutants. Notably, a strong correlation between L(3)mbt and chromatin insulator binding was also observed at the SWH target genes ban and diap1 (Supplementary Fig. S7b, c).

Since we did not observe any deregulation of the diap1-GFP4.3 reporter upon RNAi mediated knockdown of CTCF, CP190, BEAF-32 and Su(Hw), we tested the insulator protein Mod(mdg4) that binds to CP190 and Su(Hw) but not directly to DNA 56,57. Upon RNAi mediated knockdown of mod(mdg4) bantam-sensor-GFP was strongly downregulated (Fig. 7e, indicating an increase in bantam activity) whereas the diap1-GFP4.3 reporter was only mildly upregulated (data not shown). Thus, insulator protein function is necessary to control SWH target gene expression.


A new mechanism for tumor formation in Drosophila

brat, lgl and dlg were identified as Drosophila brain tumor suppressors. In all cases, defects in asymmetric cell division cause a huge expansion of the neuroblast pool 39-42,58,59. In l(3)mbt mutants, however, the neuroblast pool is expanded because an upregulation of SWH target genes results in a massive expansion of neuroepithelial tissue. Why those neuroblasts proliferate indefinitely upon transplantation 15 is currently not understood for any of those mutants.

While the SWH-pathway is essential for tumorigenesis in l(3)mbt mutants, its overactivation can not recapitulate the neuroblast tumor phenotype seen in l(3)mbt mutants (this study and Reference 11). Similar to the multifactorial origin of mammalian tumors, therefore, the combined deregulation of several signaling pathways could be required. The Notch pathway could be involved as it regulates the formation of OL neuroblasts from neuroepithelia 10,12,13 and Notch pathway genes are bound by L(3)mbt (Table S1 and data not shown). We also observe increased activity of the Jak/STAT pathway, a major regulator of OL development 5. Finally, the deregulation of germline genes in l(3)mbt mutants that has been described while this manuscript was under review 36 could provide another exciting explanation.

L(3)mbt acts on insulator elements

Our results indicate that L(3)mbt acts on insulator elements, which isolate promoters from the activity of nearby enhancers acting on other genes 37,38. Our analysis showed that L(3)mbt binding sites overlap with CP190, CTCF and BEAF-32, placing the protein into what has been called the class I of chromatin insulators 51.

The identification of a DNA consensus motif for a histone binding protein like L(3)mbt is highly unexpected as insulators are typically nucleosome free 51. Currently, the activity of these important transcriptional regulators could be explained in several ways 37,38. Either, they form physical barriers blocking the interaction between enhancers and promoters. Alternatively, they mimic promoters and compete with endogenous promoters for enhancer interaction. Finally, they could interact with each other or nuclear structures to form loop domains that regulate transcriptional activity. Our data suggests another model in which insulators interact with histones on nearby nucleosomes and influence the structure of higher order chromatin. Importantly, in the regions flanking CTCF binding sites nucleosomes are enriched for histones that are mono- and di-methylated on H3K4 or mono-methylated on H3K9 or H4K20 60, the variants to which MBT domains can bind in vitro 18,21,35,61. As the human L(3)mbt homolog L3MBTL1 was shown to compact nucleosome arrays in vitro 21, a model becomes feasible in which simultaneous binding to insulators and the surrounding nucleosomes reduces flexibility and thereby restricts the ability of nearby enhancers to interact with promoters on the other side of the insulator. However, our data could equally well be worked into the other prevalent models for insulator activity. Since L(3)mbt is currently the only chromatin insulator besides CTCF that is conserved in vertebrates, analysis of its homologs will certainly allow to distinguish between those possibilities.

A conserved role for L(3)mbt

OL development resembles vertebrate neurogenesis 4. Both processes consist of an initial epithelial expansion phase followed by neurogenesis through a series of asymmetric divisions 62. Together with previous findings 11, our data demonstrate that l(3)mbt and the SWH-pathway are crucial regulators of the initial neuroepithelial proliferation phase. Interestingly, the SWH-pathway has been implicated in regulating neural progenitors in the chicken embryo 63 and it will be exciting to test the role of mammalian L(3)mbt in this process. It is remarkable that YAP is upregulated 64 and L3MBTL3 is deleted in a subset of human medulloblastomas 65. Medulloblastoma is the leading cause of childhood cancer death and investigating the role of the SWH-pathway might contribute to the progress in fighting this disastrous disease.

