Human ERG oncoprotein represses a Drosophila LIM domain binding protein–coding gene Chip

The proximal and distal domains of the developing wing imaginal disc of Drosophila give rise to the adult thorax (notum) and wing proper, which are held together by a hinge domain. Ap, LIM–HD, and Engrailed (En), homeobox, transcription factors, respectively, specify the dorsal (D) and posterior (P) compartments of the wing primordium [Fig. 1A, for review, see ref. 26]. DV and AP boundaries of the developing wing double up as signaling centers regulating its anisotropic growth by sending out Wg (a Wnt) and Dpp (a BMP) morphogens, respectively [for recent articles, see refs. 27–29]. The presumptive adult wing is marked by a POU-domain protein, Nubbin [Nub, Fig. 1B; (30)], a target of Wg [Fig. 1C; (30–33)]. Nub also marks the inner of the two epithelial folds of the presumptive wing hinge region [blue arrowhead, Fig. 1B, also see refs. 31].

To examine the fallout of a heterologous gain of ERG oncoprotein, we drove its expression in the larval wing imaginal discs under four different Gal4 drivers individually. These were en-Gal4 [en>GFP, Fig. 1A, (35)], vg-Gal [boundary enhancer-BE, vg>GFP Fig. 1D, (36)], ci-Gal4 [ci>GFP, SI Appendix, Fig. S1A, (37)], and dpp-Gal4 [dpp>GFP, SI Appendix, Fig S1C, (38)]. Gain of ERG under vg-Gal4 (star, Fig. 1E) or en-Gal4 driver (stars, Fig. 1 F–I) induced a striking fallout, namely, ectopic expression of Nub in the presumptive notum, revealing a notum-to-wing cell fate switch or transdetermination [for review, see ref. 39]. In extreme scenarios, the transdetermined wing primordium (star, Fig. 1G) outgrew its endogenous counterpart. More than one transdetermined wing primordia were infrequently seen in en>ERG notum (stars, Fig. 1 H and I). Frequencies of transdetermined wings progressively declined in the order of the Gal4 used: namely, en-Gal4, vg-Gal4, dpp-Gal4, and ci-Gal4 (Fig. 1J). Notably, plotting the domains of these Gal4 drivers revealed a shared feature: their expression within or abutting the presumptive posterior notum of the wing imaginal disc wherein out-of-context Nub was induced (yellow stippled dots, Fig. 1K). Thus, ERG-gain under these Gal4 drivers induced ectopic Nub in only the posterior notum, even while their expressions extended far beyond this domain. The posterior notum, therefore, displayed hallmarks of a hotspot: that is, a sensitive zone for cell fate switch as seen during regeneration of the leg (40) or upon gain of Eyeless (Ey) master transcription factor in the wing, leg, eye, and haltere imaginal discs (23, 41).

Wg expression is seen in an anterior ventral wedge (42) in the distal wing imaginal disc of the early second larval instar (33). These Wg-expressing cells represent the progenitors of the future adult wing (42) and are marked by expression of Nub and, Vestigial, Vg (33), the latter being a wing cell fate selector (43). Subsequently, during third larval instar, growth and patterning of the wing primordium are driven by Wg morphogen synthesized and secreted from its DV signaling center (28, 29, 33, 42, 44). -The third larval instar wing imaginal disc, therefore, Wg displays its characteristic, spatially restricted morphogen signaling from the DV boundary, which drives wing growth and patterning (27–29, 33).

