PAF1 Complex Component Leo1 Helps Recruit Drosophila Myc to Promoters

PAF1 complex component Leo1 helps recruit Drosophila Myc to promoters

Jennifer M. Gerlacha, Michael Furrerb, Maria Gallanta, Dirk Birkela, Apoorva Baluapuria, Elmar Wolfa, and Peter Gallanta,c,1

aDepartment of Biochemistry and Molecular Biology, Biocenter, University of Würzburg, 97074 Würzburg, Germany; bZoological Institute, University of Zürich, 8057 Zürich, Switzerland; and cComprehensive Cancer Center Mainfranken, 97078 Würzburg, Germany

Edited by Robert N. Eisenman, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved September 11, 2017 (received for review April 7, 2017) The Myc oncogene is a transcriptionfactorwithapowerfulgripon We decided to search for molecular partners that contribute to cellular growth and proliferation. The physical interaction of Myc Myc’s recruitment to DNA and/or mediate transactivation by with the E-box DNA motif has been extensively characterized, but it Myc, using Drosophila as a model system. Fruit flies contain a is less clear whether this sequence-specific interaction is sufficient for single Myc protein that primarily controls cellular and organis- Myc’s binding to its transcriptional targets. Here we identify the mal growth (reviewed in ref. 30). Like its vertebrate homologs, PAF1 complex, and specifically its component Leo1, as a factor that Drosophila Myc functions by dimerizing with Max and binding to helps recruit Myc to target genes. Since the PAF1 complex is typically target genes. Most of the directly Myc-activated genes control associated with active genes, this interaction with Leo1 contributes ribosome biogenesis and function. This is consistent with Myc’s to Myc targeting to open promoters. biological properties in flies and also with the described targets of vertebrate Myc proteins expressed at physiological levels (e.g., Drosophila | Myc | transcription | growth | PAF1 refs. 7 and 10). Illustrating this evolutionary conservation of Myc function, vertebrate MYC can substitute for Drosophila Myc in he Myc family of transcription factors (MYC, MYCN, and flies (31), and Drosophila Myc can partially replace c-Myc in TMYCL) are potent oncogenes thought to contribute to the cultured murine fibroblasts (32). majority of human cancers (1, 2). Through their C-terminal basic- To identify proteins that influence Myc’s transcriptional output, helix-loop-helix zipper region (bHLHZ), Myc proteins dimerize we carried out an RNAi screen in Drosophila S2 cells (33) and with the bHLHZ protein Max and subsequently bind their target identified the PAF1 complex as a functional interactor of Myc. genes (3) at promoter-proximal sites or at distally located en- The PAF1 complex is conserved from yeast to man. It consists of hancers (4–7). High-affinity Myc targets often contain E-boxes the core subunits Paf1 (atms in Drosophila), Cdc73 (also known as (CACGTG and variants thereof) that are directly contacted by hyrax in Drosophila and Parafibromin in vertebrates), Leo1 (Atu Myc:Max dimers. Many of these genes are involved in translation, in Drosophila), Ctr9, Rtf1 (34, 35), and, in vertebrate cells, the or ribosome biogenesis or function (7–10). When expressed at more loosely associated Ski8 (36). The complex was initially supraphysiological levels, Myc also invades most active promoters identified as an RNA polymerase II-associated factor (e.g., refs. 37 (11–16), and Myc has been proposed to act as general amplifier of and 38) and subsequently shown to interact with some general transcription (11, 12). The mechanism by which Myc recognizes its factors involved in transcription elongation, including DSIF (35, different targets is currently under investigation. 39), SII (40), FACT (35), the “super elongation complex” SEC In vitro Myc:Max dimers most efficiently bind to E-box motifs, (41), P-TEFb, Cdk12, and Cyclin T (42). Consistent with these and this motif is frequently found in high-affinity Myc targets (7, observations, the PAF1 complex has been shown to positively 10). However, several lines of evidence suggest that primary affect RNA polymerase II pause release and transcriptional DNA sequence does not suffice to define Myc targets. First, in elongation (42–44). Other studies have revealed an inhibitory ef- vivo Myc does not bind to all E-boxes indiscriminately, but rather fect of the PAF1 complex on elongation (45–48). Thus, it has been preferentially associates with sites located in active promoters (5, proposed that the genetic background and physiological state of 13). Second, Myc binds to numerous promoters lacking any different cells may affect the output of the PAF1 complex (42). In common sequence motif, especially when expressed at supra- either case, depletion of the PAF1 complex affects transcript levels physiological levels (e.g., ref. 13). Third, the measured in vitro of most genes, but to only a rather small degree; for example, Myc binding constant of Myc:Max dimers for E-boxes does not suffice to explain the observed in vivo affinity for its target sites (10). Significance Thus, it has been proposed that additional proteins help recruit Myc in a sequence-independent manner to its target sites (13). One such factor is the recently discovered WDR5, which inter- We identify the PAF1 complex component Leo1 as a factor that acts specifically with the highly conserved centrally located helps recruit Myc to its target genes. In particular when Myc is MBIIIb motif in Myc (10, 17). Loss of the WDR5 interaction overexpressed, Leo1 becomes limiting for transcriptional reg- domain strongly reduces the binding of Myc:Max complexes to ulation by Myc. their targets, including sites with canonical E-boxes. Author contributions: E.W. and P.G. designed research; J.M.G., M.F., M.G., D.B., A.B., and P.G. However, WDR5 likely is not the only such factor, since Myc performed research; E.W. and P.G. analyzed data; and J.M.G. and P.G. wrote the paper. mutants lacking the WDR5 interaction region still retain partial The authors declare no conflict of interest. transforming activity in tissue culture and in mice (17). In addition, the MBIIIb region is highly conserved in Drosophila Myc, but a This article is a PNAS Direct Submission. mutant Myc derivative lacking the entire region can substitute for Published under the PNAS license. wild-type Myc in null mutant flies (18). Additional recruitment Data deposition: The sequences reported in this paper have been deposited in the Ar- rayExpress archive, https://www.ebi.ac.uk/arrayexpress (accession nos. E-MTAB-5470, factors might include various sequence-specific transcription factors E-MTAB-5471,andE-MTAB-5472). with which Myc has been reported to associate (notably Miz1, Sp1, 1To whom correspondence should be addressed. Email: peter.gallant@biozentrum. NF-Y, Smad2/3, and YY1; reviewed in ref. 19) and several com- uni-wuerzburg.de. – ponents of the core transcription machinery (15, 20 22), including This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Brf, P-TEFb, TBP, TFII-I, and RNA polymerase II (23, 24–29). 1073/pnas.1705816114/-/DCSupplemental.