Supplementary Material

Supplementary Figures

Supplementary Figure Legends, References 1-6

Supplementary Table 1

Supplementary Table 2, References 66-81


We wish to thank I. Reichardt, N. Corsini, A. Fischer and C. Jueschke for comments on the manuscript; R. Neumueller and all former and present members of the Knoblich laboratory, as well as Wu Wei, Charles Girardot and Julien Gagneur for discussions; R. Lehmann, D. Pan, L. Zhang, N. Tapon, B. Thompson, G. Halder, G.H. Baeg, M. Labrador, the Flytrap Yale, the Bloomington Drosophila Stock Center, the Vienna Drosophila RNAi Center (VDRC) and the Developmental Studies Hybridoma Bank (DSHB) for flystocks, antibodies and constructs; P. Serrano Drozdowskyj and M. Novatchkova for bio-informatic support; M. Madalinski for affinity purification; E. Kleiner for technical assistance; K. Aumayr, P. Pasierbek and G. Schmauss for bio-optics support; S. Farina Lopez, S. Wculek and C. Valenta for fly work support. Work in J.A.K.’s laboratory is supported by the Austrian Academy of Sciences, the Austrian Science Fund (FWF) and the EU FP7 network EuroSystem.



Drosophila Strains and Constructs

The l(3)mbt alleles used in this study were: l(3)mbtts1, l(3)mbtE2 16,17, l(3)mbt76 19 (original stock contained suppressive second site mutation, chromosome was cleaned during recombination to new FRT82B). All experiments and controls with l(3)mbt mutants were conducted at 29°C except if noted otherwise. Other mutant fly strains were: exe1 66; ykiB5 25; bandelta1 67; Df(3R)D605 (Bloomington). The SWH and Jak/STAT pathway reporter lines were: ex697 (ex-lacZ) (gift from N.Tapon) 66; diap1-GFP4.3 and diap1-GFP5.1 30; ban-sensor-GFP 46; Sd-GFPtrapCA07575 68; 10xStat92E-GFP 49. See below for complete list of all fly strains.

UAS-constructs for L(3)mbt and Yki were cloned using the Gateway System (Invitrogen). l(3)mbt was PCR-amplified from DGRC cDNA clone pB SK LD05287 and yki was cloned from mixed stage cDNA. L(3)mbtshmiR was cloned according to Haley et al. (2008) 44. All experiments with l(3)mbtshmiR were conducted at 32°C except if noted otherwise (over proliferation phenotype in optic lobe neuroepithelia is observed only at 32°C). The following driver lines were used: GAL41407 (insc-Gal4) 69 and GAL4C855a 70, GAL4c253, GAL4en (all Bloomington Stock Center). All other UAS lines are listed below.

For the candidate screen UAS constructs were crossed to GAL4C855a at 29°C. Phenotypes were determined from z-stacks of stained brains from wandering third instar larvae and scored manually on a scale from −10 to +10 (0 = wild type optic anlagen size; +10 = l(3)mbt76 mutant optic anlagen; −10 = strong underproliferation).

Image acquisition and live imaging

Images were acquired on a Zeiss LSM510 Meta confocal microscope using 25x (zoom 0.7x for imaginal discs and zoom 1x for brains) or 60x oil immersion objectives (zoom 3x for polytene chromosomes). For live imaging, larval brains were dissected in PBS, mounted in PBS on a round coverslip and covered with bioFOLIE25 (In Vitro Systems and Services, 96077317). For four-dimensional (4D) z-stacks of 15-20 μm at 0.8-1 μm intervals were acquired at 30sec intervals (40x oil immersion objective). A wet chamber prevented evaporation.

