dMec is a nucleosome-stimulated ATPase. (A) Reconstitution of recombinant dMec. Extracts derived from baculovirus infected Sf9 cells expressing FLAG-tagged dMEP-1 (dMEP-1F) and untagged dMi-2 were immunopurified with anti-FLAG antibodies and eluted with FLAG peptide. The first two eluates (E1, E2) were subjected to SDS–PAGE and Coomassie staining. The position of dMi-2 and dMEP-1F are indicated on the left, and molecular weight markers are indicated on the right. (B) dMec has nucleosome-stimulated ATPase activity in vitro. dMec was incubated in the absence or presence of 100 ng DNA or nucleosomes (Nucl.) and ³²P-ATP as indicated. Percentage of hydrolysed ATP was determined by thin layer chromatography and quantified by phosphoimager analysis. Three independent experiments were performed using two different protein preparations; standard deviations are indicated by error bars. (C) Schematic representation of dMEP-1 mutants used (top panel). Zinc-finger domains are indicated by black bars, and a C-terminal FLAG-tag is indicated by a grey bar. Extracts derived from Sf9 cells expressing untagged dMi-2 and a FLAG-tagged dMEP-1 mutant (ΔC-dMEP-1, ΔN-dMEP-1 and ΔNΔZn-dMEP-1), as indicated on the top, were immunoprecipitated with anti-FLAG antibodies and analysed by western blot using anti-dMi-2 and anti-FLAG mouse antibodies, as indicated on the right. The calculated molecular mass of each mutant is given in brackets. Molecular weight markers are shown on the left. IN, input; IP, immunoprecipitate.

dMec expression in ovaries. (A) Proteins extracts were immunoprecipitated with rat anti-dMEP-1 antibody and immunoprecipitates were subjected to western blotting using rabbit anti-dMEP-1 antibody. (B) Confocal micrographs of immunofluorescence-stained Drosophila ovaries. Ovaries were stained with DAPI (left panel), anti-dMi-2 antibody (second panel, red) and anti-dMEP-1 antibody (third panel, green). The fourth panel shows an overlay of dMi-2 and dMEP-1 staining. The germinal vesicle of the oocyte is indicated by an arrow. Scale bar: 50 μm. (C) Protein extracts from Drosophila ovaries were immunoprecipitated with anti-dMEP-1 antibody and subjected to western blot analysis using anti-dMi-2 (top panel) and anti-dMEP-1 (bottom panel) antibodies. Beads, antibody was omitted from precipitation.

dMec contributes to the repression of AS-C genes. (A) Top panel: schematic representation of the AS-C locus. Position of amplicons is denoted on top, genes are indicated by black boxes, the direction of transcription is shown with arrows (modified from Badenhorst et al, 2002). Middle panels: Drosophila embryos were subjected to ChIP analysis using anti-dMi-2-specific, anti-dMEP-1-specific, anti-dp66-specific and anti-GST-specific (NS Ab, used as control antibody) antibodies, as shown on top. Specific primers were used to amplify sequences derived from the AS-C locus and the hsp70 gene by end-point PCR, as indicated on the right. Bottom panels: Chromatin immunoprecipitated DNA was analysed in triplicate by qPCR with primers for promoter sequences of genes from the AS-C locus (from left to right: ac, sc, l'sc, pcl, ase) and an indicated control region (sc-p2). Mean values are expressed as percentage of input (% input). (B) Extracts from SL2 cells treated with different double-stranded RNAs (dsRNA), as indicated on top, were analysed by western blot using specific antibodies, as indicated on the left. Mock: untreated cells.

Purification of dMec. (A) Schematic representation of dMEP-1-containing complex (dMec) purification procedure. (B) The Resource Q dMi-2 peak fraction was immunopurified using anti-dMEP-1 and anti-dMi-2 antibodies, respectively, as indicated. Bound material was eluted with appropriate peptides and subjected to SDS–PAGE and colloidal Coomassie staining. Polypeptides identified by peptide mass fingerprinting are marked with solid black circles. Substoichiometric proteins were identified as degradation products of dMi-2 or dMEP-1 (Supplementary Table 1). SDS–PAGE size markers are shown on the right. dMEP-1 complex (dMec) subunits are indicated by arrows on the left. MW, molecular weight markers; IN, input. (C) Crude nuclear extracts of Kc cells (upper panel) and the Resource Q dMi-2 peak containing fraction (lower panel) were subjected to Superose 6 gel filtration. Fractions were analysed by western blot using specific antibodies as indicated on the right. Fraction numbers are denoted on top, size standards above. IN, input.

dMec expression in embryos. (A) Protein extracts were prepared from embryos at different developmental stages as indicated (lanes 1–4) and analysed by western blotting using anti-dMEP-1 (top panels), anti-dMi-2 (middle panels) and anti-tubulin (bottom panels) antibodies. Molecular weights are indicated on the left. (B) Confocal micrographs of immunofluorescence-stained embryos. Top panel: early embryo; second to fourth panels: syncytial blastoderm stage embryos; third panel: interphase nuclei; bottom panel: metaphase nuclei. Embryos were stained with DAPI (first column) and anti-dMEP-1 antibody (second column, green). The third column shows overlays of DAPI and dMEP-1 stainings. Pole cells are indicated by arrows. Scale bars in the two top panels: 100 μm; scale bars in the two bottom panels: 10 μm. (C) Confocal micrographs of immunofluorescence-stained syncytial blastoderm embryos. Embryos were stained with DAPI (first column), anti-dMi-2 antibody (second column, red) and anti-dMEP-1 antibody (third column, green). The fourth column shows an overlay of dMi-2 and dMEP-1 signals. The second panel shows an enlargement of the micrographs in the first panel. Scale bars: 5 μm. (D) Protein extracts were prepared from embryos at the developmental stages indicated and analysed by western blotting after immunoprecipitation with the indicated antibodies. Beads, antibody was omitted from precipitation; AED, after egg deposition.

dMec is the major dMi-2-containing complex in Drosophila. (A) Kc cell nuclear extract was fractionated as shown in Figure 1A, and Resource Q column eluates were analysed by western blot using specific antibodies, as indicated on the right. Fraction numbers are shown on top. IN, input. (B) Serial immunodepletion experiments with anti-dMi-2 antibody (α-dMi-2 IP) or anti-dMEP-1 antibody (α-dMEP-1 IP) from Drosophila embryo nuclear extract. Supernatants from subsequent immunodepletions (s1, s2, s3) were subjected to western blotting using antibodies, as shown on the left. (C) Kc cell nuclear extract was immunoprecipitated with anti-dp66, -dRPD3, -dMi-2 and -dMEP-1 antibodies, as indicated on top. Immunoprecipitates were analysed by western blotting using antibodies indicated on the left. IP, immunoprecipitate; Beads, antibody was omitted from the precipitation.

(C) Total RNA isolated from SL2 cells shown in (B) (as indicated on the left) was analysed by qRT–PCR for expression levels of genes from the AS-C locus (from left to right: ac, sc, l'sc, pcl, ase). RNA levels determined for untreated samples (Mock) were set to 1. (D) Total RNA isolated from SL2 cells treated for 18 h with 1 μM TSA (dissolved in DMSO) or control cells treated with DMSO, as indicated at the bottom, was analysed by qRT–PCR for expression levels of genes from the AS-C locus (from left to right: l'sc, pcl) and of the CG8399 gene as a positive control for the effectiveness of the TSA treatment. RNA levels determined for DMSO treated samples (DMSO) were set to 1.