Generation of mutant flies, transgenes and reporters
The allele Uhg1
1 (null mutant) and Uhg1
rev (wild type revertant) were generated by mobilization of P{w[+mC] = EP}Uhg1[G11659] (Bloomington Stock Center number B-28084, Indiana University; Bloomington, IN, USA), inserted after position chr2R:13’586’605 (coordinates throughout refer to genome release 5) in Uhg1 exon 1. Uhg1
1 carries a deletion of nucleotides chr2R:13’586’606 to 13’590’803 (first to last exon of Uhg1).
UAS-Uhg transgenes were generated in a ‘y w’ background by cloning appropriate PCR fragments (Additional file 1: Table S5) into pUAS-T, followed by sequence verification and standard transgenesis procedures.
Additional flies: wor-GAL4 ase-GAL80/CyO; UAS-brat-IR UAS-FLuc/TM3, Sb Ser tubulin-GAL80[ts] [5], actin-FRT-CD2-FRT-GAL4 hs-FLP (H.Stocker, ETH Zürich, Switzerland), UAS-vito::GFP [61] (Bloomington Stock Center).
In vivo analysis
To measure the time from egg deposition to adult eclosion, timed egg lays (5 to 14 hours) were performed in 10 to 50 culture vials per genotype (‘y w; Uhg1
1/CyO, y + ’, ‘y w; Uhg1
rev’, ‘y w’, ‘y w; Uhg1
1/CyO,y+’ x ‘y w’), so as to avoid overcrowding. Eclosion was monitored two to three times a day. Developmental times were combined using a weighted 10-hour floating window (perl script available upon request).
For weighing flies, one to four day old flies were dried for 20 minutes at 95° (first for 10 minutes with a closed, then with an opened lid) and then stored at room temperature. Before weighing on a Mettler UMT5 Comparator scale (Mettler Toledo, Giessen, Germany), the flies were allowed to equilibrate with ambient atmosphere for at least 30 minutes.
For clonal overexpression of Myc, the genotypes ‘y w actin-FRT-CD2-FRT-GAL4 hs-FLP/y w; Uhg1
1/Uhg1
1; UAS-vito::GFP/UAS-Myc UAS-GFP’ and ‘y w actin-FRT-CD2-FRT-GAL4 hs-FLP/y w; (Sp or CyO,y+)/+; UAS-vito::GFP/UAS-Myc UAS-GFP’ were analyzed. At two to three days of development, clones were induced by an 8- to 15-minute incubation in a 37° water bath; 48 hours later, wandering larvae were collected, fixed for 20 minutes at room temperature in 4% paraformaldehyde/PBS containing 0.1% Tween-20, stained with 10 μg/ml Hoechst 33258 (Sigma Aldrich, St. Louis, MO, USA) and 2 u/μl Alexa Fluor 546 Phalloidin (Molecular Probes, Life technologies; Carlsbad, CA, USA), and destained with PBS-Tween before mounting in Vectashield Mounting Medium (Biozol Diagnostica, Eching, Germany). Images were recorded on a Nikon Eclipse Ti-E confocal microscope, using a 20 × lens.
To determine protein translation activity in wandering larvae, 750 μl Ringer’s solution containing 15 μCi/ml 3H-labeled amino acid mix (Hartmann Analytic, Braunschweig, Germany) were added to 10 fully inverted larvae and placed on a rotating wheel for one hour at room temperature. The supernatant was then decanted and the carcasses were washed twice with cold Ringer’s solution before they were crushed in 350 μl cell lysis buffer (100 mM Tris-Cl (pH 8.0), 100 mM NaCl, 0.5% Triton X-100) with a pestle. Samples were incubated for 10 minutes on ice with periodic vortexing, followed by a 2 minute centrifugation. A total of 250 μl of the aqueous lysate was mixed with 15 μl suspended Strataclean Resin (Stratagene, Agilent Technologies; Santa Clara, CA, USA) and allowed to rest for five minutes. After removing the supernatant, resins were washed with Ringer’s solution and transferred to 3 ml scintillation buffer, rested for 30 minutes, and then counted for 1 minute per vial. The remaining aqueous lysate was processed for protein quantification [62].
Type II neuroblast tumors were induced by knocking down the tumor suppressor brat specifically in these cells with the driver system ‘worniu-GAL4 asense-GAL80.’ Co-expression of firefly luciferase allowed the quantitative determination of tumor mass [5]. Appropriate male adults (at most 12 hours old) were collected and frozen at −80° until use. Upon thawing, individual flies were lysed in Passive Lysis Buffer (Dual Luciferase Reporter Assay System; Promega, Madison, WI, USA) in a Bullet Blender homogenizer (Next Advance, Averill Park, NY, USA) and processed for luminometry in a Glomax luminometer (Promega).
Cell culture and RNA interference
Drosophila S2 and Kc167 cells were cultivated at 25°C in Schneider’s Insect Medium (Sigma) including 10% fetal bovine serum (Pan™ Biotech) and 1% penicillin/streptomycin (Sigma). For RNAi experiments, cells were plated out and washed once in serum free medium. dsRNA against Myc, or GFP for control, was added directly to the cells kept in 1/3 volume of serum free medium. After 30 minutes 2/3 volume of full medium was added. A total of 2 μg of dsRNA were added per 106 cells. Cells were harvested 24 hours after RNA addition.
