independent studies through a variety of techniques [35]. 115 (24%) of the 475 genes downregulated in the presence of Omomyc were listed in the Myc target gene database: 45 were listed as upregulated and 35 as downregulated by Myc, while the up or down regulation of the remaining 35 genes was unknown. To point out direct Myc targets within the list of genes downregulated in the presence of Omomyc, the list was crossed with those from two studies that used ChIP analysis to define genome wide promoter occupancy by Myc in embryonic stem (ES) cells [36,37]. 31.5% of the genes downregulated in the presence of Omomyc had promoters that were reported to be directly bound by Myc in ES cells (P-value: 1.3610224) as compared to 19% in its absence (P-value: 361022). Altogether, 44.4% of the Omomyc downreg- ulated genes turned out to be genuine Myc targets according to either the Myc target genes database or the listing of Myc bound promoters in ES cells (Venn diagram: Figure 4A). Genes common to all three groups are clustered in one dimension in Figure 4B. Such a large – albeit incomplete – overlap among genes whose downregulation was associated to Omomer activation in Rat1 fibroblasts and validated Myc targets is highly significant. That the overlap is incomplete is consistent with other analyses of expression and computationally derived gene sets [38,39], and it might be due in part to other effects of serum. Figure 3 (middle) shows the classification in functional categories based on GO (Gene Ontology) terms. As reported for genes associated with c- Myc activity [36,40,41], genes affected by Omomyc fall into multiple functional classes involving growth, metabolism, cell signaling pathways, cell cycle progression, and apoptosis. Notably, genes downregulated in the presence of Omomyc were enriched in categories – nucleotide and nucleic acid metabolism – that are overrepresented among targets upregulated by c-Myc [36,40–42], supporting the notion of Omomyc as an inhibitor of Myc mediated transactivation (Figure 3, bottom). Targets known to be upregulated by Myc and found to be downregulated by Omomyc include genes encoding proteins directly involved in translation and ribosome assembly – such as the translation initiation factor Eif3s9, the translation elongation factor Eef1a1, and the ribosomal protein L3 and S4 – as well as genes encoding metabolic enzymes – isocitrate dehydrogenase 2, lactate dehydro- genase B and phosphoglycerate kinase 1 –, proteins involved in cell cycle progression – the anaphase-promoting complex subunit Anapc5, Cyclin B1 and Cyclin D1 –, the ATPase/DNA helicase TIP49 (Ruvbl1), and the structural chromatin protein Hmgb1. Hmgb1 has an important role in chromatin remodeling and is a mediator of inflammation whose overexpression is associated with many tumor types [43]. TIP49 is an important Myc cofactor, involved in Myc transcriptional and oncogenic functions [44]. Several genes found to be downregulated by Omomyc and known to be Myc repressed targets encode proteins involved in signaling pathways, cell adhesion and transcription – such as Akt1, Erbb2, c-Jun and the fibroblast growth factor receptor Fgfr1 – while Mxi1 encodes a protein that interacts with Max and negatively regulates Myc function. Mxi1 is repressed by Myc [45] but activated by Hif- 1a [46,47]. Defects in this gene have been reported in prostate cancer and in a subset of glioblastomas [48,49]. To further validate our findings, we performed Real Time PCR assays in wild type and Myc Rat1 fibroblasts expressing tamoxifen inducible Omomyc (Figure 4C). We compared – at 0 and 90 min of serum stimulation – the expression levels of a select sample of genes affected by serum stimulation that are known to be either repressed – Gadd45a, Mxi1, Erbb2, Akt1 – or activated – Pgk1, Eif3s9, Ccnd1 – by Myc [40,45,50]. These genes, like any gene, are not exclusive Myc targets, and other transcription factors modulated or not by serum may contribute to their regulation. We The Action Mechanism of Omomyc found that the expression levels of the repressed targets Gadd45a, Mxi1, Erbb2 and Akt1 in wild type Rat1 cells were unaffected or even more repressed in the presence of Omomyc whereas upregulation of the activated targets Pgk1, Eif3s9 and Ccnd1 was compromised (Figure 4C, left). In the Myc cells, we found that Omomyc did not significantly affect expression of the repressed and did not impair upregulation of the activated targets (Figure 4C, right). Therefore the effect of Omomyc on transcription of Myc activated targets appears to require some activity of Myc. In sum, these findings indicate that Omomyc does not globally inhibit Myc function in transcriptional regulation of target genes and suggest that it selectively perturbs the Myc transcriptome by hampering Myc mediated transactivation and preserving Myc mediated repression. Omomyc differentially affects transactivation and repression by influencing Myc binding to target gene promoters To investigate the mechanisms involved in gene regulation by Omomyc, we performed luciferase reporter and chromatin immunoprecipitation (ChIP) assays on two genes encoding the nucleolar protein nucleolin and the cyclin-dependent kinase inhibitor p21, respectively representing genuine and direct targets of Myc mediated activation and repression [51,52]. c-Myc is able to activate nucleolin transcription via two highly conserved E-boxes in intron 1 [51]. To test the capability of Omomyc to inhibit Myc mediated transcriptional activation, we performed reporter assays in 293T cells transfected with the luciferase reporter plasmid pNucL14 [51] – containing the mouse nucleolin promoter, exon 1, intron 1 and the first 8 nt of exon 2 – together with different combinations of FLAG-c-Myc and Omomyc expression plasmids (Figure 5A). We found that Omomyc inhibited Myc mediated activation of the luciferase reporter in a dose dependent manner, while it did not affect its basal activity. To test whether the inhibition of nucleolin transactivation by Omomyc was due to a reduction of promoter binding, we measured the amount of Myc bound to the nucleolin promoter on 293T cells transfected with nucleolin reporter, FLAG-c-Myc and Omomyc expressing plasmids. Recovery of nucleolin DNA coprecipitated with Myc was quantified by real-time PCR, using primers located around the two E-boxes in nucleolin intron 1. We found that Omomyc caused a 40% reduction of the amount of promoter bound Myc (Figure 5B, left). Besides competing for Myc/Max association, Omomyc is able to form homodimers as well as Omomyc/Max dimers: only the latter bind E-boxes in vitro with high efficiency [9]. Therefore, Omomyc might affect Myc binding to DNA via two concurring mecha- nisms: inhibition of Myc/Max dimerization as well as direct competition for E-box binding. To assess the latter one, we measured the amount of Omomyc bound to the nucleolin promoter in cells transfected with c-Myc and FLAG-Omomyc expressing plasmids (Figure 5B, right). We found that Omomyc was specifically recruited to the E-box containing region in intron 1 and that it competed with c-Myc for binding to this region. Altogether, these data indicate that Omomyc can affect transacti- vation by sequestering Myc in complexes incapable of binding to E-boxes as well as by acting – presumably in association with Max – as competitive inhibitor of Myc/Max complexes for E-Box binding. To investigate how Omomyc may act to support Myc mediated repression, we conducted assays on the human p21 promoter – specifically the sequence comprised between 2194 and +16 from the transcriptional start site – on which Myc is recruited by the zinc finger protein Miz-1 [27,52]. We performed reporter assays and found that luciferase expression driven by the full-length PLoS ONE | www.plosone.org 6 July 2011 | Volume 6 | Issue 7 | e22284