PAF1 complex component Leo1 helps recruit Drosophila Myc to promoters
Edited by Robert N. Eisenman, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved September 11, 2017 (received for review April 7, 2017)
Significance
We identify the PAF1 complex component Leo1 as a factor that helps recruit Myc to its target genes. In particular when Myc is overexpressed, Leo1 becomes limiting for transcriptional regulation by Myc.
Abstract
The Myc oncogene is a transcription factor with a powerful grip on cellular growth and proliferation. The physical interaction of Myc with the E-box DNA motif has been extensively characterized, but it is less clear whether this sequence-specific interaction is sufficient for Myc’s binding to its transcriptional targets. Here we identify the PAF1 complex, and specifically its component Leo1, as a factor that helps recruit Myc to target genes. Since the PAF1 complex is typically associated with active genes, this interaction with Leo1 contributes to Myc targeting to open promoters.
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The Myc family of transcription factors (MYC, MYCN, and MYCL) are potent oncogenes thought to contribute to the majority of human cancers (1, 2). Through their C-terminal basic-helix-loop-helix zipper region (bHLHZ), Myc proteins dimerize with the bHLHZ protein Max and subsequently bind their target genes (3) at promoter-proximal sites or at distally located enhancers (4–7). High-affinity Myc targets often contain E-boxes (CACGTG and variants thereof) that are directly contacted by Myc:Max dimers. Many of these genes are involved in translation, or ribosome biogenesis or function (7–10). When expressed at supraphysiological levels, Myc also invades most active promoters (11–16), and Myc has been proposed to act as general amplifier of transcription (11, 12). The mechanism by which Myc recognizes its different targets is currently under investigation.
In vitro Myc:Max dimers most efficiently bind to E-box motifs, and this motif is frequently found in high-affinity Myc targets (7, 10). However, several lines of evidence suggest that primary DNA sequence does not suffice to define Myc targets. First, in vivo Myc does not bind to all E-boxes indiscriminately, but rather preferentially associates with sites located in active promoters (5, 13). Second, Myc binds to numerous promoters lacking any common sequence motif, especially when expressed at supraphysiological levels (e.g., ref. 13). Third, the measured in vitro binding constant of Myc:Max dimers for E-boxes does not suffice to explain the observed in vivo affinity for its target sites (10). 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 interacts specifically with the highly conserved centrally located MBIIIb motif in Myc (10, 17). Loss of the WDR5 interaction domain strongly reduces the binding of Myc:Max complexes to their targets, including sites with canonical E-boxes.
However, WDR5 likely is not the only such factor, since Myc mutants lacking the WDR5 interaction region still retain partial transforming activity in tissue culture and in mice (17). In addition, the MBIIIb region is highly conserved in Drosophila Myc, but a mutant Myc derivative lacking the entire region can substitute for wild-type Myc in null mutant flies (18). Additional recruitment factors might include various sequence-specific transcription factors with which Myc has been reported to associate (notably Miz1, Sp1, NF-Y, Smad2/3, and YY1; reviewed in ref. 19) and several components of the core transcription machinery (15, 20–22), including Brf, P-TEFb, TBP, TFII-I, and RNA polymerase II (23, 24–29).
We decided to search for molecular partners that contribute to Myc’s recruitment to DNA and/or mediate transactivation by Myc, using Drosophila as a model system. Fruit flies contain a single Myc protein that primarily controls cellular and organismal growth (reviewed in ref. 30). Like its vertebrate homologs, Drosophila Myc functions by dimerizing with Max and binding to target genes. Most of the directly Myc-activated genes control ribosome biogenesis and function. This is consistent with Myc’s biological properties in flies and also with the described targets of vertebrate Myc proteins expressed at physiological levels (e.g., refs. 7 and 10). Illustrating this evolutionary conservation of Myc function, vertebrate MYC can substitute for Drosophila Myc in flies (31), and Drosophila Myc can partially replace c-Myc in cultured murine fibroblasts (32).