N regulates Wg during second phase of its wing growth-promoting role (27–29, 44–46). We noted that the gain of a constitutively active N receptor, N^intra (47), too, induced a notum-to-wing cell fate switch (star in Fig. 1L), reminiscent of that seen upon the gain of ERG (Fig. 1 E–I). Conversely, coexpression of ERG and a dominant-negative form of N, namely, N^DN (48), or that of its downstream target, Mastermind, Mam^DN (49), extinguished wing transdetermination in the posterior notum (blue star, Fig. 1 M and N). Further, coexpression of ERG and a membrane-anchored Frizzled (Fz) receptor, GPI-dFz2—which tethers Wg ligand to the membrane arresting its signal transduction (50)—suppressed wing transdetermination (blue star, Fig. 1 O and P). Finally, ERG gain in the haltere induced metanotum-to-capitellum transdetermination (Fig. 1 Q–S), which is anticipated given that these two dorsal appendages, the wing and haltere, share common developmental ground plan and genetic tool kits (34).

ERG-expressing second instar larval wing imaginal disc, however, did not display notum-to-wing transdetermination, as revealed by the absence of Nub expression (blue star, en>ERG, Fig. 2A), while at this stage, the endogenous wing primordium displayed its characteristic expression (arrow, en>ERG, Fig. 2A). Subsequently, in a mid-third instar vg>ERG wing imaginal disc, we noticed induction of Nub (31–33) at a far posterior margin of its notum (star, Fig. 2 B and B’). Induction of Nub expression in these cells of notum by ERG was cell-autonomous (yellow arrowheads in Fig. 2 B’ and B” and XZ optical section in Fig. 2B’) as well as non-cell autonomous (red arrowhead in Fig. 2 B’ and B” and XZ section in Fig. 2B’). These characteristics were further quantified by their fluorescence intensities (Fig. 2C) and colocalization (Fig. 2D, see SI Appendix, Methods). These results suggest that Wg induced in ERG-expressing cells of the posterior notum is secreted. Indeed, ERG-expressing somatic clones in the posterior notum displayed cell-autonomous (yellow arrowhead, broken line, Fig. 2 E and E”) and extensive non-cell-autonomous notum-to-wing cell fate switches (red arrowhead, Nub, Fig. 2 E and E” ), as can be anticipated from their secretion of the long-range Wg morphogen (51). Moreover, we also noticed cell-autonomous and non-cell-autonomous Wg expressions, respectively, within (broken line, Fig. 2 F’ and F” ) and around (arrow, Fig. 2 F’ and F”) the ERG-expressing clone in the posterior notum.

Starting mid-third larval instar stage, en>ERG wing imaginal discs displayed Wg expression in the notum, straddling the anterior (A) and posterior (P) compartment boundary (broken line, Fig. 2G). In older third larval wing imaginal discs, cell-autonomous and non-cell-autonomous Wg expressions in transdetermined wing primordia were more pronounced (Fig. 2 H and I). Finally, we also noticed instances where Wg expression in the transdetermined wing matched its endogenous counterpart (Fig. 2 J and J' ). In ERG-expressing adult thorax, we noticed amorphic wing tissue growth (stars, Fig. 2 K and L) to transdetermination into wing proper that appeared patterned (stars, Fig. 2 M and N), like their endogenous counterparts. Overall, about two-thirds of the eclosed vg>ERG adults displayed notum-to-wing transdetermination (Fig. 2O). Further, transdetermined wing primordia showed growth along their AP, DV, and PD axes (SI Appendix, Fig. S1 E–H), suggesting their anisotropic growth via AP and DV morphogen-signaling centers, much like their endogenous counterpart (27, 29).

Our results reveal that ERG-triggered Wg induces both cell-autonomous and non-cell-autonomous notum-to-wing cell fate switches (Fig. 2P). A near-perfect patterning of the transdetermined wing, although infrequent, suggests the development of DV and AP signaling centers in these transdetermined wings by mechanism(s) that can only be speculated based on the emergent understanding of the growth and patterning in the endogenous wing primordium [(27–29, 33, 42, 44), see Discussion].

LIM–HD protein complexes maintain developmental domain-specific cellular signaling and cell fate during the development of Drosophila appendages (5, 52, 53). For instance, Chip–Tup (52, 54) and Chip–Ap (4, 5) are, respectively, active in the proximal (notum) and distal (wing) domains of the wing imaginal disc, maintaining their respective cell fates. Therefore, it is plausible that the loss of Chip, Tup, or both underlies the notum-to-wing cell fate switch seen upon ERG gain.