E9224–E9232 | PNAS | Published online October 16, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1705816114 Downloaded by guest on October 1, 2021 targets were shown to be up-regulated by 10% on knockdown of PNAS PLUS Cdc73 (48), and efficient depletion of Paf1 altered the average expression of a group of 4,855 direct PAF1 targets by <10% (46). Nevertheless, the PAF1 complex is essential for the survival of metazoans. The contributions of individual complex components to these biochemical activities have not yet been fully elucidated. Thus, Paf1 is essential for complex formation and for histone H3 lysine 4 methylation, but Leo1 is dispensable for both (40, 49), and no specific biochemical activity has been ascribed to this protein (44). Along with associating with general transcription factors, the PAF1 complex also interacts with the sequence-specific transcription factors β-catenin (through Cdc73; ref. 50), STAT3 (through Ctr9; ref. 51), p53 (40), Ci/Gli (through Cdc73; ref. 52), and GCN4 (39). These interactions have been proposed to contribute to recruitment of the PAF1 complex to specific genes (as in the case of GCN4) or, conversely, to the recruitment of the interacting transcription factor (as shown for STAT3). Myc was also recently shown to interact with Cdc73 and proposed to recruit the PAF1 complex and subsequently transfer it onto RNA polymerase II. Depletion of Cdc73 moderately increases the average expression of Myc targets (by ∼10%), suggesting that the PAF1 complex exerts a negative effect on Myc-dependent targets in the examined setting (48). The extent to which the PAF1 complex promotes cellular proliferation and contributes to transformation in vertebrates is unclear. On one hand, the complex component Cdc73 was originally identified as a tumor suppressor (53), and the PAF1 complex has been reported to reduce transcription and protein stability of the proto-oncogene Myc (54). On the other hand, posttranslational Fig. 1. The Paf1 complex affects Myc activity in vivo. (A) The indicated modification can convert Cdc73 to an oncogene (55), and over- components of the Paf1 complex were depleted in bristle precursor cells, and expression of Paf1 promotes growth of NIH 3T3 cells and induces sizes of dissected scutellar bristles from adult males were measured. tumor formation in vivo (56). (B) Areas of wing imaginal disk clones (in pixels) with Myc overexpression Here we show that the PAF1 complex functionally and physi- (red bars) or without Myc overexpression (blue bars), on knockdown of the cally interacts with Myc in Drosophila larvae and cells. Elimination indicated PAF1 complex components. Knockdown of LacZ served as a control of several complex components impairs Myc-dependent growth in in A and B. The numbers of bristles from independent flies (A) or of analyzed clones (B) are indicated in each bar. Error bars represent SEM. *P < 0.05, vivo, reduces the recruitment of Myc to its target genes, and affects **P < 0.001, two-tailed Student’s t test, for comparison with the appropriate their expression in S2 cells and in vivo. This suggests that the LacZ knockdowns. PAF1 complex contributes to Myc’s binding to its target promoters, thereby promoting cellular growth and proliferation. pletion of all tested PAF1 complex components strongly affected Results this overgrowth, but did not reduce the size of control clones The PAF1 Complex Contributes to Myc-Dependent Growth in Vivo. To (Fig. 1B). Simultaneous depletion of the PAF1 component identify novel transcriptional cofactors for Myc, we carried out Leo1 slightly reduced the level of ectopically expressed Myc an RNAi screen in S2 cells (a cell line derived from Drosophila protein (Fig. S1C); the reason for this is unclear, and no such embryos). Out of 752 tested transcription-associated factors, we effect of Leo1 depletion on endogenous Myc levels was ob- found 33 proteins that consistently affected the activity of the served), but had a clearly stronger impact on clonal area (Fig. Myc-dependent reporter, including the scaffold protein dHCF 1B). Strong effects on Myc-induced growth were also seen on (33) and the components of the PAF1 complex. Depletion of the depletion of other PAF1 complex components. Thus, the PAF1 proteins does not strongly affect Myc protein levels (Fig. PAF1 complex is limiting for the extra growth of imaginal disk S1A, and see below), but it does reduce the expression of various cells that is driven by Myc overexpression. Myc targets (see Fig. 6 and Fig. S1B), indicating that PAF1 acts as a positive cofactor for Myc-dependent transcription. ’ Leo1 Physically Interacts with Myc to Recruit It to Individual Targets. To assess the importance of the PAF1 complex for Myc sbi- To explore the molecular mechanism for these functional effects ological activities, we evaluated the consequences of PAF1 de- of the PAF1 complex on Myc activity, we turned back to cultured pletion in vivo. Myc primarily controls cellular and organismic cells. Since the PAF1 complex did not strongly affect Myc pro- growth in flies (30). A moderate reduction in Myc levels results in tein levels, we considered the possibility that it might physically small adult flies with disproportionally small bristles, indicating that Myc activity is particularly important for the growth of bristle interact with Myc and thereby influence its transcriptional output. We first investigated the PAF1 complex component Leo1

precursor cells (57, 58). Knockdown of the PAF1 proteins Rtf1, BIOCHEMISTRY Paf1, and Leo1 in these cells phenocopies the Myc-mutant phe- (since a corresponding epitope-tagged version was available) and notype and significantly reduces the size of adult bristles (Fig. 1A; observed a strong association with coexpressed Myc in S2 cells A the different magnitudes presumably reflect differences in knock- (Fig. 2 ). This interaction did not require any of the previously down efficiencies). This observation is consistent with participation described Myc domains, i.e., the Myc boxes 1 or 2 situated in the of the PAF1 complex in Myc-dependent growth. transactivation domain, the Myc box 3 mediating binding to To provide more direct evidence, we measured PAF1’s effect WDR5, or the C-terminally located Max-interacting bHLHZ on the areas of imaginal disk cell clones overexpressing Myc. domain. Instead, a broad central region of Myc was needed for Myc-overexpressing clones are significantly larger than controls, the binding to Leo1 (Fig. 2A). Within Leo1, a central region due to a strong increase in cell size (ref. 58 and Fig. 1B). De- between amino acids 442 and 474 was required for the associa-

Gerlach et al. PNAS | Published online October 16, 2017 | E9225 Downloaded by guest on October 1, 2021 S2 cells (Fig. 3E). These interactions may be mediated by endogenous Leo1, but we cannot exclude the possibility that additional PAF1 components besides Leo1 directly interact with Myc. This physical interaction suggests two possibilities for how the PAF1 complex might contribute to Myc’s activities: as a coactivator or as a corecruiter. According to one scenario, Myc first binds to its target genes (via specific DNA motifs as well as other chromatin factors, such as WDR5) and subsequently recruits the PAF1 complex that then promotes transcriptional elongation on these targets. In the second scenario, the PAF1 complex localizes to

Fig. 2. Physical interaction of overexpressed Myc and Leo1. The epitope- tagged proteins were transiently expressed in S2 cells (as indicated above the blots) and immunoprecipitated with the indicated antibodies. (A) None of the conserved regions in Myc (N terminus, Myc boxes 2 and 3; C terminus consisting of a bHLHZ region) is individually essential for interaction with Leo1. Instead, a central region of Myc is involved in binding to Leo1. (B)Leo1’s central region between amino acids 442 and 474 is essential for binding to Myc.

tion with Myc (Fig. 2B). No biochemical functions or specific structures have been ascribed to this region. We next examined whether this Leo1–Myc interaction also oc- curs at physiological levels of either protein. We failed to observe coimmunoprecipitation of the endogenous proteins (presumably because of the limitations of our anti-Leo1 antiserum), and thus established a stable S2 cell line that allows inducible expression of HA epitope-tagged Leo1. In the absence of inducer, these cells showed low leaky expression of HA:Leo1; on addition of CuSO4, the levels of HA:Leo1 rose to levels approaching those of endogenous Leo1 (Fig. 3C and Figs. S2A and S3A,5–8). The addition of dsRNA targeting Myc efficiently eliminated Myc from these cells (Figs. S2A and S3A,9–12). Using these cells, we carried out a proximity-ligation assay (PLA; ref. 59) and found that HA: Leo1 associates with endogenous Myc in nuclei (Fig. S3). More- over, endogenous Myc could be coimmunoprecipitated with HA: Fig. 3. Myc and Leo1 associate at physiological expression levels. Leo1 in this setup, indicating that the PLA signal reflects a physical (A) Coimmunoprecipitation of ectopically expressed HA:Leo1 with endoge- interaction between the two proteins (Fig. 3A). This interaction nous Myc in S2 cells. HA:Leo1 expression was induced to near-physiological