NE tumor volume was quantified using Amira 3D reconstruction software. Z-stacks of 40-80 μm were recorded at 2 μm intervals (25x oil immersion objective at 1x zoom). Optic lobe neuroepithelia were outlined manually (Actin positive; Deadpan negative) in each z-slice and the 3D surface and volume were determined automatically. Significance was calculated by using unpaired t-test with Welch’s correction and visualized as aligned dot plot with mean and standard error of the mean (SEM).

Antibodies and Immunohistochemistry

L(3)mbt antibodies were raised in guinea pigs against amino acids 1211 to 1477 of the L(3)mbt protein fused to Maltose Binding Protein (MBP) (affinity purified, IF: 1:200, ChIP: ~5μg (10μl for 250μl chromatin)). Other antibodies were rabbit anti-Miranda (1:200; 42), rat anti-E-Cadherin (1:100, DCAD2, Developmental Studies Hybridoma Bank, University of Iowa, DSHB), guinea pig anti-Deadpan (1:1000, gift from J. Skeath), goat anti-aPKCzeta (1:200, Santa Cruz Biotechnology), mouse and rabbit anti-phospho histone H3 (1:2000, Cell Signaling), mouse anti-Prospero (1:50, MR1A, DSHB), mouse anti-Lamin (1:50, ADL67.10, DSHB), chicken anti-beta Galactosidase (1:500, Abcam), rabbit anti-Yorkie (1:200, Duojia Pan), rabbit anti-dSfmbt531-980aa (ChIP: 20μl, 61), rabbit anti-Mod(mdg4) 71, Alexa Fluor 488 and 568 phalloidin (Invitrogen). All secondary antibodies were generated in donkey and were from Invitrogen.

Immunofluorescence experiments in larval brains and discs were carried out as described 42. Briefly, brain-disc complexes were dissected in PBS and fixed for 15-20min in 4% PFA in PBS, 0.1% TritonX-100 (1 change of fixative); antibodies were diluted in 10% normal donkey serum in PBS, 0.1% TritonX-100; brains were mounted in Vectashield containing DAPI (Vector Laboratories).

Chromatin preparation and ChIP

Chromatin immunoprecipitation (ChIP) experiments were carried out essentially as described in Oktaba el al. (2008) 72. A detailed protocol can be found on Nature Protocol exchange (; doi:10.1038/protex.2011.229). Briefly, 200 wild-type (Canton S or w118) third instar larvae were dissected in ice-cold PBS. Carcasses were stored at 4°C in wash B (maximal O/N). Brain-disc tissues were hand dissected in wash B and material of 600-800 brain-disc complexes pooled for one chromatin preparation. Sonication of dissected material was performed in 0.5 ml RIPA buffer in two steps (1st: 2x for 2 min with a tip sonicator (Omni-Ruptor 250 (Omni-International Inc.), microtip, power output: 20, pulser: 60; 2nd: 3x for 10 min in a Covaris machine (Covaris S, maximum all)). This resulted in ~500bp fragments for ~80 % of the chromatin. After centrifugation (4°C, 20min, 16000g) the soluble chromatin in the supernatant was diluted, aliquoted and quick-frozen in liquid nitrogen prior to storage at −80°C.

For ChIPs, 110 or 250 μl of chromatin were incubated with antibodies (anti-L(3)mbt1211-1477aa and anti-dSfmbt531-980aa) at 4°C O/N. DNA pellets were re-suspended in 500 μl H2O prior to storage at −20°C (10 μl per qPCR reaction, triplicates). For ChIP-Seq 12 ChIPs were pooled into 30 μl H2O and used for library preparation according to the Illumina ChIP-Seq protocol. Primer sequences are shown below.