To identify proteins that influence Myc’s transcriptional output, we carried out an RNAi screen in Drosophila S2 cells (33) and identified the PAF1 complex as a functional interactor of Myc. The PAF1 complex is conserved from yeast to man. It consists of the core subunits Paf1 (atms in Drosophila), Cdc73 (also known as hyrax in Drosophila and Parafibromin in vertebrates), Leo1 (Atu in Drosophila), Ctr9, Rtf1 (34, 35), and, in vertebrate cells, the more loosely associated Ski8 (36). The complex was initially identified as an RNA polymerase II-associated factor (e.g., refs. 37 and 38) and subsequently shown to interact with some general factors involved in transcription elongation, including DSIF (35, 39), SII (40), FACT (35), the “super elongation complex” SEC (41), P-TEFb, Cdk12, and Cyclin T (42). Consistent with these observations, the PAF1 complex has been shown to positively affect RNA polymerase II pause release and transcriptional elongation (42–44). Other studies have revealed an inhibitory effect of the PAF1 complex on elongation (45–48). Thus, it has been proposed that the genetic background and physiological state of different cells may affect the output of the PAF1 complex (42). In either case, depletion of the PAF1 complex affects transcript levels of most genes, but to only a rather small degree; for example, Myc targets were shown to be up-regulated by 10% on knockdown of 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 modification can convert Cdc73 to an oncogene (55), and overexpression of Paf1 promotes growth of NIH 3T3 cells and induces tumor formation in vivo (56).
Here we show that the PAF1 complex functionally and physically interacts with Myc in Drosophila larvae and cells. Elimination of several complex components impairs Myc-dependent growth in vivo, reduces the recruitment of Myc to its target genes, and affects their expression in S2 cells and in vivo. This suggests that the PAF1 complex contributes to Myc’s binding to its target promoters, thereby promoting cellular growth and proliferation.
Results
The PAF1 Complex Contributes to Myc-Dependent Growth in Vivo.
To identify novel transcriptional cofactors for Myc, we carried out an RNAi screen in S2 cells (a cell line derived from Drosophila embryos). Out of 752 tested transcription-associated factors, we found 33 proteins that consistently affected the activity of the Myc-dependent reporter, including the scaffold protein dHCF (33) and the components of the PAF1 complex. Depletion of the PAF1 proteins does not strongly affect Myc protein levels (Fig. S1A, and see below), but it does reduce the expression of various Myc targets (Fig. 6 and Fig. S1B), indicating that PAF1 acts as a positive cofactor for Myc-dependent transcription.
Fig. S1.
To assess the importance of the PAF1 complex for Myc’s biological activities, we evaluated the consequences of PAF1 depletion in vivo. Myc primarily controls cellular and organismic growth in flies (30). A moderate reduction in Myc levels results in small adult flies with disproportionally small bristles, indicating that Myc activity is particularly important for the growth of bristle precursor cells (57, 58). Knockdown of the PAF1 proteins Rtf1, Paf1, and Leo1 in these cells phenocopies the Myc-mutant phenotype and significantly reduces the size of adult bristles (Fig. 1A; the different magnitudes presumably reflect differences in knockdown efficiencies). This observation is consistent with participation of the PAF1 complex in Myc-dependent growth.
Fig. 1.
To provide more direct evidence, we measured PAF1’s effect on the areas of imaginal disk cell clones overexpressing Myc. Myc-overexpressing clones are significantly larger than controls, due to a strong increase in cell size (ref. 58 and Fig. 1B). Depletion of all tested PAF1 complex components strongly affected this overgrowth, but did not reduce the size of control clones (Fig. 1B). Simultaneous depletion of the PAF1 component Leo1 slightly reduced the level of ectopically expressed Myc protein (Fig. S1C); the reason for this is unclear, and no such effect of Leo1 depletion on endogenous Myc levels was observed), but had a clearly stronger impact on clonal area (Fig. 1B). Strong effects on Myc-induced growth were also seen on depletion of other PAF1 complex components. Thus, the PAF1 complex is limiting for the extra growth of imaginal disk cells that is driven by Myc overexpression.
Leo1 Physically Interacts with Myc to Recruit It to Individual Targets.
