lethal for females and toxic to males in combination with the overexpression of msl1, again highlighting the require- ment for correct concentrations of the MSL complex (12,19,21,22). Furthermore, association studies suggest that MSL2 levels are tuned to match those of the other MSL-complex components (23). Although the roX genes are important for correct tar- geting of the MSL complex, in roX1 roX2 double mutants escaping males are recovered (22,24,25). This contrasts with the absence of escaper males when the msl genes are mutated. The partial viability of roX1 roX2 double mutants could be explained by the presence of additional roX RNA species (26,27), even though unsaturated screens have failed to isolate such species (27,28). It has also been hypothesized that MSL complex may bind to additional RNAs produced by cryptic transcription as a mediator of spreading (26). Here we have used RNA-immunoprecipitation (RIP) followed by tiling array analysis to determine the associ- ation between the MSL complex and RNA moieties. We found no additional ncRNA associated to the complex. However, we observed a strong association between the msl2 mRNA and a non-chromatin-associated MSL complex. These data suggest a novel mechanism in chro- matin structure regulation in which a non-chromatin- associated form of a chromatin regulatory complex attracts an intrinsic rate-limiting mRNA and thus regu- lates the amounts of the complex available for chromatin targeting by feedback. MATERIALS AND METHODS Immunostaining and in situ hybridization of polytene chromosomes Polytene chromosomes from the salivary glands of third-instar larvae were prepared and stained essentially as previously described (29). Salivary glands were ﬁxed in 2% formaldehyde in PBS, 0.1% Triton X-100, 0.2% NP-40 for 40 s followed by 2–3 min in 50% acetic acid, 1% formaldehyde. Polytene chromosomes were squashed as previously described (30). The slides were washed for 30 min in 1 PBS, 0.1% Triton X-100, transferred to blocking solution (0.1 M maleic acid, 0.15 M NaCl, 1% Boehringer blocking reagent) and incubated for 30 min at room temperature. The slides were then incubated over- night at 4 C with primary antibody raised against MSL2 (1:300), MSL3 (1:2000) or MLE (1:2000). The slides were washed 2 10 min in 0.1 M maleic acid, 0.15 M NaCl, 0.3% Tween-20 and blocked for 30 min. Donkey anti-rabbit and anti-goat (Jackson Laboratories) conjugated with Cy3 or FITC (diluted 1:400) were used as secondary antibodies and incubated at room tempera- ture for 2 h. The squashes were counterstained with DAPI (1 mg/ml) and washed 2 10 min before mounting with Vectashield (Vector). Preparations were analyzed using a Zeiss Axiophot microscope equipped with a KAPPA DX20C charge-coupled device camera. Images were assembled and merged electronically using Adobe Photoshop. For in situ hybridization, the slides were prepared as above and hybridized and washed essentially Nucleic Acids Research, 2011, Vol. 39, No. 15 6429 as previously described (31). The slides were hybridized against a DIG-labeled antisense RNA probe against roX1 (GH10432) or msl2 (GH22488). The DIG-labeled probe was detected using sheep anti-Digoxigenin (0.4 mg/ml, Roche) followed by donkey–anti-sheep conjug- ated with AlexaFluor555 (Molecular Probes, diluted 1:500) and DAPI counterstaining. roX1 roX2 double mutant males were selected as non-GFP males from a yw roX1ex6 Df(1)roX252 P[w+4D4.3]/ FM7i,P[w+mC ActGFP]JMR3 stock. Females and males overexpressing msl2 were selected from a w; P[w+ hsp83:msl2] msl3/TM6B stock. Immunostaining and in situ hybridization of S2-cells Schneider’s line 2 cells (ATCC CRL-1963) were grown as a suspension culture at 25 C in Erlenmeyer ﬂasks to a density of 1 107 cells/ml in Drosophila SFM medium (Invitrogen), supplemented with 100 U/ml Penicillin G, 100 mg/ml Streptomycin sulfate and 2 mM of L-glutamine. 0.2 107 cells were placed on Polysine slides (VWR) and incubated at room temperature for 1 h. The cells were ﬁxed for 10 min with 2% formaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. The slides were washed twice in 70% ethanol before dehydration at 80, 95 and 99.5% ethanol and then air dried. The slides were hybridized and washed as described above using DIG-labeled antisense RNA probes against roX1 (GH10432), roX2 (GH18991), msl2 (GH22488) or CkII cDNA and immunostained in parallel with anti-MSL1 (1:500). RIP-chip For each sample treatment condition, we used 300 ml cultured D. melanogaster Schneider’s line 2 cells. The cells were pelleted, washed twice in 100 ml 10 mM HEPES pH 7.4, 140 mM NaCl and resuspended in 50 ml of lysis buffer (20 mM HEPES pH 7.4, 3 mM MgCl2, 0.1% Triton X-100, 1 mM dithiothreitol, 0.5 mM PMSF, 10 U/ml RNasin and 0.5 complete protease inhibitor cocktail, Roche). The cells were allowed to swell on ice for 10 min and then homogenized on ice with 30 strokes of a Dounce homogenizer. The nuclei were pelleted at 2 000g for 5 min and used either as native samples (non-cross- linked) or after formaldehyde cross-linking. For the cross-linked sample (FA), the pelleted nuclei were resus- pended in 50 ml lysis buffer and cross-linked by adding formaldehyde to a ﬁnal concentration of 0.5% and incubated for 10 min at room temperature. The cross- linking was stopped by adding glycine (ﬁnal concentration 0.125 M), the nuclei were washed once in lysis buffer and resuspended in 2 ml of sonication buffer (20 mM HEPES pH 7.4, 10% glycerol, 0.1 M NaCl, 1 mM MgCl2, 0.1% Triton X-100, 1 mM dithiothreitol, 0.5 mM PMSF, 10 U/ml RNasin and 0.5 Protease inhibitor cocktail) and sonicated using a Bioruptor (Diagenode) for 4 min at high setting (30 s ON, 30 s OFF). For the native samples the pelleted nuclei were resuspended in 2 ml son- ication buffer and sonicated for 1, 3 or 6 min (N1, N3 and N6, respectively). Cellular debris was removed by