BACKGROUND
growth and proliferation are fundamental for all life.
in metazoans, these processes need to be coordinated with animal development and homeostasis  <cit> .
one important factor in the control and coordination of growth is the myc family of oncogenes .
myc’s activity is essential for normal animal development in insects and in vertebrates .
a moderate reduction of myc levels in drosophila results in small cells with small nucleoli, reduced organismal growth and adult size, delayed overall development and female sterility  <cit> .
fruit flies lacking all myc activity fail to undergo normal growth and mostly die during early larval stages  <cit> .
conversely, myc overexpression in vertebrates  leads to unscheduled cellular growth and proliferation  <cit> .
consistent with this, mutational activation of myc, or of upstream signaling pathways, is frequently found in human cancers and causally contributes to this disease  <cit> .
knowledge of the relevant myc targets, therefore, is essential to understand the processes of myc-dependent growth in normal and pathological situations.
while myc can control a large number of genes in vertebrates  <cit> , the number of myc targets is still open in the simpler model drosophila.
several studies have characterized myc-regulated genes  <cit> , but they have not comprehensively addressed which of these genes are controlled by direct myc binding, nor have they included genes that do not code for proteins.
however, recent studies have revealed the abundance and variety of non-coding transcripts in the eukaryotic genome, and have emphasized the biological importance of various such transcripts  <cit> .
some non-coding rnas have already been identified as physiologically relevant myc targets in vertebrates , raising the possibility that the biological activities of myc might be mediated  by these and additional non-protein coding transcripts.
we, therefore, set out to extend the search for myc targets to non-polyadenylated transcripts, using a combination of rnaseq and chromatin immunoprecipitation sequencing  approaches in cultured drosophila cells.
this approach led to the identification of a core set of less than  <dig> myc targets.
the majority of these targets control ribosome biogenesis and translation, in good agreement with earlier reports  <cit> .
in addition to the previously identified mrnas, however, we identified small nucleolar rnas  as a novel class of myc targets.
the drosophila genome encodes  <dig> snornas , most of which fall into one of two classes: the  <dig> to  <dig> nucleotides long box c/d snornas and the  <dig> to  <dig> nucleotides long box h/aca snornas.
upon association with a small set of specific proteins, these two types of snornas form small nucleolar riboproteins  that catalyze 2’-o-methylation and pseudouridylation, respectively.
their best characterized targets are small nuclear rnas  and ribosomal rnas, where many of these post-transcriptional modifications cluster in functionally important regions and contribute to efficient ribosome biogenesis and/or function.
in addition, snornas have been shown to affect other biological processes, such as rna editing, alternative splicing, and gene silencing .
intriguingly, the snorna associated proteins all are encoded by core myc targets, as are several of the factors involved in snornp processing  <cit> .
these findings indicate that myc acts as a master regulator of snornp biogenesis, and they suggest a biological mechanism that ensures the stoichiometry of rna and protein components of snornps.
at the same time, they reinforce the notion that snornp generation, and hence ribosome biogenesis, constitutes the core function of drosophila myc.
we further provide evidence that vertebrate myc also controls snorna expression.
finally, we show that the snorna host gene uhg <dig> is important for normal animal growth, and that overexpression of different uhg genes enhances tumor growth.
RESULTS
myc directly binds a core set of sites in drosophila
in order to establish a core set of genes directly controlled by myc in drosophila, we performed chipseq experiments in s <dig> cells using a mouse monoclonal anti drosophila myc antibody  <cit> .
to control for background signal, we used non-immune mouse immunoglobulin g  in parallel experiments, and we repeated both anti-myc and control igg chips with cells that had been depleted of myc.
this set of experiments resulted in the identification of  <dig> peaks that are specifically bound by myc in naïve s <dig> cells but not in myc-depleted cells, and that are not recognized by control iggs .
since these chips relied on a monoclonal antibody, it is conceivable that some myc binding sites were missed due to epitope masking.
to exclude this possibility, we conducted another set of chipseq experiments with a rabbit polyclonal anti drosophila myc antibody  <cit>  and chromatin from s <dig> cells, again using species-matched non-immune iggs as control.
this approach yielded  <dig> specifically myc-bound peaks, most of which  overlapped with the myc-binding sites identified above .
given this coincidence between the two antibodies, we are confident that we have identified the majority of myc bound genes, and  that these genes represent bona fide myc-binding sites.figure  <dig> myc binding sites in drosophila.
a) myc binding to the fibrillarin locus as an example of a binding site in s <dig> cells.
strong binding is observed with mouse α-myc antibodies in naïve s <dig> cells , but not upon myc depletion  or with control mouse igg ; the grey box marks the myc binding peak as called by the software macs, the asterisk shows a consensus e-box .
chromosomal coordinates  are indicated below the traces, as are the extents and orientations of genes in this region.
the western blot illustrates the efficiency of myc depletion; the arrowhead points to a band presumably arising from post-translational modification of myc.
molecular weight markers  are indicated.
b) overlaps of myc binding sites from three different chipseq experiments, using either mouse monoclonal or rabbit polyclonal anti-myc antibodies, and chromatin from s <dig> or kc <dig> cells.
only peaks called by the software as ‘significant’ were considered, that is, with false discovery rate  of <10%.
c) position of myc binding peaks relative to the nearest transcription start site .
data are distributed in 100-nt bins for a distance of up to  <dig>  nt from the tss and in  <dig> -nt bins for distances between  <dig>  and  <dig>  nt, and shown as percent of all  <dig> peaks.
grey bars show all myc binding peaks, the red line only the myc binding peaks containing a canonical e-box within a 100 nt window straddling the myc binding summit.
horizontal lines above the graph illustrate windows of +/− 100 nt and +/−  <dig>  nt around the tss, containing 58% and 80% of all myc peaks, and 77% and 93% of the myc peaks with canonical e-boxes, respectively.
this number of myc-binding sites is consistent with what was found in an earlier study that used damid for assessing myc binding  <cit> , but it is considerably lower than the nearly  <dig>  sites that were recently reported for a chipseq study of drosophila kc <dig> cells  <cit> .
several possibilities can be considered for this discrepancy.
first, although both kc <dig> and s <dig> cells are hematopoietic cells of embryonic origin, it is conceivable that they differ in their molecular characteristics.
to address this possibility, we carried out chipseq experiments with rabbit anti-myc antibodies and control iggs using chromatin from kc <dig> cells.
this led to the identification of  <dig> myc-binding sites .
however, in these experiments we observed considerably higher background binding than in s <dig> cells, and, therefore, only  <dig> binding sites were statistically significant  <10%),  <dig> of which overlap peaks in s <dig> cells.
this similarity with the results in s <dig> cells argues against major differences between the cell lines .
second, yang et al.
used a different antibody  which might have higher affinity and/or recognize other epitopes on myc, thus allowing the identification of additional bona fide myc-binding sites.
alternatively, since there are no published data showing the specificity of this antibody, or of control chips with non-immune igg, or control chips from myc-depleted cells, some of the  <dig>  peaks might also reflect non-specific binding of the antiserum to chromatin.
to address such a possibility, we prepared chromatin from naïve and from myc-depleted s <dig> cells, and conducted chip experiments with this commercial antiserum.
as expected, the chip signals at the high-affinity myc targets nop <dig> and uhg <dig>  are strongly reduced upon myc depletion .
in contrast, very poor or no reduction is observed at  <dig> sequences that were identified as myc binding sites by yang et al.
but not in our experiments .
