Figure 3. MSL complex is strongly associated with nuclear msl2 mRNA. (A) High resolution enrichment proﬁling along chromosome 2 L shows that msl2 is enriched in the MSL2-RIP at similar ratios to roX1 and roX2. The high peaks within the yellow boxes correspond to msl2 (left box) and msl1 (right box). (B) Average calculated enrichment levels of genes (x-axis) plotted against the average amount of nuclear transcript (y-axis, log2 scale) for all genes with at least 10 probes within exons. roX1, roX2 and msl2 clearly ordinate as highly enriched. Note that the absolute amount of msl2 RNA pulled down is similar to the absolute amounts of the roX RNAs. (C) The entire msl2 mRNA is associated with a complete or partial MSL complex. High resolution enrichment proﬁles of MSL2-RIP (blue) and MOF-RIP (green) at the msl2 locus. The msl2 gene is transcribed from right to left and the different splice forms are indicated. Numbers on the x-axis denote chromosomal position along the X chromosome in kilobase. The y-axis shows the RIP enrichment as the log2 ratio. Nucleic Acids Research, 2011, Vol. 39, No. 15 6433 (Figure 3B and Supplementary Figure S3). We asked whether the msl2 and msl1 RNAs were pulled down by free MSL2 protein, or by MSL2 associated with the MSL complex. Notably, roX1 and roX2 RNAs are enriched at similar levels in both MSL2-RIP and MOF-RIP. In contrast, msl2 RNA is enriched in both RIPs but at higher levels in the MSL2-RIP than in the MOF-RIP (compare Figure 2D–E with Figure 3C). The results suggest that msl2 RNA is associated with a less stable MSL complex. Still, since the MOF-RIP also shows high enrichment of msl2 and msl1 RNA, we conclude that msl2 and msl1 RNAs are associated with a partial or complete nuclear MSL complex (Figure 3C and Supplementary Figure S4). We have not found any signiﬁcant sequence identities or predicted secondary structure similarities between msl2 and roX1 or roX2. msl2 RNA is bound by a non-chromatin-associated MSL complex We considered two plausible models to account for a po- tential function for the strong association of a nuclear complete or partial MSL complex with the msl2 RNA. First, msl2 may aid the correct targeting of the MSL complex, by functioning similarly to roX. Second, since the local concentration of MSL complex is important for spreading and therefore for the correct regulation of most X-linked genes, we can hypothesize that the non-chromatin bound fraction of the MSL complex titrates the msl2 RNA. A combination of these two models is also possible. Recalling that the roX RNAs coat the male X chromosome, co-localizing with MSL complex, we performed in situ hybridization with RNA probes against roX1 and msl2. As previously shown, roX1 decorates the male X chromosome in a similar pattern to the MSL complex. However, we did not observe chromatin-associated msl2 RNA in the wild-type (Figure 4A). Previously, a co-transcriptional assembly of the MSL complex at the roX loci has been suggested (1,27,36). In contrast, the MSL complex is not found at the msl2 locus in published ChIP-chip analysis (Supplementary Figure S5). This indicates that the associ- ation of MSL complex with msl2 RNA is not co-transcriptional. We speculated that a loss of roX1 roX2 and/or overexpression of msl2 may enhance an intrinsic property of the msl2 RNA to be included in the chromatin-associated MSL complex. In roX1 roX2 mutant males, MSL complex is still detected at a reduced number of sites on the X chromosome, but strong binding is also reproducibly found in the Figure 2. Continued 400 kb region from chromosome 3 R (C). The plots show the mean enrichment ratios obtained using a bandwidth of 300 bp for the MSL1 ChIP-chip and a bandwidth of 90 bp for the MSL2 RIP-chip. Numbers on the x-axis denote chromosomal position along the chromosome in kb. The y-axis shows the ChIP and RIP enrichments, respectively, as the log2 ratio. Genes expressed from left to right are shown above the horizontal line and genes expressed in the opposite direction are shown below the line. The roX1 and roX2 loci are indicated by yellow boxes. The MSL1 ChIP data is from (32). (D and E) High resolution enrichment proﬁles of MSL2-RIP and MOF-RIP at the rox1 (D) or roX2 (E) locus. Exons are indicated in black and introns in grey. The different transcript forms of roX1 and roX2 are indicated. The described DNAse hypersensitive regions and roX-boxes suggested to be of functional importance (36,51–53) are indicated as deep-purple and yellow boxes, respectively. We observed a general enrichment of the entire mRNA and no obvious speciﬁcity to parts of the RNAs.