Western blot analysis for phosphorylation of ERK

lead us to propose the existence of a dRB complex consisting of dBRE1 and dRYBP. The dRRK and dRB complexes may have distinct effects on gene expression. dRYBP genetically interacts with Sce, dkdm2 and dBre1 We hypothesized that dRYBP genetically interacts with Sce and dkdm2 to control gene silencing and with dBre1 to control gene activation. To test this, we analyzed the PcG genes inactivation related homeotic phenotypes such as: the transformation of meta- and meso-thoracic legs into pro-thoracic legs, the transformation of wings into halteres and the transformation of the forth abdominal segment towards the fifth one in males. Furthermore, we analyzed the trxG genes inactivation related homeotic phenotype such as the transformation of segment A5 towards segment A4. We first determined the penetrance of these homeotic phenotypes in dRYBP and dkdm2 double mutant flies. Neither loss of dRYBP function nor loss of dkdm2 function or the double mutant combination show homeotic phenotypes. Therefore, we investigated the interaction phenotypes in flies with a sensitized mutant genetic background i.e. in Polycomb heterozygous mutant flies and in trithorax heterozygous mutant flies flies. The use of these sensitized mutant 6 / 17 dRYBP Counteracts Activation and Repression 7 / 17 dRYBP Counteracts Activation and Repression genetic backgrounds has been previously probed very useful to detect interactions between PcG and trxG genes. Polycomb heterozygous mutant flies present Vorapaxar PcG-related mutant homeotic phenotypes. Flies dkdm2KG04325/Pc3 show an increase in the penetrance of these phenotypes, suggesting that dkdm2 is an enhancer of Pc. When dRYBP and dkdm2 are concomitantly inactivated the penetrance of the phenotypes was significantly reduced suggesting that dRYBP is a suppressor of dkdm2 repressor effect. Moreover, trithorax heterozygous mutant flies present the A5 to A4 transformation with 33% penetrance and this frequency is highly increased PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19689163 in the absence of dRYBP function indicating that dRYBP is an enhancer of trx. Conversely, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19692133 the frequency of the A5 to A4 phenotype of trxE2/+ is decreased in the absence of dkdm2 function indicating that dkdm2 is a suppressor of trx. When, dRYBP and dkdm2 are simultaneously inactivated, the trx enhancer effect of dRYBP decreases and the trx suppressor effect of dkdm2 decreases. Thus, these results indicate that dRYBP and dkdm2 antagonize each other activities. Next, we analyzed the genetic interactions with Sce. The heterozygous Sce1/+ mutant flies show neither PcG- nor trxG-related homeotic phenotypes even when dkdm2 expression is reduced. However, the simultaneous inactivation of dRYBP and Sce produces both PcG- and trxG-related homeotic phenotypes suggesting that dRYBP and Sce are enhancers of Pc and, interestingly that dRYBP and Sce are enhancers of trx. Notably, the frequency of both of these phenotypes is significantly decreased when the levels of dkdm2 expression are reduced. Therefore, dkdm2 is a suppressor of the dRYBP and Sce repressor effects and of the dRYBP and Sce enhancer effects. Finally, we studied the genetic interaction between dRYBP and dBre1. As indicated in 8 / 17 dRYBP Counteracts Activation and Repression and indicate that dRYBP associates with dBRE1 protein to counteract and alleviate the dBRE1-mediated activation. dRYBP modulates levels of H2Aub, H2Bub and H3K36me2 Our biochemical and functional results showing interactions of dRYBP with dkdm2, Sce and dBre1 raised the possibility