We first used in situ hybridization to directly visualize actively growing msl2 transcripts on polytene chromosomes. Most salivary gland cells have completed their endoreplication cycles at the wandering larval stage used in all our experiments, but a few cells are still actively replicating. When males carrying both the wild type endogenous msl2 gene and the H83M2 transgene were treated with antisense msl2 riboprobes, all nuclei showed strong hybrid- ization to a cloud of msl2 RNA over the H83M2 transgene inserted at 87A (Figure 6A). By contrast, a weaker hybridization signal was observed over the endogenous msl2 gene at 23F in only about 20% of the nuclei. Most nuclei lacked detectable msl2 transcripts at the endogenous locus. We tested whether the H83M2 transgene might somehow inhibit expression of endogenous msl2 gene by testing nontransgenic, wild type males. We found that again, only about 20% of male cells actively transcribed msl2 (data not shown). This shows that while wild type males have abundant MSL2 protein painting the X chromosome in all cells, few cells are making new msl2 mRNA. To test if the sporadic transcription of endogenous msl2 might coincide with the replication phase of the cell cycle, we briefly incubated dissected salivary glands in EdU to label newly replicated DNA, fixed the glands, and processed them for msl2 FISH. We found a strong bias (Fisher’s exact test p,0.0001) for replicating cells to also be transcribing msl2, but the overlap was not perfect (Figure 6A). For comparison we also examined histone H2A transcription, but even outside S phase every nucleus contained an intense hybridization signal over the endogenous histone gene cluster at polytene band 39DE (Figure S7). Only a portion of histone mRNA regulation occurs at transcription initiation. Most regulation is posttranscriptional [38,39]. To directly test whether the sporadic msl2 transcription pattern is associated with S phase, we treated our salivary glands with 1 mM of hydroxyurea (HU) to inhibit replication. EdU incorpo- ration was completely blocked (Figure 6B9) but more significantly, transcripts were specifically lost only from the endogenous msl2 locus at 23F while msl2 transcripts made from the H83M2 transgene at 87A remained strong (Figure 6B, 6B0). Likewise, HU treatment had no effect on bulk nascent transcripts as shown by strong incorporation of BrUTP on all chromosome arms (Figure 6C). These results show that msl2 transcription normally occurs around S phase and is blocked by replication inhibitors. By contrast, H83M2 does not show any cell cycle regulation. This difference might impact roX1 expression. To test whether the replication associated msl2 transcription might be limited to only polytene cells with unusual cell cycles, we examined mitotically dividing diploid S2 cell in culture. Actively growing S2 cells were sorted into G1, S, and G2 (Figure 6G) via FACS (Fluorescence Activated Cell Sorting). RNA from these populations was assayed for msl2, histone H2A, and PKA transcripts by qPCR. As previously reported [38], histone RNA accumulates during S phase but is low in G1 and G2 (Figure 6E). Importantly, msl2 transcripts are most abundant during S while lowest during G2 phase (Figure 6F). These results show that msl2 RNA accumulation and synthesis normally occur predominantly in S phase, but the H83M2 transgene lacks this coordination with replication. We attempted to determine whether roX1 transcription was also linked to S phase as might be expected, but were unsuccessful. We used both BrUTP and EU (Ethynyl Uridine) to label all new transcripts followed by a cold chase. We anticipated that the bulk of mRNA would leave the nucleus while newly labeled roX1 RNA remained behind on the X. However, we were unable to detect any labeled RNA localized on the male X after chase (data not shown). We do not know if the signal was too weak to detect, or if Autoregulation of a Noncoding RNA Gene the substitutions at the 5 position of uridine interferes with the function of RNA [40], leading to problems in roX1 RNA folding, stability, MSL protein assembly, and/or targeting to the X. Discussion Previous studies of roX1 transcriptional control argued that either MSL2 alone or with a full set of MSL proteins was sufficient to drive male-specific expression [31–33]. Here, we present evidence that the expression of roX1 gene is instead controlled through an autoregulatory loop. Pre-existing roX RNA, presum- ably in mature MSL complex, is required to drive new transcription. The reason we reached a different conclusion is largely attributed to removing the functionally redundant roX2 in most of our experiments and assaying transcription only from the wild type roX1 locus at its normal location on the X. The pathway we describe shares some elements with the negative regulatory loop between TFIIIA binding 5S rDNA to drive transcription or 5S rRNA for storage during Xenopus oogenesis [41]. A model for autoregulation of roX1 Figure 7 shows a model for how such an autoregulatory loop might operate. Because roX RNA is not maternally deposited into embryos, one problem is how male embryos could build their first MSL complex needed to initiate the cycle. Meller has shown that roX1 transcription switches on in both sexes around blastoderm, just as general zygotic transcription begins [15,16]. This suggests that an embryonic roX1 promoter is active without MSL complex and could supply the first roX1 RNA molecules to males, but these RNAs are eventually degraded in females. Early RNA assembles with MSL proteins and then drive roX1 transcription from the known male- specific MSL-dependent promoters, setting up a positive autoreg- ulatory loop necessary for the future maintenance of roX1 expression in males. In our experiments, we showed that roX2 RNA or truncated roX1 RNA can also initiate endogenous roX1 expression late in development after the early roX1 transcripts are gone. This model requires that male embryos preferentially sequester newly assembled MSL complex at the roX1 gene to drive sustained transcription instead of allowing it to diffuse away to the vastly larger pool of ordinary X linked genes that must be dosage compensated. Only after roX1 transcription is successfully upregulated can MSL complexes be released to the target genes along the X chromosome. Such behavior has been previously documented in cells containing abundant free MSL subunits and low levels of roX1 transcription, exactly the conditions we believe occur as young male embryos initiate dosage compensation [13]. Examining cells shortly after MSL2 first turned on roX1 transcription showed the earliest roX1 transcripts remained near the site of synthesis (Figure 2B and 2C) consistent with the idea that newly formed MSL complexes preferentially act on the roX1 gene. At later times, such as seen in H83roX1-D39 animals that have had five days to drive endogenous roX1 expression, every cell painted the entire X with roX1 RNA (Figure 3G). While massive local cis spreading of the MSL complex has been reported for roX1 and roX2 transgenes inserted into autosomes [13,18,42], the physiological relevance of this is not widely accepted [25,26,43,44]. Here we instead report striking local cis spreading of newly made MSL complex from the wild type roX1 gene in its normal X chromosome environment (Figure S1E–S1F). Our data agree well with previous reports of local MSL spreading along the X [19] and support a role for cis spreading in the normal process of dosage compensation. We have not directly determined what region of the roX1 gene is necessary for autoregulation. However, a strong candidate is the PLoS Genetics | www.plosgenetics.org 8 March 2012 | Volume 8 | Issue 3 | e1002564