ABSTRACT

Here, we report novel tumour suppressor activity for the Drosophila Argonaute family RNA-binding protein AGO1, a component of the miRNA-dependent RNA-induced silencing complex (RISC). The mechanism for growth inhibition does not, however, involve canonical roles as part of the RISC; rather, AGO1 controls cell and tissue growth by functioning as a direct transcriptional repressor of the master regulator of growth, Myc. AGO1 depletion in wing imaginal discs drives a significant increase in ribosome biogenesis, nucleolar expansion and cell growth in a manner dependent on Myc abundance. Moreover, increased Myc promoter activity and elevated Myc mRNA in AGO1-depleted animals requires RNA polymerase II transcription. Further support for transcriptional AGO1 functions is provided by physical interaction with the RNA polymerase II transcriptional machinery (chromatin remodelling factors and Mediator Complex), punctate nuclear localisation in euchromatic regions and overlap with Polycomb Group transcriptional silencing loci. Moreover, significant AGO1 enrichment is observed on the Myc promoter and AGO1 interacts with the Myc transcriptional activator Psi. Together, our data show that Drosophila AGO1 functions outside of the RISC to repress Myc transcription and inhibit developmental cell and tissue growth.

This article has an associated ‘The people behind the papers’ interview.

INTRODUCTION

Tightly coordinated regulation of cell and tissue growth is essential for animal development. Decreased growth leads to small organs and diminished body size, whereas heightened proliferative growth is associated with genomic instability and cancer. The MYC transcription factor and growth regulator has been studied extensively since its identification as an oncogene in the early 1980s (Vennstrom et al., 1982), when MYC overexpression caused by chromosomal translocation was found to drive malignant transformation in Burkitt's lymphoma (Dalla-Favera et al., 1982; Taub et al., 1982). Research in subsequent decades implicated increased MYC in progression of most tumours (Dang, 2012; Liao and Dickson, 2000; Meyer and Penn, 2008). In normal adult tissues, MYC expression is relatively low and generally restricted to cells with regenerative and proliferative potential (Marcu et al., 1992). Even small increases in MYC abundance are sufficient to promote proliferative cell growth (reviewed by Dang, 2010; Levens, 2010; Zaytseva and Quinn, 2017); thus, understanding the molecular control of MYC expression can provide crucial insight into the mechanisms of MYC dysregulation in cancer.

In normal cells, MYC is regulated by signalling inputs from a diverse array of developmental and growth signalling pathways (Zaytseva and Quinn, 2017). The many cellular signalling inputs converging on MYC transcription are integrated by FUBP1, a KH domain protein that binds single-stranded DNA and interacts with the general transcription factor complex TFIIH to modulate MYC promoter output (Chung and Levens, 2005; Chung et al., 2006; He et al., 2000; Liu et al., 2006; Zhang and Chen, 2013). The mammalian FUBP family comprises three proteins (FUBP1-3) (Zhang and Chen, 2013), which are represented by one orthologue in Drosophila, Psi. Like FUBP1, Psi also interacts with the RNA polymerase II (RNA Pol II) transcriptional machinery, particularly the transcriptional Mediator (MED) complex, to pattern Myc transcription and cell and tissue growth in the Drosophila wing epithelium (Guo et al., 2016). In addition to roles in transcription, Psi binds RNA via the KH domains and interacts with the spliceosome to regulate mRNA splicing (Labourier et al., 2001; Wang et al., 2016). Although co-immunoprecipitation (co-IP) mass spectrometry detected Psi in complex with the Argonaute protein AGO1 (Guo et al., 2016), the potential significance of this interaction is unknown.

Argonaute proteins comprise the core of the RNA-induced silencing complex (RISC), which uses noncoding RNA as a guide to target mRNAs for post-transcriptional gene silencing. Drosophila AGO2 is best characterised as part of the siRNA-induced silencing complex (siRISC) (Okamura et al., 2004), whereas AGO1 predominantly functions in microRNA-induced silencing complexes (miRISCs) and post-transcriptional mRNA silencing (Förstemann et al., 2007). Of importance to this study, AGO1-mediated mRNA silencing has been implicated in transcript destabilisation and translational repression of Myc in flies (Daneshvar et al., 2013) and humans (Challagundla et al., 2011). Here, we report a novel role for AGO1 as a direct Myc transcriptional repressor and demonstrate that this underlies cell growth inhibition. AGO1 depletion not only increases Myc promoter activity, mRNA and protein abundance, but the elevated Myc expression requires RNA Pol II transcriptional activity. Localisation to the nucleus, together with interaction with transcriptional machinery and significant AGO1 enrichment on the Myc promoter suggests, in addition to the established roles in miRNA silencing in the cytoplasm, AGO1 constrains Myc transcription to control cell and tissue growth during Drosophila development.

RESULTS

AGO1 interacts with Psi and RNA Pol II transcriptional machinery

The single-stranded DNA/RNA binding protein Psi has essential roles in Myc transcriptional control and RNA processing in Drosophila. In addition to physically and genetically interacting with the RNA Pol II transcriptional machinery, the Drosophila Protein Interaction Map (DPiM) large scale co-IP mass spectrometry (Guruharsha et al., 2011) suggested association between Psi and AGO1 (Guo et al., 2016). Our analysis of the DPiM identified Psi as the most frequent AGO1 interacting partner (Fig. 1A). Ontological class analysis for the top 70 AGO1 interactors revealed RNA processing factors (49%), as expected; however, most (59%) interactors had ascribed functions in RNA Pol II transcription (Fig. 1A-C, note 10 factors are implicated in both transcription and RNA processing). As the DPiM studies were performed in vitro with overexpressed tagged proteins in Drosophila S2 cell lines, we validated the interaction between endogenous AGO1 and Psi in vivo using co-IP from wild-type third instar larval imaginal tissue. Immunoprecipitation using anti-Psi antibody, followed by anti-AGO1 western blot detected a 110 kDa band for AGO1 (Fig. 1D), whereas reciprocal IP with anti-AGO1 antibody precipitated the 97 kDa Psi band (Fig. 1E). The observation that endogenous AGO1 and Psi form a complex in vivo led us to investigate potential genetic interactions between AGO1 and Psi.

Fig. 1.

AGO1 interacts with RNA Pol II transcriptional machinery and RNA processing factors. (A) List of top 70 AGO1 interactors from the Drosophila Protein Interaction Map (DPiM) dataset (Guruharsha et al., 2011). (B) Summary of ontology classes for the top 70 AGO1 interactors. (C) Intersection of interactors with functions in RNA Pol II transcription and/or RNA processing. (D,E) Co-IP of endogenous Psi and AGO1 from wild-type third instar larvae. (D) IP with anti-Psi and western blot with anti-AGO1 (110 kDa). (E) IP with anti-AGO1 and western blot with anti-Psi (97 kDa). Mock IP refers to no-antibody control.

Fig. 1.