takes place on DNA, as shown by sequential chromatin immuno- levels (C) with CuSO4 for 24 h before cell lysis. Immunoprecipitates were precipitation (Re-ChIP) experiments; previously identified Myc incubated with the indicated antibodies to detect endogenous Myc and HA: targets were specifically recovered when chromatin from HA: Leo1. (B) Re-ChIP experiments were performed from HA:Leo1-expressing Leo1-expressing cells was immunoprecipitated with an anti-HA (Left, HA:Leo1) or naïve S2 cells (Right, control). Bound chromatin from an serum, followed by immunoprecipitation with anti-Myc anti- anti-HA chromatin immunoprecipitate was eluted with HA peptides and incubated with either anti-Myc (blue bars) or IgG as a control (red bars). bodies (Fig. 3B). No such enrichment was seen in naïve S2 cells B C (C) Western blot displaying the successful induction of HA:Leo1 was probed or for control DNA sequences (Fig. 3 ). Fig. 3 shows the ex- with antibodies against HA, endogenous Leo1, and endogenous Myc; α-tubulin pression levels of HA:Leo1 and endogenous Myc. Together, served as a loading control. (D) Leo1 was translated in vitro in a rabbit re- these experiments show that Leo1 and Myc physically associate ticulocyte lysate in the presence of 35S-labeled methionine; 10% of the trans- in cells. This interaction is likely direct, since it also can be seen lation mixture was then loaded directly on a gel (lane “input”), and the between in vitro synthesized 35S-labeled Leo1 and a GST:Myc remainder was incubated with a GST:Myc fusion protein (amino acids 46–507) fusion protein purified from Escherichia coli (Fig. 3D). We purified from E. coli, or with GST as a control. After SDS/PAGE, the gel was fixed – and dried, and radiolabeled Leo1 was visualized by autoradiography. (E)The consider it likely that this Leo1 Myc interaction serves to asso- indicated epitope-tagged PAF1 complex components were transiently coex- ciate the entire PAF1 complex with Myc. Indeed, epitope-tagged pressed with either HA-tagged Myc (Left) or Myc carrying a 9E10 tag (Right), versions of the other PAF1 complex components Paf1, Ctr9, and followed by immunoprecipitations with anti-AU1 or anti-HA antibodies. As- Cdc73 can also be coimmunoprecipitated with coexpressed Myc in terisks in the lower input panel denote the PAF1 complex proteins.

E9226 | www.pnas.org/cgi/doi/10.1073/pnas.1705816114 Gerlach et al. Downloaded by guest on October 1, 2021 open promoters (notably via contacts with the general transcription PNAS PLUS machinery) and thereby acts as one of the chromatin-associated factors that helps recruit Myc to its targets. Subsequently, Myc stimulates target gene expression through other coactivators and/or by relieving the negative effects of the PAF1 complex on transcriptional elongation. The first scenario predicts that recruitment of Leo1 to Myc target genes is dependent on the presence of Myc. Therefore, we assessed the binding of Leo1 to Myc targets. Since our affinity-purified anti-Leo1 antibody is not suitable for ChIP, we used anti-HA antiserum to assess the binding of HA:Leo1 to Myc targets with or without Myc knockdown. Quantitative depletion of Myc (Fig. S2B) did not strongly reduce binding of HA:Leo1 to Myc targets, indicating that Myc contributes only moderately to Leo1 recruitment to target genes (Fig. 4A). To address the alternative scenario, we knocked down Leo1 and determined the binding of endogenous Myc to the same targets described above. Although Leo1 was only partially eliminated (Fig. S2C), Myc binding to these genes was significantly reduced (Fig. 4B). Reduced Myc binding was also observed when any of the other PAF1 complex components were depleted, although the effects were less prominent than after Leo1 knockdown (Fig. 4C; Fig. S2D shows depletion efficiencies). These data show that Leo1 and most likely the entire PAF1 complex are required for efficient binding of Myc to certain target genes. To investigate the impact of the PAF1 complex on Myc binding on a global scale, we carried out ChIPseq experiments, focusing on Myc’s direct interactor Leo1, which had also shown the strongest effect on Myc binding. In naïve S2 cells, we found Myc to bind 714 regions (Table S1), including 296 localized in promoter- proximal regions and 166 overlapping with enhancers (as defined experimentally in ref. 60). As expected, Myc knockdown strongly impaired these interactions; as examples, the genes analyzed in Fig.4areshowninFig.5A (Fig. S2E shows exemplary depletion efficiencies). Myc binding to promoter and enhancer sites was similarly reduced (Fig. 5 B and C). In contrast, Leo1 knockdown did not affect the interaction of Myc with enhancer sites (98% of control; P = 0.7907) (Fig. 5 B and C), attesting to the comparability of the different ChIPseq samples, but it significantly reduced the binding of Myc to promoter sites (to 82% of control; P < 0.0001). The difference between enhancer and promoter sites is not related to the differential presence of Leo1 at the two types of sites, since HA:Leo1 was chipped with comparable efficiency to promoter and Fig. 4. Mutual influence of Myc and Leo1 on chromatin binding. (A) Anti- enhancer sites (Fig. 5B; Fig. S2F shows protein levels). Many of HA ChIP from cells expressing HA:Leo1 or naïve S2 cells, with and without these promoters contain E-boxes (115 out of 296), which are di- Myc depletion. Cells were harvested and processed at 24 h after the addition μ rectly bound by Myc:Max complexes. The association of Myc with of Myc-dsRNA and 125 M CuSO4 to induce expression of HA:Leo1. Shown such E-box–containing promoters is somewhat more strongly im- are averages and SEMs from two biologically independent experiments, paired by Leo1 depletion compared with the association with normalized to the signal obtained for Uhg1 from HA:Leo1-expressing con- promoters lacking E-boxes (P = 0.055) (Fig. 6D). trol cells. (B) ChIP of control cells or cells incubated with Leo1-dsRNA for 48 h, obtained with rabbit anti-Myc or control rabbit IgGs. Shown are averages and SEMs from two to three biologically independent experiments, nor- Leo1 Is Required for Efficient Expression of Myc Targets. The fore- malized to the signal obtained for Uhg1 from control cells. (C) ChIP from going data indicate a requirement for Leo1 for efficient re- control cells or cells incubated with the indicated dsRNA for 48 h and pro- cruitment of Myc to its target promoters, suggesting a role for cessed as described for B. Data are the average of two biologically in- Myc’s transcriptional output. Therefore, we investigated the dependent experiments for each knockdown (six for control ChIPs), each impact of Leo1 on the cellular transcriptome of S2 cells. Despite with SEM. fairly efficient reduction of Leo1 protein levels (Fig. S2G), the effects on global transcription were modest (Fig. S4A and Table S2). However, Leo1 knockdown significantly reduced the ex- Consistent with such small effects on gene expression in naïve pression of genes with E-boxes immediately downstream of their S2 cells, depletion of Leo1 (and other PAF1 complex compo-

transcription start sites (the majority of these genes were pre- nents) in control clones in vivo had little effect on clonal size BIOCHEMISTRY viously shown to be Myc targets; ref. 61), as well as of genes that (Fig. 1B). In contrast, the clonal overgrowth induced by Myc were bound by Myc over an E-box situated in the promoter and overexpression was strongly impaired by simultaneous depletion whose expression decreased on Myc knockdown (presumably of PAF1 complex components, suggesting that effects on Myc direct Myc targets; ref. 7), consistent with the idea that Leo1- target gene expression become more pronounced in such a set- mediated Myc binding to target promoters is required for the full ting. Thus, we set out to investigate the impact of Leo1 depletion transactivation of these genes by Myc (Fig. 6A and Fig. S4B). on the transcriptomes of Myc-overexpressing imaginal disks, The magnitude of these effects was small, however, possibly using the same animals and analogous conditions as for the because PAF1’s positive effect on Myc recruitment is partially clonal analysis shown in Fig. 1B. In addition, we assessed the offset by its negative effects on transcription (48). consequences of loss of Max on Myc-dependent transcription