To explore the molecular mechanism for these functional effects of the PAF1 complex on Myc activity, we turned back to cultured cells. Since the PAF1 complex did not strongly affect Myc protein levels, we considered the possibility that it might physically interact with Myc and thereby influence its transcriptional output. We first investigated the PAF1 complex component Leo1 (since a corresponding epitope-tagged version was available) and observed a strong association with coexpressed Myc in S2 cells (Fig. 2A). This interaction did not require any of the previously described Myc domains, i.e., the Myc boxes 1 or 2 situated in the transactivation domain, the Myc box 3 mediating binding to WDR5, or the C-terminally located Max-interacting bHLHZ domain. Instead, a broad central region of Myc was needed for the binding to Leo1 (Fig. 2A). Within Leo1, a central region between amino acids 442 and 474 was required for the association with Myc (Fig. 2B). No biochemical functions or specific structures have been ascribed to this region.
Fig. 2.
We next examined whether this Leo1–Myc interaction also occurs 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). Moreover, endogenous Myc could be coimmunoprecipitated with HA:Leo1 in this setup, indicating that the PLA signal reflects a physical interaction between the two proteins (Fig. 3A). This interaction takes place on DNA, as shown by sequential chromatin immunoprecipitation (Re-ChIP) experiments; previously identified Myc targets were specifically recovered when chromatin from HA:Leo1-expressing cells was immunoprecipitated with an anti-HA serum, followed by immunoprecipitation with anti-Myc antibodies (Fig. 3B). No such enrichment was seen in naïve S2 cells or for control DNA sequences (Fig. 3B). Fig. 3C shows the expression levels of HA:Leo1 and endogenous Myc. Together, these experiments show that Leo1 and Myc physically associate in cells. This interaction is likely direct, since it also can be seen between in vitro synthesized 35S-labeled Leo1 and a GST:Myc fusion protein purified from Escherichia coli (Fig. 3D). We consider it likely that this Leo1–Myc interaction serves to associate the entire PAF1 complex with Myc. Indeed, epitope-tagged versions of the other PAF1 complex components Paf1, Ctr9, and Cdc73 can also be coimmunoprecipitated with coexpressed Myc in 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.
Fig. 3.
Fig. S2.
Fig. S3.
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 open promoters (notably via contacts with the general transcription 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).
Fig. 4.
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. 4 are shown in Fig. 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 enhancer sites (Fig. 5B; Fig. S2F shows protein levels). Many of these promoters contain E-boxes (115 out of 296), which are directly bound by Myc:Max complexes. The association of Myc with such E-box–containing promoters is somewhat more strongly impaired by Leo1 depletion compared with the association with promoters lacking E-boxes (P = 0.055) (Fig. 6D).
Fig. 5.
Fig. 6.
Leo1 Is Required for Efficient Expression of Myc Targets.
The foregoing data indicate a requirement for Leo1 for efficient recruitment of Myc to its target promoters, suggesting a role for Myc’s transcriptional output. Therefore, we investigated the impact of Leo1 on the cellular transcriptome of S2 cells. Despite 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 expression of genes with E-boxes immediately downstream of their transcription start sites (the majority of these genes were previously shown to be Myc targets; ref. 61), as well as of genes that were bound by Myc over an E-box situated in the promoter and whose expression decreased on Myc knockdown (presumably direct Myc targets; ref. 7), consistent with the idea that Leo1-mediated Myc binding to target promoters is required for the full transactivation of these genes by Myc (Fig. 6A and Fig. S4B). The magnitude of these effects was small, however, possibly because PAF1’s positive effect on Myc recruitment is partially offset by its negative effects on transcription (48).
Fig. S4.
Consistent with such small effects on gene expression in naïve S2 cells, depletion of Leo1 (and other PAF1 complex components) in control clones in vivo had little effect on clonal size (Fig. 1B). In contrast, the clonal overgrowth induced by Myc overexpression was strongly impaired by simultaneous depletion of PAF1 complex components, suggesting that effects on Myc target gene expression become more pronounced in such a setting. Thus, we set out to investigate the impact of Leo1 depletion on the transcriptomes of Myc-overexpressing imaginal disks, using the same animals and analogous conditions as for the clonal analysis shown in Fig. 1B. In addition, we assessed the consequences of loss of Max on Myc-dependent transcription (either alone or in combination with Leo1 down-regulation), since our data suggested potentially related roles for Leo1 and Max in Myc recruitment.