Western blotting
S2 cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris (pH 8.0), 5 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0), 1% Nonidet-P40), mixed with an equal amount of Laemmli buffer (containing 20% β − mercaptoethanol) and an extract from 1.25 × 106 cells was separated on a 10% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and incubated with appropriate antibodies (mouse anti-Myc (described in [25]), rabbit anti-Myc [26], rabbit anti-Myc (Santa Cruz, Dallas, TX, USA), mouse anti-α-Tubulin (Sigma)).
RNAseq
Total RNA was isolated from Drosophila S2 cells using Trizol (Invitrogen, Life Technologies; Carlsbad, CA, USA) and purified with miRNeasy (Qiagen, Venlo, Netherlands), followed by on-column DNase treatment to eliminate genomic DNA, all according to the manufacturer’s instructions. Samples were treated once with RiboMinus™ (Life Technologies, Carlsbad, CA, USA) to selectively deplete ribosomal RNA (rRNA). Sequencing libraries were prepared with the NEBnext® mRNA Library Prep Master Mix set for Illumina (E6100S/L) following the instruction manual. Briefly, ribominus-treated RNA was fragmented, first and second cDNA strands were synthesized, and the resulting duplex was end-repaired, ligated to NEBnext adaptors and gel purified with the Qiagen gel extraction kit selecting 200 bp fragments. cDNA was then amplified with 15 cycles of PCR and the resulting library was subjected to Illumina GAIIx sequencing. For both Myc-knockdown (experimental) and GFP-knockdown (control) samples, three biologically independent replicates were prepared and analyzed. Quality and quantity of RNAs and resulting cDNAs were assessed at several steps of the procedure through an Experion™ Automated Electrophoresis System (Bio-Rad, Hercules, CA, USA).
Sequence data were processed through the bioinformatics pipeline OLB_v1.9, then through a Perl script to eliminate reads with perfect matches to Drosophila rRNA. The non-rRNA reads were mapped to the Drosophila genome release 5 with bowtie-0.12.7. (between 2,082,423 and 14,466,659 mapped reads per condition and repeat), converted from sam to bam format with SAMtools, and statistically analyzed with the BioconductoR work package. For subsequent analysis we normalized the total read number from each RNAseq experiment to 1,000,000. Only genes with ≥10 reads combined in all six samples and with predicted transcript sizes ≥ 125 nt were kept for final analysis (for a total of 8,019 genes). RNAseq and ChIPseq data are available in the ArrayExpress database [63] under accession number E-MTAB-3209.
ChIPseq
For chromatin immunoprecipitation (ChIP), cells were cross-linked with 1% formaldehyde at 37°C for 10 minutes and the reaction was stopped with 50 mM glycine. Cells were lysed and nuclei were resuspended in radioimmunoprecipitation assay (RIPA) buffer. Sonication with a Branson sonifier was carried out until the majority of fragments showed nucleosomal size. Cells were immunoprecipitated with Myc antibody (mouse α-Myc, rabbit α-Myc, Santa Cruz rabbit α-Myc) or control IgG from mouse or rabbit (Sigma) which were coupled to Protein A/G-dynabeads (Invitrogen). DNA was purified with phenol-chloroform after elution of the bound chromatin with 1% SDS and reversion of the crosslink.
ChIP DNA was end repaired and A tailed. Illumina adaptors were ligated to the ChIP DNA fragments. Fractions with a size of 175 to 225 bp were cut out from a 2% agarose gel, extracted by Qiagen gel extraction kit and enriched by 18 cycles of PCR amplification. The library-size was controlled with the Experion-system (BioRad) and quantified using a picogreen assay. The library was sequenced on a Illumna GAIIx sequencer.
Sequence data were processed with OLB_v1.9 and mapped to the Drosophila genome release 5 with bowtie-0.12.7. For each condition, we obtained between 12,569,801 and 33,927,814 reads, for which between 47% and 75% aligned to a single position in the Drosophila genome and were further considered, 2% to 31% did not align anywhere, and the remainder showed multiple alignments (corresponding typically to transposons and heterochromatic regions). Peaks were identified with MACS14, using the same number of reads with single alignment for all conditions (6,874,000) and the default settings (with the switch ‘-g dm’). Number of called peaks for the different conditions: 260 and 27 (mouse α-Myc and non-immune mouse IgG in naïve S2 cells), 22 and 24 (mouse α-Myc and non-immune mouse IgG in Myc-depleted S2 cells), 263 and 31 (rabbit α-Myc and non-immune rabbit IgG in S2 cells), 308 and 187 (rabbit α-Myc and non-immune rabbit IgG in Kc167 cells). Subsequently, we eliminated from the Myc-ChIP lists all unmapped peaks (mostly assigned to chromosome ‘Uextra’; 3 to 17 peaks per condition) and all peaks that were called in any of the ChIPs with non-immune IgGs or in Myc-ChIPs from Myc-depleted S2 cells, resulting in 240 (mouse α-Myc in S2 cells; 240 with FDR < 10%), 243 (rabbit α-Myc in S2 cells; 98 with FDR <10%), 279 peaks (rabbit α-Myc in Kc167 cells; 21 with FDR < 10%). Additional file 1: Table S1 lists all 265 peaks that are significant (FDR <10%) in at least one condition, and includes non-significant peaks (FDR ≥10%) if they overlap a significant peak.