Gerlach et al. PNAS | Published online October 16, 2017 | E9227 Downloaded by guest on October 1, 2021 structure, translation, and mitochondrial function; Table S3)and show an excellent overlap with the Myc target genes identified in S2 cells (e.g., Fig. S5A). The number of Myc-repressed genes is surprisingly large; Table S3 presents a Gene Ontology (GO) analysis of these genes. We and others have previously identified fewer than 500 Myc-repressed genes in S2 cells or in vivo, and typically the number of repressed genes is smaller than the number of activated genes (7, 61–63). It also has been noted that the Myc- repressed transcriptomes are less concordant across different experimental settings, suggesting a greater impact of uncontrollable experimental conditions than for the Myc-activated transcriptomes (7). Nevertheless, previously reported lists of Myc-repressed genes coincide to some extent with our present list of Myc-repressed genes (Fig. S5B). Depletion of Leo1 reduced the median induction of the 977 Myc activated genes from 176% to 146% and restored the expression of the 1,175 Myc-repressed genes from 57% to 77% of control (Fig. 6D). In particular, the effect on gene repression (a relative relief of 47%) was greater than the slight reduction in Myc levels caused by Leo1 knockdown (Fig. S1C), indicating that Leo1 is required for efficient binding and control of target genes by Myc. The lack of Max affected Myc-repressed genes to a slightly greater degree (restoring expression to 85% of control), and almost completely eliminated Myc-mediated activation (re- ducing expression to 107% of control). A combination of the two lesions did not suppress Myc-dependent transcriptional control any further (Fig. 6D). Very similar effects were observed when this analysis was restricted to a narrowly defined set of putative direct Myc targets, 221 induced genes and 25 repressed genes that were also bound by Myc in promoter regions in S2 cells (Fig. 6E); we did not include Myc-bound enhancers, since we could not unambiguously assign transcripts to these enhancers. The lack of Max completely eliminated Myc-dependent transactivation of Fig. 5. Global effects of Leo1 or Myc depletion on Myc binding in S2 cells E-box–containing targets, but allowed for some transactivation of (ChIPseq). (A) ChIPseq traces of Myc over the genomic regions that were also E-box–lacking Myc targets (Fig. S5C). These data show that Myc analyzed by manual ChIP in Fig. 5. Asterisks indicate the positions of canonical retained substantial transactivation potential in a Max null-mutant E-boxes. Lengths of the vertical axes (i.e., maximal read numbers) are 800 for α background in which essentially no Max transcripts were present Uhg1, 500 for Uhg2, and 200 for NAC .(B) Myc binding is significantly reduced (ref. 28 and Fig. 6C), and that even though the Leo1 knockdown on Leo1 depletion compared with naïve S2 cells for sites located in promoters C (296 sites; average reduction of the median, 22%; P < 0.0001, Wilcoxon signed- did not completely remove Leo1 transcripts (Fig. 6 ), its conse- rank test), but not for sites located in enhancers (166 sites; average reduction quences on global Myc-dependent gene induction and repression of the median, 2%; P = 0.7907). The difference between the effects on pro- were significant. These analyses demonstrate a clear requirement moter and enhancer sites is highly significant (P < 0.0001, two-tailed t test), for Leo1 (and, by inference, the entire PAF1 complex) for Myc- whereas Myc depletion affects both types of sites to the same extent (78% and dependent gene regulation, particularly in conditions of supra- 79% reductions relative to the median of control cells, respectively; P = 0.2940, physiological Myc activity. two tailed t test). (Lower) Relative read number of anti-HA ChIPs. Both promoter and enhancer regions are significantly enriched for reads in HA:Leo1- Genetic Interaction of Leo1 and Max in Vivo. As an additional test to < expressing cells compared with naïve S2 cells (P 0.0001, Wilcoxon signed-rank phenotypically assess the effects of Leo1 and/or Max on Myc- test), but the two regions do not significantly differ with respect to enrichment dependent processes, we turned to a previously described in vivo (7.9× and 6.6× median enrichment, respectively; P = 0.1895, two tailed t test). (C) Average distribution of reads over Myc-binding sites located in promoters assay for Myc activity. Myc overexpression in developing eyes or enhancers (labeled “P” and “E”, respectively) in control cells or on depletion promotes the growth of individual ommatidia and also increases of Myc or Leo1. Reads were counted over 50-nt windows for the indicated the rate of apoptosis in this tissue (64). The resulting adult eyes conditions, followed by subtraction of input reads for the corresponding are larger overall and contain larger ommatidia, but their ar- window. (D) Leo1 depletion affects Myc binding to enhancer sites to the same rangement is disturbed, causing a rough appearance. The apo- extent regardless of whether or not these sites contain canonical E-boxes ptosis of pigment cells produces an overall lighter eye coloration (median binding relative to naïve S2 cells, 99% and 97%, respectively; P = with numerous interspersed dark ommatidia (Fig. 7A). De- 0.2234, two-tailed t test), whereas promoter sites containing canonical E-boxes pletion of Max in this context abrogates Myc’s growth-promoting are affected more strongly by Leo1 depletion compared with promoter sites ability but does not affect its proapoptotic effects, thereby lacking E-boxes (median binding relative to naïve S2 cells, 74% and 83%, respectively; P = 0.0564, two-tailed t test). producing a small, very rough-looking eye (28). In contrast, Leo1 knockdown does not impair Myc-dependent overgrowth, but does reduce eye roughness (i.e., Myc’s proapoptotic activity). Codepletion of Max and Leo1 eliminated much of this roughness (either alone or in combination with Leo1 down-regulation), and Myc-dependent eye overgrowth (Fig. 7; compare the Max since our data suggested potentially related roles for Leo1 and knockdown with or without Leo1 knockdown). Importantly, in Max in Myc recruitment. the absence of Myc overexpression, neither Leo1 nor Max In an otherwise wild-type background, Myc overexpression sig- knockdown had a strong effect on eye size or morphology, nificantly induced 977 genes and repressed 1,176 genes (Fig. 6B). demonstrating that all of the described effects are mediated by The Myc-induced genes largely fall into the previously recognized Myc. These observations illustrate the importance of both categories of Myc targets (notably ribosome biogenesis, ribosome Leo1 and Max for Myc-dependent transcriptional control, and

