6434 Nucleic Acids Research, 2011, Vol. 39, No. 15 Figure 4. In polytene chromosomes, msl2 RNA is not associated with the chromatin bound MSL complex. (A) In situ hybridization shows that roX1 decorates the male X chromosome (top row of images). No msl2 RNA associated with the wild-type male X chromosome is detected (bottom row of images). (B) In roX1 roX2 mutant males MSL2 and MSL3 target the chromocenter and three speciﬁc sites on the fourth chromosome. Merged images of immunostainings showing DAPI in blue, MSL3 in green and MSL2 in red. The chromocenter is indicated by an arrow and the speciﬁc sites on the fourth chromosome with arrowheads (Supplementary Figure S6). (C) Merged images from in situ hybridization with msl2 RNA antisense probe on males overexpressing msl2 (left panel), roX1 roX2 mutant males (middle) and roX1 roX2 mutant males overexpressing msl2 (right) (Supplementary Figure S8). chromocenter and at a few speciﬁc sites on the fourth chromosome (Figure 4B and Supplementary Figure S6) (24,37). Notably, the speciﬁc sites on the fourth chromo- some targeted by the MSL complex in roX1 roX2 mutants are also targeted weakly by the MLE protein alone in wild-type, both in males and females (Supplementary Figure S7). We hybridized roX1 roX2 mutant male larvae with and without overexpression of msl2 and wild-type males with overexpression of msl2. In none of these cases did we detect msl2 RNA linked to the X chromosome, in the chromocenter, or the highly speciﬁc sites on the fourth chromosome (Figure 4C and Supplementary Figure S8). In the RIP experiments performed on S2 cells a strong enrichment of msl2 RNA in the MSL complex was detected. To test if msl2 is included in an X chromosome-associated MSL complex in S2 cells, we per- formed in situ hybridizations on these cells (Figure 5).