Autoregulation of a Noncoding RNA Gene Figure 1. MSL proteins alone cannot drive roX1 expression late in development. A) 4 day old larvae were heatshocked to induce expression of Flp, resulting in the removal of the blocking sequence from GAL4 and subsequent expression of both MSL2 and GFP. MSL2 is expected to initiate roX transcription and MSL complex assembly. (B) GFP+ clones mark imaginal disc cells that have successfully removed the blocking sequences from GAL4 (B9–B0). Induction of MSL2 results in punctate subnuclear foci in imaginal disc cells. (C) MSL2 immunostaining of polytene chromosome shows late MSL2 paints the entire X chromosome. (D) Whole salivary gland showing GFP induced in some cells. (E–E9) roX1 FISH of whole mount of similar GFP+ salivary glands or (F) polytene squashes shows successful induction of roX1 expression in a subset of cells. (G) roX1 FISH of wildtype males (H) The same experiment was repeated in roX1+roX22 larvae. However, in the absence of roX2, MSL2 fails to drive roX1 expression. (I) Despite the presence of GFP+ (late MSL2 expressing) cells, MSL2 is not detectable over the X in (I9–I0) imaginal disc cells or (J) polytene chromosomes. (K) Whole salivary gland showing successful GFP expression in roX1+roX22 larvae. (L–L9) Expression of roX1 is never observed painting the X or as nascent transcripts at band 3F in separately processed GFP+ glands or on (M) polytene squashes. doi:10.1371/journal.pgen.1002564.g001 of these negative cells showed very weak staining for MSL2, similar to the situation found in roX1 roX2 double mutant animals [17]. Males also exhibited a similar mosaic phenotype when their only source of MSL2 was the H83M2 transgene demonstrating that failed dosage compensation cannot be due to SXL or some other female factor (Figure 3B). It is unclear how two adjacent cells that are genetically identical containing a full set of MSL subunits adopt opposite dosage compensation fates. However, this fortuitous observation allowed us to test whether failure to activate the endogenous roX1 gene might explain lack of dosage compensation in some cells. In order to test whether pre-existing roX1 RNA is necessary to drive continued transcription of the roX1 gene, we used a 1.2 kb roX1 minigene, H83roX1-D39, that is able to form partially active MSL complexes (Figure 3C) [13]. We designed a probe that recognizes only the internal sequence of the endogenous roX1 RNA missing from H83roX1-D39. In this way, we could selectively visualize the expression of only the endogenous roX1 RNA in Figure 2. Late induction of roX1 expression requires roX2 RNA. In nuclei where dosage compensation was successfully turned on after late msl2 induction, extensive roX2 was observed painting the entire X chromosome. (A) However, only 1% of the chromosomes showed extensive roX1 painting. 34% and 59% of chromosomes showed roX1 expression confined to several Mbp around (B) or just at the endogenous roX1 locus (C), respectively. The remaining chromosomes (6%) had no roX1 expression despite the presence of roX2 (data not shown). roX1 and roX2 were detected by biotin (green, A–C) and digoxigenin (red, A9–C9) labeled antisense riboprobes, respectively. The merged figure is shown in A0–C0. White arrows denote the endogenous roX1 locus at band 3F. doi:10.1371/journal.pgen.1002564.g002 PLoS Genetics | www.plosgenetics.org 4 March 2012 | Volume 8 | Issue 3 | e1002564