ChIP-Seq mapping and data analysis

All uniquely mappable reads from two biological replicates were mapped to the genome (FlyBase 5.27) using Bowtie 0.12.5 73. Since our data set contained an unusually high number of reads (73 million reads), we developed new peakfinder software called PyPeak that was optimized for rich data sets. For low-ranking peaks PyPeak was comparable to the commonly used MACS software 74 whereas it was superior in identifying high ranking peaks (Supplementary Fig. S7d). PyPeak used the high number of sequences to refine the identified peaks and often identified two independent binding sites where MACS predicted one wide binding region (data not shown). The false discovery rate (FDR) was estimated as the ratio of the number of peaks called in the control to the number of peaks called for the ChIP data for a given threshold. To define a cut off for “true bound regions” we plotted the calculated false discovery rate (FDR) against the peak score (Fig. S6b). We identified a highly stringent set of 3314 L(3)mbt bound regions at a 0.5% FDR, as well as a larger set of 4572 bound regions at a 3% FDR, including more low occupancy peaks. Analysis of the binding site distribution showed that L(3)mbt bound preferentially between 700bp upstream and 200bp downstream of transcriptional start sites (Fig. 6c). Within this promoter proximal section the most frequent L(3)mbt binding site was at 250bp upstream of the TSS. Remarkably, this “peak” contained two maxima with a distance of approximately 150bp.

All ChIP-Seq datasets have been deposited to the GEO repository (GSE29206). The peak finding software is available under the terms of the GNU General Public License and can be downloaded at The frequency of peak distances with respect to the closest TSS was computed.

Data analyses were done using the Drosophila melanogaster BDGP Release 5 (UCSC dm3) genome assembly and the Flybase 5.26 genome annotation release. ChIP-Seq tracks were visualized with the Integrated Genome Browser (IGB) 75. As background for all L(3)mbt target gene analyses all annotated D.melanogaster genes were used.

We assigned to each of the 3314 and 4572 L(3)mbt bound regions (0.5% FDR and 3% FDR) the gene with closest TSS from the peak summit of the region. Bound regions further than ±5kb from a TSS were left without assigned gene (311of 3314 or 9.3% and 415 of 4572 or 9%). 2730 (0.5% FDR) and 3543 (3% FDR) unique genes were assigned as target genes (table S1). The 2730 (0.5% FDR) target genes were tested for enriched GO slim term annotations ( with a hypergeometric distribution test. As the number of tested terms is small, p-values were not adjusted for multiple testing.

The Ontologizer application 76 was adapted to test the 3543 (3 % FDR) target genes for enrichment in KEGG pathways (, Release 53.0) and for enriched Gene Ontology (GO) term annotations ( (biological process subontology)). The significance (posterior probability) of this enrichment is based on a Bayesian model-based gene set analysis (MGSA) 48.

Motif analysis

De novo motif discovery was performed on 100bp long regions centered on the peak summit of the 3314 L(3)mbt bound regions (0.5% FDR) using RSAT (Oligo-analysis tool, Pattern-assembly tool (occ_sig>2 and maximum substitution 1) and Convert-matrix tool for position weight matrix (PWM) conversions) 77 and MEME (parameters: distribution of zero or one occurrence per sequence model, allowing sites on + and − strands, 6 as minimum and 25 as maximum motif width, and as background model a 2-order Markov model) 78 tools. The background in these and all following analyses consisted of repeat-masked sequences around the TSS (−700 to +200bp) of all annotated D.melanogaster genes. The GGTT motif was only found using MEME.

Binding site predictions were generated with the Patser tool 79 using the PWMs. The Patser score cut-offs varied for each motif, from ls2 to ls0. Motif enrichment was computed as the ratio of the observed motif frequency in the 3314 L(3)mbt bound regions (0.5% FDR) and in the background. Significance was assessed using a Fisher’s exact test. The fraction of regions with motif was assessed in the ranked 4572 L(3)mbt bound regions (3% FDR) and the number of motifs identified in each of the 3314 L(3)mbt bound regions (0.5% FDR) was counted.

For Venn diagram counts two or more regions that overlap with at least one base were merged and defined as a ‘common’ region. Overlaps were generated using the 3314 L(3)mbt bound regions (0.5% FDR) and regions bound by insulator-associated proteins CP190 (6648), BEAF-32 (4706), CTCF (2487, overlap CTCF-N and CTCF-C) and Su(Hw) (3258, overlap of Su(Hw)-1 and Su(Hw)-2 51).


Competing financial interests

The authors declare no competing financial interests.


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