AGO1 interacts with RNA Pol II transcriptional machinery and RNA processing factors. (A) List of top 70 AGO1 interactors from the Drosophila Protein Interaction Map (DPiM) dataset (Guruharsha et al., 2011). (B) Summary of ontology classes for the top 70 AGO1 interactors. (C) Intersection of interactors with functions in RNA Pol II transcription and/or RNA processing. (D,E) Co-IP of endogenous Psi and AGO1 from wild-type third instar larvae. (D) IP with anti-Psi and western blot with anti-AGO1 (110 kDa). (E) IP with anti-AGO1 and western blot with anti-Psi (97 kDa). Mock IP refers to no-antibody control.

Psi-dependent growth is sensitive to AGO1 abundance

Psi knockdown in the dorsal wing compartment results in a ‘wings up’ phenotype as impaired cell and tissue growth of the top layer of the wing results in torsional strain and wing bending (Guo et al., 2016). We therefore tested whether this was modulated by AGO1 depletion, using two independent P-element insertion mutants (AGO1k00208 and AGO104845). Interestingly, AGO1 heterozygosity alone was sufficient to increase wing size, suggesting that AGO1 normally constrains growth. Moreover, heterozygosity for either AGO1 mutant suppressed impaired tissue growth caused by Psi depletion (Fig. 2A-C). Thus, AGO1 normally functions as a negative growth regulator and the wing overgrowth associated with AGO1 reduction is dependent upon Psi. However, we need to be somewhat cautious in our interpretation of these data as either genotype alone results in a phenotype, where the outcome may be an intermediate phenotype between the larger wings from AGO1 heterozygotes and impaired growth from Psi knockdown.

Fig. 2.

Impaired cell and tissue growth in the Psi knockdown wing is sensitive to AGO1 levels. (A,B) Adult flies (A) and wings (B), with compartment below vein L5 outlined in red (genotypes in the ser-GAL4 background as marked). (C) Quantification of compartment area below vein L5 in individual adult wings in the ser-GAL4 background. Data points are measurements per individual wing, with mean±s.d. shown. Both AGO1 mutants were compared with the control; AGO1 mutants in Psi RNAi background were compared with Psi knockdown alone. Unpaired two-tailed t-test was used for all comparisons. ****P<0.0001.

Fig. 2.

Impaired cell and tissue growth in the Psi knockdown wing is sensitive to AGO1 levels. (A,B) Adult flies (A) and wings (B), with compartment below vein L5 outlined in red (genotypes in the ser-GAL4 background as marked). (C) Quantification of compartment area below vein L5 in individual adult wings in the ser-GAL4 background. Data points are measurements per individual wing, with mean±s.d. shown. Both AGO1 mutants were compared with the control; AGO1 mutants in Psi RNAi background were compared with Psi knockdown alone. Unpaired two-tailed t-test was used for all comparisons. ****P<0.0001.

AGO1 depletion drives cell growth in a Myc-dependent manner

To further examine the cellular basis of the tissue overgrowth associated with AGO1 depletion in specific compartments of the larval wing, we used two independent non-overlapping AGO1 RNAi lines. We first demonstrated efficient mRNA knockdown for both AGO1 lines in the wing (Fig. S1A) and dorsal compartment-specific protein knockdown 24 h after induction of ser-GAL4 (Fig. S1B). As pupal lethality and dorsal compartment cell death were associated with constitutive ser-GAL4 driven AGO1 knockdown (Fig. S2), the baculoviral caspase inhibitor p35 was co-expressed to prevent apoptosis (Hay et al., 1994) and enable investigation of potential changes to cell growth.

Cell growth requires ribosomal RNA (rRNA) synthesis, processing and assembly with ribosomal proteins (RPs) into 40S and 60S ribosomal subunits in the nucleolus. Thus, the size of this structure, as measured by nucleolar-specific fibrillarin antibody, provides an indirect measure of ribosome biogenesis (Mitchell et al., 2015). AGO1 depletion significantly increased nucleolar size (Fig. 3A,B), and cell size (Fig. 3C), suggesting that AGO1 normally functions to inhibit cell growth. Consistent with AGO1 depletion driving nucleolar expansion, at least in part a result of increased rDNA transcription, transient AGO1 knockdown significantly increased pre-rRNA levels and Polr1c (RNA polymerase I (subunit) mRNA (Fig. 3D). AGO1 depletion also significantly increased levels of the ribosomal protein subunits RpS19 and RpS24 (Fig. 3D). Together, these data suggest that AGO1 normally inhibits ribosome biogenesis and cell growth in the wing imaginal disc. Chromatin immunoprecipitation (ChIP) sequencing studies have previously identified AGO2 binding throughout the 47S region of the consensus rRNA gene in human cell lines (Atwood et al., 2016), suggesting direct roles for AGO proteins in rDNA transcription and/or rRNA processing. However, these observations do not explain the increase in expression of RNA Pol II-transcribed ribosomal proteins and RNA Pol I subunits, nor the increase in overall cell growth, which requires coordinated activity from all three RNA polymerases, RNA Pol I, II and III (the last required for transcription of 5S rRNA).

Fig. 3.

AGO1 knockdown increases ribosome biogenesis. (A) Third instar wing discs with ser-GAL4-driven AGO1 RNAi in the UAS-p35 background compared with control (ser-GAL4, p35 alone), marked with UAS-RFP and stained with anti-fibrillarin (green) and DAPI (blue). Far right panel shows magnification of the boxed areas. (B) Quantification of average nucleolar area of approximately 50-100 nucleoli within the dorsal compartment, taken from a confocal z-section through the wing discs with ser-GAL4 expression in p35 background (data points are average measurements per image, with mean±s.d. shown). AGO1 RNAi was compared with control. (C) Quantification of cell area marked by CD8-RFP, calculated as average of approximately 50-100 cells per wing disc, in the dorsal compartment of wing ser-GAL4 in p35 background (data points are average measurements per image, with mean±s.d. shown). AGO1 RNAi was compared with control. (D) qPCR for 47S pre-RNA (ETS), RNA Pol I subunit (Polr1c) and ribosomal proteins (RpS19 and RpS24) in wing discs (20 discs pooled for each data point, mean±s.d. shown). Expression was performed for 2 days using tub-GAL4 in the background of tub-GAL80ts. AGO1 knockdown in the wing significantly increased abundance of Polr1c, 47S rRNA 5′-ETS, RpS19 and RpS24 (P=0.0004 and P=0.0006, respectively) compared with the corresponding control. Unpaired two-tailed t-test was used for all comparisons. **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 3.