this non myc-specific chip signal presumably corresponds to a background reactivity of the commercial antibody, which is unaffected by the myc knock-down even though myc protein is depleted highly efficiently .
by inference, we consider it most likely that most of the sequences that were not identified in our experiments do not specifically bind to myc in drosophila.
in our following analysis we, therefore, focus on the  <dig> sites we have identified in our combined chipseq experiments as the core set of myc binding sites in drosophila .
ribosome biogenesis and ribosome protein genes are the core myc targets
in a first step, we determined the position of the binding sites relative to annotated genes.
fifty-eight percent of all sites map within 100 bp from the nearest transcription start site , and 80% of the sites are less than  <dig>  bp from a tss, suggesting that myc preferentially binds to promoter-proximal sequences.
this preference is even more striking when only myc peaks are considered that cover a canonical e-box, with 77% of these peaks localizing to within 100 bp of a tss .
due to the high gene density in drosophila, several myc binding sites are located close to more than one tss, so that altogether  <dig> genes are potentially affected by myc binding .
to determine which of these  genes indeed require myc for their correct expression we conducted rnaseq experiments in s <dig> cells.
for this purpose, we transfected dsrna directed against myc  into drosophila s <dig> cells and harvested the cells 24 hours later for total rna isolation, by which time myc protein levels were strongly reduced .
in order to assess both polyadenylated and non-adenylated transcripts, we then depleted the samples of ribosomal rna and processed the remainder for deep sequencing.
the bioinformatic analysis of biologically independent triplicates  resulted in  <dig>  genes with detectable expression in s <dig> cells and a minimal transcript length of 125 nt.
of these,  <dig> genes were differentially expressed upon myc depletion .
the majority of these genes,  <dig>  is downregulated upon myc depletion.
among these, the poly-adenylated transcripts show a good overlap with previously established lists of myc-activated genes that were obtained in different settings .
note that, in contrast to the myc-activated genes, myc-repressed genes show only poor overlap between different data sets .
taken together, these observations validate the current rnaseq data set.
a comparison of the binding and expression data shows that  <dig> of the myc-bound genes are downregulated by at least one third upon myc-knockdown and  <dig> are upregulated by at least one third .
the remaining transcripts were either not detected in our rnaseq experiments  or they showed lower responses to altered myc levels .
the following analysis focuses on the  <dig> genes that are bound by myc and require myc for their full expression and that we, therefore, consider to be the core set of directly myc-activated genes.
the overwhelming majority of these genes  functions in ribosome biogenesis and in translation .
myc controls several steps in this pathway, starting with the synthesis of ribosomal rna by rna polymerase i .
consistent with earlier reports  <cit> , we did not observe direct binding of myc to rrna loci in our chipseq experiments, but myc increases the activity of rna polymerase i by inducing some of its components or cofactors .
numerous myc targets then control the post-transcriptional modifications of rrna in general , and the maturation of the 40s subunit  or 60s subunit .
myc further induces the expression of ribosomal protein genes , of translation factors  and of genes involved in trna maturation .
myc has also been reported to promote rna polymerase iii activity via an interaction with its co-factor brf and, therefore, increases the amounts of trnas and other pol iii products  <cit> ; because of their small size these transcripts were not contained in our analysis.
we also did not observe any significant enrichment of myc at the  <dig> annotated trna loci in our chipseq data .
presumably, myc does not contact dna directly at these sites; indeed, myc does not require its dna binding domain nor its classical dimerization partner max to activate rna polymerase iii  <cit> , and most trna loci  also do not contain any consensus myc binding site .table  <dig>
categories of directly myc-activated genes
function
genes
total ribi and translation
114
ribi, ribosome biogenesis; snornp, small nucleolar ribonucleoproteins.
the observation that the majority of myc-activated genes controls ribosome biogenesis and translation indicates that these are the main and presumably primordial functions of myc proteins.
at the same time, they emphasize myc’s central role for the production  of ribosomes.
indeed, of  <dig> genes that were recently identified as essential for rrna processing in human cells  <cit> , the homologs of  <dig> count among these core myc targets in drosophila.
the situation is particularly striking for snornps, whose best-characterized activities reside in the post-transcriptional modifications of rrnas.
all protein components of snornps are known to be myc targets, and our analysis now uncovers a large and novel class of myc targets: the non-coding snornas.
a novel class of myc targets: snorna and uhg genes
after myc depletion  <dig> genes are significantly downregulated  – and  <dig> of these do not code for proteins, but instead for snornas.
the drosophila genome is predicted to encode  <dig> snornas , ranging in size from  <dig> to 316 nt.
strikingly, all of the  <dig> snornas that pass our 125 nt size cutoff and are detectably expressed in s <dig> cells are strongly downregulated upon myc depletion .
this includes most of the box h/aca snornas, but the majority of the box c/d snornas are smaller than 125 nt and, therefore, not detectable in these rnaseq experiments.
however, qpcr and northern blot analysis of several small box c/d snornas confirmed that they, too, are significantly depleted upon myc knockdown , suggesting that myc affects snorna levels in general.
this dramatic impact of myc on snorna levels can be explained by a combination of different mechanisms.
first, most drosophila snorna genes are encoded in introns of other genes, from which the mature snornas are excised  <cit> .
many of the  <dig> different host genes are bona fide myc targets , and myc’s impact on host gene expression is likely to be reflected in reduced levels of the intron-encoded snornas.
second, snornas function as part of snornps, whose known protein components all are myc targets .
hence, myc downregulation is expected to deplete snornps and, as a consequence, destabilize the corresponding snornas  <cit> .
third, myc also controls the expression of genes that are dedicated to the synthesis of snornas, the so-called uhg genes .
the following analysis focuses on myc’s transcriptional effects on these uhg genes.figure  <dig> effect of altered myc levels on uhg and snorna expression.
a) gene set enrichment analysis  of snornas.
the  <dig>  detectable genes are ordered by relative expression  from left to right, and all detectable snornas are mapped onto these genes .
nes indicates the normalized enrichment score, p the nominal p-value and q the false discovery rate.
b and c) rna levels were assayed 24 hours after addition of myc-dsrna to s <dig> cells by quantitative pcr  and northern blotting ; of ten investigated snornas, five gave no signal or such a weak signal that a reduction in response to myc-knockdown could not be reliably quantified, four were clearly reduced upon myc-knockdown and one was unaffected .
d) qpcr assays carried out six hours after addition of 125 μm cuso <dig> to s2-myc cells to induce myc overexpression.
error bars show standard deviations of biologically independent duplicates.
selected snornas are grouped with the transcripts of their host uhg genes.
panel c, arrowheads point to mature snornas; the identity of the cross-reactive slower migrating band in the me28s-g2703c blot  is unclear.
the locations of molecular weight dna markers are indicated.
the same rna samples were analyzed by reverse transcription and quantitative pcr for the reference genes snm <dig> and rab <dig>  as well as for the snornas.
c’, qpcr results and quantification of the northern blot bands.
c”, part of each sample that was not used for rna isolation  was analyzed by western blotting with mouse anti-myc antibody  and mouse anti-α-tub84b .
the experiment shown in panels c, c’ and c” is representative of biologically independent duplicates.
snorna, small nucleolar rna; uhg, u-snorna host genes.
seven such genes have been identified in drosophila, numbered  <dig> to  <dig>   <dig>   <dig> ; the existence of an additional uhg gene is suggested by our data .
these genes do not contain recognizable open reading frames, and no translation products have been detected .
instead, they are dedicated to the generation of snornas: uhg genes contain up to  <dig> introns, each of which codes for a single snorna –  <dig> snornas for the previously described uhg genes combined .