We noticed a transcriptional downregulation of Chip in en>ERG wing imaginal discs (Fig. 3 A–C). Therefore, ERG-induced notum-to-wing transdetermination is likely linked to Chip loss. In agreement, we noticed that compromising Chip activity by expressing a dominant-negative form of Chip, Chip^Δoid—which partially lacks its other interacting domain, OID (55)—in somatic clones (Fig. 3D) or under the en-Gal4 driver (Fig. 3E and SI Appendix, Fig. S4C)induced ectopic Nub expression in the notum. We also recovered vg-Gal4>Chip^Δoid adults, which showed de novo wing development in the thorax (Fig. 3F). Further, knockdown of Chip (en>Chip-RNAi, Fig. 3G), too, phenocopied ERG gain (see Figs. 1 and 2). Conversely, a simultaneous gain of Chip and ERG abrogated de novo Nub (Fig. 3H) or Wg (Fig. 3I) expressions in the notum. These results reveal that ERG-induced notum-to-wing transdetermination entails a downregulation of Chip.

ERG-mediated repression of Chip is also likely to affect Chip–Ap tetramers formed in the dorsal wing pouch (4, 5). We noted that the ap expression revealed by its ap-lacZ reporter in the dorsal wing primordium was not perturbed by ERG gain (SI Appendix, Fig. S2A). In the notum, ap displays a non-uniform pattern of expression (ap-lacZ, SI Appendix, Fig. S2 B–D). We reasoned that ERG-induced Chip downregulation—and consequently, loss of Chip–Ap tetramers in the dorsal wing—might compromise Ap activity, which likely culminates in the development of de novo Ap⁺/Ap⁻ boundary: that is, DV signaling center (57, 58). Surprisingly, a ubiquitous gain of ERG (Fig. 1) or loss of Chip (Fig. 3) under different Gal4 drivers did not display signs of ectopic DV signaling center development in the endogenous wing primordium. By contrast, ERG-expressing somatic clones induced ectopic DV signaling centers in only select spatial domain of dorsal wing primordium (SI Appendix, Fig. S2 E–F) while Chip loss-of-function clones invariably induce ectopic DV boundaries in the dorsal wing pouch (59). Thus, ERG-expressing clones might cause a partial and incomplete depletion of Chip levels. Alternatively, it is also plausible that the Chip–Tup complex in the notum is more sensitive to ERG gain than Chip–Ap.

Ets21C [synonym Ets6, (60)] is a Drosophila ortholog of ERG that displays 86% and 26% identity of its ETS- and PNT-domains with those of its human counterpart, respectively (60–63). Ets21C expression is triggered during regeneration and tumorigenesis (64, 65), while its gain in oncogenically targeted cells induces cooperative carcinogenesis in Drosophila (62, 63, 65, 66). We noted that ectopic gain of Ets21C induced Nub and Wg expressions in the posterior notum (Fig. 3 J–K), albeit far less strikingly than that seen upon ERG gain (Figs. 1 and 2) or Chip loss (Fig. 3 A–F). Moreover, a gain of Ets21C under en-Gal4 driver displayed suppression of N target, Cut (Fig. 3L) at the wing primordium's DV boundary, culminating in characteristic notched wing phenotypes in adult flies (Fig. 3M).