E9228 | www.pnas.org/cgi/doi/10.1073/pnas.1705816114 Gerlach et al. Downloaded by guest on October 1, 2021 raise the possibility that these two proteins influence different PNAS PLUS sets of Myc target genes. However, our RNAseq analysis did not identify any apoptosis-related Myc targets that were differentially affected by Leo1 and Max, and thus we cannot molecularly explain these different phenotypes. It remains possible that they are caused by small transcripts that were not investigated in our transcriptome studies (e.g., miRNAs, tRNAs), or that they in- volve nontranscriptional functions of Myc (Discussion). Discussion Although the Myc:Max complex binds to specific DNA sequences in vitro, this interaction is not sufficiently strong to allow efficient interaction with these motifs when they are embedded in chromatin context (10, 13). Additional proteins are needed for the observed binding of Myc to its targets in vivo, such as WDR5 (17) and the PAF1 complex identified here. We specifically show that most components of the PAF1 complex can associate with Myc, and that Leo1 does so in vivo and in vitro, suggesting that it is Leo1 that mediates the interaction between PAF1 and Myc (possibly in addition to other complex components). By virtue of its association with the general transcription machinery, the PAF1 complex is preferentially localized to active promoters and thus ideally placed to help recruit Myc to relevant binding sites. We assume that additional factors participate in the recruitment of Myc to chromatin, and that the relative contributions of these factors vary in different cellular backgrounds. Nevertheless, the PAF1 complex is essential for full Myc binding to its endogenous targets in S2 cells. It may appear surprising that knockdown of PAF1 components does not affect the expression of Myc targets to the same extent as Myc binding does; however, such mild consequences of PAF1 depletion on cellular transcriptomes despite stronger effects on chromatin-associated proteins and chromatin marks have been consistently reported (e.g., refs. 46 and 48). This can be rationalized by the combination of positive (e.g., recruitment of Myc) and negative (e.g., inhibition of elongation; ref. 48) contributions of the PAF1 complex to gene expression. As a result, the net effect on mature transcript levels is dampened—overall, it may appear to be either positive or negative (42). Depletion of PAF1 proteins has a substantial impact on the Fig. 6. Effects of Leo1 and Max on the expression of Myc targets (RNAseq). extra tissue growth induced by high levels of Myc in vivo. This may (A) Distribution of relative expression levels in S2 cells [kernel density of log2 be caused in part by a moderate reduction of Myc protein levels (Leo1-KD/GFP-KD)] for different gene groups: all 9,123 expressed genes (the reason for which is unknown), as well as by impaired re- (black curve; median, 0.001838), 58 direct Myc targets (i.e., genes that are cruitment of Myc to a large number of target genes. In addition, significantly down-regulated after Myc depletion and bound by Myc over an we cannot exclude the possibility that the PAF1 complex also af- E-box situated in the promoter region; red curve; median, −0.02579), and 170 genes with a downstream E-box (see text; blue curve; median, fects transcription-independent processes. Although Myc is best −0.03935). The latter two groups differ significantly from the former, with known for its role in controlling transcription, it also affects other P = 0.00067 and 0.024, respectively (nonparametric Spearman correlation). cellular processes independent of transcription, such as DNA (B) Dot plots showing log2 read numbers of the indicated imaginal disk replication (27), mRNA cap methylation and translation (65), and genotypes (averages of three biologically independent replicates) for 8,251 α-tubulin acetylation (66). A role for the PAF1 complex in the first genes expressed above a minimal threshold in our samples (Methods). Dots two processes (which obviously contribute to cellular proliferation marked in red are significantly (P < 0.05), by >1.5-fold, deregulated by Myc and/or growth) is conceivable, but this has not been addressed overexpression in the corresponding genotype. (C) Relative read numbers so far. for the Leo1 and Max transcripts in imaginal disks. Each bar is derived from In contrast to cells with high expression of Myc, the growth of six biologically independent RNAseq samples of the indicated genotype, where wt indicates Max+/+ and control corresponds to no Leo1 knockdown. wild-type eyes or control wing imaginal disk clones is not reduced Values for wt control are set to 100%. Error bars represent SEM. (D) Effect of by depletion of the PAF1 complex. Indeed, ubiquitous depletion Myc overexpression in different genotypes. The x-axis shows the log2 of the of the PAF1 complex throughout the animal allows most flies to ratio (expression on Myc overexpression in genotype X/expression in the develop to the pharate adult stage, and some escapers even SI Materials and absence of Myc overexpression, in genotype X), whereas the y-axis shows the complete development and eclose as adults ( BIOCHEMISTRY effect of Myc overexpression for control wing disks (i.e., Max wt and no Methods). This effect may be explained in part by insufficient Leo1 knockdown). Genes were sorted according to their relative Myc effect knockdown efficiencies (the available null mutants in Paf1, in control wing disks and pooled into bins of 40. (E) Fold expression changes Cdc73, or Ctr9 do show a stronger phenotype), but it also sug- in response to Myc overexpression in the indicated genotypes. Shown are gests that the PAF1 complex is less essential under normal 221 and 25 genes that are bound by Myc in promoter regions in S2 cells and that are significantly induced or repressed, respectively, by Myc overgrowth conditions. On the other hand, in tissues undergoing expression in an otherwise wild-type background (a subset of the genes rapid growth (notably imaginal disks experiencing Myc over- marked in red in the upper left plot of B). All bars show median expression expression), depletion of Leo1 clearly reduces the ability of Myc ratios, and all bars differ significantly from one another (P < 0.01, Mann– to regulate targets and impedes the associated overgrowth. Both Whitney U test), except where indicated by “ns” (nonsignificant). Myc-repressed and Myc-activated genes are affected, consistent

Gerlach et al. PNAS | Published online October 16, 2017 | E9229 Downloaded by guest on October 1, 2021 absence of Max (28), Myc retains the ability to regulate a significant number of target genes. Since Myc is unlikely to bind to these genes on its own, this begs the question of what other partner can substitute for Max in such a situation. Materials and Methods Flies. The following flies were used in our analyses: sca-GAL4 (Bloomington Drosophila Stock Center; 6479), UAS-Max-IR (line 2-7; ref. 28), UAS-GFP (E. Hafen), UAS-LacZ (B. Edgar), actin-FRT-CD2-FRT-GAL4 (K. Basler), and GMR- GAL4 3×(UAS-Myc) (characterized in ref. 28). Additional UAS lines for RNAi were obtained from the Vienna Drosophila Resource Center: UAS-Rtf1-IR (27341), UAS-atms-IR (20876), and UAS-atu-IR (17490). Relevant genotypes for Fig. 6 included hs-FLP actin5C-FRT-stop-FRT-GAL4 UAS-GFP UAS-p35 (wt ctr), hs- FLP actin5C-FRT-stop-FRT-GAL4 UAS-GFP UAS-Myc UAS-p35 (wt ctr Myc- overexpression), hs-FLP actin5C-FRT-stop-FRT-GAL4 UAS-GFP UAS-p35 UAS- Leo1-IR (wt Leo1-KD), hs-FLP actin5C-FRT-stop-FRT-GAL4 UAS-GFP UAS-Myc UAS-p35 UAS-Leo1-IR (wt Leo1-KD Myc-overexpression), hs-FLP actin5C-FRT- − − stop-FRT-GAL4 UAS-p35 Max / (Max ctr), hs-FLP actin5C-FRT-stop-FRT-GAL4 − − UAS-Myc UAS-p35 UAS-GFP Max / (Max ctr Myc-overexpression), hs-FLP actin5C-FRT-stop-FRT-GAL4 UAS-GFP UAS-p35 UAS-Leo1-KD Max−/− (Max Leo1-KD), and hs-FLP actin5C-FRT-stop-FRT-GAL4 UAS-GFP UAS-Myc UAS-p35 − − UAS-Leo1-KD Max / (Max Leo1-KD Myc-overexpression). Relevant genotypes for Fig. 7 included GMR-GAL4 UAS-GFP UAS-LacZ, GMR-GAL4 UAS-GFP UAS- Leo1-IR, GMR-GAL4 UAS-Max-IR UAS-GFP, GMR-GAL4 UAS-Max-IR UAS-Leo1- IR, GMR-GAL4 3×(UAS-Myc) UAS-GFP UAS-LacZ, GMR-GAL4 3×(UAS-Myc) UAS- GFP UAS-Leo1-IR, GMR-GAL4 3×(UAS-Myc) UAS-Max-IR UAS-GFP, and GMR- GAL4 3×(UAS-Myc) UAS-Max-IR UAS-Leo1-IR.