In an otherwise wild-type background, Myc overexpression significantly induced 977 genes and repressed 1,176 genes (Fig. 6B). The Myc-induced genes largely fall into the previously recognized categories of Myc targets (notably ribosome biogenesis, ribosome 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).
Fig. S5.
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 (reducing 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 E-box–containing targets, but allowed for some transactivation of E-box–lacking Myc targets (Fig. S5C). These data show that Myc retained substantial transactivation potential in a Max null-mutant background in which essentially no Max transcripts were present (ref. 28 and Fig. 6C), and that even though the Leo1 knockdown did not completely remove Leo1 transcripts (Fig. 6C), its consequences on global Myc-dependent gene induction and repression were significant. These analyses demonstrate a clear requirement for Leo1 (and, by inference, the entire PAF1 complex) for Myc-dependent gene regulation, particularly in conditions of supraphysiological Myc activity.
Genetic Interaction of Leo1 and Max in Vivo.
As an additional test to phenotypically assess the effects of Leo1 and/or Max on Myc-dependent processes, we turned to a previously described in vivo assay for Myc activity. Myc overexpression in developing eyes promotes the growth of individual ommatidia and also increases the rate of apoptosis in this tissue (64). The resulting adult eyes are larger overall and contain larger ommatidia, but their arrangement is disturbed, causing a rough appearance. The apoptosis of pigment cells produces an overall lighter eye coloration with numerous interspersed dark ommatidia (Fig. 7A). Depletion of Max in this context abrogates Myc’s growth-promoting ability but does not affect its proapoptotic effects, thereby 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 and Myc-dependent eye overgrowth (Fig. 7; compare the Max knockdown with or without Leo1 knockdown). Importantly, in the absence of Myc overexpression, neither Leo1 nor Max knockdown had a strong effect on eye size or morphology, demonstrating that all of the described effects are mediated by Myc. These observations illustrate the importance of both Leo1 and Max for Myc-dependent transcriptional control, and raise the possibility that these two proteins influence different 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 involve nontranscriptional functions of Myc (Discussion).
Fig. 7.
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 extra tissue growth induced by high levels of Myc in vivo. This may be caused in part by a moderate reduction of Myc protein levels (the reason for which is unknown), as well as by impaired recruitment of Myc to a large number of target genes. In addition, we cannot exclude the possibility that the PAF1 complex also affects transcription-independent processes. Although Myc is best known for its role in controlling transcription, it also affects other cellular processes independent of transcription, such as DNA replication (27), mRNA cap methylation and translation (65), and α-tubulin acetylation (66). A role for the PAF1 complex in the first two processes (which obviously contribute to cellular proliferation and/or growth) is conceivable, but this has not been addressed so far.
In contrast to cells with high expression of Myc, the growth of wild-type eyes or control wing imaginal disk clones is not reduced by depletion of the PAF1 complex. Indeed, ubiquitous depletion of the PAF1 complex throughout the animal allows most flies to develop to the pharate adult stage, and some escapers even complete development and eclose as adults (SI Materials and Methods). This effect may be explained in part by insufficient knockdown efficiencies (the available null mutants in Paf1, Cdc73, or Ctr9 do show a stronger phenotype), but it also suggests that the PAF1 complex is less essential under normal growth conditions. On the other hand, in tissues undergoing rapid growth (notably imaginal disks experiencing Myc overexpression), depletion of Leo1 clearly reduces the ability of Myc to regulate targets and impedes the associated overgrowth. Both Myc-repressed and Myc-activated genes are affected, consistent with the idea that Myc recruitment is impaired in this situation. This suggests that supraphysiological levels of Myc saturate the available PAF1 complex and possibly also other “recruitment factors.” Thus, such settings, as are notably encountered in many human tumors, might be particularly sensitive to inhibition of PAF1 activity. Taken together, our observations emphasize the importance of Leo1 for the biological activity of Myc, but they do not demonstrate that all PAF1 activities are mediated by Myc. Indeed, the PAF1 complex interacts with several transcription factors besides Myc, and it is conceivable that some of these factors also contribute to the growth-related functions of PAF1.
