Author Summary In fruit flies, the male undergoes dosage compensation to increase the transcriptional output of its single X chromosome to match that of the females’ two copies. A large ribonucleoprotein complex that consists of two large noncoding RNA, roX1 and roX2, and at least five MSL (male specific lethal) chromatin-modifying proteins is responsi- ble. We were surprised to find that transcription of new roX1 RNA failed in the absence of pre-existing roX RNA. When we introduced either roX2 or the truncated but functional version of roX1, expression of endogenous wild- type roX1 gene was restored. We propose that this mechanism is essential for ensuring sustained dosage compensation in males and perhaps also for bursts of new MSL complex at each cell cycle. Thousands of large noncoding RNAs have recently been discovered in vertebrates, many of which are associated with chromatin remodeling enzymes. We postulate that some of these will face similar regulatory and functional demands as the roX RNAs and may have evolved comparable strategies to control their production. compensation that is toxic to females [15,30]. These observations led to the idea that MSL2 protein alone, or acting with the other MSL proteins drives transcription of roX1 RNA [31–33]. We reexamined the question of how MSL proteins regulate transcription of the roX1 gene using flies missing the roX2 locus. Deleting roX2 allowed us to study expression of the wild type endogenous roX1 gene without the confounding effects of a second functionally redundant RNA species. In this way we found an RNA-dependent autoregulatory loop controlling roX1 expression. We propose that the early burst of roX1 transcription at blastoderm initiates this cycle. Furthermore, production of the key male-limited MSL2 protein subunit is not only regulated at the translational level as has been extensively documented, but we find msl2 transcription is associated with DNA replication. We propose a model where pre-existing roX RNA, assembled in mature MSL complexes, drives bursts of roX1 transcription during S phase when its chromatin target is doubling. Results roX1 transcription is dependent on roX2 RNA Male embryos normally establish dosage compensation by the onset of gastrulation [16,34]. Ectopic expression of MSL2 in females leads to roX1 transcription and dosage compensation [15]. We asked whether dosage compensation can only be initiated during early embryogenesis, or could it be artificially started later during larval development. To achieve that, we used an inducible Flp-out system [35] to create clones of GAL4 expressing cells on day 4 AEL (after egg laying) that in turn drove expression of UAS- GFP and UAS-MSL2 in female larvae (Figure 1A and Methods). The purpose of this was to assay females in which the early burst of roX1 RNA had decayed away leaving cells devoid of any roX RNA. When no clones were induced, we never observed GFP or MSL painting in any cell showing that expression was tightly blocked prior to induction (data not shown). Third instar larvae in which late GAL4+ clones were induced displayed overlapping expression of GFP and MSL2 (Figure 1B–1B0). More importantly, MSL2 appeared as subnuclear punctate staining in imaginal disc cells suggesting that it was concentrated on the X chromosome (Figure 1B0). MSL2 immunostaining of polytene squashes confirmed binding along the X (Figure 1C). Unfortunately, we Autoregulation of a Noncoding RNA Gene were unable to perform GFP immunostaining and reliable roX1 FISH (Fluorescence in situ hybridization) in the same glands as the proteinase K treatment necessary to expose roX RNA often destroyed protein epitopes. We took comparable salivary glands from GFP positive larvae (Figure 1D) and processed them for roX1 FISH. The results show that roX1 transcription was successfully induced (Figure 1E–1F), consistent with previous reports that MSL2 drives roX1 transcription [15], and confirms an earlier report that dosage compensation can be initiated long after it normally occurs [13]. Figure 1G shows a wild type male X for comparison. We repeated the experiment in roX1+ roX22 females (Figure 1H). While GFP+ clones were recovered at similar frequencies indicating successful MSL2 induction (Figure 1I, 1K), no MSL2 accumulation was observed in imaginal discs (Figure 1I9) or on polytene chromosomes (Figure 1J). We will later demonstrate that the failure to detect MSL2 is due to reduced protein stability in the absence of roX RNA. More importantly, we also could not detect roX1 expression in any cell. Absence of roX1 RNA might be attributed to poor RNA stability or transcription failure. We favor the latter since even minute amounts of roX1 transcription can be readily detected when MSL complex accumulates over the roX1 gene [13]. Moreover, we could not detect nascent transcripts from the roX1 locus in these animals although such nascent roX1 transcripts were easily detected in other genotypes (Figure 1L–1L9, 1M). This argues that although late MSL2 readily switches on roX2, it is not sufficient alone or with the other MSL subunits to drive expression of the roX1 gene when roX2 is absent. Without any roX RNA, cells rapidly destroy the ectopic MSL2 as well. To further test our hypothesis that late expression of the endogenous roX1 locus depends on roX2 RNA, we returned to wildtype (roX1+ roX2+) females to perform RNA in situ hybridiza- tion for both RNAs in the same nuclei. Late induction of MSL2 results in roX2 RNA painting the length of X chromosomes in many cells (Figure 2A9). By contrast, roX1 painting over the full X chromosome was seen in only a small minority of nuclei (Figure 2A). A much more common pattern was roX2 over the entire X while roX1 expression confined to either several Mbp (Figure 2B–2B9) around or just at the endogenous roX1 locus at polytene band 3F (Figure 2C–2C9). This suggests that roX2 expression reliably follows MSL2 induction, but roX1 expression lags. Delayed roX1 expression might be explained if fully functional MSL complex containing roX2 RNA must first assemble before transcription of roX1 can occur. We conclude that roX2 RNA, presumably packaged in mature MSL complexes, is necessary to initiate transcription from the endogenous roX1 gene late in development. Autoregulation of roX1 expression Finding unusually late activation of the roX1 gene required preexisting roX2 RNA, we wondered if roX1 RNA could also perform the same role leading to a positive autoregulatory loop. To answer this question, we used a fly stock displaying an unusual mosaic pattern of dosage compensation. The H83M2 transgene makes MSL2 constitutively using the hsp83 promoter [30]. It lacks the regulatory 59 and 39 msl2 UTRs and drives ectopic dosage compensation in 100% of female cells. Females carrying a roX1 deletion also showed MSL X chromo- some painting utilizing roX2 RNA in all nuclei (data not shown). However, an entirely different result was obtained in H83M2 females missing only roX2. Roughly half the nuclei adopted a fully male-like pattern of dosage compensation utilizing roX1 RNA, while the other half lacked dosage compensation (Figure 3A and personal communication Art Alekseyenko). The X chromosomes PLoS Genetics | www.plosgenetics.org 2 March 2012 | Volume 8 | Issue 3 | e1002564