The Action Mechanism of Omomyc Figure 5. Omomyc inhibits Myc binding and transactivation of the nucleolin promoter. A) Luciferase activity of the mouse nucleolin promoter reporter plasmid – pNucL14 – transfected in 293T cells together with FLAG-c-Myc and Omomyc expressing vectors. Data were normalized by cotransfection of pRL-TK Renilla luciferase. The basal activity of the reporter was set to a value of 100. B) Quantitative ChIP assay of Myc and Omomyc binding to the nucleolin promoter region (Ncl; grey bars). Left: FLAG-c-Myc binding in 293T cells transfected with the nucleolin reporter pNucL14 together with FLAG-c-Myc and Omomyc expressing plasmids. Right: FLAG-Omomyc binding in 293T cells transfected with the nucleolin reporter together with FLAG-Omomyc and c-Myc expressing plasmids. A region of the luciferase coding sequence (Luc; black bars) was used as control. Bars represent the percentage of input DNA immunoprecipitated, after background subtraction. ChIP values are expressed as % of input DNA. doi:10.1371/journal.pone.0022284.g005 transfected with FLAG-Omomyc and increasing amount of c-Myc plasmid indicated that Omomyc was capable of interacting with the same p21 promoter region (Figure 6B, bottom). In this case, Myc overexpression did not compete with Omomyc binding. These data support the hypothesis that transrepression of Myc targets is sustained by Omomyc. Epigenetic modifications associated to Myc activity are affected by Omomyc Myc proteins are involved in the widespread maintenance of active chromatin. Disruption or downregulation of Myc expression leads to decreased H3 and H4 acetylation at selected histone residues accompanied by increased chromatin repressive marks, an effect that is reversed upon Myc reactivation [3,38,54,55]. If Omomyc indeed targets Myc function, its overexpression should entail changes in the histone modifications associated with Myc activity. To test this hypothesis, we performed immunohistochem- istry assays to detect global levels of histone H3 lysine 9 acetylation and dimethylation – representing marks of active and repressive chromatin – reported to be respectively decreased and increased upon Myc loss of function in neuronal progenitors and other cell types [54,56]. N-Myc genomic binding is strongly linked to H3AcK9 while loss of N-Myc decreases the pool of this active chromatin mark [54]; 90% to 95% of the H3AcK9 mark in a human neuroblastoma cell line was reported to be N-Myc dependent [55]. We assessed levels of H3acK9 and H3diMeK9 in parental Rat1 cells (w. t.), in Rat1 cells harboring an inducible Omomyc (Omomer) and in Rat1 cells harboring the inducible Omomyc and an overexpressed c-Myc (Myc+Omomer) (Figure 7). For a comparison, H3K9 acetylation and dimethylation was also measured in Myc Rat1 cells (Myc 2/2) and Myc cells harboring the tamoxifen inducible Omomyc (Myc 2/2 Omomer) (Figure 8). We found that wild type Rat1 cells displayed significant staining of H3acK9 and faint staining of H3diMeK9. Treatment with tamoxifen triggered Omomer translocation to the nucleus, as expected (Figures 7, top panels), which was accompanied by a dramatic reduction of the H3acK9 signal, not affected by a concomitant Myc overexpression (Figure 7, middle panels). Omomyc caused an approximately four-fold reduction in H3acK9 according to the densitometric analysis (Figure 7). Myc Rat1 cells displayed the opposite pattern to parental cells with significant H3diMeK9 staining and a quasi-complete loss of H3acK9 staining, similarly to what was reported for N-Myc neuroprogenitor cells [54]. This pattern was unaffected upon Omomer induction (Figure 8). Of note, H3acK9 levels in Omomyc overexpressing cells were even lower than observed in Myc cells, possibly due to compensatory events that occurred in the latter. The H3K9 hypoacetylation observed upon Omomer induction in the Omomer and Myc+Omomer expressing Rat1 cells was associated with a two-fold enhancement in dimethylation of the same histone residue (Figure 7, bottom panels). In Myc fibroblasts, instead, Omomer induction did not affect the levels of H3K9 dimethylation (Figure 8, bottom panels). The data demonstrate that Omomyc impacts histone H3K9 acetylation and methylation in an opposite way to Myc, leading to decreased active and increased repressive chromatin marks. Omomyc affects proliferation and survival of cells in culture Omomyc was shown to strongly potentiate Myc induced apoptosis in murine myoblasts [10], and to slow growth and trigger death of the tumor cells while sparing the surrounding normal tissue in a lung adenocarcinoma model [16]. To further assess its effects on cell growth and death, we determined PLoS ONE | www.plosone.org 8 July 2011 | Volume 6 | Issue 7 | e22284