AGO1 knockdown increases ribosome biogenesis. (A) Third instar wing discs with ser-GAL4-driven AGO1 RNAi in the UAS-p35 background compared with control (ser-GAL4, p35 alone), marked with UAS-RFP and stained with anti-fibrillarin (green) and DAPI (blue). Far right panel shows magnification of the boxed areas. (B) Quantification of average nucleolar area of approximately 50-100 nucleoli within the dorsal compartment, taken from a confocal z-section through the wing discs with ser-GAL4 expression in p35 background (data points are average measurements per image, with mean±s.d. shown). AGO1 RNAi was compared with control. (C) Quantification of cell area marked by CD8-RFP, calculated as average of approximately 50-100 cells per wing disc, in the dorsal compartment of wing ser-GAL4 in p35 background (data points are average measurements per image, with mean±s.d. shown). AGO1 RNAi was compared with control. (D) qPCR for 47S pre-RNA (ETS), RNA Pol I subunit (Polr1c) and ribosomal proteins (RpS19 and RpS24) in wing discs (20 discs pooled for each data point, mean±s.d. shown). Expression was performed for 2 days using tub-GAL4 in the background of tub-GAL80ts. AGO1 knockdown in the wing significantly increased abundance of Polr1c, 47S rRNA 5′-ETS, RpS19 and RpS24 (P=0.0004 and P=0.0006, respectively) compared with the corresponding control. Unpaired two-tailed t-test was used for all comparisons. **P<0.01, ***P<0.001, ****P<0.0001.

MYC drives cell growth by stimulating transcription of all three RNA polymerases to upregulate ribosome production (Poortinga et al., 2014). MYC directly stimulates the initiation of RNA Pol I-mediated transcription in mammals (Arabi et al., 2005; Grandori et al., 2005; Shiue et al., 2009) and activates transcription of RNA Pol II-transcribed genes encoding the ribosomal proteins (RPs), rRNA processing factors and components of the nucleolus essential for ribosome biogenesis (Grandori et al., 2005; Grewal et al., 2005; Poortinga et al., 2011). Furthermore, MYC directly activates RNA Pol III transcription to increase 5S rRNA expression (for assembly of the large 60S ribosomal subunit) and tRNA for translation of mRNA into protein (Fernandez et al., 2003; Gomez-Roman et al., 2006; Oskarsson and Trumpp, 2005). In Drosophila, Myc depletion reduced nucleolar expansion in AGO1 knockdown wing cells to the control range (Fig. 4A,B). Co-depletion of the Myc-regulator Psi also significantly decreased nucleolar size compared with AGO1 knockdown alone (Fig. 4A,B). Importantly, given the intermediate effect between ser-GAL4 driven Psi knockdown and AGO1 heterozygotes in the adult wing (Fig. 2), Psi depletion alone did not modify nucleolar size. Thus, the increased ribosome biogenesis and cell overgrowth associated with AGO1 depletion is dependent on both Psi and Myc.

Fig. 4.

Increased nucleolar size as a result of AGO1 knockdown is dependent on Psi and Myc. (A) ser-GAL4-driven RNAi in RFP-labelled cells for the indicated genotypes in wing discs stained with anti-fibrillarin (green) and DAPI (blue). Far right panel shows magnification of regions in the white squares. (B) Quantification of average nucleolar area of approximately 50-100 nucleoli within the dorsal compartment, taken from a confocal z-section through the wing discs with ser-GAL4 expression in p35 background (data points are average measurements per image, mean±s.d. shown). Myc RNAi reduced nucleolar size compared with control. Psi or Myc co-depletion significantly reduced nucleolar size in the AGO1 RNAi background compared with AGO1 knockdown alone (P<0.0001 and P=0.0065 for Myc and Psi, respectively). Unpaired two-tailed t-test was used for all comparisons. NS, not significant; **P<0.01, ****P<0.0001.

Fig. 4.

Increased nucleolar size as a result of AGO1 knockdown is dependent on Psi and Myc. (A) ser-GAL4-driven RNAi in RFP-labelled cells for the indicated genotypes in wing discs stained with anti-fibrillarin (green) and DAPI (blue). Far right panel shows magnification of regions in the white squares. (B) Quantification of average nucleolar area of approximately 50-100 nucleoli within the dorsal compartment, taken from a confocal z-section through the wing discs with ser-GAL4 expression in p35 background (data points are average measurements per image, mean±s.d. shown). Myc RNAi reduced nucleolar size compared with control. Psi or Myc co-depletion significantly reduced nucleolar size in the AGO1 RNAi background compared with AGO1 knockdown alone (P<0.0001 and P=0.0065 for Myc and Psi, respectively). Unpaired two-tailed t-test was used for all comparisons. NS, not significant; **P<0.01, ****P<0.0001.

AGO1 depletion increases Myc abundance and function

The overgrowth observed in AGO1 knockdown wing imaginal disc cells (Figs 3,4) was associated with a significant increase in Myc mRNA, which was reduced by Psi co-knockdown (Fig. 5A). AGO1 knockdown also increased Psi mRNA (Fig. 5B) and protein abundance (Fig. S3), suggesting that AGO1 depletion might increase Myc (at least in part) by increasing abundance of Psi. Together, these data suggest that AGO1 represses Myc expression in larval wing discs in a manner partially dependent on Psi. In accordance with AGO1 normally being required for Myc repression, AGO1 depletion also increased Myc protein levels (Fig. 5C). To determine whether increased Myc mRNA and protein resulted in heightened Myc function (i.e. transcriptional activity) we monitored abundance of two established Myc target genes in mammalian and Drosophila systems, Polr1c (polymerase I polypeptide C) and Cad (carbamoyl-phosphate synthetase 2) (Mitchell et al., 2015; Poortinga et al., 2014, 2011). Polr1c and Cad mRNAs were significantly increased following AGO1 depletion, and co-knockdown of Psi or Myc reduced abundance of these Myc target mRNAs (Fig. 5D,E). Together, these data suggest that AGO1 is essential for restraining Myc levels and preventing cell overgrowth.

Fig. 5.

AGO1 knockdown increases Myc abundance and function in larval wing discs. (A) Myc qPCR following AGO1 RNAi and/or Psi RNAi knockdown (20 discs pooled for each data point, mean±s.d. shown). Expression was performed for 2 days using tub-GAL4 in the background of tub-GAL80ts. AGO1 knockdown significantly increased Myc mRNA, compared with control. Psi co-knockdown significantly reduced Myc mRNA compared with AGO1 RNAi alone (P=0.0134). Myc RNAi, either alone or combined with AGO1 RNAi, significantly decreased Myc mRNA compared with control (P<0.0001 for both comparisons. (B) Psi qPCR for AGO1 RNAi and/or Psi RNAi knockdown wing discs (20 discs pooled for each data point, mean±s.d. shown). Expression was performed for 2 days using tub-GAL4 in the background of tub-GAL80ts. AGO1 knockdown increased Psi mRNA compared with control (P=0.0002). Psi RNAi, either alone or combined with AGO1 RNAi, significantly decreased Psi mRNA compared with control (P<0.0001 for both comparisons). (C) Anti-Myc antibody (purple) on ser-GAL4-driven AGO1 RNAi compared with control. White asterisk indicates a region with elevated Myc protein in non-AGO1 knockdown cells. (D,E) qPCR for Myc-target genes Polr1c (D) and CAD (E) in wing discs following AGO1 RNAi and/or Psi RNAi knockdown (20 discs pooled for each data point, mean±s.d. shown). Expression was performed for 2 days using tub-GAL4 in the background of tub-GAL80ts. AGO1 knockdown significantly increased Polr1c and CAD mRNA, compared with control (P=0.0005 and P=0.0043, respectively). Psi co-knockdown significantly reduced Polr1c and CAD mRNA compared with AGO1 RNAi alone (P<0.0001 and P=0.0005 respectively). Myc co-knockdown significantly reduced Polr1c and CAD mRNA compared with AGO1 RNAi alone (P=0.0003 and P=0.0205, respectively). Unpaired two-tailed t-test was used for all comparisons. NS, not significant; *P<0.05, **P<0.01, ***P<0.001 , ****P<0.0001.