all of these uhg  transcripts are downregulated upon myc-depletion in the rnaseq data set.
qpcr on biologically independent samples confirmed this downregulation .
since all but three of these uhg-hosted snornas are shorter than  <dig> nucleotides and, therefore, not contained in our rnaseq data set, we investigated several of these transcripts by qpcr and found that they were also downregulated .
such a downregulation can also be seen on northern blots, which further confirms that the mature snornas are affected by myc depletion and not just their immature precursors .
myc is not only required for the full expression of these uhg-genes and snornas hosted within their exons, but transient overexpression of myc is also sufficient to increase their expression to supraphysiological levels .
thus, myc controls the expression of many snornas independently of their stability or the induction of their protein-coding host genes – and it does so by direct binding to four of the seven uhg genes.
for uhg-genes  <dig>   <dig> and  <dig> the myc peaks are centered on consensus e-boxes, for uhg <dig> the myc peak is located in the vicinity of an e-box .
in addition, myc binds to a cluster of three snorna genes on chromosome 2r, that are arranged in tandem and flanked by a single strongly myc-bound consensus e-box .
since only seven other snorna genes are independently transcribed in drosophila, we surmise that this cluster might constitute a novel uhg locus.
the binding of myc to uhg sequences was confirmed by chips on biologically independent samples .
it presumably involves myc:max complexes , since knockdown of myc’s dimerization partner max reduces myc recruitment to these uhg promoters .
to further corroborate myc’s transcriptional effect on uhg genes, we cloned a 983 bp genomic dna fragment, comprising the basal promoter and e-box of the uhg <dig> locus, in front of firefly luciferase.
this uhg <dig> promoter fragment drives strong expression of luciferase upon transfection into s <dig> cells.
rna-mediated knockdown of myc reduces relative luciferase expression to a similar extent as that of a reporter driven by the promoter of the previously characterized myc target cg <dig>  conversely, myc overexpression significantly increases luciferase expression .
similar results were obtained with a luciferase reporter driven by the uhg <dig> promoter region .
note that reporter activities change more moderately after myc overexpression than after knockdown.
this suggests that myc levels in naïve s <dig> cells may be sufficiently high to saturate the myc binding sites in the reporter plasmids; alternatively, essential co-factors of myc may become limiting in conditions of increased myc expression.figure  <dig> myc directly binds to and controls uhg promoters.
a) chipseq profile for the uhg <dig> locus in s <dig> cells.
labeling conventions are as in figure 1a.
b) chip-qpcr of uhg loci.
chromatin was isolated from naïve s <dig> cells or from s <dig> cells depleted of myc, precipitated with rabbit anti-myc  or control igg antibodies and assayed by qpcr for enrichment of the indicated loci.
similar results were obtained with mouse anti-myc antibodies .
c) activity of cg5033- and uhg1-luciferase reporters in s <dig> cells upon myc-overexpression or myc-knockdown.
as a control for dsrna against myc , dsrna against gfp was used ; for myc-overexpression tubulin-gal <dig> was co-transfected with uas-myc, and control samples were transfected with tubulin-gal <dig> and uas-t .
error bars show standard deviations from six transfections.
*p < <dig> ; **p < <dig>  .
myc control of snorna levels in vertebrates
taken together, these data demonstrate that drosophila myc directly controls the expression of snornas.
to determine whether this activity of myc is evolutionarily conserved, we re-evaluated chipseq and rna expression data from vertebrate cells.
the human genome encodes  <dig> snornas,  <dig> of which reside in introns of  <dig> different host genes  <cit> , including  <dig> non protein-coding snhg loci .
to address their regulation by vertebrate myc, we first examined published data from human u2os cells  <cit> .
when all genes were sorted by the level of myc binding to their promoters, we observed a significant enrichment for snorna host genes among the highly myc-bound genes .
thus, as was described for genes involved in ribosome biogenesis and translation  <cit> , snorna host genes are bound by the low physiological levels of c-myc in u2os cells.
these genes are also activated by c-myc, as can be seen in murine t-cell lymphomas caused by expression of a tet-controllable myc transgene  <cit> .
silencing of the myc transgene in this system leads to tumor regression.
this is accompanied by the rapid up- and down-regulation of  <dig>  and  <dig> genes, respectively, as was determined by agilent microarrays  <cit> .
more than 70% of the latter genes are bound by myc and, therefore, have been proposed to correspond to direct transcriptional targets.
snorna host genes are highly enriched among myc-activated genes .
together with the chipseq data above, this shows that myc directly controls the expression of these host genes, and it suggests that myc also globally activates the levels of many intronic snornas.
the impact of vertebrate myc on snorna synthesis can also be observed at the so-called snhg loci – genes that are dedicated to the production of snornas, analogously to the drosophila uhg genes.
as an example, the snhg <dig> promoter is strongly bound by the low levels of endogenous c-myc in u2os cells ; this association is at least as strong as that between myc and its well-established target nucleolin .
induction of ectopic myc expression increases the binding to both snhg <dig> and nucleolin further , but only slightly augments the expression of these genes , probably because they are already maximally transactivated by endogenous myc  <cit> .
in contrast, in murine t cells expression of endogenous myc and binding to the snhg <dig> promoter are virtually undetectable .
in these cells, derepression of transgenically expressed myc triggers strong association with the snhg <dig> promoter, and concomitantly induces snhg <dig> mrna expression by  <dig> -fold .
importantly, myc is not only recruited to snhg <dig>  but to the majority of snhg promoters in murine and human cells, most often to positions containing canonical e-boxes .
these data show that the control of non-coding snorna host genes, in particular, and presumably of snorna levels, in general, are an evolutionarily conserved function of myc proteins.figure  <dig> myc binds to vertebrate non-coding snorna host genes .
a) chipseq traces of snhg <dig>  and ncl  in human u2os cells, both shown at the same vertical scale.
b) chipseq traces of snhg <dig> in murine t cells; the expression level of snhg <dig> drops by  <dig> -fold after silencing of transgenic myc expression.
c) chipseq traces for all  <dig> snhg loci in u2os cells.
each window is 2 kb wide.
horizontal bars below the input samples denote canonical e-boxes.
samples ‘ectopic myc’ contain overexpressed myc, samples ‘-‘ only endogenous myc; overexpression of myc was induced  or repressed  by the addition of doxycyclin.
chipseq, chromatin immunoprecipitation sequencing; snorna, small nucleolar rna.
uhg-genes promote growth in vivo in drosophila
to address the importance of snornas for growth, we focused on the uhg <dig> locus since it encodes  <dig> snornas, but no other potentially growth-relevant transcripts.
using imprecise excision of a p-element we generated a null allele that eliminates the coding regions for all snornas, but does not affect the neighboring genes .
this mutation is expected to affect 2’-o ribose-methylation of 18s- and 28s-rrna at five positions that are exclusively targeted by uhg1-snornas  <cit> , and as a consequence ribosomal assembly and/or activity.
interestingly, 66% of the resulting uhg11/uhg <dig> mutant females and 50% of the males survive to adulthood .
these adults are not significantly lighter than control animals .
similarly, uhg <dig> mutant wandering larvae contain at least as much protein as control animals .
however, the uhg <dig> grow at a significantly slower rate  and accordingly reach corresponding developmental stages later than wild type flies – adult eclosion is delayed by about 12 hours in uhg <dig> mutants .
in addition, uhg <dig> females, but not males, show a strongly reduced fertility .
