The Action Mechanism of Omomyc Figure 4. Upregulation of Myc activated targets is compromised whereas downregulation of Myc repressed targets is supported by Omomyc. A) Venn diagram illustrating the overlap among genes downregulated by Omomyc in the present study, genes listed as Myc targets in the Myc target gene database (www.myccancergene.org), and genes reported to be directly bound by c-Myc in ES cells [29,38]. Within the Myc target gene database and the ES cell datasets, only genes that had a probe on the Affymetrix U34A array were taken into account. B) Overlapping, bona fide Myc target genes were extracted from our dataset and clustered in one dimension. C) Relative expression level (Fold Change) – measured by Real Time PCR at 90 min following serum stimulation in the presence or absence of 4-OHT – of Ccnd1, Eif3s9, Pgk1, Akt1, Erbb2, Gadd45a, Mxi1 mRNAs in wild type (left) and Myc (right) Rat1 cells expressing tamoxifen inducible Omomyc (Omomer). Ccnd1, Eif3s9, Pgk1 represent Myc activated targets; Akt1, Erbb2, Gadd45a, Mxi1 represent Myc repressed targets. Expression at the time of serum addition (time 0) was taken as reference for calculating the Fold Change. doi:10.1371/journal.pone.0022284.g004 human p21 promoter – p21Cip1-Luc [53] – was inhibited by c- Myc and that Omomyc was synergic with c-Myc (Figure 6A). Interestingly – even in the absence of a cotransfected c-Myc – Omomyc provoked p21 promoter repression to levels similar to the ones caused by c-Myc. Therefore Omomyc is able to support p21 promoter repression both in the presence and absence of an over- expressed c-Myc. To investigate whether this was associated to increased promoter binding, we performed ChIP assays on 293T cells transfected with the p21 reporter together with FLAG-Myc and increasing amount of Omomyc plasmid. We observed that Omomyc markedly increased c-Myc binding to the 2194 to +16 region (Figure 6B, top). Interestingly, the reciprocal assay on cells PLoS ONE | www.plosone.org 7 July 2011 | Volume 6 | Issue 7 | e22284