Fig. 5.

AGO1 knockdown increases Myc abundance and function in larval wing discs. (A) Myc qPCR following AGO1 RNAi and/or Psi RNAi knockdown (20 discs pooled for each data point, mean±s.d. shown). Expression was performed for 2 days using tub-GAL4 in the background of tub-GAL80ts. AGO1 knockdown significantly increased Myc mRNA, compared with control. Psi co-knockdown significantly reduced Myc mRNA compared with AGO1 RNAi alone (P=0.0134). Myc RNAi, either alone or combined with AGO1 RNAi, significantly decreased Myc mRNA compared with control (P<0.0001 for both comparisons. (B) Psi qPCR for AGO1 RNAi and/or Psi RNAi knockdown wing discs (20 discs pooled for each data point, mean±s.d. shown). Expression was performed for 2 days using tub-GAL4 in the background of tub-GAL80ts. AGO1 knockdown increased Psi mRNA compared with control (P=0.0002). Psi RNAi, either alone or combined with AGO1 RNAi, significantly decreased Psi mRNA compared with control (P<0.0001 for both comparisons). (C) Anti-Myc antibody (purple) on ser-GAL4-driven AGO1 RNAi compared with control. White asterisk indicates a region with elevated Myc protein in non-AGO1 knockdown cells. (D,E) qPCR for Myc-target genes Polr1c (D) and CAD (E) in wing discs following AGO1 RNAi and/or Psi RNAi knockdown (20 discs pooled for each data point, mean±s.d. shown). Expression was performed for 2 days using tub-GAL4 in the background of tub-GAL80ts. AGO1 knockdown significantly increased Polr1c and CAD mRNA, compared with control (P=0.0005 and P=0.0043, respectively). Psi co-knockdown significantly reduced Polr1c and CAD mRNA compared with AGO1 RNAi alone (P<0.0001 and P=0.0005 respectively). Myc co-knockdown significantly reduced Polr1c and CAD mRNA compared with AGO1 RNAi alone (P=0.0003 and P=0.0205, respectively). Unpaired two-tailed t-test was used for all comparisons. NS, not significant; *P<0.05, **P<0.01, ***P<0.001 , ****P<0.0001.

Neither miR-996 nor miR-308 drive Myc mRNA turnover

Because AGO1 induces miRNA-dependent mRNA degradation as part of the RISC complex (Hutvagner and Simard, 2008), we tested whether AGO1 depletion altered Myc mRNA levels post transcriptionally. We screened miRBase (Griffiths-Jones, 2004), which contains published mature miRNA sequences from 223 species (Kozomara and Griffiths-Jones, 2014), for miRNAs predicted to target the Myc 3′UTR by sequence similarity (http://www.mirbase.org). miR-308 and miR-996 were the only miRNAs predicted to target Myc (Fig. S4A) that were also expressed in third instar larval tissues based on the modENCODE database (Contrino et al., 2012). In Drosophila embryos, miR-308 drives Myc mRNA and protein depletion (Daneshvar et al., 2013); however, overexpression of miR-308 did not reduce Myc mRNA in the larval wing imaginal disc (Fig. S4B), suggesting that the capacity of miR-308 to target Myc depends on the developmental context. In contrast, miR-996 overexpression significantly increased Myc mRNA abundance (Fig. S4B), indicating that Myc mRNA is not a target for miR-996-driven degradation in the wing. Moreover, the capacity of AGO1 knockdown to increase Myc was not altered by overexpression of either miR-308 or miR-996 (Fig. S4B), suggesting that AGO1 repression of Myc is not dependent on the function of either of the miRNAs predicted to target Myc.

AGO1 protein localises to the cytoplasm and the nucleus

AGO proteins, together with some components of the RISC, have been reported to enter the nucleus and regulate transcription (Catalanotto et al., 2016; Gosline et al., 2016; Kalantari et al., 2016; Shimada et al., 2016; Thomson et al., 2015; Woolnough et al., 2015). In early stage Drosophila blastoderm embryos, AGO1 protein localises to both the nucleus and cytoplasm (Pushpavalli et al., 2012). Biochemical fractionation and confocal immunofluorescence have also detected AGO proteins in the nuclear compartment of mammalian cells (Ahlenstiel et al., 2012). We therefore investigated the localisation of AGO1 in Drosophila wing imaginal disc cells using an anti-AGO1 antibody and an AGO1-GFP protein trap, which generates a GFP fusion with endogenous AGO1 (Buszczak et al., 2007). As expected, given miRNA silencing functions, AGO1 and the AGO1-GFP fusion localised predominantly to the cytoplasm in both the wing discs (Fig. 6A,B) and salivary glands (Fig. S5). In addition, co-staining with lamin to mark the nuclear envelope revealed punctate AGO1 staining within the nucleus.

Fig. 6.

AGO1 protein localises to both cytoplasmic and nuclear compartments. (A) RFP-marked control and AGO1 RNAi flip out clones in wing imaginal discs 2 days after heat shock in the AGO1-GFP protein trap background, stained with AGO1 antibody (purple). (B) Wild-type third instar wing imaginal discs stained with AGO1 antibody (green) and nuclear lamin (red). (C) Single 1 μm z-section from Zeiss Airyscan images for AGO1 antibody (green) and Pc-GFP (false coloured red). Blue arrow marks AGO1 puncta with PcG overlap, white arrow marks AGO1 without clear PcG overlap. Asterisks mark heterochromatin with intense DAPI staining. (D) AGO1 (green) and CTCF-GFP (false coloured red) for single 1 μm z-sections. Blue arrow marks three AGO1 puncta, the closest two without CTCF overlap and furthest one with overlap. White arrow marks AGO1 with weak CTCF overlap. Asterisks mark heterochromatin with intense DAPI staining.

Fig. 6.

AGO1 protein localises to both cytoplasmic and nuclear compartments. (A) RFP-marked control and AGO1 RNAi flip out clones in wing imaginal discs 2 days after heat shock in the AGO1-GFP protein trap background, stained with AGO1 antibody (purple). (B) Wild-type third instar wing imaginal discs stained with AGO1 antibody (green) and nuclear lamin (red). (C) Single 1 μm z-section from Zeiss Airyscan images for AGO1 antibody (green) and Pc-GFP (false coloured red). Blue arrow marks AGO1 puncta with PcG overlap, white arrow marks AGO1 without clear PcG overlap. Asterisks mark heterochromatin with intense DAPI staining. (D) AGO1 (green) and CTCF-GFP (false coloured red) for single 1 μm z-sections. Blue arrow marks three AGO1 puncta, the closest two without CTCF overlap and furthest one with overlap. White arrow marks AGO1 with weak CTCF overlap. Asterisks mark heterochromatin with intense DAPI staining.