both such a developmental delay and female-specific sterility are typical manifestations of mutations in pathways affecting growth, and they have been observed for hypomorphic mutations in myc  <cit> .
consistent with a contribution of uhg <dig> to myc-dependent growth, the documented ability of myc to promote nuclear growth is blunted in uhg <dig> mutant salivary gland cells .
together, these data demonstrate that uhg <dig> is required for protein synthesis and growth.
nevertheless, the complete absence of uhg <dig> has comparatively mild consequences, and in some tissues uhg <dig> seems not to be limiting for growth and proliferation.
we did not observe any impact of the uhg <dig> mutation on the area of imaginal disc cell clones overexpressing myc .
similarly, larval brain tumors were not obviously influenced by the complete loss of uhg1: knockdown of the tumor suppressor brat in type ii neuroblast stem cell lineages leads to tumors that critically depend on myc  <cit> .
however, homozygosity for uhg <dig> in such a setting did not reduce tumor mass, nor did it prevent the brat knock-down induced reversion of intermediate progenitor cells to supernumerary neuroblasts .
it is possible, though, that the extension of larval development that is caused by the loss of uhg <dig>  masks a potential reduction in tumor growth rate.
as a complementary approach that does not suffer from confounding effects on developmental timing, we therefore investigated the consequences of excessive uhg levels.
we generated various uhg transgenes and expressed them in different settings.
overexpression of uhg genes on their own did not increase the size of normal diploid or polyploid cells, nor did this treatment affect the size of cells co-expressing myc .
however, independent transgenes for uhg <dig> and uhg <dig> increased the size of brat-knockdown induced brain tumors, as assayed with a recently described luciferase-based system .
thus, overexpression of at least two different uhg-genes promotes the growth of tumors.figure  <dig> biological effects of uhg genes.
a) uhg <dig> locus with adjacent genes.
black arrows indicate direction of transcription, blue rectangles and lines correspond to exons and introns, respectively, red triangles show snornas.
below are indicated the genomic regions used for the luciferase reporter  and the extent of the deletion in the uhg1
<dig> null allele.
b) amino acid incorporation rates in wandering larvae.
ratios  and standard deviations are shown for three biological replicates each with ten larvae for each genotype.
statistical significance of the difference p =  <dig>  .
c) average time from egg deposition to adult eclosion for male flies; numbers of analyzed flies are indicated in parentheses.
‘mut’ corresponds to ‘uhg1
1’, ‘rev’ to ‘uhg1
rev’, ‘+’ to the standard lab strain ‘y w’.
d) average area of salivary gland nuclei overexpressing myc  relative to neighboring nuclei without myc overexpression .
each bar represents  <dig> to  <dig> nuclei from  <dig> separate salivary glands; error bars indicate sem.
e) luciferase assays from single adult males overexpressing the indicated uhg-transgenes under brat-knockdown  or brat-wildtype  conditions in type ii neuroblast lineages.
# <dig> and # <dig> correspond to independent transgenes; numbers in parentheses indicate the numbers of individually assayed flies .
genotypes: in addition to the indicated uas-uhg transgene, the flies carried ‘worniu-gal <dig> asense-gal80/+; uas-brat- uas-luciferase/+’.
difference to control is < <dig>   and < <dig>  , respectively .
sem, standard error of the mean; snorna, small nucleolar rna.
discussion
in this study, we combine rnaseq and chipseq approaches to identify a core set of direct myc targets in drosophila s <dig> cells.
we find that drosophila myc controls less than  <dig> genes, substantially fewer than have been proposed to be controlled by vertebrate myc  <cit> .
the overwhelming part of drosophila myc targets control ribosome biogenesis and protein synthesis, which, therefore, constitute the core and presumably primordial function of myc proteins.
among these core targets we find an entire novel gene class that has previously not been recognized as myc targets: the non protein-coding snornas.
some of myc’s effect on snorna levels may be indirect , but a sizable fraction of snornas encoded in uhg genes is directly transcriptionally activated by myc .figure  <dig> schematic of myc function in drosophila.
the majority of rna polymerase ii transcribed direct myc targets  control ribosome biogenesis  or code for ribosomal proteins.
the area of each group is proportional to the number of genes it contains; intronic snornas are counted as direct myc targets if their host genes are direct myc targets.
previously, myc has been shown to activate rna polymerases i and iii and hence the transcription of rrnas and trnas.
thus, the major function of drosophila myc resides in the control of protein synthesis.
snorna, small nucleolar rna.
snornas mostly fall into either the box h/aca or the box c/d class, depending on the structural elements they contain .
upon binding to specific protein partners they form snornps that process rrnas  and contribute to their folding, thereby playing an important role in ribosome biogenesis.
in addition, some snornas have been shown to regulate alternative splicing  <cit> , affect rna editing  <cit> , mediate apoptosis in response to lipotoxic stress  <cit> , serve as progenitors of mirnas , and they may contribute to the maintenance of open chromatin structure  <cit> .
most animal snornas are encoded in introns of other genes, many of which code for ribosome- and translation-related proteins  <cit> .
this arrangement is thought to help adapt snorna production to a cell’s biosynthetic needs.
here, we identify a new means by which the balanced synthesis of the different components of snornp complexes is achieved.
the protein partners of snornas are well established direct transcriptional targets of myc, that is, the box c/d snornp components fibrillarin, nop <dig>  nop58/nop <dig>   <dig> k/hoi-polloi, and the box h/aca snornp components gar <dig>  dyskerin/nop60b, nhp <dig>  nop10/cg <dig>  moreover, several factors required for snornp assembly or localization are direct myc targets  or show close genetic and physical interaction with myc  .
thus, a single transcription factor, myc, controls all components that make up snornp particles, as well as several factors that contribute to their function, thereby acting as a master regulator of snornp production.
at the same time, the production of snornps and rrna modifications constitute the core function of myc, as 48% of its mostly highly controlled target genes are dedicated to this task.
our data further show that this control of snorna levels by myc is biologically relevant.
the loss of uhg1-encoded snornas impairs normal growth, whereas overexpression of a subset of uhg genes promotes tumor growth in a myc-dependent brain tumor system.
while our studies have focused on drosophila, we also found that myc’s control of snorna host genes is conserved in vertebrates.
indeed, recent publications provide support for myc’s role in snorna expression.
a bioinformatic analysis of  <dig> intergenic human snorna promoters found an enrichment of e-boxes, that is, potential myc binding sites  <cit> .
this suggests that, in addition to intronic snornas , independently transcribed snornas might be activated by direct myc binding.
moreover, several snornas were found to be significantly overexpressed in neuroblastomas with high-level n-myc expression , as well as in cultured cell lines in response to n-myc activation  <cit> .
this observation raises the possibility that snornas contribute to the growth of n-myc dependent tumors, analogously to what we observed upon overexpression of uhg genes in drosophila type ii neuroblast tumors.
several publications point to a possible role for snorna deregulation in other cancers as well .
thus, the levels of snornas are elevated in non-small-cell lung cancer , acute leukemias, and metastatic prostate cancer  <cit> , and individual snornas have been shown to contribute to cellular proliferation and/or transformation .
on the other hand, certain snornas were also shown to have a negative impact on proliferation , breast cancer  <cit> , and some tumors display reduced snorna levels .