We further noted that the DNA-binding sequence of ERG (Fig. 3N) and Ets21C (Fig. 3O) are highly conserved [CCGGAA (Fig. 3 N and O)]. To examine if Chip is a transcriptional target of ERG and Ets21C, we examined a −3.0kb region upstream of its TSS (transcription start site) for putative Ets21C/ERG binding site (EBS). Such an upstream region of Chip may serve as its CRM (cis-regulatory module). We found nine putative Ets21C-binding sites in this −3.0-kb CRM of Chip; eight of these were putative ERG binding sites, (SI Appendix, Fig. S2C). Next, we looked for conservation of the EBS containing CRM of Chip across different insect species. Two standard bioinformatics analysis methods were used for the estimation of evolutionary conservation: phyloP (44, 67) and phastCons [(68), see SI Appendix, Methods] across 27 insect species available at the UCSC genome browser portal. We noticed positive phyloP scores for most nucleotides in this CRM of Chip (SI Appendix, Fig. S3A). Likewise, phastCons analysis, too, revealed stretches of nucleotide runs with a positive score (SI Appendix, Fig. S3A), indicating their conservation across all 27 insect species. We noted that six of the predicted EBSs were located within “Conserved Elements” based on phastCons scores (Fig. 3P and SI Appendix, Fig. S3B). Finally, chromatin immunoprecipitation (ChIP) of en>ERG imaginal discs confirmed the binding of ERG on three (EBS1, EBS6, and EBS7) of the nine EBSs in the CRM of Chip (Fig. 3 Q and R).

While the expression of Chip is ubiquitous throughout the wing imaginal discs (69), its LIM–HD-binding partner, Tup, is selectively expressed in the notum of the wing primordium [Fig. 4A, see refs. 54], that largely overlaps with the domain of expression of the posterior cell fate-specifying selector, En (also see Fig. 1A). Consequently, the Chip–Tup tetrameric complex remains restricted to the Tup-expressing cell of the notum. Not surprisingly, tup knockdown induced selective notum-to-wing transdetermination in the Tup-expressing cells of the notum (Nub, Fig. 4 B and C; Cut, SI Appendix, Fig. S4D, also see Fig. 1 E and F”), reminiscent of that seen upon ERG-induced Chip loss. Loss of Tup in the distal wing did not down-regulate N-Wg signaling (SI Appendix, Fig. S4 D and D”)—unlike that seen upon loss of Chip (Fig. 3 D–G)—which is consistent with the fact that the Chip–Tup complex is formed only in the posterior notum. Further, neither Chip loss (Fig. 4D) nor ERG gain (Fig. 4E) repressed Tup expression. Finally, tup gain—unlike that of Chip (see Fig. 3 H and I )—failed to arrest ERG-induced notum-to-wing transdetermination (Fig. 4F). These results reveal that spatial regulation of Tup (Fig. 4G) dictates the fallout of heterologous ERG, although it is not a direct target of repression by the latter.

By a direct binding on EZH2 (19), ERG upregulates its expression in different cancers [for review, see ref. 70]. In ERG-expressing wing epithelium, E(z) (71), a Drosophila homolog of mammalian EZH2, was seen upregulated (en>ERG, Fig. 5A). Further, in a −3.0kb CRM upstream of E(z) TSS, we notice three putative Ets21C-binding sites, two of which were also putative ERG binding sites (Fig. 5B and SI Appendix, Fig. S5C). Notably, this E(z) CRM was conserved across 27 insect species (SI Appendix, Fig. S5 A and B). Finally, we confirmed the binding of ERG on EBS1 through ChIP from en>ERG imaginal discs using ERG antibody (Fig. 5C).

E(z) is a transcriptional repressor, a core component of the Polycomb repressive complex 2, PRC2, which binds to the PREs of its targets [see refs. 72 and 73 and SI Appendix, Methods]. ERG-induced E(z) upregulation may thus contribute to the repression of ERG targets, such as Chip. Indeed, we noticed three PREs within the -3.0 kb CRM of Chip (Fig. 5D) that also harbored the EBSs (see Fig. 3P). These PREs were enriched in the ChIP of ERG-expressing wing imaginal discs [E(z)-GFP, en>ERG, Fig. 5 E and F] and displayed the trimethylation of lysine 27 on histone H3, H3K27me3 (Fig. 5G), a hallmark of E(z)-mediated epigenetic silencing (73).