As previous analysis in Drosophila antennal discs reported substantial overlap between AGO1 protein and Polycomb (Pc) body foci (42% colocalisation) (Grimaud et al., 2006), we examined whether AGO1 localises to regions of Polycomb group (PcG) mediated silencing in wing imaginal discs by staining with anti-AGO1 in the Pc-GFP background to mark PcG foci (Dietzel et al., 1999). In contrast to earlier studies using lower resolution microscopy, our high-resolution analysis separated PcG bodies from AGO1 puncta, revealing limited direct overlap (Fig. 6C). Indeed, quantification revealed overlap of just 8% and close proximity of 8.4% between AGO1 and PcG complexes, with the majority (83.6%) of staining occurring independently (Fig. S6A). To confirm that PcG bodies overlap euchromatin, as previously reported (Pirrotta and Li, 2012), DAPI was used to distinguish heterochromatin by intense staining, which revealed both PcG bodies and AGO1 puncta in regions of weaker DAPI staining; that is, AGO1 and PcG localise with euchromatin (Fig. 6C).

The observation that AGO1 puncta and PcG bodies localise to euchromatin, but generally do not directly overlap (83.6%), lends support to the idea that multiprotein and RNA complexes containing AGO1 might serve as a scaffold for assembly of the PcG supercomplexes that underlie both PcG and insulator bodies (Pirrotta and Li, 2012; Shevtsov and Dundr, 2011). Although AGO2 has been reported to enable insulator function independent of RNAi activity through physical association with CTCF binding sites in Drosophila (Moshkovich et al., 2011), such roles have not been reported for AGO1. We therefore tested proximity between AGO1 and chromatin insulator bodies, and the localisation of AGO1 and CTCF chromatin insulator complexes in the nucleus using anti-AGO1 and CTCF-GFP (Fig. 6D). As expected, based on DAPI staining, AGO1 complexes were detected in regions of euchromatin; however, only 15% of the AGO1 puncta were found overlapping or in close proximity with CTCF-marked insulator domains (Fig. 6D; quantified in Fig. S6B). Together these data suggest that AGO1 complexes interact with a small subpopulation of PcG transcriptional silencing loci and CTCF insulator domains in the nucleus.

AGO1 knockdown increases Myc transcription

Recent studies demonstrated transcriptional regulation of the MYC oncogene involving looping of super-enhancers and that the MYC promoter requires a conserved CTCF site (Schuijers et al., 2018). This, together with our observations that AGO1 interacts with the RNA Pol II machinery, localises to euchromatic regions of DNA and overlaps PcG and CTCF complexes, led us to investigate whether AGO1 regulates Myc mRNA abundance at the level of transcription. Indeed, AGO1 is required to constrain the Myc promoter, as Myc-lacZ enhancer trap (Mitchell et al., 2010; Peter et al., 2002) activity was significantly increased in the AGO1 knockdown wing disc compartment (Fig. 7A). To further investigate whether increased Myc mRNA associated with AGO1 loss of function was caused by altered transcription, we designed primers to the first intron of Myc to measure pre-mRNA levels. Quantitative PCR revealed an increase in the immature Myc message following AGO1 knockdown in wing discs (Fig. 7B). Together, these data suggest that the increased Myc expression associated with AGO1 depletion is a result of activation of the Myc promoter and increased transcription.

Fig. 7.

AGO1 represses Myc at the level of transcription. (A) Myc-lacZ enhancer trap activity, marked with anti-β-Gal antibody (green) for ser-GAL4-driven AGO1 RNAi in the UAS-p35 background compared with UAS-p35 alone control, marked with RFP and stained for DNA (blue). (B) qPCR for Myc pre-mRNA following AGO1 RNAi knockdown in larval wing discs (20 discs pooled for each data point, mean±s.d. shown). Expression was performed for 2 days using tub-GAL4 in the background of tub-GAL80ts. AGO1 RNAi was compared with control. (C) qPCR for Myc pre-mRNA following AGO1 knockdown compared with control, performed for 2 days using tub-GAL4 in the background of tub-GAL80ts, in larval head tissues treated with α-amanitin for 20 or 40 min or untreated (five heads pooled for each data point, mean±s.d. shown). P=0.0014 for untreated AGO1 RNAi compared with untreated control. P<0.0001 for 20 min α-amanitin-treated control compared with untreated control. P=0.0003 for 40 min α-amanitin-treated control compared with untreated control. P=0.0006 for AGO1 RNAi 20 min α-amanitin-treated compared with AGO1 RNAi untreated. P=0.0003 for AGO1 RNAi 40 min α-amanitin-treated compared with AGO1 RNAi untreated. (D) qPCR for mature Myc mRNA; genotypes and treatment with α-amanitin as described in C. P=0.0002 for untreated AGO1 RNAi compared with untreated control. P=0.0004 for 20 min α-amanitin treated control compared with untreated control. (E) Schematic of Myc showing the position of amplicons used for qPCR. (F) AGO1 ChIP on wild-type larval tissues compared with no-antibody (mock IP) controls (n=4 independent experiments, mean±s.d. shown). P=0.0095 at MYC1, P=0.0003 at MYC2, P=0.0011 at MYC3 and P=0.0031 at MYC4 for AGO1 antibody compared with control. Unpaired two-tailed t-test was used for all comparisons. NS, not significant; **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 7.

AGO1 represses Myc at the level of transcription. (A) Myc-lacZ enhancer trap activity, marked with anti-β-Gal antibody (green) for ser-GAL4-driven AGO1 RNAi in the UAS-p35 background compared with UAS-p35 alone control, marked with RFP and stained for DNA (blue). (B) qPCR for Myc pre-mRNA following AGO1 RNAi knockdown in larval wing discs (20 discs pooled for each data point, mean±s.d. shown). Expression was performed for 2 days using tub-GAL4 in the background of tub-GAL80ts. AGO1 RNAi was compared with control. (C) qPCR for Myc pre-mRNA following AGO1 knockdown compared with control, performed for 2 days using tub-GAL4 in the background of tub-GAL80ts, in larval head tissues treated with α-amanitin for 20 or 40 min or untreated (five heads pooled for each data point, mean±s.d. shown). P=0.0014 for untreated AGO1 RNAi compared with untreated control. P<0.0001 for 20 min α-amanitin-treated control compared with untreated control. P=0.0003 for 40 min α-amanitin-treated control compared with untreated control. P=0.0006 for AGO1 RNAi 20 min α-amanitin-treated compared with AGO1 RNAi untreated. P=0.0003 for AGO1 RNAi 40 min α-amanitin-treated compared with AGO1 RNAi untreated. (D) qPCR for mature Myc mRNA; genotypes and treatment with α-amanitin as described in C. P=0.0002 for untreated AGO1 RNAi compared with untreated control. P=0.0004 for 20 min α-amanitin treated control compared with untreated control. (E) Schematic of Myc showing the position of amplicons used for qPCR. (F) AGO1 ChIP on wild-type larval tissues compared with no-antibody (mock IP) controls (n=4 independent experiments, mean±s.d. shown). P=0.0095 at MYC1, P=0.0003 at MYC2, P=0.0011 at MYC3 and P=0.0031 at MYC4 for AGO1 antibody compared with control. Unpaired two-tailed t-test was used for all comparisons. NS, not significant; **P<0.01, ***P<0.001, ****P<0.0001.