CONCLUSIONS
in transformed vertebrate cells, the oncogene myc is thought to act as a general transcriptional amplifier.
in contrast, our present study shows that at physiological levels in drosophila cells, myc directly binds and controls only a limited number of transcriptional targets – less than  <dig>  in addition to previously described myc targets that contribute to ribosome biogenesis and protein translation, we find that myc controls the levels of all snornas.
most of these small rnas function as part of ribonucleoproteins to post-transcriptionally modify rrnas, which then presumably affects ribosome activity.
indeed, several observations in vertebrate tumors  suggest a correlation between the levels of certain snornas and the rate of normal and/or pathological growth.
it remains to be seen whether this reflects a global alteration of protein synthesis or differential effects on specific proteins.
the accessibility of the drosophila system, both for loss-of-function and overexpression approaches, raises the possibility of experimentally addressing these issues in the future.
in short, our data highlight the non-coding snornas as a novel and evolutionarily conserved class of direct myc targets that are likely to play an important role both in normal development, as well as in myc-dependent pathological growth.

 growth and proliferation are fundamental for all life. in metazoans, these processes need to be coordinated with animal development and homeostasis. one important factor in the control and coordination of growth is the myc family of oncogenes. myc’s activity is essential for normal animal development in insects and in vertebrates. a moderate reduction of myc levels in drosophila results in small cells with small nucleoli, reduced organismal growth and adult size, delayed overall development and female sterility. fruit flies lacking all myc activity fail to undergo normal growth and mostly die during early larval stages. conversely, myc overexpression in vertebrates leads to unscheduled cellular growth and proliferation. consistent with this, mutational activation of myc, or of upstream signaling pathways, is frequently found in human cancers and causally contributes to this disease. knowledge of the relevant myc targets, therefore, is essential to understand the processes of myc-dependent growth in normal and pathological situations. while myc can control a large number of genes in vertebrates, the number of myc targets is still open in the simpler model drosophila. several studies have characterized myc-regulated genes, but they have not comprehensively addressed which of these genes are controlled by direct myc binding, nor have they included genes that do not code for proteins. however, recent studies have revealed the abundance and variety of non-coding transcripts in the eukaryotic genome, and have emphasized the biological importance of various such transcripts. some non-coding rnas have already been identified as physiologically relevant myc targets in vertebrates, raising the possibility that the biological activities of myc might be mediated by these and additional non-protein coding transcripts. we, therefore, set out to extend the search for myc targets to non-polyadenylated transcripts, using a combination of rnaseq and chromatin immunoprecipitation sequencing approaches in cultured drosophila cells. this approach led to the identification of a core set of less than myc targets. the majority of these targets control ribosome biogenesis and translation, in good agreement with earlier reports. in addition to the previously identified mrnas, however, we identified small nucleolar rnas as a novel class of myc targets. the drosophila genome encodes snornas, most of which fall into one of two classes: the to nucleotides long box c/d snornas and the to nucleotides long box h/aca snornas. upon association with a small set of specific proteins, these two types of snornas form small nucleolar riboproteins that catalyze 2’-o-methylation and pseudouridylation, respectively. their best characterized targets are small nuclear rnas and ribosomal rnas, where many of these post-transcriptional modifications cluster in functionally important regions and contribute to efficient ribosome biogenesis and/or function. in addition, snornas have been shown to affect other biological processes, such as rna editing, alternative splicing, and gene silencing. intriguingly, the snorna associated proteins all are encoded by core myc targets, as are several of the factors involved in snornp processing. these findings indicate that myc acts as a master regulator of snornp biogenesis, and they suggest a biological mechanism that ensures the stoichiometry of rna and protein components of snornps. at the same time, they reinforce the notion that snornp generation, and hence ribosome biogenesis, constitutes the core function of drosophila myc. we further provide evidence that vertebrate myc also controls snorna expression. finally, we show that the snorna host gene uhg is important for normal animal growth, and that overexpression of different uhg genes enhances tumor growth.  myc directly binds a core set of sites in drosophila in order to establish a core set of genes directly controlled by myc in drosophila, we performed chipseq experiments in s cells using a mouse monoclonal anti drosophila myc antibody. to control for background signal, we used non-immune mouse immunoglobulin g in parallel experiments, and we repeated both anti-myc and control igg chips with cells that had been depleted of myc. this set of experiments resulted in the identification of peaks that are specifically bound by myc in naïve s cells but not in myc-depleted cells, and that are not recognized by control iggs. since these chips relied on a monoclonal antibody, it is conceivable that some myc binding sites were missed due to epitope masking. to exclude this possibility, we conducted another set of chipseq experiments with a rabbit polyclonal anti drosophila myc antibody and chromatin from s cells, again using species-matched non-immune iggs as control. this approach yielded specifically myc-bound peaks, most of which overlapped with the myc-binding sites identified above. given this coincidence between the two antibodies, we are confident that we have identified the majority of myc bound genes, and that these genes represent bona fide myc-binding sites.figure myc binding sites in drosophila. a) myc binding to the fibrillarin locus as an example of a binding site in s cells. strong binding is observed with mouse α-myc antibodies in naïve s cells, but not upon myc depletion or with control mouse igg; the grey box marks the myc binding peak as called by the software macs, the asterisk shows a consensus e-box. chromosomal coordinates are indicated below the traces, as are the extents and orientations of genes in this region. the western blot illustrates the efficiency of myc depletion; the arrowhead points to a band presumably arising from post-translational modification of myc. molecular weight markers are indicated. b) overlaps of myc binding sites from three different chipseq experiments, using either mouse monoclonal or rabbit polyclonal anti-myc antibodies, and chromatin from s or kc cells. only peaks called by the software as ‘significant’ were considered, that is, with false discovery rate of <10%. c) position of myc binding peaks relative to the nearest transcription start site. data are distributed in 100-nt bins for a distance of up to  nt from the tss and in -nt bins for distances between and  nt, and shown as percent of all peaks. grey bars show all myc binding peaks, the red line only the myc binding peaks containing a canonical e-box within a 100 nt window straddling the myc binding summit. horizontal lines above the graph illustrate windows of +/− 100 nt and +/−  nt around the tss, containing 58% and 80% of all myc peaks, and 77% and 93% of the myc peaks with canonical e-boxes, respectively. this number of myc-binding sites is consistent with what was found in an earlier study that used damid for assessing myc binding, but it is considerably lower than the nearly sites that were recently reported for a chipseq study of drosophila kc cells. several possibilities can be considered for this discrepancy. first, although both kc and s cells are hematopoietic cells of embryonic origin, it is conceivable that they differ in their molecular characteristics. to address this possibility, we carried out chipseq experiments with rabbit anti-myc antibodies and control iggs using chromatin from kc cells. this led to the identification of myc-binding sites. however, in these experiments we observed considerably higher background binding than in s cells, and, therefore, only binding sites were statistically significant <10%), of which overlap peaks in s cells. this similarity with the results in s cells argues against major differences between the cell lines. second, yang et al. used a different antibody which might have higher affinity and/or recognize other epitopes on myc, thus allowing the identification of additional bona fide myc-binding sites. alternatively, since there are no published data showing the specificity of this antibody, or of control chips with non-immune igg, or control chips from myc-depleted cells, some of the peaks might also reflect non-specific binding of the antiserum to chromatin. to address such a possibility, we prepared chromatin from naïve and from myc-depleted s cells, and conducted chip experiments with