It remains to be seen whether Leo1 and Max are involved in recruiting Myc to different functional sets of targets. The analysis of Myc-overexpressing adult eyes suggests that Leo1 and Max predominantly affect different Myc-dependent processes: apoptosis and growth, respectively. Indeed, individual genes are differentially affected by Max or Leo1 knockdown, but no gene sets obviously marked as “growth-related” or “apoptosis-related” behave in the expected manner. It is conceivable that the gene(s) responsible for apoptosis in the eye specifically disrupt pupal eye development (a 4-d-long process that mostly involves cellular growth and differentiation, but little proliferation), and thus might not be recognized as being generally apoptosis-related. Alternatively, the relevant genes code for small transcripts (e.g., miRNAs, tRNAs) that have not been included in our transcriptome analyses. Finally, we stress that even in the complete 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 disks, flies of the appropriate genotypes were raised under standard conditions at 25 °C. At the age of 53–66 h (139–143 h for the Max−/− genotypes), they were subjected to a 2-h heat shock at 37 °C to induce ubiquitous expression of the GAL4-dependent transgenes. Then, 48 h later, wing imaginal disks were dissected into Qiazol and immediately stored at −80 °C until further processing.
To determine ommatidial size, flies were raised under noncrowding conditions. Adult males were collected at 1–7 d after eclosion and killed by freezing. Eyes were photographed with a Zeiss Discovery V8 stereomicroscope fitted with a 1.5× lens and processed with Axiovision Extended Focus software. For each genotype, the area of 20 centrally located ommatidia was measured from at least seven eyes from independent individuals.
RNAi Screen and S2 Cell Culture.
Molecular Biology.
Antibodies.
Antibodies were mouse anti-Drosophila Myc (7), rabbit anti-Drosophila Myc (Santa Cruz Biotechnology), mouse anti–α-tubulin (Sigma-Aldrich), rabbit anti-HA (Abcam or ICL), rabbit anti-AU1 (Bethyl Laboratories), mouse anti-AU1 (Covance), rat anti-Cdc73, and rabbit anti-Rtf1 (gifts from J. Lis). A polyclonal rabbit antiserum was raised against the peptide RDKVESQVESAPKEC (amino acids 356–369 of Drosophila Leo1) and affinity-purified (New England Peptide or ImmunoGlobe).
Plasmids for Expression in S2 Cells.
Wild-type Myc and mutant derivatives were cloned in-frame with an N-terminal hemagglutinin (HA) tag in the vector pUASattB. Numbered deletions (created by site-directed mutagenesis) retain the indicated regions of the Myc protein, e.g., amino acids 403–717. Mutants lacking specific Myc domains have been described previously (18, 28, 33). In brief, they carry the following modifications: ΔN-term lacks amino acids 1–293, ΔMB2 has the amino acid “GP” instead of amino acids 68–84 (Myc box 2), ΔMB3 has amino acid “F” instead of amino acids 405–422 (Myc box 3), ΔC-term lacks amino acids 626–717 (C terminus: bHLHZ), and ΔZ lacks amino acids 676–717 (leucine zipper). Analogously, the Leo1 coding region was cloned behind an HA or AU1 epitope tag in pUASattB, and mutant derivatives were generated and numbered as for the Myc mutants.
To obtain stable expression of HA:Leo1 (wild-type), the corresponding coding region was inserted under control of the metallothionein promoter in the vector pMT181 carrying a puromycin resistance marker (a gift of M. Tiebe and A. Telemann, Zentrum für Molekulare Biologie der Universität Heidelberg). On puromycin selection of stable cell pools, HA:Leo1 expression was induced by incubation with 125 µM CuSO4 for 24 h.
In Vitro Interaction.
The E. coli strain BL21 was transformed with constructs coding for GST or a GST:Myc (amino acids 46–507) fusion, and protein expression was induced by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside for 3 h at 37 °C. On bacterial lysis, GST proteins were purified by incubation with glutathione Sepharose beads (Amersham Biosciences). Leo1 was expressed in vitro in a coupled rabbit reticulocyte lysate in the presence of 35S-labeled methionine (TNT Kit; Promega), and incubated with the GST/GST:Myc bound to glutathione beads in GST Binding Buffer (200 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.5% Nonidet P-40, 10% glyerol, and 0.05% BSA) containing protease inhibitors (Roche). Bead-bound proteins were then analyzed by SDS/PAGE followed by autoradiography, as described previously (33); 10% of the in vitro translation mix was directly loaded on the gel and served as input control.