Increased Myc due to AGO1 depletion requires RNA Pol II transcription

To determine whether AGO1 regulates Myc expression at the transcriptional level, we used α-amanitin to block RNA Pol II activity. Consistent with observations using wing imaginal discs (Fig. 5), Myc pre- and processed mRNA levels were significantly increased in untreated larval head tissues following AGO1 depletion (Fig. 7C,D). Interestingly, although Myc pre-mRNA was significantly decreased 20 and 40 min after α-amanitin treatment (Fig. 7C), mature mRNA was significantly increased in control tissues at the 20 min time point (Fig. 7D), suggesting that Myc mRNA stability might increase in response to transcriptional inhibition. In the AGO1 knockdown background, Myc pre-mRNA levels were dramatically decreased following α-amanitin treatment (Fig. 7C). Thus, the AGO1 knockdown-induced increase in Myc pre-mRNA was dependent on RNA Pol II transcriptional activity. Abundance of the mature Myc transcript was not significantly altered in the AGO1 knockdown background following α-amanitin treatment, again suggesting that feedback mechanisms might result in increased mRNA stability in response to RNA Pol II inhibition. Together with the observation that AGO1 knockdown increased Myc promoter activity in the wing discs, these data suggest that AGO1 is normally required for repression of Myc transcription.

AGO1 is enriched on Myc

The increased Myc promoter activity following AGO1 depletion, and increased Myc mRNA abundance requiring RNA Pol II activity, led us to test whether AGO1 directly regulates Myc transcription. We therefore performed ChIP using the anti-AGO1 antibody followed by qPCR with amplicons flanking the Myc transcription start site (Fig. 7E). In wild-type larval tissues, significant AGO1 enrichment was observed in Myc regulatory regions compared with the mock-IP control, with highest enrichment observed at the transcription start site (Fig. 7F), suggesting that AGO1 normally inhibits Myc transcription through direct interaction with the Myc promoter.

DISCUSSION

Here, we demonstrate a novel role for AGO1 as a growth inhibitor in Drosophila. AGO1 depletion was sufficient to increase Myc (mRNA and protein) to drive ribosome biogenesis, nucleolar expansion and cell growth in a Myc and Psi-dependent manner. The increased Myc promoter activity in AGO1 knockdown wing discs, together with the α-amanitin-dependent increase in Myc pre-mRNA abundance, suggests that AGO1 represses Myc at the level of transcription. In accordance with the observed growth inhibitory capacity of AGO1, the increased Myc mRNA and protein abundance in AGO1 knockdown wings were associated with increased Myc function (i.e. activation of established Myc targets). Interestingly, although Psi co-knockdown only modestly decreased Myc mRNA levels in AGO1-depleted wings, Psi co-depletion strongly reduced expression of Myc targets. This observation suggests that Psi is not only required for Myc transcription (Guo et al., 2016) but may also be required for activation of Myc growth targets in the context of AGO1 depletion. Thus, future studies are required to determine whether Psi and Myc bind common targets and whether Psi is required for transcriptional activation of Myc target genes.

Recent genome-wide functional RNAi screens in Drosophila S2 cells, identifying AGO1 as a modifier of Polycomb foci, suggested extra-miRNA functions for AGO1 (Gonzalez et al., 2014). PcG mediates epigenetic repression of key developmental genes to control cell fate, and PcG repression is stabilised via aggregation of PcG foci in the nucleus. AGO1 depletion disrupted nuclear organisation and reduced the intensity of Pc foci, suggesting that AGO1 negatively regulates PcG-mediated silencing (Grimaud et al., 2006). The Drosophila PcG complex has been characterised for roles in silencing homeotic genes by binding PcG response elements (PREs), including the Fab-7 PRE-containing regulatory element from the Hox gene, Abdominal-B. Components of the RNAi machinery, including AGO1 and Dicer-2, have been implicated in driving PcG-dependent silencing between remote copies of the Fab-7 element, engineered throughout the genome to monitor long-distance gene contacts. Interactions between Hox genes silenced by PcG proteins were decreased in AGO1 mutants, suggesting that AGO1 regulates nuclear organisation, at least in part, by stabilising PcG protein recruitment to chromatin (Grimaud et al., 2006).

Myc transcriptional autorepression, modelled in the Drosophila embryo via overexpression of Myc from an exogenous promoter, leads to repression of the endogenous Myc locus in a Pc-dependent manner (Goodliffe et al., 2005). This, together with the partial overlap between AGO1 and PcG in wing imaginal disc cells (Fig. 6C), suggests that Pc mediates transcriptional autorepression of Myc via AGO1. In contrast to our studies, where AGO1 depletion phenotypes are associated with a moderate (>three- to fivefold) increase in Myc, autoregulation in the embryo was investigated in response to non-physiological increases in Myc (over 100 times endogenous levels) (Goodliffe et al., 2005). Thus, our data suggest that AGO1 binds the Myc promoter under normal conditions and is required for repression of endogenous Myc transcription (Fig. 5A), but whether AGO1 is required for Pc-dependent Myc autorepression requires further investigation. In a similar vein, super-enhancers control human MYC transcription via CTCF in the context of high-MYC cancers (Schuijers et al., 2018). Thus, failed Pc-dependent autorepression and/or defective repression of super-enhancers via CTCF could further elevate MYC to promote cancer progression. Given the observed overlap between AGO1 and Pc/CTCF in the Drosophila wing, future studies determining whether AGO1 interacts with Pc and/or CTCF to control autoregulatory feedback on Myc transcription in the context of tumorigenesis will be of great interest.

The question remains regarding how AGO1 targets Myc transcription. The physical and genetic interaction between Psi and AGO1, and the observation that AGO1 loss-of-function mutants restore cell and tissue growth in the Psi knockdown wing, suggests that AGO1 inhibits growth that is dependent on this Myc transcriptional regulator. AGO2 has been implicated in insulator-dependent looping interactions defining 3D transcriptional domains (TADs) through association with CTCF binding sites in Drosophila (Moshkovich et al., 2011). Although similar roles for AGO1 have not been reported, the cancer-related super-enhancers for the MYC oncogene lie within the 2.8 Mb TAD and control MYC transcription via a common and conserved CTCF binding site located 2 kb upstream of the MYC promoter; that is, in proximity with the FUSE (1.7 kb upstream) bound by FUBP1. Moreover, gene disruption of the enhancer-docking site reduces CTCF binding and super-enhancer interaction, which results in reduced MYC expression and proliferative cell growth (Schuijers et al., 2018). AGO1 ChIP revealed significant enrichment on the Myc promoter, suggesting that AGO1 probably interacts with Psi and the RNA Pol II machinery to directly regulate Myc transcription. Given the high level of conservation between AGO and CTCF proteins throughout evolution, it is of great interest to determine whether human AGO1 also interacts with FUBP1 to regulate transcription of the MYC oncogene.