this commercial antiserum. as expected, the chip signals at the high-affinity myc targets nop and uhg are strongly reduced upon myc depletion. in contrast, very poor or no reduction is observed at sequences that were identified as myc binding sites by yang et al. but not in our experiments. this non myc-specific chip signal presumably corresponds to a background reactivity of the commercial antibody, which is unaffected by the myc knock-down even though myc protein is depleted highly efficiently. by inference, we consider it most likely that most of the sequences that were not identified in our experiments do not specifically bind to myc in drosophila. in our following analysis we, therefore, focus on the sites we have identified in our combined chipseq experiments as the core set of myc binding sites in drosophila. ribosome biogenesis and ribosome protein genes are the core myc targets in a first step, we determined the position of the binding sites relative to annotated genes. fifty-eight percent of all sites map within 100 bp from the nearest transcription start site, and 80% of the sites are less than  bp from a tss, suggesting that myc preferentially binds to promoter-proximal sequences. this preference is even more striking when only myc peaks are considered that cover a canonical e-box, with 77% of these peaks localizing to within 100 bp of a tss. due to the high gene density in drosophila, several myc binding sites are located close to more than one tss, so that altogether genes are potentially affected by myc binding. to determine which of these genes indeed require myc for their correct expression we conducted rnaseq experiments in s cells. for this purpose, we transfected dsrna directed against myc into drosophila s cells and harvested the cells 24 hours later for total rna isolation, by which time myc protein levels were strongly reduced. in order to assess both polyadenylated and non-adenylated transcripts, we then depleted the samples of ribosomal rna and processed the remainder for deep sequencing. the bioinformatic analysis of biologically independent triplicates resulted in genes with detectable expression in s cells and a minimal transcript length of 125 nt. of these, genes were differentially expressed upon myc depletion. the majority of these genes, is downregulated upon myc depletion. among these, the poly-adenylated transcripts show a good overlap with previously established lists of myc-activated genes that were obtained in different settings. note that, in contrast to the myc-activated genes, myc-repressed genes show only poor overlap between different data sets. taken together, these observations validate the current rnaseq data set. a comparison of the binding and expression data shows that of the myc-bound genes are downregulated by at least one third upon myc-knockdown and are upregulated by at least one third. the remaining transcripts were either not detected in our rnaseq experiments or they showed lower responses to altered myc levels. the following analysis focuses on the genes that are bound by myc and require myc for their full expression and that we, therefore, consider to be the core set of directly myc-activated genes. the overwhelming majority of these genes functions in ribosome biogenesis and in translation. myc controls several steps in this pathway, starting with the synthesis of ribosomal rna by rna polymerase i. consistent with earlier reports, we did not observe direct binding of myc to rrna loci in our chipseq experiments, but myc increases the activity of rna polymerase i by inducing some of its components or cofactors. numerous myc targets then control the post-transcriptional modifications of rrna in general, and the maturation of the 40s subunit or 60s subunit. myc further induces the expression of ribosomal protein genes, of translation factors and of genes involved in trna maturation. myc has also been reported to promote rna polymerase iii activity via an interaction with its co-factor brf and, therefore, increases the amounts of trnas and other pol iii products; because of their small size these transcripts were not contained in our analysis. we also did not observe any significant enrichment of myc at the annotated trna loci in our chipseq data. presumably, myc does not contact dna directly at these sites; indeed, myc does not require its dna binding domain nor its classical dimerization partner max to activate rna polymerase iii, and most trna loci also do not contain any consensus myc binding site.table categories of directly myc-activated genes function genes total ribi and translation 114 ribi, ribosome biogenesis; snornp, small nucleolar ribonucleoproteins. the observation that the majority of myc-activated genes controls ribosome biogenesis and translation indicates that these are the main and presumably primordial functions of myc proteins. at the same time, they emphasize myc’s central role for the production of ribosomes. indeed, of genes that were recently identified as essential for rrna processing in human cells, the homologs of count among these core myc targets in drosophila. the situation is particularly striking for snornps, whose best-characterized activities reside in the post-transcriptional modifications of rrnas. all protein components of snornps are known to be myc targets, and our analysis now uncovers a large and novel class of myc targets: the non-coding snornas. a novel class of myc targets: snorna and uhg genes after myc depletion genes are significantly downregulated – and of these do not code for proteins, but instead for snornas. the drosophila genome is predicted to encode snornas, ranging in size from to 316 nt. strikingly, all of the snornas that pass our 125 nt size cutoff and are detectably expressed in s cells are strongly downregulated upon myc depletion. this includes most of the box h/aca snornas, but the majority of the box c/d snornas are smaller than 125 nt and, therefore, not detectable in these rnaseq experiments. however, qpcr and northern blot analysis of several small box c/d snornas confirmed that they, too, are significantly depleted upon myc knockdown, suggesting that myc affects snorna levels in general. this dramatic impact of myc on snorna levels can be explained by a combination of different mechanisms. first, most drosophila snorna genes are encoded in introns of other genes, from which the mature snornas are excised. many of the different host genes are bona fide myc targets, and myc’s impact on host gene expression is likely to be reflected in reduced levels of the intron-encoded snornas. second, snornas function as part of snornps, whose known protein components all are myc targets. hence, myc downregulation is expected to deplete snornps and, as a consequence, destabilize the corresponding snornas. third, myc also controls the expression of genes that are dedicated to the synthesis of snornas, the so-called uhg genes. the following analysis focuses on myc’s transcriptional effects on these uhg genes.figure effect of altered myc levels on uhg and snorna expression. a) gene set enrichment analysis of snornas. the detectable genes are ordered by relative expression from left to right, and all detectable snornas are mapped onto these genes. nes indicates the normalized enrichment score, p the nominal p-value and q the false discovery rate. b and c) rna levels were assayed 24 hours after addition of myc-dsrna to s cells by quantitative pcr and northern blotting; of ten investigated snornas, five gave no signal or such a weak signal that a reduction in response to myc-knockdown could not be reliably quantified, four were clearly reduced upon myc-knockdown and one was unaffected. d) qpcr assays carried out six hours after addition of 125 μm cuso to s2-myc cells to induce myc overexpression. error bars show standard deviations of biologically independent duplicates. selected snornas are grouped with the transcripts of their host uhg genes. panel c, arrowheads point to mature snornas; the identity of the cross-reactive slower migrating band in the me28s-g2703c blot is unclear. the locations of molecular weight dna markers are indicated. the same rna samples were analyzed by reverse transcription and quantitative pcr for the reference genes snm and rab as well as for the snornas. c’, qpcr results and quantification of the northern blot bands. c”, part of each sample that was not used for rna isolation was analyzed by western blotting with mouse anti-myc antibody and mouse anti-α-tub84b. the experiment shown in panels c, c’ and c” is representative of biologically independent duplicates. snorna, small nucleolar rna; uhg, u-snorna host genes. seven such genes have been identified in drosophila, numbered to; the existence of an additional uhg gene is suggested by our data. these genes do not contain recognizable open reading frames, and no translation products have been detected. instead, they are dedicated to the generation of snornas: uhg genes contain up to introns, each of which codes for a single snorna – snornas for the previously described uhg genes combined. all of these uhg transcripts are downregulated upon myc-depletion in the rnaseq data set. qpcr on biologically independent samples confirmed this downregulation. since all but three of these uhg-hosted snornas are shorter than nucleotides and, therefore, not contained in our rnaseq data set, we investigated several of these transcripts by qpcr and found that they were also downregulated. such a downregulation can also be seen on northern