Western Blot and Immunoprecipitation Analyses.
For transient expression in S2 cells, appropriate UAS plasmids were cotransfected with tubulin-GAL4, and cells were harvested at 24–48 h after transfection. Pelleted cells were washed once with cold 1× PBS and then lysed on ice for 30 min in lysis buffer [150 mM NaCl, 50 mM Tris⋅HCl pH 8.0, 5 mM EDTA pH 8.0, 0.5% Nonidet P-40, containing Protease Inhibitor Mixture tablets (Roche)]. Insoluble contents were precipitated by centrifuging for 15 min at 16,200 × g. The lysates were precleared for 1 h at 4 °C with protein G Sepharose bead suspension (GE Healthcare) and 5% of the lysate was set aside as input control. Incubations with 0.2–1 μg of the primary antibodies were performed at 4 °C for 3 h, followed by a 1-h precipitation of the epitope antibody complexes with protein G Sepharose beads. Some immunoprecipitations were performed with Dynabeads (Life Technologies GmbH) which were incubated with 1 µg of the primary antibody for 6–8 h at 4 °C. 10% of the lysate were set aside as input control. Lysates were incubated over night at 4 °C with the antibody coupled beads. For all immunoprecipitations, the immunoprecipitated material was washed three times for 5 min in lysis buffer on ice, SDS sample buffer was added, and the samples were analyzed by SDS/PAGE and immunoblotting as described previously (33).
For coimmunoprecipitations of endogenous Myc with HA:Leo1, cells with a stably integrated MT-HA:Leo1 plasmid were induced with 125 µM CuSO4. At 24 h later, 1.5 × 108 cells were harvested, washed once with cold 1× PBS, lysed in Hepes-EDTA-glycerol-Nonidet P-40 (HEGN) buffer with 140 mM KCl, and sonicated for 40 s at 20% amplitude (Digital Sonifier Cell Disruptor; Branson). Five percent of the lysate was set aside as input control. Dynabeads (Life Technologies) were preincubated with 8 µg of rabbit anti-HA (Abcam) primary antibody or control rabbit IgG for 6–8 h at 4 °C, and cell lysates were incubated overnight at 4 °C with the antibody-coupled beads. The immunoprecipitate was washed three times with HEGN buffer containing protease inhibitors, SDS sample buffer was added, and the samples were analyzed by SDS/PAGE and immunoblotting 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, permeabilized with 0.3% Triton-X 100 and then treated with blocking solution (10% goat serum, 2% BSA, and 5% sucrose in PBS) for 45 min after washing. Cells were incubated overnight at 4 °C with primary antibodies in blocking solution [rabbit anti-HA (Santa Cruz Biotechnology), 1:500 and mouse anti-Myc, at 0.3 µg/mL], washed in TBS with 0.1% Tween-20, incubated for 1 h with the secondary antibodies at room temperature, and washed again. Cells were mounted on glass slides using aqua-fluoromount (Sigma-Aldrich) and imaged with a confocal microscope (Nikon Ti-Eclipse) with a 60× objective. Images were processed with ImageJ 1.50h (67).
Re-ChIP.
Cell fixation, lysis, and sonication were carried out as described previously for manual ChIP (7). On cell lysis, 1% of the lysate was set aside as input 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 with the chromatin overnight at 4 °C. Dynabeads (Thermo Fisher Scientific) for the secondary ChIP were similarly washed and incubated with 3 µg of anti-Myc antibody or control rabbit IgGs overnight. The precipitates were washed as described for manual ChIP and then eluted twice with 0.8 mg/mL Pierce HA peptides (Thermo Fisher Scientific) in 1× RIPA buffer for 15 min at 37 °C. Five percent of the combined eluates were set aside as input control; the remainder was incubated with the antibody-coupled Dynabeads for 6 h at 4 °C. Washing, elution, and extraction of the precipitates, as well as analysis by quantitative real-time PCR, were carried out as described for manual ChIP.