Here, we have shown that AGO1 behaves as a growth inhibitor during Drosophila development, through the ability to suppress Myc transcription, ribosome biogenesis and cell growth in the wing disc epithelium. Consistent with AGO1 having tumour suppressor activity, across a wide range of human cancers, large scale genomics data in cBioPortal (Gao et al., 2013) identified AGO1 as frequently mutated or deleted in a diverse variety of tumours (e.g. reproductive, breast, intestinal, bladder, and skin cancers). Region 1p34–35 of chromosome 1, which includes AGO1, is frequently deleted in Wilms' tumours and neuro-ectodermal tumours (Parisi et al., 2011). In neuroblastoma cell lines, AGO1 behaves as a tumour suppressor, with overexpression heightening checkpoint sensitivity and reducing cell cycle progression. GEO Profile microarray data inversely correlates AGO1 expression with proliferative index (Parisi et al., 2011); that is, AGO1 levels are significantly lower in tumorigenic cells than in differentiated cells (Barrett et al., 2011). In the context of cancer, it is important to determine whether AGO1 loss of function alters MYC-dependent cancer progression and vice versa. As increased abundance of the MYC oncoprotein is associated with the pathogenesis of most human tumours (Dang, 2012; Levens, 2010), deciphering such novel mechanisms of MYC repression is fundamental to understanding MYC-dependent cancer initiation and progression.

MATERIALS AND METHODS

Drosophila strains

The UAS-AGO1 RNAi 1 (BL53293), UAS-AGO1 RNAi 2 (BL33727), UAS-Myc (BL9675), AGO-GFP (BL50805), UAS-miR-308 (BL41809), UAS-miR-996 (BL60653), Myc-lacZ (BL12247), CTCF-GFP (BL64810), Pc-GFP (BL9593), ser-GAL4 (BL6791), tub-GAL4 (BL5138), tub-GAL80ts (BL7019), hsflp (BL1929) and Act<CD2<GAL4 (BL30558) were obtained from the Bloomington Drosophila Stock Center. The UAS-Myc RNAi (V2947), UAS-Psi RNAi (V105135), AGO1k00208 (V10470) and AGO104845 (V11388) lines were obtained from the Vienna Drosophila RNAi Center.

Co-immunoprecipitation and western blotting

Co-IP was performed using 25 wild-type third instar larval heads dissociated in cold lysis buffer (50 mM Tris pH 7.5, 1.5 mM MgCl2, 125 mM NaCl, 0.2% NP40, 5% glycerol, 1× protease inhibitor cocktail). Following homogenisation, protein was collected by centrifugation at 13,000 g for 10 min at 4°C. The extract was pre-cleared by incubation with nProtein A Sepharose beads (GE Healthcare Life Science) for 1 h at 4°C with rotation and the supernatant collected by centrifugation at 13,000 g. Equal amounts of pre-cleared protein lysate were incubated with either anti-AGO1 antibody (Abcam, ab5070, 1:70), anti-Psi antibody (custom generated rabbit polyclonal antibody, Biomatik, 1:100) or without antibody (mock IP control) overnight at 4°C. Beads were washed five times with lysis buffer and the eluent resolved using 10% SDS PAGE/western blot with appropriate primary antibody before detection with Li-Cor Odyssey IR.

Immunofluorescence, microscopy and image analysis

Because of the high level of cell death and lethality associated with constitutive ser-GAL4-driven AGO1 depletion, analysis was conducted with co-expressed baculoviral caspase inhibitor p35 to prevent apoptosis (Hay et al., 1994); crosses were maintained at 18°C, unless otherwise stated. Wandering third instar larvae were dissected and fixed for 20 min in 4% paraformaldehyde (PFA), washed in PBS containing 0.1% Tween (PBT), blocked in 5 mg/ml bovine serum albumin (BSA) prior to incubation overnight at 4°C with primary antibody. Primary antibodies used for immunofluorescence were Myc N (rabbit, 1:500; Santa Cruz d46-507), fibrillarin (rabbit, 1:500; Abcam ab5821), AGO1 (rabbit, 1:500; Abcam ab5070), Psi (rabbit, 1:500; custom-made via Biomatik), Lamin (mouse, 1:20; DSHB ADL 101), β-galactosidase (chicken, 1:1000; Abcam ab9361). After incubating with appropriate fluorophore-tagged secondary antibodies (Jackson ImmunoResearch Laboratories; anti-rabbit 488, 1:1000, 711-545-152; anti-chicken 488, 1:1000, 703-485-155; anti-mouse 649, 1:1000, 715-495-151; and anti-rabbit 647, 1:1000, 711-605-152), samples were counterstained with DAPI solution and wing imaginal discs imaged using a Zeiss LSM800 confocal microscope (Zen Blue software). Overlapping 1 μm z-sections were collected at 40× magnification. Fluorophores were imaged using band-pass filters to remove cross-detection between channels. Images were processed and prepared using ImageJ and Adobe Photoshop CS5. Fibrillarin- and CD8-RFP-marked cell sizes were quantified in FIJI on confocal z-sections of wing columnar epithelial cells, merged to display maximum projections (two or three sections). Thresholding was performed and images were used to measure average fibrillarin area or cell size in the dorsal compartment marked by serrate-GAL4>UAS-RFP expression. About 50-100 nucleoli were selected using the freeform selection tool and analysed with the ‘Analyse Particles’ tool, with minimum particle size of 0.5 μm2 applied in order to exclude noise and out-of-focus nucleoli. For cell size, 50-100 cells per wing disc were analysed, with a minimum particle size of 3 μm2 applied. The output used image metadata to calculate average area for each wing disc analysed. Analysis of percentage overlap between AGO1 and PcG/CTCF was performed in FIJI by thresholding to isolate individual puncta and overlaying channels to detect co-occurrence or adjacency, which was counted and expressed as a proportion of total AGO1 puncta per individual nuclei.

Adult wing size analysis

Adult wing size was determined for male wings imaged with an Olympus SZ51 binocular microscope, at 4.5× magnification using the Olympus DP20 camera. Wing size was measured by pixel count for the area posterior to wing vein L5, using Photoshop CS5. For wing hair counts, adult male wings were imaged with an Olympus BX 61 microscope at 20× magnification using the Olympus DP70 camera. Wing cell size was determined using wing hair counts in a defined area (200×100 pixels) at the central region posterior to wing vein L5. Then, the hair number was converted to relative single cell size by dividing the area of the fixed region by hair numbers.