blots, which further confirms that the mature snornas are affected by myc depletion and not just their immature precursors. myc is not only required for the full expression of these uhg-genes and snornas hosted within their exons, but transient overexpression of myc is also sufficient to increase their expression to supraphysiological levels. thus, myc controls the expression of many snornas independently of their stability or the induction of their protein-coding host genes – and it does so by direct binding to four of the seven uhg genes. for uhg-genes and the myc peaks are centered on consensus e-boxes, for uhg the myc peak is located in the vicinity of an e-box. in addition, myc binds to a cluster of three snorna genes on chromosome 2r, that are arranged in tandem and flanked by a single strongly myc-bound consensus e-box. since only seven other snorna genes are independently transcribed in drosophila, we surmise that this cluster might constitute a novel uhg locus. the binding of myc to uhg sequences was confirmed by chips on biologically independent samples. it presumably involves myc:max complexes, since knockdown of myc’s dimerization partner max reduces myc recruitment to these uhg promoters. to further corroborate myc’s transcriptional effect on uhg genes, we cloned a 983 bp genomic dna fragment, comprising the basal promoter and e-box of the uhg locus, in front of firefly luciferase. this uhg promoter fragment drives strong expression of luciferase upon transfection into s cells. rna-mediated knockdown of myc reduces relative luciferase expression to a similar extent as that of a reporter driven by the promoter of the previously characterized myc target cg conversely, myc overexpression significantly increases luciferase expression. similar results were obtained with a luciferase reporter driven by the uhg promoter region. note that reporter activities change more moderately after myc overexpression than after knockdown. this suggests that myc levels in naïve s cells may be sufficiently high to saturate the myc binding sites in the reporter plasmids; alternatively, essential co-factors of myc may become limiting in conditions of increased myc expression.figure myc directly binds to and controls uhg promoters. a) chipseq profile for the uhg locus in s cells. labeling conventions are as in figure 1a. b) chip-qpcr of uhg loci. chromatin was isolated from naïve s cells or from s cells depleted of myc, precipitated with rabbit anti-myc or control igg antibodies and assayed by qpcr for enrichment of the indicated loci. similar results were obtained with mouse anti-myc antibodies. c) activity of cg5033- and uhg1-luciferase reporters in s cells upon myc-overexpression or myc-knockdown. as a control for dsrna against myc, dsrna against gfp was used; for myc-overexpression tubulin-gal was co-transfected with uas-myc, and control samples were transfected with tubulin-gal and uas-t. error bars show standard deviations from six transfections. *p <; **p <. myc control of snorna levels in vertebrates taken together, these data demonstrate that drosophila myc directly controls the expression of snornas. to determine whether this activity of myc is evolutionarily conserved, we re-evaluated chipseq and rna expression data from vertebrate cells. the human genome encodes snornas, of which reside in introns of different host genes, including non protein-coding snhg loci. to address their regulation by vertebrate myc, we first examined published data from human u2os cells. when all genes were sorted by the level of myc binding to their promoters, we observed a significant enrichment for snorna host genes among the highly myc-bound genes. thus, as was described for genes involved in ribosome biogenesis and translation, snorna host genes are bound by the low physiological levels of c-myc in u2os cells. these genes are also activated by c-myc, as can be seen in murine t-cell lymphomas caused by expression of a tet-controllable myc transgene. silencing of the myc transgene in this system leads to tumor regression. this is accompanied by the rapid up- and down-regulation of and genes, respectively, as was determined by agilent microarrays. more than 70% of the latter genes are bound by myc and, therefore, have been proposed to correspond to direct transcriptional targets. snorna host genes are highly enriched among myc-activated genes. together with the chipseq data above, this shows that myc directly controls the expression of these host genes, and it suggests that myc also globally activates the levels of many intronic snornas. the impact of vertebrate myc on snorna synthesis can also be observed at the so-called snhg loci – genes that are dedicated to the production of snornas, analogously to the drosophila uhg genes. as an example, the snhg promoter is strongly bound by the low levels of endogenous c-myc in u2os cells; this association is at least as strong as that between myc and its well-established target nucleolin. induction of ectopic myc expression increases the binding to both snhg and nucleolin further, but only slightly augments the expression of these genes, probably because they are already maximally transactivated by endogenous myc. in contrast, in murine t cells expression of endogenous myc and binding to the snhg promoter are virtually undetectable. in these cells, derepression of transgenically expressed myc triggers strong association with the snhg promoter, and concomitantly induces snhg mrna expression by -fold. importantly, myc is not only recruited to snhg but to the majority of snhg promoters in murine and human cells, most often to positions containing canonical e-boxes. these data show that the control of non-coding snorna host genes, in particular, and presumably of snorna levels, in general, are an evolutionarily conserved function of myc proteins.figure myc binds to vertebrate non-coding snorna host genes. a) chipseq traces of snhg and ncl in human u2os cells, both shown at the same vertical scale. b) chipseq traces of snhg in murine t cells; the expression level of snhg drops by -fold after silencing of transgenic myc expression. c) chipseq traces for all snhg loci in u2os cells. each window is 2 kb wide. horizontal bars below the input samples denote canonical e-boxes. samples ‘ectopic myc’ contain overexpressed myc, samples ‘-‘ only endogenous myc; overexpression of myc was induced or repressed by the addition of doxycyclin. chipseq, chromatin immunoprecipitation sequencing; snorna, small nucleolar rna. uhg-genes promote growth in vivo in drosophila to address the importance of snornas for growth, we focused on the uhg locus since it encodes snornas, but no other potentially growth-relevant transcripts. using imprecise excision of a p-element we generated a null allele that eliminates the coding regions for all snornas, but does not affect the neighboring genes. this mutation is expected to affect 2’-o ribose-methylation of 18s- and 28s-rrna at five positions that are exclusively targeted by uhg1-snornas, and as a consequence ribosomal assembly and/or activity. interestingly, 66% of the resulting uhg11/uhg mutant females and 50% of the males survive to adulthood. these adults are not significantly lighter than control animals. similarly, uhg mutant wandering larvae contain at least as much protein as control animals. however, the uhg grow at a significantly slower rate and accordingly reach corresponding developmental stages later than wild type flies – adult eclosion is delayed by about 12 hours in uhg mutants. in addition, uhg females, but not males, show a strongly reduced fertility. both such a developmental delay and female-specific sterility are typical manifestations of mutations in pathways affecting growth, and they have been observed for hypomorphic mutations in myc. consistent with a contribution of uhg to myc-dependent growth, the documented ability of myc to promote nuclear growth is blunted in uhg mutant salivary gland cells. together, these data demonstrate that uhg is required for protein synthesis and growth. nevertheless, the complete absence of uhg has comparatively mild consequences, and in some tissues uhg seems not to be limiting for growth and proliferation. we did not observe any impact of the uhg mutation on the area of imaginal disc cell clones overexpressing myc. similarly, larval brain tumors were not obviously influenced by the complete loss of uhg1: knockdown of the tumor suppressor brat in type ii neuroblast stem cell lineages leads to tumors that critically depend on myc. however, homozygosity for uhg in such a setting did not reduce tumor mass, nor did it prevent the brat knock-down induced reversion of intermediate progenitor cells to supernumerary neuroblasts. it is possible, though, that the extension of larval development that is caused by the loss of uhg masks a potential reduction in tumor growth rate. as a complementary approach that does not suffer from confounding effects on developmental timing, we therefore investigated the consequences of excessive uhg levels. we generated various uhg transgenes and expressed them in different settings. overexpression of uhg genes on their own did not increase the size of normal diploid or polyploid cells, nor did this treatment affect the size of cells co-expressing myc. however, independent transgenes for uhg and uhg increased the size of brat-knockdown induced brain tumors, as assayed with a recently described luciferase-based system. thus, overexpression of at least two different uhg-genes promotes