In Vivo Analysis. UAS-RNAi-transgenes were targeted to bristle precursor cells using the sca-GAL4 driver. Adult scutella were then dissected and mounted on glass slides in glycerol. Pictures were taken using a 5× lens, and bristle size was determined in Adobe Photoshop as the total pixel count a bristle covers in a picture. Clones expressing GFP or Myc + GFP were induced and analyzed (7) at 48 h after clone induction in wandering larvae. For RNAseq of imaginal Fig. 7. Effect of Leo1 and Max depletion on the Myc overexpression phe- disks, flies of the appropriate genotypes were raised under standard con- − − notype in the eye. (A) Photomicrographs of male adult eyes of the indicated ditions at 25 °C. At the age of 53–66 h (139–143 h for the Max / genotypes), genotypes. (B) Average ommatidial size of flies with the genotypes shown in they were subjected to a 2-h heat shock at 37 °C to induce ubiquitous ex- A. The number of analyzed independent eyes per genotype are indicated. pression of the GAL4-dependent transgenes. Then, 48 h later, wing imaginal Error bars represent SEM. Significance of deviation from the corresponding disks were dissected into Qiazol and immediately stored at −80 °C until genotype without knockdown transgene according to Student’s two-tailed further processing. t test: *P < 0.05; **P < 0.001. All genotypes within one series contain the To determine ommatidial size, flies were raised under noncrowding condi- same number of UAS transgenes, to rule out titration of GAL4 (relevant tions. Adult males were collected at 1–7 d after eclosion and killed by freezing. genotypes are described in Materials and Methods). Eyes were photographed with a Zeiss Discovery V8 stereomicroscope fitted with a1.5× lens and processed with Axiovision Extended Focus software. For each genotype, the area of 20 centrally located ommatidia was measured from at with the idea that Myc recruitment is impaired in this situation. least seven eyes from independent individuals. This suggests that supraphysiological levels of Myc saturate the available PAF1 complex and possibly also other “recruitment RNAi Screen and S2 Cell Culture. Culture and transfection of S2 cells and the factors.” Thus, such settings, as are notably encountered in many RNAi screen for Myc cofactors have been described previously (7, 33). human tumors, might be particularly sensitive to inhibition of PAF1 activity. Taken together, our observations emphasize the Molecular Biology. dsRNA-mediated knockdowns, quantitative real-time PCR, and manual chromatin-immunoprecipitations were carried out as described importance of Leo1 for the biological activity of Myc, but they do previously (7). For Primers see Table S4. Dual luciferase assays were con- not demonstrate that all PAF1 activities are mediated by Myc. ducted as described previously at 48–60 h after transfection of reporters and Indeed, the PAF1 complex interacts with several transcription dsRNA (7). factors besides Myc, and it is conceivable that some of these factors also contribute to the growth-related functions of PAF1. Antibodies. Antibodies were mouse anti-Drosophila Myc (7), rabbit anti- It remains to be seen whether Leo1 and Max are involved in Drosophila Myc (Santa Cruz Biotechnology), mouse anti–α-tubulin (Sigma- recruiting Myc to different functional sets of targets. The analysis Aldrich), rabbit anti-HA (Abcam or ICL), rabbit anti-AU1 (Bethyl Laborato- of Myc-overexpressing adult eyes suggests that Leo1 and Max ries), mouse anti-AU1 (Covance), rat anti-Cdc73, and rabbit anti-Rtf1 (gifts from J. Lis). A polyclonal rabbit antiserum was raised against the peptide predominantly affect different Myc-dependent processes: apo- RDKVESQVESAPKEC (amino acids 356–369 of Drosophila Leo1) and affinity- ptosis and growth, respectively. Indeed, individual genes are purified (New England Peptide or ImmunoGlobe). differentially affected by Max or Leo1 knockdown, but no gene sets obviously marked as “growth-related” or “apoptosis-related” Plasmids for Expression in S2 Cells. Wild-type Myc and mutant derivatives behave in the expected manner. It is conceivable that the gene(s) were cloned in-frame with an N-terminal hemagglutinin (HA) tag in the responsible for apoptosis in the eye specifically disrupt pupal eye vector pUASattB. Numbered deletions (created by site-directed mutagenesis) development (a 4-d-long process that mostly involves cellular retain the indicated regions of the Myc protein, e.g., amino acids 403–717. growth and differentiation, but little proliferation), and thus Mutants lacking specific Myc domains have been described previously (18, 28, 33). In brief, they carry the following modifications: ΔN-term lacks amino might not be recognized as being generally apoptosis-related. acids 1–293, ΔMB2 has the amino acid “GP” instead of amino acids 68–84 Alternatively, the relevant genes code for small transcripts (Myc box 2), ΔMB3 has amino acid “F” instead of amino acids 405–422 (Myc (e.g., miRNAs, tRNAs) that have not been included in our box 3), ΔC-term lacks amino acids 626–717 (C terminus: bHLHZ), and ΔZ lacks transcriptome analyses. Finally, we stress that even in the complete amino acids 676–717 (leucine zipper). Analogously, the Leo1 coding region

E9230 | www.pnas.org/cgi/doi/10.1073/pnas.1705816114 Gerlach et al. Downloaded by guest on October 1, 2021 was cloned behind an HA or AU1 epitope tag in pUASattB, and mutant secondary antibodies at room temperature, and washed again. Cells were PNAS PLUS derivatives were generated and numbered as for the Myc mutants. mounted on glass slides using aqua-fluoromount (Sigma-Aldrich) and imaged To obtain stable expression of HA:Leo1 (wild-type), the corresponding with a confocal microscope (Nikon Ti-Eclipse) with a 60× objective. Images were coding region was inserted under control of the metallothionein promoter in processed with ImageJ 1.50h (67). the vector pMT181 carrying a puromycin resistance marker (a gift of M. Tiebe and A. Telemann, Zentrum für Molekulare Biologie der Universität Heidelberg). Re-ChIP. Cell fixation, lysis, and sonication were carried out as described pre- On puromycin selection of stable cell pools, HA:Leo1 expression was induced by viously for manual ChIP (7). On cell lysis, 1% of the lysate was set aside as input μ incubation with 125 MCuSO4 for 24 h. control. Anti-HA magnetic beads (Thermo Fisher Scientific) were prepared by three washes with 1× PBS containing BSA (5 mg/mL), and 60 μL was incubated In Vitro Interaction. The E. coli strain BL21 was transformed with constructs with the chromatin overnight at 4 °C. Dynabeads (Thermo Fisher Scientific) for coding for GST or a GST:Myc (amino acids 46–507) fusion, and protein expression the secondary ChIP were similarly washed and incubated with 3 μgofanti-Myc was induced by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside for antibody or control rabbit IgGs overnight. The precipitates were washed as 3 h at 37 °C. On bacterial lysis, GST proteins were purified by incubation with described for manual ChIP and then eluted twice with 0.8 mg/mL Pierce HA glutathione Sepharose beads (Amersham Biosciences). Leo1 was expressed in peptides (Thermo Fisher Scientific) in 1× RIPA buffer for 15 min at 37 °C. Five vitro in a coupled rabbit reticulocyte lysate in the presence of 35S-labeled me- percent of the combined eluates were set aside as input control; the remainder thionine (TNT Kit; Promega), and incubated with the GST/GST:Myc bound to was incubated with the antibody-coupled Dynabeads for 6 h at 4 °C. Washing, glutathione beads in GST Binding Buffer (200 mM NaCl, 1 mM EDTA, 1 mM DTT, elution, and extraction of the precipitates, as well as analysis by quantitative 0.5% Nonidet P-40, 10% glyerol, and 0.05% BSA) containing protease inhibitors real-time PCR, were carried out as described for manual ChIP. (Roche). Bead-bound proteins were then analyzed by SDS/PAGE followed by autoradiography, as described previously (33); 10% of the in vitro translation RNAseq, ChIPseq, and Bioinformatic Analysis. RNAseq, ChIPseq, and bioinformatic mix was directly loaded on the gel and served as input control. analyses were carried out as described previously (7), with the following modifications. Antibodies for ChIPseq were rabbit anti-Myc (Santa Cruz Biotechnology) Western Blot and Immunoprecipitation Analyses. For transient expression in and rabbit anti-HA (Abcam). Sequencing was done on an Illumina NextSeq 500. S2 cells, appropriate UAS plasmids were cotransfected with tubulin-GAL4, For each ChIPseq condition, as well as input control, 7,847,000 reads were and cells were harvested at 24–48 h after transfection. Pelleted cells were mapped onto the reference genome dm6 (bowtie 2.2.4). Peaks were called with washed once with cold 1× PBS and then lysed on ice for 30 min in lysis buffer macs 1.4.0 and statistically analyzed with R and GraphPad Prism. · [150 mM NaCl, 50 mM Tris HCl pH 8.0, 5 mM EDTA pH 8.0, 0.5% Nonidet Promoter regions were defined as the ±100 nucleotides flanking the an- P-40, containing Protease Inhibitor Mixture tablets (Roche)]. Insoluble con- notated transcription start sites (FlyBase FB2015_4) for a total of 17,716 pro- × tents were precipitated by centrifuging for 15 min at 16,200 g. The lysates moters. Enhancers (5,499 regions) were derived from ref. 60, with coordinates were precleared for 1 h at 4 °C with protein G Sepharose bead suspension adapted to the reference genome dm6. Myc-binding sites were defined as (GE Healthcare) and 5% of the lysate was set aside as input control. Incu- those identified by MACS in naïve S2 cells that did not overlap background – μ bations with 0.2 1 g of the primary antibodies were performed at 4 °C for sites (as called by anti-HA ChIP from naïve S2 cells) and did not have increased 3 h, followed by a 1-h precipitation of the epitope antibody complexes with read numbers on Myc depletion (714 sites); 166 of these were located in en- protein G Sepharose beads. Some immunoprecipitations were performed hancer regions (as defined by ref. 60) and did not overlap promoters, whereas μ with Dynabeads (Life Technologies GmbH) which were incubated with 1 g 296 overlapped promoters. For the analysis shown in Fig. 6, reads were – of the primary antibody for 6 8 h at 4 °C. 10% of the lysate were set aside as counted in 300-nt windows centered on the Myc-bound summits (as called by input control. Lysates were incubated over night at 4 °C with the antibody MACS) using the bedtools v2.17.0 suite, reads for the input sample over the coupled beads. For all immunoprecipitations, the immunoprecipitated ma- same window were subtracted, and read ratios were calculated relative to terial was washed three times for 5 min in lysis buffer on ice, SDS sample ChIPs from naïve control cells. ChIPseq profiles for Fig. 6A were generated with buffer was added, and the samples were analyzed by SDS/PAGE and im- the genome browser IGB, using a MACS output retaining all duplicate reads munoblotting as described previously (33). (parameter “keep-dup all”). For coimmunoprecipitations of endogenous Myc with HA:Leo1, cells with a For RNAseq of S2 cells, RNA was isolated in biologically independent tripli- stably integrated MT-HA:Leo1 plasmid were induced with 125 μM CuSO .At 4 cates at 48 h after Leo1 or control depletion, depleted of rRNA using the 24 h later, 1.5 × 108 cells were harvested, washed once with cold 1× PBS, Ribominus Kit (Invitrogen), and processedforsequencingtoadepthof6,757,000 lysed in Hepes-EDTA-glycerol-Nonidet P-40 (HEGN) buffer with 140 mM KCl, (non-rRNA) reads. For final analysis, 9,123 genes were kept with at least one read and sonicated for 40 s at 20% amplitude (Digital Sonifier Cell Disruptor; in each of the six samples and at least one read per million on average for either Branson). Five percent of the lysate was set aside as input control. Dyna- the control or Leo1 knockdown condition. Statistical analysis was performed beads (Life Technologies) were preincubated with 8 μg of rabbit anti-HA with the Bioconductor tools in R and with GraphPad Prism. For RNA isolation (Abcam) primary antibody or control rabbit IgG for 6–8 h at 4 °C, and cell from larvae, between 16 and 27 imaginal disks per sample were dissected into lysates were incubated overnight at 4 °C with the antibody-coupled beads. Qiazol (Qiagen). Further processing was done as described previously (7), except The immunoprecipitate was washed three times with HEGN buffer con- that cDNAs were prepared with NEBNext Poly(A) mRNA Magnetic Isolation taining protease inhibitors, SDS sample buffer was added, and the samples Module (New England BioLabs). For each of the 24 samples (eight different were analyzed by SDS/PAGE and immunoblotting as described above. genotypes × three replicates), an average of 6.3 million mapped reads were obtained and analyzed as described above. Immunostaining in Drosophila S2 Cells. Drosophila S2 cells were plated on poly-

