and roX2 we did not ﬁnd any additional roX RNA that could explain the escape of male lethality seen in roX1 roX2 double mutants. It should be stressed that it is still possible that a roX-like encoding gene is harbored in the heterochromatic regions which are not fully covered by the tiling arrays. Alternatively, several different RNAs may substitute for roX1 and roX2 but with much lower afﬁnity or the MSL complex can exert its function without an RNA molecule. In contrast, in the formaldehyde ﬁxed sample we detected enrichments of transcripts from genes targeted by the MSL complex. We conclude that RIP using formaldehyde cross-linked extracts will be enriched in RNA species directly linked to the targeted protein as well as nascent RNA molecules linked via chromatin and active transcription. This is important to consider when using formaldehyde ﬁxation in RNA immunoprecipitation experiments. We describe two potential functions for the strong as- sociation of a nuclear MSL complex with the msl2 RNA. First, msl2 may aid the correct targeting of the MSL complex, by functioning similarly to roX. This could have been a function in a more ancient form of the MSL complex and may in part have been retained during evolution. In favour of this model is the fact that roX1 roX2 double mutations are not completely male lethal, in contrast to msl mutations. The elevation of cellular levels of MSL1 and MSL2 that partially suppress roX1 roX2 male lethality, also supports this model. Second, since the local concentration of MSL complex is important for spreading and, therefore, also 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. Thus, this process would regulate the amount of MSL2 protein and assembled MSL complex, as a feedback mechanism. Since the msl2 RNA is highly enriched in a nuclear but not chromatin-associated MSL complex, we hypothesize that this interaction functions as a feedback control that avoids elevated MSL-complex levels and compensates for overshooting (Figure 6D). It has been observed that ectopic expression of an msl2-GFP transgene led to reduc- tion of the endogenous MSL2 protein levels (23). This is in line with what would be predicted from a feedback mech- anism as hypothesized here. The classical example of self-mRNA targeting of proteins is from Escherichia coli, in which the ribosomal proteins bind to their mRNA and inhibit translation. The ribosome biogenesis can thus be regulated solely through the amount of rRNA which syn- thesis rate becomes rate limiting for ribosome assembly. As long as the process of ribosome assembly requires ribo- somal proteins, the corresponding mRNAs will escape translation inhibition (38). Intrinsic association of RNA in chromatin regulation has been reported for the yeast SET1C histone methyl transferase complex. In this case, the association is linked to co-translational assembly of the complex (39). The dosage compensation system is under strong evo- lutionary pressure to respond as new genes, chromosome regions or chromosome arms join the X chromosome and it has been shown that the MSL proteins are under adaptive evolution (40). Interestingly, strong positive Nucleic Acids Research, 2011, Vol. 39, No. 15 6437 selection was detected in MSL1 and MSL2 protein domains shown to be responsible for their speciﬁc target- ing to the X chromosome (41,42). Positive selection has also been shown to act on the MSL binding sites (43). This means that the dosage compensation system is under constant pressure to adapt to emerging changes. The target sequences are under constant selection as are the targeting proteins, in particular MSL1 and MSL2. Thus, the concentration requirements will also need constant ad- justments. A feedback module based on association of a rate limiting mRNA would provide an optimal target for evolution to act on in order to provide a dynamic ﬁne-tuning of complex concentration, and therefore, correct targeting. This may explain the afﬁnity of the MSL complex to both msl1 and msl2 RNAs which encode the core proteins for a functional complex. At limiting concentrations of freely diffusible proteins and protein complexes only the sites with highest afﬁnity will bind while at high concentrations even sites with low afﬁnity will bind. It should be stressed that chromatin- associated factors are often described to be highly sensitive to correct dose (44,45). This is observed for the Polycomb-group proteins involved in maintaining repres- sion of homeotic genes (46,47) as well as for suppressors of variegation, important for heterochromatin formation (48–50). In both these cases many loci exhibit dosage effects, indicating strict dosage needs. As more RNA immunoprecipitation data are produced it is likely that the msl2 titration reported here is just one example of a general self-afﬁnity module important for feedback regulation. ACCESSION NUMBER The microarray data reported in this article have been deposited at http://www.ncbi.nlm.nih.gov/geo/ (Accession: GSE20249). SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank M. Kuroda for antibodies, Y. Park and V. Meller for ﬂy stocks, K. Ekstro¨ m for technical assist- ance and members of the Larsson and Stenberg labs for stimulating discussions. FUNDING This work was supported by the Carl Tryggers and Philip So¨ renssen Foundations (to P.S.); the Kempe Foundations (A.M.J. and A.A.); and the Swedish Research Council and Magnus Bergvalls Foundation (J.L.). Funding for open access charge: The Swedish Research Council. Conﬂict of interest statement. None declared.