The preceding observations thus suggest an E(z)-mediated silencing of Chip in ERG-expressing imaginal discs. To further test this interpretation, we fed vg>ERG larvae on food supplemented with a well-characterized inhibitor of EZH2, GSK126 (74). These animals display a progressive, concentration-dependent suppression of notum-to-wing transdetermination in vg>ERG animals (Fig. 5H). Likewise, a knockdown of E(z) in ERG-expressing notal epithelium (Fig. 5 I and J) extinguished notum-to-wing transdetermination. Thus, ERG directly represses Chip (Fig. 5K) on the one hand, and on the other, its upregulation of E(z) leads to epigenetic silencing of the former (Fig. 5L).

Drosophila displays a well-known two-hit (75, 76) paradigm of cooperative carcinogenesis (76) in select developmental lineages (77–79). In cells displaying loss of a tumor suppressor, like Lgl, tumor progression is often driven via the recruitment of endogenously active signaling pathways (78). Given these developmental underpinnings of cooperative carcinogenesis, we reasoned that ERG gain might display lineage-restricted tumor cooperation in the posterior notum. Indeed, in the posterior notum, lgl^-; ERG⁺ clones displayed Nub expression (star, Fig. 6A)—reminiscent of that seen upon ERG gain in the posterior notum (see Fig. 2 B and B’). Further, lgl^-; ERG⁺ somatic clones displayed synthesis and secretion of Wg (Fig. 6 B and B’ )—reminiscent of morphogen-sending neoplastic clones (80, 81)— besides inducing non-cell-autonomous hyperproliferation in the tumor microenvironment (arrow, PH3, red, Fig. 6 C and D). In the rest of the mosaic wing imaginal disc, lgl^-; ERG⁺ clones largely failed to display neoplastic transformation (white arrowheads, Fig. 6 A–C). Notably, a gain of N signaling in lgl clones, too, induced notum-to-wing cell fate switch and neoplastic transformation in the posterior notum (lgl^-; N ^intra, SI Appendix, Fig. S6 A and B) phenocopying their lgl^-; ERG⁺ counterpart (Fig. 6A). Finally, we note that Chip gain (Fig. 6 E and E'), E(z) knockdown (Fig. 6 F and F’), or sequestration of the Wg ligand (Fig. 6 G and G' ) in lgl^-; ERG⁺ clones arrested their neoplastic transformation (white arrowheads, Fig. 6 E'–G' ). Comparable results were also obtained by feeding host larvae with lgl^-; ERG⁺ mosaic discs on food supplemented with an E(z) inhibitor, GSK126 (white arrowheads, Fig. 6 H and H').

Together, these results reveal that an ERG-induced, lineage-specific, cooperative carcinogenesis stems from its downregulation of Chip, leading to loss of Chip–Tup, LIM–HD complex, triggering an out-of-context N-mediated Wg signaling (Fig. 6I).