L-lysine (Sigma-Aldrich)–coated coverslips and exposed to 125 μM CuSO4 and/or Myc-dsRNA (2 μg/106 cells) for 24 h, fixed with 4% paraformaldehyde, per- ACKNOWLEDGMENTS. We thank André Kutschke and Reinhold Krug for tech- nical support; the Bloomington Drosophila Stock Center, the Vienna Drosophila meabilized with 0.3% Triton-X 100 and then treated with blocking solution Resource Center, and the fly community for various fly lines; K. Basler, M. Tiebe, (10% goat serum, 2% BSA, and 5% sucrose in PBS) for 45 min after washing. and A. Teleman for plasmids; and J. Lis for antibodies. Funding for this project Cells were incubated overnight at 4 °C with primary antibodies in blocking was provided by the Swiss National Science Foundation (Grant 3100A0-120458/1, solution [rabbit anti-HA (Santa Cruz Biotechnology), 1:500 and mouse anti-Myc, to P.G.) and the German Research Foundation (Grants WO 2108/1-1, to E.W.; GA at 0.3 μg/mL], washed in TBS with 0.1% Tween-20, incubated for 1 h with the 1553/1-1, to P.G.; and GA 1553/2-1, to P.G.).

1. Conacci-Sorrell M, McFerrin L, Eisenman RN (2014) An overview of MYC and its in- 7. Herter EK, et al. (2015) snoRNAs are a novel class of biologically relevant Myc targets. teractome. Cold Spring Harb Perspect Med 4:a014357. BMC Biol 13:25. BIOCHEMISTRY 2. Gabay M, Li Y, Felsher DW (2014) MYC activation is a hallmark of cancer initiation and 8. Chandriani S, et al. (2009) A core MYC gene expression signature is prominent in basal- maintenance. Cold Spring Harb Perspect Med 4:a014241. like breast cancer but only partially overlaps the core serum response. PLoS One 4:e6693. 3. Lüscher B, Vervoorts J (2012) Regulation of gene transcription by the oncoprotein 9. Ji H, et al. (2011) Cell-type independent MYC target genes reveal a primordial sig- MYC. Gene 494:145–160. nature involved in biomass accumulation. PLoS One 6:e26057. 4. Fernandez PC, et al. (2003) Genomic targets of the human c-Myc protein. Genes Dev 10. Lorenzin F, et al. (2016) Different promoter affinities account for specificity in MYC- 17:1115–1129. dependent gene regulation. eLife 5:e15161. 5. Guccione E, et al. (2006) Myc-binding site recognition in the human genome is de- 11. Lin CY, et al. (2012) Transcriptional amplification in tumor cells with elevated c-Myc. termined by chromatin context. Nat Cell Biol 8:764–770. Cell 151:56–67. 6. Perna D, et al. (2012) Genome-wide mapping of Myc binding and gene regulation in 12. Nie Z, et al. (2012) c-Myc is a universal amplifier of expressed genes in lymphocytes serum-stimulated fibroblasts. Oncogene 31:1695–1709. and embryonic stem cells. Cell 151:68–79.

