The Action Mechanism of Omomyc analysis of H3AcK9 and H3diMeK9 staining. Values in the graphs represent fold change in histone H3 acetylation and dimethylation, relative to parental wild-type fibroblasts. Data were collected by densitometric analysis of nuclear fluorescence from three independent biological repeats. doi:10.1371/journal.pone.0022284.g007 in its absence, and these genes were enriched in genuine, direct Myc targets. Our data also suggest that Omomyc by itself may directly contribute to downregulation of Myc repressed targets by associating with Miz-1. It is presently unclear whether the enhancement of transrepression by Omomyc may be solely explained via Miz-1, as other transcription factors as well – e.g. NFY and SP1 – have been reported to interact with Myc to promote promoter binding and transrepression [4]. Further experiments – like the design of Omomyc variants that hamper the interaction with Miz-1 – will be required to assess these points. Another contribution to the pro-repressive action is given by the decrease of activating and the increase of repressive chromatin marks – respectively H3K9 and H3diMeK9 – in the presence of Omomyc. Myc elicits changes in the histone acetylation and methylation patterns by two distinct mechanisms [54], both of which can be affected by Omomyc. Specifically, Myc was shown to recruit a number of proteins with chromatin modification activity – histone acetyltransferases and methyltransferases, a H3- specific kinase, a histone deacetylase, a histone H3-K4 dimethylase [38,59] – and to activate target genes encoding chromatin modification enzymes like the histone acetyltransferase GCN5 [54] and the Lysine-specific demethylase 5C [38]. While the upregulation of these target genes by Myc is presumably hampered by Omomyc, the direct influence of Omomyc on the Figure 8. Omomyc does not influence the pattern of histone H3 lysine 9 acetylation and methylation in Myc cells. Immunofluorescence staining (in green) of ER (top), H3acK9 (middle) and H3diMeK9 (bottom panels) in parental (w.t.), c-Myc (Myc 2/2), and c- Myc expressing Omomer (Myc 2/2 Omomer) Rat1 fibroblasts [12] grown for 48 h with or without 4-OHT. DAPI staining (blue) was used for visualizing cell nuclei. The graphs at the right side of the immunofluorescence pictures display the quantitative analysis of H3AcK9 and H3diMeK9 staining: values represent fold change in histone H3 acetylation and dimethylation, relative to parental wild-type fibroblasts. Data were collected by densitometric analysis of nuclear fluorescence from three independent biological repeats. doi:10.1371/journal.pone.0022284.g008 PLoS ONE | www.plosone.org 11 July 2011 | Volume 6 | Issue 7 | e22284