We noticed that a protein–protein interaction map centered on the Chip–Tup complex of Drosophila was comparable with its mammalian counterparts (SI Appendix, Fig. S7A and also see ref. 82, (83), suggesting its ancient origin. We thus further asked if ERG targets the LDB genes, the mammalian homologs of Drosophila Chip; namely, LDB1 and LDB2, its two isoforms (84). ERG transcription and protein levels are minimal or absent in the healthy prostatic epithelium, whereas LDB1 and LDB2 show robust levels (SI Appendix, Fig. S7 B–G). Thus, we reasoned that ERG gain might target the repression of these LDBs. We chose to test this hypothesis in ERG-positive or -negative PCa cell lines. ChIP-Seq data of ERG-positive VCaP cell line [GSE28950, (84)] revealed binding peaks of ERG on the LDB1 promoter (Fig. 7A) but not on that of LDB2 (Fig. 7B). This binding peak was no longer seen in ChIP-Seq data of the VCaP cell line that displays knockdown of ERG [Fig. 7C, (GSE110655)]. We further noted the presence of a putative ERG-binding site (EBS) on the LDB1 promoter (Fig. 7D), reminiscent of that seen in its Drosophila counterpart, Chip (see Fig. 3P). In agreement, in ChIP-qPCR using ERG-positive VCaP cells, we noticed the binding of ERG on the LDB1 promoter (ERG_LDB1 Fig. 7 E and F), suggesting a possible causal underpinning of transcriptional downregulation of the latter in this cell line. Conversely, a knockdown of ERG in the VCaP cells up-regulated LDB1 expression (Fig. 7G). Gene expression data of an ERG-negative benign RWPE-1 prostate cell line revealed LDB1 downregulation upon the gain of ERG (Fig. 7H). In contrast, gene expression data of an ERG-positive VCaP cells, ERG-knockdown up-regulated LDB1 expression (Fig. 7I), further reconfirming an inverse association between ERG and LDB1. Finally, we also noticed the downregulation of EZH2 upon ERG-knockdown in ERG-positive VCaP cells (Fig. 7J). Thus, EZH2 is a transcriptional target of ERG in mammalian cancers (19, 84) reminiscent of that seen in Drosophila epithelium (Fig. 4). Together, these results reveal that ERG targets identified from Drosophila are conserved and functionally relevant for ERG-driven carcinogenesis in human.

Our results show that heterologous ERG oncoprotein targets repression of Drosophila Chip. One of the most striking fallouts of ERG gain is registered in the developing posterior notum of the wing imaginal disc, wherein disruption of Chip–Tup, LIM–HD complex leads to out-of-context N-Wg signaling. That ERG could be a transcriptional repressor of Chip/LDB1 was not predictable from the large body of literature directed at identifying ERG targets in diverse cancers [for reviews, see refs. 25 and 85]. Moreover, although we could identify EBS on human LDB1 promoter from ChIP-seq data (Fig. 7) published previously (84), its relevance in cancer progression was not evident in the absence of a display of causal association. Finally, the shared consensus binding sequence of human ERG and fly Ets21C on the CRMs of Chip and E(z) reveal a rationale for discovering functionally relevant human oncoprotein targets in Drosophila. Given the deep homology of essential genetic tool kits of development, it is also not surprising that ERG-positive prostate cancer cells display suppression of LDB1, an ERG target revealed in Drosophila.

Our results further show that the spatial limits of expression of the LIM–HD complexes underlie lineage-specific ERG-induced carcinogenesis. By extension, the developmental history of an oncoprotein-targeted cell prefigures its propensities to become cancer cells-of-origin (see refs. 78 and 86). This essential principle of lineage-specific carcinogenesis may hold for cancers that entail disruption of the LIM–HD complexes. For instance, LDB1-mediated Wnt signaling appears to play a more significant role in proximal colorectal cancer than in distal (87). Likewise, different LIM–HD complex-dependent regulations of N and Wnt signaling could underpin cell-type specificity of ERG-induced cancers (16–18).

Our results show that ERG-induced Wg synthesis initially specifies wing cell fate-specification in the notum and subsequently drives its growth (33, 42, 44). Notably, de novo Wg (this study) or Dpp (81, 88) morphogen signaling centers thus drive tumor progression in cooperation with an oncogenic lesion. An ERG-induced Wg-signaling underlies its diverse, context-specific fallouts: cell fate switch alone or cell fate switch-linked tumor development in the posterior notum (Fig. 8). These findings reaffirm the maxim that carcinogenesis is essentially development gone awry (89, 90).

