6428–6439 Nucleic Acids Research, 2011, Vol. 39, No. 15 doi:10.1093/nar/gkr236 Published online 6 May 2011 msl2 mRNA is bound by free nuclear MSL complex in Drosophila melanogaster Anna-Mia Johansson 1 , Anders Allgardsson 1 , Per Stenberg1,2 and Jan Larsson1,* 1Department of Molecular Biology, Umea˚ University, SE-90187 Umea˚ and 2Computational Life Science Cluster (CLiC), Umea˚ University, Sweden Received February 4, 2011; Revised March 14, 2011; Accepted April 4, 2011 ABSTRACT In Drosophila, the global increase in transcription from the male X chromosome to compensate for its monosomy is mediated by the male-specific lethal (MSL) complex consisting of five proteins and two non-coding RNAs, roX1 and roX2. After an initial sequence-dependent recognition by the MSL complex of 150–300 high affinity sites, the spread to the majority of the X-linked genes depends on local MSL-complex concentration and active transcription. We have explored whether any add- itional RNA species are associated with the MSL complex. No additional roX RNA species were found, but a strong association was found between a spliced and poly-adenylated msl2 RNA and the MSL complex. Based on our results, we propose a model in which a non-chromatin- associated partial or complete MSL-complex titrates newly transcribed msl2 mRNA and thus regulates the amount of available MSL complex by feedback. This represents a novel mechanism in chromatin structure regulation. INTRODUCTION Dosage compensation in Drosophila provides one of the best examples of chromosome-wide gene regulation (1,2). The compensation mechanism must fulﬁl two require- ments. It must balance the relative expression between sex chromosomes and autosomes and also equalize the transcriptional activities of the two X chromosomes in the homogametic sex with that of the single X chromo- some in the heterogametic sex. Examination of X-linked and autosomal gene expression in Drosophila, Caenorhabditis elegans and mammals has revealed that X-linked genes are expressed, on average, twice as strongly as autosomal genes (3–5), implying that dosage compensation involves compensation of X chromosome expression not only between the two sexes, but also in relation to the expression of autosomes. Thus, X inactiva- tion in mammals might be considered as a response in females to the overexpression of the X chromosome. This overexpression is required by males because they have just one X chromosome. The male restricted 2-fold increase of X chromosome gene expression in Drosophila fulﬁls both compensation requirements described above. This seems to be mediated by a general buffering mechanism for all mono- somic regions, resulting in an 1.4-fold expression increase (6–8) followed by an 1.35-fold expression increase restricted to the X chromosome, mediated by the MSL complex (7–10). In Drosophila melanogaster, two non-coding RNAs, roX1 and roX2, have been shown to be essential components of the dosage compen- sation system. Together with at least ﬁve MSL proteins, roX1 and roX2 form male-speciﬁc lethal (MSL) complexes that ‘paint’ the male X chromosome, and mediate acetyl- ation of H4 at lysine 16 on the male X chromosome, which partly explains its subsequent hypertranscription (2,11). Recent data suggest that the MSL complex, in addition, have intrinsic properties that constrain the activation po- tential of MOF (males absent on the ﬁrst) to end up with the required 2-fold activation (12). The prevailing model of the targeting process is that the MSL complex is re- cruited to a set of 150–300 high-afﬁnity MSL recognition elements (MRE) in a sequence-depending mechanism (13). Subsequent spreading to neighboring genes with lower afﬁnities depends on the local MSL-complex concentra- tion (14), X chromosome location (15) and active tran- scription (16,17). The sequence dependence of the spreading remains poorly understood. Irrespective of dif- ferences in transcript levels of genes and the transcription requirements of different cells, a 2-fold transcriptional up-regulation is required. MSL2 protein is the limiting component for the forma- tion of the MSL complex. In females, translation of msl2 mRNA is inhibited by the binding of the Sex lethal (Sxl) protein in both the 50 and the 30-UTRs (18–20). Overexpression of msl2 is toxic to females and becomes *To whom correspondence should be addressed. Tel: +46 (0)90 7856785, Fax: +46 (0)90 778007, Email: jan.larsson@molbiol.umu.se The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.