Gerlach et al. PNAS | Published online October 16, 2017 | E9231 Downloaded by guest on October 1, 2021 13. Guo J, et al. (2014) Sequence specificity incompletely defines the genome-wide oc- 43. Stevens JR, et al. (2011) FACT, the Bur kinase pathway, and the histone co-repressor cupancy of Myc. Genome Biol 15:482. HirC have overlapping nucleosome-related roles in yeast transcription elongation. 14. Sabò A, et al. (2014) Selective transcriptional regulation by Myc in cellular growth PLoS One 6:e25644. control and lymphomagenesis. Nature 511:488–492. 44. Tomson BN, Arndt KM (2013) The many roles of the conserved eukaryotic Paf1 complex in 15. Rahl PB, et al. (2010) c-Myc regulates transcriptional pause release. Cell 141:432–445. regulating transcription, histone modifications, and disease states. Biochim Biophys Acta 16. Walz S, et al. (2014) Activation and repression by oncogenic MYC shape tumour- 1829:116–126. specific gene expression profiles. Nature 511:483–487. 45. Bai X, et al. (2010) TIF1gamma controls erythroid cell fate by regulating transcription 17. Thomas LR, et al. (2015) Interaction with WDR5 promotes target gene recognition elongation. Cell 142:133–143. and tumorigenesis by MYC. Mol Cell 58:440–452. 46. Chen FX, et al. (2015) PAF1, a molecular regulator of promoter-proximal pausing by – 18. Schwinkendorf D, Gallant P (2009) The conserved Myc box 2 and Myc box 3 regions RNA polymerase II. Cell 162:1003 1015. are important, but not essential, for Myc function in vivo. Gene 436:90–100. 47. Crisucci EM, Arndt KM (2012) Paf1 restricts Gcn4 occupancy and antisense transcrip- – 19. Sabò A, Amati B (2014) Genome recognition by MYC. Cold Spring Harb Perspect Med tion at the ARG1 promoter. Mol Cell Biol 32:1150 1163. 4:a014191. 48. Jaenicke LA, et al. (2016) Ubiquitin-dependent turnover of MYC antagonizes MYC/ – 20. Roy AL, Carruthers C, Gutjahr T, Roeder RG (1993) Direct role for Myc in transcription PAF1C complex accumulation to drive transcriptional elongation. Mol Cell 61:54 67. 49. Tenney K, et al. (2006) Drosophila Rtf1 functions in histone methylation, gene ex- initiation mediated by interactions with TFII-I. Nature 365:359–361. pression, and Notch signaling. Proc Natl Acad Sci USA 103:11970–11974. 21. Hateboer G, et al. (1993) TATA-binding protein and the retinoblastoma gene product 50. Mosimann C, Hausmann G, Basler K (2006) Parafibromin/Hyrax activates Wnt/Wg bind to overlapping epitopes on c-Myc and adenovirus E1A protein. Proc Natl Acad Sci target gene transcription by direct association with beta-catenin/Armadillo. Cell 125: USA 90:8489–8493. 327–341. 22. Koch HB, et al. (2007) Large-scale identification of c-MYC–associated proteins using a 51. Youn MY, et al. (2007) hCTR9, a component of Paf1 complex, participates in the tran- combined TAP/MudPIT approach. Cell Cycle 6:205–217. scription of interleukin 6-responsive genes through regulation of STAT3-DNA interac- 23. Eberhardy SR, Farnham PJ (2001) c-Myc mediates activation of the cad promoter via a tions. J Biol Chem 282:34727–34734. – post-RNA polymerase II recruitment mechanism. J Biol Chem 276:48562 48571. 52. Mosimann C, Hausmann G, Basler K (2009) The role of Parafibromin/Hyrax as a nu- 24. Eberhardy SR, Farnham PJ (2002) Myc recruits P-TEFb to mediate the final step in the clear Gli/Ci-interacting protein in Hedgehog target gene control. Mech Dev 126: – transcriptional activation of the cad promoter. J Biol Chem 277:40156 40162. 394–405. 25. Gargano B, Amente S, Majello B, Lania L (2007) P-TEFb is a crucial co-factor for Myc 53. Carpten JD, et al. (2002) HRPT2, encoding parafibromin, is mutated in hyperparathyroidism- – transactivation. Cell Cycle 6:2031 2037. jaw tumor syndrome. Nat Genet 32:676–680. 26. Rahl PB, Young RA (2014) MYC and transcription elongation. Cold Spring Harb 54. Lin L, Zhang JH, Panicker LM, Simonds WF (2008) The parafibromin tumor suppressor Perspect Med 4:a020990. protein inhibits cell proliferation by repression of the c-myc proto-oncogene. Proc 27. Gomez-Roman N, Grandori C, Eisenman RN, White RJ (2003) Direct activation of RNA Natl Acad Sci USA 105:17420–17425. polymerase III transcription by c-Myc. Nature 421:290–294. 55. Takahashi A, et al. (2011) SHP2 tyrosine phosphatase converts parafibromin/Cdc73 from a 28. Steiger D, Furrer M, Schwinkendorf D, Gallant P (2008) Max-independent functions of tumor suppressor to an oncogenic driver. Mol Cell 43:45–56. Myc in Drosophila melanogaster. Nat Genet 40:1084–1091. 56. Moniaux N, et al. (2006) The human homologue of the RNA polymerase II-associated 29. Marshall L, Rideout EJ, Grewal SS (2012) Nutrient/TOR-dependent regulation of RNA factor 1 (hPaf1), localized on the 19q13 amplicon, is associated with tumorigenesis. polymerase III controls tissue and organismal growth in Drosophila. EMBO J 31:1916–1930. Oncogene 25:3247–3257. 30. Gallant P (2013) Myc function in Drosophila. ColdSpringHarbPerspectMed3:a014324. 57. Gallant P, Shiio Y, Cheng PF, Parkhurst SM, Eisenman RN (1996) Myc and Max ho- 31. Benassayag C, et al. (2005) Human c-Myc isoforms differentially regulate cell growth mologs in Drosophila. Science 274:1523–1527. and apoptosis in Drosophila melanogaster. Mol Cell Biol 25:9897–9909. 58. Johnston LA, Prober DA, Edgar BA, Eisenman RN, Gallant P (1999) Drosophila myc 32. Trumpp A, et al. (2001) c-Myc regulates mammalian body size by controlling cell regulates cellular growth during development. Cell 98:779–790. number but not cell size. Nature 414:768–773. 59. Söderberg O, et al. (2008) Characterizing proteins and their interactions in cells and 33. Furrer M, et al. (2010) Drosophila Myc interacts with host cell factor (dHCF) to activate tissues using the in situ proximity ligation assay. Methods 45:227–232. transcription and control growth. J Biol Chem 285:39623–39636. 60. Arnold CD, et al. (2013) Genome-wide quantitative enhancer activity maps identified 34. Mueller CL, Jaehning JA (2002) Ctr9, Rtf1, and Leo1 are components of the Paf1/RNA by STARR-seq. Science 339:1074–1077. polymerase II complex. Mol Cell Biol 22:1971–1980. 61. Hulf T, et al. (2005) Whole-genome analysis reveals a strong positional bias of con- – 35. Squazzo SL, et al. (2002) The Paf1 complex physically and functionally associates with served dMyc-dependent E-boxes. Mol Cell Biol 25:3401 3410. transcription elongation factors in vivo. EMBO J 21:1764–1774. 62. Orian A, et al. (2003) Genomic binding by the Drosophila Myc, Max, Mad/Mnt tran- – 36. Zhu B, et al. (2005) The human PAF complex coordinates transcription with events scription factor network. Genes Dev 17:1101 1114. downstream of RNA synthesis. Genes Dev 19:1668–1673. 63. Bonke M, et al. (2013) Transcriptional networks controlling the cell cycle. G3 (Bethesda) 3: – 37. Shi X, et al. (1996) Paf1p, an RNA polymerase II-associated factor in Saccharomyces 75 90. 64. Montero L, Müller N, Gallant P (2008) Induction of apoptosis by Drosophila Myc. Genesis cerevisiae, may have both positive and negative roles in transcription. Mol Cell Biol 46:104–111. 16:669–676. 65. Cowling VH, Cole MD (2007) The Myc transactivation domain promotes global 38. Phatnani HP, Jones JC, Greenleaf AL (2004) Expanding the functional repertoire of phosphorylation of the RNA polymerase II carboxy-terminal domain independently of CTD kinase I and RNA polymerase II: Novel phosphoCTD-associating proteins in the direct DNA binding. Mol Cell Biol 27:2059–2073. yeast proteome. Biochemistry 43:15702–15719. 66. Conacci-Sorrell M, Ngouenet C, Eisenman RN (2010) Myc-nick: A cytoplasmic cleavage 39. Qiu H, Hu C, Wong CM, Hinnebusch AG (2006) The Spt4p subunit of yeast DSIF product of Myc that promotes alpha-tubulin acetylation and cell differentiation. Cell stimulates association of the Paf1 complex with elongating RNA polymerase II. Mol 142:480–493. – Cell Biol 26:3135 3148. 67. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image 40. Kim J, Guermah M, Roeder RG (2010) The human PAF1 complex acts in chromatin analysis. Nat Methods 9:671–675. transcription elongation both independently and cooperatively with SII/TFIIS. Cell 68. Prober DA, Edgar BA (2000) Ras1 promotes cellular growth in the Drosophila wing. 140:491–503. Cell 100:435–446. 41. He N, et al. (2011) Human Polymerase-Associated Factor complex (PAFc) connects the 69. Bolte S, Cordelieres FP (2006) A guided tour into subcellular colocalization analysis in Super Elongation Complex (SEC) to RNA polymerase II on chromatin. Proc Natl Acad light microscopy. J Microsc 224:213–232. Sci USA 108:E636–E645. 70. Wood A, Schneider J, Dover J, Johnston M, Shilatifard A (2005) The Bur1/Bur2 com- 42. Yu M, et al. (2015) RNA polymerase II-associated factor 1 regulates the release and plex is required for histone H2B monoubiquitination by Rad6/Bre1 and histone phosphorylation of paused RNA polymerase II. Science 350:1383–1386. methylation by COMPASS. Mol Cell 20:589–599.

E9232 | www.pnas.org/cgi/doi/10.1073/pnas.1705816114 Gerlach et al. Downloaded by guest on October 1, 2021