While the ERG-induced Wg in the posterior notum leads to amorphous wing tissue growth from the adult thorax (see Fig. 2), the shapes and sizes of some of these transdetermined wing primordia (Fig. 1) or the adult wings also suggest their acquisition of a near-perfect orthogonal positioning of AP (Dpp morphogen) and DV (Wg morphogen) signaling centers [Fig. 1 and see ref. 27]. We speculate that an initial non-cell-autonomous wing cell fate specification via secreted Wg from ERG-expressing cells of the posterior notum may underlie this phenomenon of near-perfect, albeit infrequent, positioning of morphogen-signaling centers. For instance, when a transdetermined wing primordium straddles an AP boundary (see Fig. 2B), the latter could provide the source of Dpp morphogen for a comprehensive wing patterning. A far more complex scenario may underlie the development of a Wg-morphogen-sending DV signaling center in a transdetermined wing primordia. For instance, since Ap expression is non-uniform in the notum (SI Appendix, Fig. S2), a non-cell-autonomous wing cell fate specification via secreted Wg may juxtapose domains of high and low Ap, reminiscent of that seen in the DV boundary of the endogenous wing (57). Alternatively, Wg signaling from a spatially aligned center in the posterior notum may be prefigured by an initial ERG-induced activation along polar coordinates of the transdetermined wing (42, 91). Further, Wg expression in the transdetermined wing may also display a feed-forward propagation of signals for its growth and patterning (29, 92).

Drosophila provides unparalleled advantages in the genetic identification of tumor suppressors and oncogenes that control cellular functions ranging from maintenance of apicobasal polarity, chromatin architecture, and vesicular trafficking, to name a few (93). The genetic tractability of Drosophila has helped unravel elusive cancer mechanisms (78, 81, 94). Drosophila also displays the two-hit model of carcinogenesis seen in mammals (75, 76, 79). Further, modeling of human cancer in Drosophila is often based on a heterologous gain of an activated oncogene (95). Results presented here reveal an approach based on the deep homology of essential genetic tool kits and their crosstalks aided by the conservation of transcription factor-binding sites, like EBS shown here, on the oncoprotein targets. In turn, phenotypic fallouts of heterologous gain of an oncoprotein offer clues to its crosstalk with a diverse set of functionally relevant pathways that could double up as oncogenic signaling nodes. For instance, activation of Wg signaling in the notum was also reported earlier upon loss of Osa (32) or subunits of BAP (Brm-associated protein) (96), which are members of a highly conserved chromatin remodeling complex. Osa/BAP, therefore, could be part of the ERG signaling network. Indeed, the binding of ERG to the BAP/BAF chromatin remodeling complex has been reported in prostate cancer (97).

Drosophila stocks and method for generation of transgenic fly line and clones (78) are described in SI Appendix, Table S1.

Method and antibodies are described in SI Appendix.

ChIP was performed using LowCell# ChIP kit protein A (Diagenode, C01010072). For details, see SI Appendix. RNA was extracted using TRIZOL followed by cDNA synthesis (Invitrogen). Fold changes for individual genes was quantified using ΔΔCt method, with GAPDH as internal control. Primer details in SI Appendix, Table S2.

Publicly available ChIP-Seq data (GSE28950, GSE116055) were used to determine the recruitment of ERG on LDB1 and LDB2 promoter. For details, see SI Appendix.

JASPAR was used to look for putative Ets21C/ERG binding sites (EBSs) in E(z) and Chip promoter, and PREs, in the Chip promoter (for further details, see SI Appendix). Evolutionary conservation of Ets21C/ERG binding on Chip and E(z) upstream regulatory sequence was done using PHAST package at the USCS genome browser. Further details are presented in SI Appendix.

Datasets (GSE86232, GSE110656) were analyzed for LDB1 expression. For detail, see SI Appendix.

M.B., A.B., B.A., and P.S. designed research; M.B. and N.M. performed research; M.B. and U.R. contributed new reagents/analytic tools; M.B., A.B., and N.M. analyzed data; B.A. and P.S. conceptualization; and M.B., A.B., B.A., and P.S. wrote the paper.

The authors declare no competing interest.