The mosaic expression of roX1 is not limited to the polytene chromosomes The mosaic pattern of dosage compensation described above has been reported before. Certain hypomorphic alleles of Sxl unreliably initiate sex determination and so contain a mixture of XXAA cells that either correctly adopt a female fate repressing dosage compensation, or mistakenly choose a male fate and paint their X chromosomes with MSL complex [37]. The mechanism underlying the mosaic pattern of dosage compensation found in roX2 H83M2 animals studied here must be different, and we set out to understand its basis. We first tested if the mosaic pattern was a peculiarity of polytene cells or a general feature in most tissues of these animals. When we performed MSL2 immunostaining on diploid imaginal discs, MSL2 protein decorated a subnuclear domain presumed to be the X chromosome in all the imaginal disc cells when both roX RNAs were present in female cells carrying the H83M2 transgene (Figure 4A). Again, roX12 roX2+ animals also had no trouble establishing dosage compensation in all the cells (data not shown). However, when roX2 was deleted, only a fraction of the cells displayed dosage compensation utilizing roX1 RNA (Figure 4B– 4B0), similar to the spotty pattern observed in salivary glands (Figure 3A, 3B and 3F). When both roX genes were deleted, no MSL2 staining was detected (Figure 4C–4C0). We had expected Autoregulation of a Noncoding RNA Gene that removing roX RNA would produce a diffuse nuclear cloud of MSL2 protein unable to bind to the X chromosome. Either diffuse MSL2 protein stains too weakly to detect with our antibodies, or MSL2 protein is unstable without roX RNA. The latter is likely to be the case since MSL2 was previously shown to be unstable when not packaged into MSL complexes [29]. To directly test this interpretation, we incubated the imaginal discs and salivary glands with MG132, a proteasome inhibitor, before performing MSL2 immunostaining. Strong nuclear MSL2 staining reappeared (Figure 4D–4D0) in the imaginal disc, showing that MSL2 is synthesized in the absence of roX RNA, but fails to accumulate due to rapid turnover. MSL2 protein stabilized by MG132 showed a dramatically different staining pattern covering all polytene chromosomes, rather than being restricted to the X (Figure S2). This shows that free MSL2 subunits have a general affinity for all chromatin in agreement with earlier biochemical work [22]. Apparently flies have evolved a mechanism to efficiently destroy any MSL2 subunits that fail to assemble into complexes with roX RNA. A somewhat similar situation was reported for the MSL1 subunit. Massively over expressed MSL1 transiently paints all chromosome arms but is quickly lost [36]. Our data suggest that many cells lacking roX2 RNA were unable to carry out dosage compensation because the remaining wild type roX1 gene failed to switch on. If true, this should affect the viability Figure 4. Dosage compensation fails in many diploid cells relying exclusively on roX1 and H83M2. (A–A0) Males and females relying entirely on H83M2 showed subnuclear MSL2 staining in all wing imaginal disc cells when both roX1 and roX2 RNAs were present. (B–B0) Many cells lacked dosage compensation if roX1+ was the only source of roX RNA. (C–C0) MSL2 did not accumulate over the X when both roX1 and roX2 were absent. (D–D0) Nuclear staining of MSL2 was easily detected in the absence of roX RNA after treatment with MG132, a proteasome inhibitor. doi:10.1371/journal.pgen.1002564.g004 PLoS Genetics | www.plosgenetics.org 6 March 2012 | Volume 8 | Issue 3 | e1002564