Figure 4: UNR facilitates the interaction of MLE with roX2 in vivo.

(a) Nuclear extracts from S2 cells depleted of UNR (+, RNA interference (RNAi)) or GFP (−, Ctrl) were used to immunoprecipitate MLE (IP), carrying empty beads as control (mock). The amount of roX2 associated to MLE was determined with RT–qPCR, and values were normalized to MLE binding in control RNAi cells (right panel). Error bars represent the s.d. of six experiments. The efficiency of UNR immunodepletion and MLE immunoprecipitation was assessed using western blot analysis (left panels), and the levels of input roX2 were monitored with RT–qPCR (middle panel). Full-size blots corresponding to this Figure are shown in Supplementary Fig. 6. (b) Left panel, UNR promotes MLE binding to HAS. Binding of MLE to Set2, CG11943, roX1 and CES11D1 HAS was measured using ChIP in control (GFP) or UNR-depleted cells. Amplification of nearby regions within the same genes was carried out as internal control and used for correction. Values represent the ChIP/input ratio normalized to the GFP control. The binding of MLE to promoters was assessed as negative control. The average of at least four independent experiments is shown. Right panel, UNR does not affect the genomic localization of MOF. Binding of MOF to Set2, CG11943 and CG5885 HAS and/or promoters in control (GFP) or UNR-depleted cells, measured as described above. The average of at least three independent experiments is shown. Asterisks indicate level of statistical significance using the Student’s t-test (*P<0.05; **P<0.005; ***P<0.001).