qPCR of S2 cells
For ‘standard’ qPCR, total RNA was isolated from Myc and control depleted Drosophila S2 cells as described for RNAseq. The miScript II RT Kit (Qiagen) was used to generate cDNA which allows the conversion of all RNA species. cDNA was analyzed as described for ChIP DNA, using the ΔΔCT method for evaluation of the results. Every qRT-PCR was performed in triplicate for at least two biologically independent samples. The averages of two or three of the reference genes rab6, snm158 and α-Tubulin were set to 100%, since these genes were found to be unaffected by Myc-knockdown in our RNAseq, as well as earlier experiments.
Northern blot
Total RNA was extracted from Drosophila S2 cells as described above. A total of 20 μg RNA was loaded per lane on a 10% acrylamide 8 M urea gel and then transferred to a Hybond N+ nylon membrane (GE Healthcare, Chalfont St Giles, Great Britain (Frankfurt)). After UV crosslinking at 254 nm the membrane was pre-hybridized in 10 ml Church buffer (1 mM EDTA pH 8.0, 0.17% phosphoric acid, 0.5 M Na2HPO4, 7% SDS) for one hour at 62°C. DNA oligonucleotide probes were 5′ end-labeled with γ-32P-ATP using T4 polynucleotide kinase (NEB) and hybridized overnight in Church buffer at 62°C. The blots were washed in 2 × SSC (300 mM NaCl, 30 mM sodium citrate) and 0.2 × SSC for 30 minutes at 62°C, dried, exposed and developed on a Typhoon 9200 phosphorimager (GE healthcare). Quantification was performed using ImageJ software.
Uhg1 reporter analysis
A 983 bp genomic fragment (chr2R: 13,585,896 to chr2R: 13,586,878) encompassing the TSS of Uhg1 (chr2R:13,586,602) and the E-box at chr2R:13,586,588 was cloned in front of the firefly luciferase coding sequence and the SV40 polyadenylation signal, yielding pGL-Uhg1WT. For luciferase assays, 1.3 × 106 S2 cells were plated into each well of a 24-well plate and transfected with 0.2 μg of reporter plasmid (pGL-Uhg1WT or pGL-CG5033WT) and 200 ng of reference plasmid pRL-CG5033ΔE, using Effectene (Qiagen) according to the manufacturer’s instructions (for CG5033-plasmids, dsRNA and assay protocol, see [38]). Where indicated, 30 ng of dsRNA against Myc or GFP, or 200 ng pTub-GAL4 plus 200 ng pUAS-Myc or pUAS-T, were included in the transfection mix. One day after the transfection, cells were harvested, lysed and firefly and Renilla luciferase activities were measured using the Dual Luciferase Assay System (Promega) in a Glomax luminometer. Each experiment was carried out at least twice (on separate days), and every transfection within each experiment was done in triplicate.
Further bioinformatic analysis
To calculate distances of the 265 peaks to the nearest transcription start sites (TSS) we used closestBed from the Bedtools suite v.2.17.0. and the Drosophila genome annotation FlyBase fb_2013_05. To assign genes to mapped ChIP peaks, we used intersectBed and closestBed and subsequently manually pruned the gene lists. For ChIPseq peaks overlapping at least one gene with statistically significant expression change upon Myc depletion (P < 0.05), only transcriptionally affected genes were retained. When a peak mapped within 100 bp of the TSS of a transcriptionally unaffected gene, other transcriptionally unaffected genes with TSS at more than 300 bp distance from the peak were eliminated (20 instances). For intronic snoRNAs, only the host genes were retained.
For the analysis of peaks from Yang et al. [27] we used the data published by these authors and the program intersectBed (bedtools v.2.17.0.) to determine the ratio of mouse α-Myc ChIPseq reads from naïve S2 cells versus Myc-depleted S2 cells for each of the 3,993 peaks. In total, 1,936 regions with a ratio ‘naïve/Myc-depleted’ ≤ 1.2 that did not overlap any of our peaks were retained and sorted by the number of reads that were recorded by Yang et al. From this sorted list we selected every 50th peak (for a total of 20 peaks), starting with the region with the highest number of reads in the Yang analysis, and designed primers covering the summit coordinate indicated by Yang et al. (one region was omitted since no acceptable primers could be generated, and two primer pairs did not function, that is, did not produce the expected product).
For GO term analysis we used the online resource GOrilla [64] and all Drosophila genes according to FlyBase annotation fb_2013_05 as the background set. Identification of binding sites was carried out with the online resource MEME-ChIP [65]. Gene Set Enrichment Analysis was performed with the GSEA software [66]. ChIPseq reads were visualized with the IGB browser [67]. Venn diagrams were drawn with the help of eulerAPE v.1.0 [68].