Quantitative PCR

qPCR was performed 2 days after induction of transgene expression using tub-GAL4 in tub-GAL80ts background. RNA was isolated from equivalent numbers of wing imaginal discs (10 pairs for each genotype) using the Promega ReliaPrep RNA Cell miniprep system and eluted in 20 μl nuclease-free water. RNA purity and integrity were assessed using an automated electrophoresis system (2200 TapeStation, Agilent Technologies). Each cDNA synthesis used 6 μl of RNA (GoScript Reverse Transcription System kit, Promega). qPCR was performed using Fast SYBR Green Master Mix (Applied Biosystems) using the StepOnePlus Real-Time PCR System and Sequence Detection Systems in 96-well plates (Applied Biosystems; 95°C for 2 min, 40 cycles 95°C 1 s and 60°C 20 s). Amplicon specificity was verified by melt curve analysis. Average Ct values for two technical replicates were calculated for each sample. Multiple candidate reference genes were analysed for stability across AGO1 RNAi and control samples using RefFinder. Target gene expression was normalized to the mean of cyp1 and tubulin, selected for having high expression and smallest sample-to-sample variability. Fold change was determined using the 2-ΔΔCT method.

Primers used for qPCR

Myc, 5′-GTGGACGATGGTCCCAATTT-3′ and 5′-GGGATTTGTGGGTAGCTTCTT-3′; Myc pre-mRNA, 5′-TTCAAAATAGAATTTCTGGGAAAGGT-3′ and 5′-GCGGCCATGATCACTGATT-3′; Psi, 5′-CGATGGCATCCCATTTGTTTGT-3′ and 5′-GGTGGTCAAGACTACTCGGC-3′; AGO1, 5′-ACTCTACGGTCTGTCCGTTC-3′ and 5′-CCCGCTCAGATGCAATCATTC-3′; 5′-ETS, 5′-GGCAGTGGTTGCCGACCTCG-3′ and 5′-GCGGAGCCAAGTCCCGTGTT-3′; Tubulin, 5′-TGGGCCCGTCTGGACCACAA-3′ and 5′-TCGCCGTCACCGGAGTCCAT-3′; CYP1, 5′-TCGGCAGCGGCATTTCAGAT-3′ and 5′-TGCACGCTGACGAAGCTAGG-3′; Polr1c, 5′-TGTATCCCGCCATTGCAA-3′ and 5′-GGGCACATCGCTGAGCAT-3′; Cad, 5′-CATTGGCAGTTTCAAGCACAA-3′ and 5′-TCTTGGCCAGATCCCGTATG-3′.

α-Amanitin treatment

α-Amanitin inhibits RNA Pol II-dependent transcription, therefore interfering with mRNA production (Lindell et al., 1970). The α-amanitin (Sigma #A2263) was diluted in 1 ml of Nanopure water to make a 1 mg/ml stock solution, which was stored at −20°C in the dark. Samples were collected 2 days after induction of transgene expression using tub-GAL4 in tub-GAL80ts background. Third instar larval heads (anterior imaginal tissues) were dissected and incubated with freshly made 20 μg/ml α-amanitin in Schneider's medium at 25°C for 0, 20 or 40 min. After α-amanitin treatment, samples were washed for 5 min using fresh Schneider's medium and snap frozen in 250 μl LBA+TG lysis buffer from the Promega ReliaPrep RNA Cell Miniprep kit. Following RNA extraction and cDNA synthesis, qPCR was performed and analysed with Myc cDNA primers and Myc pre-mRNA primers.

Chromatin immunoprecipitation

ChIP assays were carried out as described previously (Lee et al., 2015). Briefly, for each ChIP sample, 30 larval heads were collected from wild-type mid-third instar larvae and fixed in 4% PFA for 20 min. Larval heads were dissociated and chromatin sheared in 0.4% sodium dodecyl sulphate (SDS) using a Covaris S2 (10 min duration, 10% DUTY, 200 cycles per burst, intensity 4, achieving average DNA fragment sizes of 200-600 bp). ChIP was performed in IP buffer containing 0.1% SDS and 3 µg of anti-RNA Pol II phospho-S5 antibody (Abcam, ab5131) or anti-RNA Pol II phospho-S2 (Abcam, ab5095) for each IP. As a control, mock IP was performed without the use of an antibody. Analysis was performed in triplicate using Fast SYBR Green Master Mix (Applied Biosystems) on a StepOnePlus Real-Time PCR System and Sequence Detection Systems in 384-well plates (Applied Biosystems). To calculate the percentage of total DNA bound, non-immunoprecipitated input samples from each condition were used as the qPCR reference for all qPCR reactions.

Primers for ChIP qPCR

The following primers were used for ChIP qPCR: MYC 1, 5′-GGCGATCGTTTCTGGCCTACGG-3′ and 5′-GCAGGCGCATTTGACTCGGC-3′; MYC 2, 5′-ACTACTACTAACAACTGTCACAAGCCAAGT-3′ and 5′-TTTATGTATTTGCGCGGTTTTAAG-3′; MYC 3, 5′-TTCAAAATAGAATTTCTGGGAAAGGT-3′ and 5′-GCGGCCATGATCACTGATT-3′; MYC 4, 5′-GGTTTTCCTTTTATGCCCTTG-3′ and 5′-CTATTAACCATTTGAACCCGAAATC-3′.

Statistical analysis

All statistical tests were performed with Graphpad Prism 6 using an unpaired two-tailed t-test with 95% confidence interval. In all figures, error bars represent s.d. and, according to the Graphpad classification of significance points, *P=0.01-0.05, **P=0.001-0.01, ***P=0.0001-0.001 and ****P<0.0001.

Acknowledgements

The authors acknowledge the facilities and the scientific and technical assistance of the Centre for Advanced Microscopy and Microscopy Australia at the Advanced Imaging Precinct, Australian National University, a facility that is funded by the University, and State and Federal Governments.

Footnotes

Author contributions

Conceptualization: O.Z., N.C.M., L.G., D.L.L., L.M.Q.; Methodology: O.Z., N.C.M., L.G., L.M.Q.; Validation: O.Z., N.C.M., L.G.; Formal analysis: O.Z., N.C.M., L.G., L.M.Q.; Investigation: O.Z., N.C.M., L.G., O.J.M.; Resources: O.J.M., L.M.Q.; Writing - original draft: O.Z., N.C.M., L.G., D.L.L., L.M.Q.; Writing - review & editing: O.Z., N.C.M., O.J.M., L.M.P., R.D.H., D.L.L., L.M.Q.; Supervision: L.M.P., R.D.H., L.M.Q.; Project administration: L.M.Q.; Funding acquisition: L.M.Q.

Funding

This work was supported by the National Health and Medical Research Council (APP1143008 to L.M.Q.). Deposited in PMC for immediate release.

Peer review history

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Competing interests

The authors declare no competing or financial interests.

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