Figure 4

Distribution of male-specific lethal (MSL) proteins in the presence of Flag-tagged maleless (MLE)(ΔG) protein. Indirect immunofluorescence was performed on salivary-gland polytene chromosomes from (A, B) y w¹¹¹⁸/w; pr mle¹/pr mle¹; H83msl2/hsp83-Flag-mle(ΔG)w⁺ females and (C, D) control y w¹¹¹⁸/w; pr mle¹/pr mle¹; H83msl2/H83msl2 females. Although MSL1 was present as expected at the high-affinity sites in the control females, it seemed to be completely absent in the females expressing the Flag-mle(ΔG)w⁺ transgene. Indirect immunofluorescence was performed on S2 cells overproducing the Flag-MLE(ΔG) protein and treated with either (E, G, I) green fluorescent protein (GFP)-tagged double-stranded (ds)RNA or (F, H, L) dsRNA complementary to the region encoding the amino acids in the ΔG deletion (E, F). The presence of endogenous wild-type MLE in the GFP dsRNA-treated cells resulted in the presence of MSL complexes in the X chromosome nuclear compartment, while the Flag-MLE(ΔG) deficient in the nuclear localization sequence remained in the cytoplasm. In the absence of wild-type MLE, a substantial amount of (F) MSL1, (H) MSL2 and MSL3 and (L) MOF (males absent on the first) remained in the cytoplasm. In these cells, MSL1, MSL2 and MOF were present in the nuclei, but were not confined to the X-chromosome compartment. For the wild-type distribution of H4K16ac please see Figure 2H.