RNAseq, ChIPseq, and Bioinformatic Analysis.
RNAseq, ChIPseq, and bioinformatic analyses were carried out as described previously (7), with the following modifications. Antibodies for ChIPseq were rabbit anti-Myc (Santa Cruz Biotechnology) and rabbit anti-HA (Abcam). Sequencing was done on an Illumina NextSeq 500. For each ChIPseq condition, as well as input control, 7,847,000 reads were mapped onto the reference genome dm6 (bowtie 2.2.4). Peaks were called with macs 1.4.0 and statistically analyzed with R and GraphPad Prism.
Promoter regions were defined as the ±100 nucleotides flanking the annotated transcription start sites (FlyBase FB2015_4) for a total of 17,716 promoters. Enhancers (5,499 regions) were derived from ref. 60, with coordinates adapted to the reference genome dm6. Myc-binding sites were defined as those identified by MACS in naïve S2 cells that did not overlap background sites (as called by anti-HA ChIP from naïve S2 cells) and did not have increased read numbers on Myc depletion (714 sites); 166 of these were located in enhancer regions (as defined by ref. 60) and did not overlap promoters, whereas 296 overlapped promoters. For the analysis shown in Fig. 6, reads were counted in 300-nt windows centered on the Myc-bound summits (as called by MACS) using the bedtools v2.17.0 suite, reads for the input sample over the same window were subtracted, and read ratios were calculated relative to ChIPs from naïve control cells. ChIPseq profiles for Fig. 6A were generated with the genome browser IGB, using a MACS output retaining all duplicate reads (parameter “keep-dup all”).
For RNAseq of S2 cells, RNA was isolated in biologically independent triplicates at 48 h after Leo1 or control depletion, depleted of rRNA using the Ribominus Kit (Invitrogen), and processed for sequencing to a depth of 6,757,000 (non-rRNA) reads. For final analysis, 9,123 genes were kept with at least one read in each of the six samples and at least one read per million on average for either the control or Leo1 knockdown condition. Statistical analysis was performed with the Bioconductor tools in R and with GraphPad Prism. For RNA isolation from larvae, between 16 and 27 imaginal disks per sample were dissected into Qiazol (Qiagen). Further processing was done as described previously (7), except that cDNAs were prepared with NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs). For each of the 24 samples (eight different genotypes × three replicates), an average of 6.3 million mapped reads were obtained and analyzed as described above.
SI Materials and Methods
Drosophila S2 cells were plated on poly-l-lysine (Sigma-Aldrich)-coated coverslips and exposed to 125 µM CuSO4 to induce HA:Leo1 expression and/or Myc-dsRNA (2 µg/106 cells) for 24 h, followed by fixation with 4% paraformaldehyde. Fixed cells were incubated overnight at 4 °C with primary antibodies in blocking solution [rabbit anti-HA, 1:500 dilution (Santa Cruz Biotechnology) and mouse anti-Myc, 0.3 µg/mL (P4C4-B10; ref. 68] and washed with TBS with 0.1% Tween-20 before the start of the PLA.
A Duo-Link in situ proximity ligation assay (Sigma-Aldrich) was performed as follows. The cells were incubated for 1 h at 37 °C with equal volumes of secondary antibodies directed against rabbit and mouse primary antibodies and covalently coupled to unique DNA sequences. Then a DNA ligation reaction was performed for 30 min at 37 °C, and covalent ligation products were amplified in situ by rolling circle-based PCR amplification for 2 h at 37 °C. The amplified DNA circles were then detected by hybridization with a complementary Alexa Fluor 561-labeled oligonucleotide. The cells were counterstained with Hoechst 33452 to visualize nuclei. Imaging was performed with a confocal microscope (Nikon Ti-3 Eclipse) using a 60× objective, with the same step size and number for all samples. The channels in the captured image stacks were separated, and the Alexa Fluor 561 channel was used to count the number of dots along with their intensities, using the ObjectCounter3D plug-in in ImageJ (69). The dots thus measured were normalized for intensities and plotted using GraphPad Prism 6.