the growth of tumors.figure biological effects of uhg genes. a) uhg locus with adjacent genes. black arrows indicate direction of transcription, blue rectangles and lines correspond to exons and introns, respectively, red triangles show snornas. below are indicated the genomic regions used for the luciferase reporter and the extent of the deletion in the uhg1 null allele. b) amino acid incorporation rates in wandering larvae. ratios and standard deviations are shown for three biological replicates each with ten larvae for each genotype. statistical significance of the difference p = . c) average time from egg deposition to adult eclosion for male flies; numbers of analyzed flies are indicated in parentheses. ‘mut’ corresponds to ‘uhg1 1’, ‘rev’ to ‘uhg1 rev’, ‘+’ to the standard lab strain ‘y w’. d) average area of salivary gland nuclei overexpressing myc relative to neighboring nuclei without myc overexpression. each bar represents to nuclei from separate salivary glands; error bars indicate sem. e) luciferase assays from single adult males overexpressing the indicated uhg-transgenes under brat-knockdown or brat-wildtype conditions in type ii neuroblast lineages. # and # correspond to independent transgenes; numbers in parentheses indicate the numbers of individually assayed flies. genotypes: in addition to the indicated uas-uhg transgene, the flies carried ‘worniu-gal asense-gal80/+; uas-brat- uas-luciferase/+’. difference to control is < and <, respectively. sem, standard error of the mean; snorna, small nucleolar rna. discussion in this study, we combine rnaseq and chipseq approaches to identify a core set of direct myc targets in drosophila s cells. we find that drosophila myc controls less than genes, substantially fewer than have been proposed to be controlled by vertebrate myc. the overwhelming part of drosophila myc targets control ribosome biogenesis and protein synthesis, which, therefore, constitute the core and presumably primordial function of myc proteins. among these core targets we find an entire novel gene class that has previously not been recognized as myc targets: the non protein-coding snornas. some of myc’s effect on snorna levels may be indirect, but a sizable fraction of snornas encoded in uhg genes is directly transcriptionally activated by myc.figure schematic of myc function in drosophila. the majority of rna polymerase ii transcribed direct myc targets control ribosome biogenesis or code for ribosomal proteins. the area of each group is proportional to the number of genes it contains; intronic snornas are counted as direct myc targets if their host genes are direct myc targets. previously, myc has been shown to activate rna polymerases i and iii and hence the transcription of rrnas and trnas. thus, the major function of drosophila myc resides in the control of protein synthesis. snorna, small nucleolar rna. snornas mostly fall into either the box h/aca or the box c/d class, depending on the structural elements they contain. upon binding to specific protein partners they form snornps that process rrnas and contribute to their folding, thereby playing an important role in ribosome biogenesis. in addition, some snornas have been shown to regulate alternative splicing, affect rna editing, mediate apoptosis in response to lipotoxic stress, serve as progenitors of mirnas, and they may contribute to the maintenance of open chromatin structure. most animal snornas are encoded in introns of other genes, many of which code for ribosome- and translation-related proteins. this arrangement is thought to help adapt snorna production to a cell’s biosynthetic needs. here, we identify a new means by which the balanced synthesis of the different components of snornp complexes is achieved. the protein partners of snornas are well established direct transcriptional targets of myc, that is, the box c/d snornp components fibrillarin, nop nop58/nop k/hoi-polloi, and the box h/aca snornp components gar dyskerin/nop60b, nhp nop10/cg moreover, several factors required for snornp assembly or localization are direct myc targets or show close genetic and physical interaction with myc. thus, a single transcription factor, myc, controls all components that make up snornp particles, as well as several factors that contribute to their function, thereby acting as a master regulator of snornp production. at the same time, the production of snornps and rrna modifications constitute the core function of myc, as 48% of its mostly highly controlled target genes are dedicated to this task. our data further show that this control of snorna levels by myc is biologically relevant. the loss of uhg1-encoded snornas impairs normal growth, whereas overexpression of a subset of uhg genes promotes tumor growth in a myc-dependent brain tumor system. while our studies have focused on drosophila, we also found that myc’s control of snorna host genes is conserved in vertebrates. indeed, recent publications provide support for myc’s role in snorna expression. a bioinformatic analysis of intergenic human snorna promoters found an enrichment of e-boxes, that is, potential myc binding sites. this suggests that, in addition to intronic snornas, independently transcribed snornas might be activated by direct myc binding. moreover, several snornas were found to be significantly overexpressed in neuroblastomas with high-level n-myc expression, as well as in cultured cell lines in response to n-myc activation. this observation raises the possibility that snornas contribute to the growth of n-myc dependent tumors, analogously to what we observed upon overexpression of uhg genes in drosophila type ii neuroblast tumors. several publications point to a possible role for snorna deregulation in other cancers as well. thus, the levels of snornas are elevated in non-small-cell lung cancer, acute leukemias, and metastatic prostate cancer, and individual snornas have been shown to contribute to cellular proliferation and/or transformation. on the other hand, certain snornas were also shown to have a negative impact on proliferation, breast cancer, and some tumors display reduced snorna levels.  in transformed vertebrate cells, the oncogene myc is thought to act as a general transcriptional amplifier. in contrast, our present study shows that at physiological levels in drosophila cells, myc directly binds and controls only a limited number of transcriptional targets – less than in addition to previously described myc targets that contribute to ribosome biogenesis and protein translation, we find that myc controls the levels of all snornas. most of these small rnas function as part of ribonucleoproteins to post-transcriptionally modify rrnas, which then presumably affects ribosome activity. indeed, several observations in vertebrate tumors suggest a correlation between the levels of certain snornas and the rate of normal and/or pathological growth. it remains to be seen whether this reflects a global alteration of protein synthesis or differential effects on specific proteins. the accessibility of the drosophila system, both for loss-of-function and overexpression approaches, raises the possibility of experimentally addressing these issues in the future. in short, our data highlight the non-coding snornas as a novel and evolutionarily conserved class of direct myc targets that are likely to play an important role both in normal development, as well as in myc-dependent pathological growth. 
among these genes we find the non-coding snorna genes as a large novel class of myc targets. this work shows that myc acts as a master regulator of snornp biogenesis. myc proteins are essential regulators of animal growth during normal development, and their deregulation is one of the main driving factors of human malignancies. the loss of snornas impairs growth during normal development, whereas their overexpression increases tumor mass in a model for neuronal tumors. we combine chromatin immunoprecipitation-sequencing and rnaseq approaches in drosophila tissue culture cells to identify a core set of less than myc target genes, whose salient function resides in the control of ribosome biogenesis.
 myc proteins are essential regulators of animal growth during normal development, and their deregulation is one of the main driving factors of human malignancies. they function as transcription factors that control many growth- and proliferation-associated genes, and in some contexts contribute to global gene regulation.  we combine chromatin immunoprecipitation-sequencing and rnaseq approaches in drosophila tissue culture cells to identify a core set of less than myc target genes, whose salient function resides in the control of ribosome biogenesis. among these genes we find the non-coding snorna genes as a large novel class of myc targets. all assayed snornas are affected by myc, and many of them are subject to direct transcriptional activation by myc, both in drosophila and in vertebrates. the loss of snornas impairs growth during normal development, whereas their overexpression increases tumor mass in a model for neuronal tumors.  this work shows that myc acts as a master regulator of snornp biogenesis. in addition, in combination with recent observations of snorna involvement in human cancer, it raises the possibility that myc’s transforming effects are partially mediated by this class of non-coding transcripts. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords myctranscriptionsnornaribosomegrowthdrosophilaissue-copyright-statement© the author 2015 