that the ability of dRYBP to counteract both transcriptional repression and activation is a result of the modulation of post-translational histones modifications. 
To investigate this possibility, we focused the analysis on the levels of H2Aub, H3K36me2 and H2Bub as these are the main targets of dKDM2, SCE and dBRE1. We also studied H3K27me3 levels to investigate the dRYBP role on PRC2-mediated recruitment in Drosophila. To test this, we analyzed histone modifications levels in S2 cells depleted of dRYBP, SCE, dKDM2, dBRE1 and PC by RNAi-mediated knockdown . In dRYBP-KD cells, the levels of H3K36me2, H3K27me3, H2Aub, H2Bub and H3K4me were examined by Western Blot analysis using specific antibodies. dRYBP depletion caused a strong decrease in H2Aub and H3K4me and slight decrease in H3K36me2, whereas H3K27me3 and H2Bub remain unchanged. Thus, dRYBP stimulates directly or indirectly H2Aub, H3K4me and H3K36me2. Next, we analyzed H3K27me3 levels in S2 cells were either dRYBP, SCE, dKDM2 and PC were inactivated. We found that in any of the analyzed cases H3K27me3 levels were affected. This results suggest that dRYBP, SCE and dKDM2, all subunits of the RYBP-PRC1 complex variant, are not required for H3K27me3 levels and therefore for PRC2 activity and/or PRC2 recruitment. Further, we analyzed H2Aub levels in S2 cells where either dRYBP and SCE or dRYBP and dKDM2 were individually or concomitantly inactivated. Reduced levels of H2Aub were observed in the single knockdowns dRYBP-KD, in SCE-KD, and in dKDM2-KD. This compares to reduction of H2Aub levels in the double knockdowns dRYBP + Mertansine SCE-KD and dRYBP + dKDM2KD . We also checked H3K36me2 levels. Depletion of dRYBP has a weak effect on H3K36me2 levels while depletion of dKDM2 results, as previously described, in increased H3K36me2 levels. Interestingly, the double knockdown dRYBP + dKDM2-KD strongly decreases H3K36me2 levels when compared to dKDM2-KD. Thus, dRYBP counteracts the repressor effect of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19689163 dKDM2 through the modulation of the H3K36me2 levels. Finally, we investigated whether PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19692133 dRYBP modulates dBRE1-dependent H2B monoubiquitylation. Depletion of dRYBP does not have an effect on H2Bub while, as previously described depletion of dBRE1 abolishes H2Bub. Curiously, the simultaneous depletion of both dRYBP and dBRE1 results in increased levels of H2Bub compared to H2Bub levels 9 / 17 dRYBP Counteracts Activation and Repression 10 / 17 dRYBP Counteracts Activation and Repression . Levels of H3K27me3 in the indicated KDs. Note that H3K27me3 levels do not change and the efficiency of the inactivation. Levels of the indicated proteins in the corresponding KDs. Efficiency of dRYBP reduction in dRYBP KD: 97%; in dRYBP + SCE KD: 98%; in dRYBP + dKDM2 KD: 100%; in dRYBP + PC KD: 100%. Efficiency of SCE reduction in SCE KD: 66%; in dRYBP + SCE KD: 62%. Efficiency of dKDM2 reduction in dKDM2 KD: 93%; in dRYBP + dKDM2 KD: 98%. Efficiency of PC reduction in PC KD: 62%; in dRYBP + PC KD: 68%. Levels of H2Aub in the indicated KDs. The intensity of the bands corresponding to H2Aub and H2A was measured and quantified using Fiji imaging software calculating the different H2Aub/H2A ratios. H2Aub levels reduction in dRYBP KD: 65%, in SCE KD: 95%, in dKDM2 KD: 56%, in PC KD: 5%, in dRYBP + SCE KD: 90%, in dRYBP + dKDM2 KD: 49%, in dRYBP + PC KD: 46%. Note that the decrease in H2Aub levels in dRYBP KD shown in and seem to be slightly different probably due to experimental conditions. Also note the efficiency of the indi