SI Results
Flies carrying a UAS-x-IR transgene (where “x” represent for Cdc73/hyrax, Rtf1, Leo1/Atu, or Paf1/Atms) or no transgene (control) were crossed to flies carrying actin5C-FRT-CD2-FRT-GAL4 hs-FLP and 72 h later subjected to a 2-h heat shock at 37 °C to induce ubiquitous expression of GAL4 and the dsRNA targeting a particular PAF1 component. The transgene targeting Cdc73 was balanced over a CyO chromosome in the parental line; thus, the described cross was expected to produce equal proportions of offspring carrying the CyO balancer and of offspring with the Cdc73-knockdown. The other transgenes were homozygous in the parental lines, and thus all offspring of the described cross experienced the indicated depletions. Each genotype was analyzed in 4–12 separate egg lays (except for control, with two experiments, and Leo1, with six experiments each with UAS-Leo1-IR ID17490 and ID106074), and the pharate and eclosed adults were counted at a time when all CyO-carrying offspring had already eclosed as adults. For the Paf1 knockdown, the number of dead (immobile) larvae was determined as well.
| Observation | Control | Cdc73 | Paf1 | Rtf1 | Leo1 |
|---|---|---|---|---|---|
| Number of egg lays | 2 | 4 | 4 | 6 | 12 |
| Dead larvae | nc | nc | 170 | nc | nc |
| Pupae | 54 | 161 | 2 | 232 | 553 |
| Pharate adults | 0 | 87 | 0 | 165 | 418 |
| Eclosed adults KD | 54 | 1 | 0 | 8 | 135 |
| Eclosed adults CyO | — | 73 | — | — | — |
nc, not counted. Only the Cdc73 cross included the CyO chromosome.
Data Availability
Data deposition: The sequences reported in this paper have been deposited in the ArrayExpress archive, https://www.ebi.ac.uk/arrayexpress (accession nos. E-MTAB-5470, E-MTAB-5471, and E-MTAB-5472).
Acknowledgments
We thank André Kutschke and Reinhold Krug for technical support; the Bloomington Drosophila Stock Center, the Vienna Drosophila Resource Center, and the fly community for various fly lines; K. Basler, M. Tiebe, and A. Teleman for plasmids; and J. Lis for antibodies. Funding for this project was provided by the Swiss National Science Foundation (Grant 3100A0-120458/1, to P.G.) and the German Research Foundation (Grants WO 2108/1-1, to E.W.; GA 1553/1-1, to P.G.; and GA 1553/2-1, to P.G.).
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Cover image: Pictured is a developing Arabidopsis thaliana lateral root labeled with a fluorescent transcriptional reporter, with cell outlines shown in blue. Yujuan Du and Ben Scheres found that the PLETHORA transcription factors PLT3, PLT5, and PLT7 regulate the initiation of cell divisions at the start of Arabidopsis lateral root development. According to the authors, PLETHORA transcription factors are key molecular triggers for the establishment of new meristems and tissue differentiation associated with a new growth axis. See the article by Yujuan Du and Ben Scheres on pages 11709–11714. Image courtesy of Yujuan Du.
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© 2017. Published under the PNAS license.
Data Availability
Data deposition: The sequences reported in this paper have been deposited in the ArrayExpress archive, https://www.ebi.ac.uk/arrayexpress (accession nos. E-MTAB-5470, E-MTAB-5471, and E-MTAB-5472).
Submission history
Published online: October 16, 2017
Published in issue: October 31, 2017
Keywords
Acknowledgments
We thank André Kutschke and Reinhold Krug for technical support; the Bloomington Drosophila Stock Center, the Vienna Drosophila Resource Center, and the fly community for various fly lines; K. Basler, M. Tiebe, and A. Teleman for plasmids; and J. Lis for antibodies. Funding for this project was provided by the Swiss National Science Foundation (Grant 3100A0-120458/1, to P.G.) and the German Research Foundation (Grants WO 2108/1-1, to E.W.; GA 1553/1-1, to P.G.; and GA 1553/2-1, to P.G.).
Notes
This article is a PNAS Direct Submission.
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The authors declare no conflict of interest.
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