In addition, in the double RNAi ovary with the transgene, egg chambers in 25% of the ovarioles were able to develop beyond stage 9 with morphologically normal nurse cells (Fig. regulation through modulating PCNA levels on chromatin. partially rescued the defective nurse cell endoreplication observed in the Elg1-depleted germline. Therefore, our results suggest that Enok may down-regulate PCNA unloading from DNA by interacting with the Elg1 complex and may promote the G1/S transition of the cell cycle. Results Enok activity in vivo requires Br140, Eaf6, and Ing5 While the composition of complexes formed by the human and yeast KAT6 has been characterized (Doyon et al. 2006; Taverna et al. 2006; Gilbert et al. 2014), Rabbit Polyclonal to CCRL1 information regarding the Enok complex is usually lacking. We sought to identify core components of the Enok complex and assess their roles in mediating the HAT function of this complex. To this end, the Enok complex was isolated using Flag affinity purification from S2 cell nuclear extracts (NEs) with Flag-tagged Enok as the bait protein, and the composition of purified complex was determined by multidimensional protein identification technology (MudPIT) (Florens and Washburn 2006). Peptides from the homologs of three subunits in the human MOZ/MORF complexes were identified: Br140, Eaf6, and CG9293 (Fig. 1A). Furthermore, MudPIT analysis of Flag affinity-purified complexes using Br140, Eaf6, or CG9293 as the bait protein consistently identified peptides from Enok, Br140, Eaf6, and CG9293 (Fig. 1A). These results indicate that this Enok complex is composed of these four proteins and is homologous to the human MOZ/MORF complex. Based on the conserved composition of the Enok complex and the specific sequence similarity VU6001376 between CG9293 and human ING5, CG9293 is usually referred to here as Ing5. Open in a separate window Physique 1. Enok forms a quartet complex homologous to the human MOZ complex. (panel), acid extraction of histones (four VU6001376 panels), and nuclear extraction (two panels) followed by Western blotting. (panel) Four percent of NEs from S2 cells treated with LacZ dsRNA (control) or dsRNA against or were used as input. Rabbit VU6001376 -Enok serum and protein A-conjugated resin were used to immunoprecipitate endogenous Enok, and the corresponding preimmune serum was used as a control. Input and 30% of immunoprecipitates were subjected to Western blot analysis using guinea pig -Enok and -Elg1 antibodies. (panel) Four percent of the NE from S2 cells were used as input. Rabbit -Elg1 serum and protein A-conjugated resin were used to immunoprecipitate endogenous Elg1, and the corresponding preimmune serum was used as a control. Input and 50% of immunoprecipitates were subjected to Western blotting using guinea pig -Enok and -Elg1 antibodies. VU6001376 (panel) Of the whole-cell lysates from Sf9 cells expressing the indicated recombinant proteins, 3.75% (-Flag) and 1.25% (-HA) were used as input. Anti-HA antibody-conjugated resin was used to pull down HA-tagged Elg1. Input and 85% (-Flag)/15% (-HA) of pull-down were subjected to Western blot analysis. (panel) Five percent (-Flag) and 1% (-HA) of whole-cell lysates from Sf9 cells expressing the indicated recombinant proteins were used as input. Anti-Flag antibody-conjugated resin was used to pull down Flag-His-Enok or Flag-His-Br140. Input and 40% (-Flag)/50% (-HA) of pull-down were subjected to Western blot analysis. (panel) A schematic representation of the interaction between the Enok and Elg1 complexes. To confirm the in vivo conversation between Enok and Elg1, coimmunoprecipitation (co-IP) was performed in S2 cells using Enok- or Elg1-specific antibodies (Supplemental Fig. S1; Huang et al. 2014). Consistent.
