Nucleic Acids Research, 2011, Vol. 39, No. 15 6435 Figure 5. In S2 cells, msl2 RNA is not associated with the chromatin bound MSL complex. Immunostaining and in situ hybridization show that roX2 (yellow) decorates the X chromosome identiﬁed by MSL1 (green). Note that roX1 decorates the X chromosome in a small fraction of cells (<0.5%). Nuclei are visualized with DAPI. Neither the msl2 RNA nor the negative control CkII RNA are enriched on the X chromosome. In each row, the location of one X chromosome in a representative cell is indicated with an arrow. As expected, roX2 RNAs co-localize with the MSL1 protein on the X chromosome. In agreement with the results from salivary glands we did not ﬁnd any enrich- ment of msl2 RNA on the X chromosome in S2 cells, indicating that the msl2 RNA associates to a non- chromatin bound MSL complex. Strong roX2 hybridiza- tion was observed on the X chromosome territories in all cells. Interestingly, roX1 hybridization was as strong, but only detected in a small fraction of cells (<0.5%). This suggests that the observed low expression of roX1 in S2 cells is due to expression in few individual cells and not a general low expression level in all cells. Next, we performed a crude fractionation of nuclei into a chromatin fraction and a nucleoplasmic fraction and determined the relative levels of roX1, roX2 and msl2 by qPCR. The results show that the majority of both roX1 and roX2 RNAs are located in the chromatin fraction while the msl2 transcripts are primarily found in the nu- cleoplasm (Figure 6A–C). MSL2-RIP experiments using the nucleoplasmic fractions show very strong enrichments of roX1, roX2 and msl2 RNA, verifying the nuclear RIP-chip data. Gel electrophoresis of the RIP experiment qPCR products shows that the MSL complex-associated msl2 RNAs are spliced. The results also show that the msl2 RNA associated to the MSL complex is poly-adenylated, since msl2 RNA is enriched to the same extent when using poly-adenylated RNA (Figure 6C) as when using total RNA (Figure 6A–B) in the analysis. To exclude that the enrichment of msl2 RNA in the nucleo- plasm is caused by contaminating cytoplasm, we per- formed MSL2-RIP also on corresponding cytoplasmic fractions. Weak enrichments of roX and msl2 RNAs were detected in the cytoplasm but much lower than the enrichments found in corresponding nucleoplasm (Supplementary Figure S9). We conclude that a spliced poly-adenylated msl2 RNA associates with a nucleoplasmic complete or partial MSL complex. DISCUSSION For many chromatin-associated factors, the relative con- centrations direct their binding. Thus, correct targeting will strictly depend on the concentration of these protein